Specifications for pesticides used in public health: insecticides, rodenticides, molluscicides, repellents, methods

SPECIMEN COPY SPECIFICATIONS FOR PESTICIDES USED IN PUBLIC HEAL TH Insecticides - Rodenticides - Molluscicides Repellents - Methods A clothbound edition is available at £2 $6.75 Sw. fr. 20.- For list of national distributors, see back cover • World Health Organization Geneva WORLD HEALTH ORGANIZATION SPECIFICATIONS FOR PESTICIDES USED IN PUBLIC HEALTH THIRD EDITION CORRIGENDA Page 15, section l. 2: Chloral hydrate content Delete 0.005 Insert 0.025 Page 4 7, section 2. l. 7 Calculation Delete x 100 in the formula Page 105, section 2. 2. 4 Calculation Insert a minus sign between b and a in the formula Page 117, section 2. I . 6 Calculation Delete x 100 in the formula Page 147, section 2.1.6 Calculation Delete x 0.3 and x 100 in the formula Page 181, section 2. I . 6 Calculation Delete x 100 in the formula Page 226, footnote Delete l l Insert l Page 227, section 2.1.4 Standardization of perchloric acid in glacial acetic acid Delete carbinol Insert methanol Page 261, section 2.2 Pure gamma-isomer Delete benzene hexachloride Insert HCH Page 295, section 3. Procedure, last line Delete 47 Insert 24 SPECIFICATIONS FOR PESTICIDES USED IN PUBLIC HEALTH SPECIFICATIONS FOR PESTICIDES USED IN PUBLIC HEALTH Insecticides - Rodenticides - Molluscicides Repellents - Methods THIRD EDITION WORLD HEALTH ORGANIZATION GENEVA 1967 First edition, 1956 Second edition, 1961 Third edition, 1967 © World Health Organization 1967 Publications of the World Health Organization enjoy copyright protection in accord- ance with the provisions of Protocol 2 of the Universal Copyright Convention. Never- theless governmental agencies or learned and professional societies may reproduce data or excerpts or illustrations from them without requesting an authorization from the World Health Organization. For rights of reproduction or translation of WHO publications in toto, application should be made to the Division of Editorial and Reference Services, World Health Organization, Geneva, Switzerland. The World Health Organization welcomes such applications. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Director- General of the World Health Organization concerning the legal status of any country or territory or of its authorities, or concerning the delimitation of its frontiers. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature which are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. PRINTED IN SWITZERLAND Preface ... Introduction CONTENTS PART I. INSECTICIDES Technical products DDT .. HCH .. Lindane . Dieldrin. Pyrethrum . Diazinon . Malathion . Parathion . Trichlorfon Parathion-methyl . Fenthion . Dichlorvos . . . . Water-dispersible powders DDT ......... . DDT for overseas shipment . HCH ... Dieldrin .. Diazinon . Malathion . Emulsion concentrates DDT .. HCH ... Dieldrin .. Diazinon . Malathion . Parathion . Trichlorfon Parathion-methyl . Fenthion ... Dusting-powders DDT .. . HCH .. . Malathion . -5- Specification Number (WHO/SIT/1.R3) (WHO/SIT/2.R3) (WHO/SIT/3.R3) (WHO/SIT/6.R3) (WHO/SIT/7.R1) (WHO/SIT /9.R2) (WHO/SIT/10.R2) (WHO/SIT/11.R2) (WHO/SIT/13.R1) (WHO/SIT/14) (WHO/SIT/15) (WHO/SIT/16) (WHO/SIF/1.R3) (WHO/SIF/26) (WHO/SIF/2.R3) (WHO/SIF/3.R3) (WHO/SIF/9.R2) (WHO/SIF/10.R2) (WHO/SIF/4.R3) (WHO/SIF/5.R3) (WHO/SIF/6.R3) (WHO/SIF/13.R2) (WHO/SIF/14.R2) (WHO/SIF/15.R2) (WHO/SIF/20.R1) (WHO/SIF/27) (WHO/SIF/28) (WHO/SIF/16.R2) (WHO/SIF/17.R2) (WHO/SIF/22.R1) Page 9 11 15 22 25 29 34 40 44 49 54 59 64 69 76 82 90 96 106 113 120 126 130 137 143 148 154 159 165 171 175 178 Specification Number Page Larvicidal oils Larvicidal oils without insecticide (WHO/SIF/'2.3) 183 Larvicidal oils with added insecticide (WHO/SIF/'2.4) 187 PART II. RODENTICIDES Technical products Coumachlor (WHO/SRT/3.R1) 195 Warfarin (WHO/SRT/6.R1) 198 Pindone . (WHO/SRT/9.R1) 200 Concentrates Coumachlor (WHO/SRF/1.R1) 204 Warfarin (WHO/SRF/'2..R1) 208 Pindone . (WHO/SRF/4) 211 PART Ill. MOLLUSCICIDES Technical products Copper sulfate . (WHO/SMT/1.R1) 217 Pentachlorophenol (WHO/SMT/'2..R1) 220 2' ,5-Dichloro-4'-nitrosalicylanilide ethanolamine salt. (WHO/SMT/4) 222 N-Triphenylmethylmorpholine (WHO/SMT/5) 226 Water-dispersible powders 2' ,5-Dichloro-4'-nitrosalicylanilide ethanolamine salt. (WHO/SMF/1) 232 PART IV. REPELLENTS Technical products Deet (WHO/SRpT/1) 241 PART V. METHODS Method Number Sampling procedures . (WHO/M/1) 247 Visual suspensibility test for 75% DDT water-dispersible powders . (WHO/M/'2.) 249 Determination of acidity and alkalinity . . . . (WHO/M/3) 250 Sieving test after accelerated storage treatment . (WHO/M/4) 253 Determination of melting point and mixed melting point (WHO/M/5) 255 -6- Method Number Page Infra-red spectrophotometric method for determination of gamma-isomer content ofHCH . . . . . . . . . . . Karl Fischer electrometric titration method for determina- tion of water content . . . . . . . . . . . . . . . Dean & Stark distillation method for determination of water content ...... . Determination of material insoluble in dichlorodifluoro- methane .................... . TAG Closed Tester method for determination of flash-point Cleveland Open Tester method for determination of flash- point ........... . Determination of crystallizing-point Emulsion stability test . . . . . . Toxicity test for rodenticides . . . Parr peroxide-bomb (total chlorine) method Revised Stepanow (total organic chlorine) method. Hydrolysable chlorine method for determination of HCH content ........ . Toxicity test for larvicidal oils . (WHO/M/6) (WHO/M/7) (WHO/M/8) (WHO/M/9) (WHO/M/10) (WHO/M/11) (WHO/M/12) (WHO/M/13) (WHO/M/14) (WHO/M/15) (WHO/M/16) (WHO/M/17) (WHO/M/18) Annex 1. Common names, trade names and chemical names of 258 266 270 272 276 279 281 284 285 286 288 291 294 pesticides . . . . . . . . . . . . . 296 Annex 2. Composition of the fifteenth WHO Expert Committee on Insecticides . . . . . . . . . . . . . . . . . . . . . 299 -7-

PREFACE Specifications for insecticides and for spraying and dusting apparatus were.first published by WHO in 1953. These were based on the fourth report of the WHO Expert Committee on Insecticides, published the previous year, and on two earlier reports, and were intended to cover all the principal com- pounds used in controlling insects of public health importance. Developments in this field were so rapid, however, that only one year after publication it was necessary to convene a new Expert Committee to expand and revise the speci- fications. This was due both to the considerable increase in the use of pesticides in public health and to the accumulation of knowledge on procedures for quality control arising out of collaborative studies. The recommendations of the Committee resulted in the publication in 1956 of the first edition of Specifica- tions for Pesticides. This incorporated specifications for molluscicides and rodenticides as well as for insecticides and for spraying and dusting equipment. A second edition appeared in 1961, following revisions recommended by a further Expert Committee in 1958.1 In 1963, a meeting of the WHO Expert Committee on Insecticides was convened to review the question of the equipment used for the application and dispersal of pesticides ; the specifications and procedures recommended by this Committee were published in 1964.2 The task of bringing up to date the specifications for the pesticides themselves was entrusted to the fifteenth WHO Expert Committee on Insecticides, which met in 1965.3 This Committee recommended extensive revisions of the specifications ; at the same time it emphasized that the specifications are designed to meet the requirements of public health programmes and may not be applicable to pesticides used in agriculture. To bring this restriction to the attention of readers, the title of the third edition has been amended to Specifications for Pesticides used in Public Health. Only those pesticides and pesticide formulations for which revised speci- fications were recommended by the Committee are included in this new edition. A certain number of new specifications have been added. In practice, this 1 For the membership of the various Committees involved in the first and second editions, see Annex 3 to the second edition. 2 World Health Organization (1964) Equipment for vector control, Geneva. 3 For the membership of this Committee, see Annex 2, page 299. -9- means that the present volume contains specifications for all pesticides at present in widespread use in public health programmes. These fall into three groups: insecticides, rodenticides, and molluscicides. One insect repellent has also been included. The various methods,formerly given as annexes, have been grouped in Part V and have also been revised. The names of pesticides used in the specifications are either the names recommended by the International Organization for Standardization (ISO) or the chemical names. The latter are in accordance with the rules of the International Union of Pure and Applied Chemistry (IUPAC) as interpreted by the American Chemical Society in Chemical Abstracts. An alphabetical list of common names, trade names and chemical names, with cross references, will be found in Annex I. -10 - INTRODUCTION A specification almost always represents a compromise between what the manufacturer can make, what the user wants, and what he is prepared to pay for the product. Specifications for pesticides and pesticide formulations are no exception to this rule. In some instances, it may be technically impossible to meet the user's requirements ; in others, it may be technically p9ssible but very costly to do so. Sometimes, desirable requirements in re'spect of physical or chemical properties conflict with criteria governing biological efficacy. It is therefore very necessary for all concerned-and particularly for manufacturers and those responsible for quality control or inspection of supplies-to know (a) for what purpose the material is required, how it is to be used, and what abnormal conditions of storage might have to be met; and (b) the reasons for inclusion of the different requirements and test methods in the specification. The specifications for pesticides included in this volume are designed specifically to meet the requirements of public health programmes, which differ in many respects from the requirements for pesticides used in agri- culture, at least as regards the various formulations. On the other hand, the specifications for the technical products, which are intended to define the quality of the material to be used in preparing the formulations, are of broader application. Indeed, in the selection of the analytical methods for inclusion in these specifications, one of the guiding principles has been that they should be acceptable alike to the manufacturer, the public health worker, and the agricultural user. For public health purposes, the majority of insecticides are procured in the form of water-dispersible powders. As many campaigns take place in remote areas with difficult lines of communication, these insecticide for- mulations may have to be stored for considerable periods under tropical conditions, and during this time they must remain in a satisfactory state for immediate use. The active ingredient must not deteriorate and the physical properties, particularly the suspensibility, must not become impaired. In this connexion, attention is drawn to the new specification for DDT powder for overseas shipment (WHO/SIF /26, page 82), which includes a requirement for long-term storage life. This requirement is particularly important when ordering powders for use in malaria eradication programmes, as it ensures that the suspensibility of the powder will remain acceptable after it has been shipped, stored, and brought to the field in readiness for application. Emulsion concentrates may deteriorate if exposed for considerable periods to low temperatures. It is therefore desirable that procurement agencies placing orders for such products should give indications of any special requirements of this kind that will have to be met. -11 - In order to make the best use of the specifications, it is suggested that the following steps be adopted when procuring pesticides : 1. The material required should be adequately defined by giving : (a) the name of the insecticide; (b) the WHO specification number; and (c) the concentration or percentage of active ingredient required in the formulation. 2. The supplier or manufacturer should be required to submit a report on an analysis of the batch showing compliance with the specification. This is particularly important when ordering DDT powders. 3. Before acceptance of the consignment, samples should be taken by the purchaser or his agent and submitted to an independent laboratory to be analysed for compliance with the specification. This step is also particularly important when ordering DDT powders. The sampling procedure to be used must in all cases be clearly specified by the purchaser. 4. The packaging required should be specified by the purchaser. Ship- ping and handling procedures may profoundly affect the properties of the material, especially where weak packages are subjected to excessive heat or pressure.1 It is therefore important to describe to the manufacturer the conditions to which the material will be subjected before it is used. If long- term storage is anticipated, this should be stated in the order. As many pesticides are highly toxic to man and animals, it is essential that the labels on the containers should carry a clear warning of the hazards, with instructions for safe handling and, where appropriate, an indication of the measures to be taken in the event of suspected intoxication. Minimum cautionary notices are included in the specifications. More detailed recom- mendations on the safe handling of pesticides, the protection of operators, the detection of exposure, and the treatment of intoxication will be found in the sixteenth report of the WHO Expert Committee on Insecticides.2 Recommendations on the most suitable pesticide formulations for the control of particular vectors and on the appropriate methods of applying them will be found in Annex 17 to the thirteenth report of the WHO Expert Committee on Insecticides 3 and a guide to the major items of equipment used in the application of pesticides is given in Equipment for Vector Con- trof.4 The latter publication, which is a companion volume to this one, also contains specifications and use descriptions for many of the items. 1 In the case of DDT 75% water-dispersible powder, experience has shown that these conditions have contributed to loss of suspensibility. Specifications for boxes that have been found convenient for shipping these DDT powders are obtainable on request from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. 2 Wld Hlth Org. techn. Rep. Ser., 1967, 356. 3 Wld Hlth Org. techn. Rep. Ser., 1963, 265. 4 World Health Organization (1964) Equipment for vector control, Geneva. -12- Part I INSECTICIDES

WHO/SIT/1.R3 TECHNICAL DDT 1. SPECIFICATIONS 1.1 Material TECHNICAL DDT Specification WHO/SIT/1.RJ Approved 25 October 1965 The material shall comprise essentially 1, 1-di-(p-chlorophenyl)-2,2,2- trichloroethane and shall be in the form of white or cream-coloured granules, flakes, or powder, free from extraneous impurities or added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1.1 and with the following requirements: Setting-point (section 2.1) . . . . . . . Total organic chlorine content (section 2.2), % by weight ........... . Hydrolysable chlorine content (section 2.3), % by weight . . . ..... p,p'-Isomer content (section 2.4), % by weight .............. . Melting-point of separated p,p'-isomer (sec- tion 2.4) ............. . Chloral hydrate content (section 2. 5), % by weight ..............• Acidity (Method WHO/M/3, page 250) % by weight, calculated as H2S04 • • • • • • Solid material insoluble in acetone (section 2.6), % by weight ........ . Water content (section 2. 7), % by weight. Minimum 89°C 49.0 9.5 70.0 104°C Maximum 51.0 11.5 0.005 0.3 1.0 1.0 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. -15 - TECHNICAL DDT WHO/SIT/1.R3 1. 3 Packing and Marking of Packages The technical DDT shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical DDT to Specification WHO/SIT/1.R3 p,p'-Isomer content, ... % w/w Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : "Keep well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Setting Point Place in a 20-cm boiling-tube of 2.5 cm internal diameter with a wall thickness of 2 ± 0.1 mm, a sufficient amount of the sample to give, when melted, a depth of liquid of approximately 7 .5 cm. Melt carefully by immersing the tube to a depth of 10 cm in an oil-bath at a maximum tem- perature of I00°C. If hydrogen chloride is evolved during the melting, another portion of the sample must be taken and melted in a bath at a lower temperature. Fit this boiling-tube with a cork collar and insert it to within 1.3 cm of the bottom of a 15-cm boiling-tube of approximately 4 cm diameter ; then immerse the two tubes to a depth of 10 cm in a water-bath maintained at a temperature 5°C to 8°C below the anticipated setting point. Place in the inner boiling-tube a stirrer, consisting of a stainless steel rod 1.5 mm in diameter bent in the form of a helix with six turns of 1.8 cm outside diameter. A thermometer graduated in one-tenths of a degree is then clamped in a central position with its bulb 2.5 cm from the bottom of the tube. Stir at a rate of about two strokes per second, by moving the stirrer up and down, until the material begins to thicken, at which point stir vigorously to work into the melt the material that has solidified on the walls of the tube. Stop stirring when the temperature ceases dropping and remains constant for some time.1 Record this temperature as the setting- point. 1 In some instances, there may be a slight rise in the temperature of the material; if this occurs, record the highest steady temperature as the setting-point. -16- WHO/SIT/1.R3 TECHNICAL DDT 2. 2 Total Organic Chlorine Content (Stepanow Method, Revised) 1 2.2.1 Procedure 2. 2. 1 . 1 Determination of total chlorine Weigh accurately about 1 g of the sample, transfer to a 250-ml volumetric flask, add 10 ml of chlorine-free and thiophene-free benzene to dissolve the sample, and then make up to the mark with 99% isopro- panol. Transfer a 25-ml aliquot to a 250-ml flask 2 and add 2.5 g of metallic sodium, in the form of ribbon or small pieces. Connect to a reflux con- denser and boil gently for at least 1 hour, shaking the flask occasionally. Remove the excess metallic sodium by cautiously adding 10 ml of 50% aqueous isopropanol through the condenser at the rate of 1-2 drops per second. Boil for an additional 10 minutes and then add 60 ml of distilled water. Cool, add 2 or 3 drops of phenolphthalein indicator solution, neutralize by adding 50% nitric acid dropwise, and then add 10 ml in excess. Cool, if necessary, to room temperature, add a slight excess of O .1 N silver nitrate solution, and coagulate the precipitated silver chloride by digesting on a steam-bath for Y2 hour, with frequent stirring. Cool, filter through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate solution. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the total chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate solution is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. 2. 2. 1 . 2 Determination of inorganic chlorine Weigh accurately about 1 g of the sample, dissolve in 10 ml of acetone, and add 100 ml of distilled water. Keep at room temperature for 10 minutes and filter. Acidify the filtrate with 50% nitric acid and proceed as described in section 2. 2. 1. 1. 1 The Parr peroxide-bomb method (see Method WHO/M/15, page 286), or any other currently used method, may be employed for routine purposes, but in the event of a dispute the Stepanow method shall govern. 2 Direct weighing of the sample may be substituted for the aliquoting, provided the weighing does not introduce an error of more than 0.1 % in the weight of the sample. -17 - 2 TECHNICAL DDT WHO/SIT/1.R3 2.2.2 Calculation Total organic chlorine content(% w/w) = (a x lO _ !!___) x 0.3546 x f W1 W2 where a = volume (ml) of 0.1 N silver nitrate equivalent to the total chlorine b = volume (ml) of 0.1 N silver nitrate equivalent to the inorganic chlorine w1 = weight (g) of sample used for the total chlorine determination w2 = weight (g) of sample used for the inorganic chlorine determi- nation 50.01 f=-A where A = value found for total chlorine in recrystallized DDT, when determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2. 3 Hydrolysable Chlorine Content 2.3.1 Procedure 2. 3. l . l Determination of hydrolysable plus inorganic chlorine Weigh accurately about 0.5 g of the sample into a 250-ml flask and add 50 ml of acetone and 20 ml of l N ethanolic potassium hydroxide. Keep at 20-25°C for 15 minutes and add 50 ml of distilled water. Add 20 ml of 2 N nitric acid and exactly 25 ml of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam- bath for Y2 hour, with frequent stirring. Cool, filter the coagulated silver chloride through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the hydrolysable plus inorganic chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. -18- WHO/SIT/1.R3 TECHNICAL DDT 2. 3. 1 . 2 Determination of inorganic chlorine Weigh accurately about 1 g of the sample, dissolve in 10 ml of acetone, and add 100 ml of distilled water. Keep at room temperature for 10 minutes and filter. Acidify the filtrate with 50% nitric acid, add exactly 25 ml of 0.1 N silver nitrate, and proceed as described in section 2.3.1.1. 2. 3. 2 Calculation Hydrolysable chlorine content(% w/w) = (~ - !!____) x 0.3546 x f W1 W2 where a = volume (ml) of 0.1 N silver nitrate equivalent to the hydroly- sable plus inorganic chlorine b = volume (ml) of 0.1 N silver nitrate equivalent to the inorganic chlorine w1 = weight (g) of sample used for the hydrolysable plus inorganic chlorine determination w2 = weight (g) of sample used for the inorganic chlorine determi- nation 10.0 f =T where A = value calculated for hydrolysable chlorine in recrystallized DDT, when determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2.4 p,p'-lsomer Content 2. 4 .1 Special reagent p,p'-Isomer saturated solution. Prepare a saturated solution of pure p,p'-isomer of DDT 1 in 75% (v/v) aqueous ethanol at 25°C (or other suitable temperature), and keep at this temperature in a thermostatically controlled bath constant to ± 0.5°C. 1 Samples of pure p,p'-isomer of DDT may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. -19- TECHNICAL DDT WHO/SIT/1.R3 2.4.2 Procedure Weigh accurately about 2 g of the sample into a 250-300-ml flask equipped with a reflux condenser, add 150 ml of p,p'-isomer saturated solution, and heat under reflux until solution is complete. Stopper the flask and allow the solution to cool slowly in the air to about 26-30°C. Crystals of p,p'-isomer are precipitated during this process. If oily material separates, redissolve by heating again under reflux; if necessary, add a seed of p,p'-isomer during the subsequent cooling process. Place the flask in the thermostatically controlled bath for 4 hours, shaking occasionally. Filter the crystals by suction into a tared Gooch crucible provided at the bottom with a disc of filter paper. As little air as possible should be sucked through the wet crystals during the filtration. Dry the crucible and contents to constant weight at 78-80°C, cool, and weigh. Calculate the percentage of p,p'-isomer, adding an empirical correction of 1.4% to give results in agreement with known mixtures. Then determine the melting-point of the separated crystals by method WHO/M/5, page 255. 2. 5 Chloral Hydrate Content 2. 5 .1 Special reagent Standard chloral hydrate solution. Dissolve 5 mg of chloral hydrate in 100 ml of distilled water. 2.5.2 Procedure Place 20 g of the sample and 200 ml of carbon-dioxide-free distilled water in a 500-ml round-bottomed flask equipped with a mercury-sealed mechanical stirrer. Heat in an oil-bath at a temperature between 140°C and 160°C, with rapid stirring to prevent superheating, and distil the mixture through a well-cooled condenser, at such a rate that 100 ml of distillate are obtained in a period of not less than 3 minutes and not more than 1 hour. Collect exactly 100 ml of distillate in a centrifuge tube and centrifuge to effect complete separation of the water-insoluble material. Place 2 ml of a 40% (w/v) sodium hydroxide solution in a test-tube and add 1 ml of colourless pyridine and 4 ml of the distillate. In another test-tube, place 2 ml of the same 40% (w/v) sodium hydroxide solution and add 1 ml of colourless pyridine and 4 ml of standard chloral hydrate solution. Shake the two tubes and heat in a boiling water-bath for 1 minute. The red colour which develops in the pyridine layer shall not be darker in the sample solution than in the standard solution. -20- WHO/SIT/1.R3 TECHNICAL DDT 2. 6 Solid Material Insoluble in Acetone Weigh accurately about 10 g of the sample into a clean, dry 250-ml flask, add 150 ml of anhydrous acetone, and warm under reflux until all soluble material has dissolved. Filter the solution through a tared Gooch or sintered-glass crucible of porosity No. 3, and wash well with more solvent. Dry at 110°C for 30 minutes, cool, and weigh. 2. 7 Water Content Determine the water content by the Karl Fischer electrometric titration method (see WHO/M/7, page 266), or, where practicable,1 by the Dean & Stark distillation method (see WHO/M/8, page 270). In the event of a dispute the Karl Fischer method shall govern. 1 At very low water contents, the Dean & Stark method is not reliable. - 21 - TECHNICAL AND REFINED HCH WHO/SIT/2.R3 TECHNICAL AND REFINED HCH Specification WHO/SIT/2.RJ Approved 25 October 1965 l. SPECIFICATIONS 1.1 Material The material shall comprise essentially a mixture of isomers of l ,2,3,4,5,6-hexachlorocyclohexane and shall be in the form of white to light-brown granules, flakes, or powder, free from extraneous impurities or added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l . l and with the following requirements : Minimum Maximum Gamma-isomer content (section 2.1), % by weight: Technical HCH . . . . . Refined HCH . . . . . . Acidity (Method WHO/M/3, page 250), % by weight, calculated as H2S04 • • • • • • . • Solid material insoluble in acetone (section 2.2), % by weight ........... . Water content (section 2.3), % by weight .. 12.0 16.l 1. 3 Packing and Marking of Packages 16.0 98.9 0.15 1.0 1.0 The technical HCH and the refined HCH shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical HCH 2 } • • Refined HCH 2 to Specification WHO/SIT/2.R3 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 Whichever is appropriate. - 22 - WHO/SIT/2.R3 TECHNICAL AND REFINED HCH Gamma-isomer content, . . . % w /w Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Keep well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Gamma-Isomer Content 1 The gamma-isomer is determined by infrared spectrophotometry on material that has been separated from interfering substances by chromato- graphy. The method is given in WHO/M/6 on page 258 and the sample is prepared as follows: Grind 20-30 g of the sample 2 in a mortar. Weigh accurately an amount of the powdered sample containing 60-80 mg of gamma-isomer into a 50-ml conical flask, add 15 ml of light petroleum,3 connect to a reflux condenser and boil for 3 minutes on a water-bath maintained at 60°C. Cool, and transfer the liquid and solid residues on to the column. Proceed with the chromatographic separation and spectrophotometric determination as described in Method WHO/M/6, page 258. 2. 2 Solid Material Insoluble in Acetone Weigh accurately about 10 g of the sample into a clean, dry 250-ml flask, add 150 ml of anhydrous acetone, and warm under reflux until all soluble material has dissolved. Filter the solution through a tared Gooch or sintered-glass crucible of porosity No. 3, and wash well with more solvent. Dry at l 10°C for 30 minutes, cool, and weigh. 1 The revised Stepanow (total organic chlorine) method and the hydrolysable chlor- ine method, described respectively in WHO/M/16, page 288 and WHO/M/17, page 291 are not specific. However, this does not preclude a purchaser or user from applying these methods for routine purposes when their use has been agreed to by all parties concerned. 2 If oily ingredients are present, treat the sample with 95% nitric acid for 30 minutes at 85°C and then pour into ice-water, filter, and dry before weighing. 3 Light petroleum, as specified in WHO/M/6, page 260, should be used. -23- TECHNICAL AND REFINED HCH WHO/SIT/2.R3 2. 3 Water Content Determine the water content by the Karl Fischer electrometric titration method (see WHO/M/7, page 266) or, where practicable,1 by the Dean & Stark distillation method (see WHO/M/8, page 270). In the event of a dispute the Karl Fischer method shall govern. 1 At very low water contents, the Dean & Stark method is not reliable. -24- WHO/SIT /3.R3 LINDANE I. SPECIFICATIONS 1.1 Material LIN DANE Specification WHO/SIT/3.RJ Approved 25 October 1965 The material shall comprise essentially the gamma-isomer of 1,2,3,4,5,6- hexachlorocyclohexane and shall be in the form of white or near-white granules, flakes, or powder, free from extraneous impurities or added modifying agents.1 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,2 shall comply with the requirements of section 1 . 1 and with the following requirements : Gamma-isomer content (section 2.1), % by weight ........... . Melting-point (Method WHO/M/5, page 255) ............... . Mixed melting-point with pure gamma- isomer3 of HCH (Method WHO/M/5, page 255) ............. . Acidity (Method WHO/M/3, page 250) % by weight, calculated as H2S04 •• Solid material insoluble in acetone (section 2. 2), % by weight . . . . . . . . . Water content (section 2.3), % by weight Minimum Maximum 99.0 112°C Not lower than melting- point of sample alone 0.15 0.1 0.1 1 If substantial freedom from odour is required, this should be specified in the order. 2 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. Samples of pure gamma-isomer of HCH may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. -25- LIN DANE WHO/SIT/3.R3 1. 3 Packing and Marking of Packages The lindane shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Lindane to Specification WHO /SIT /3. R3 Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : II " Keep well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 Gamma-Isomer Content FIG.1. LOOP STIRRER - --:--5.0 :t 1.0 mm - 0.55 ± 0.05 mm / I : I I o-A 2 .1.1 Special apparatus 1. Loop stirrer, made from capil- lary glass tubing, of 5 .0 ± 1.0 mm external diameter and 0.55 ± 0.05 mm internal diameter, as shown in Fig. I. The external diameter of the loop should be 18.0 ± 1.0 mm. 2. Drying-train for nitrogen, as shown in Fig. 2. 2.1.2 Procedure I = SIDE VIEW II= PLAN Place in a 20-cm boiling-tube of 2.5 cm internal diameter a sufficient amount of the sample to give, when melted, a depth of liquid of approxi- mately 7.5 cm (about 25 g). Melt carefully by immersing the tube to a depth of 10 cm in an oil-bath at approximately 120°C. By means of the loop stirrer connected to the nitro-A = pin-hole blown at end of loop -26- WHO/SIT/3.R3 LIN DANE gen drying-train, pass nitrogen as a fine stream of bubbles through the melt for 1 hour. FIG. 2. DRYING-TRAIN FOR NITROGEN Fit the boiling-tube with a cork collar and insert it to within 1.3 cm of the bottom of a 15-cm boiling- tube of approximately 4 cm diamet- er ; then immerse the two tubes to a depth of 10 cm in an oil-bath at 106°C,1 maintaining the flow of nitrogen through the stirrer. A ther- mometer graduated in one-tenths of a degree 2 is clamped in a central position with its bulb 2.5 cm from the bottom of the tube. Stir at the rate of about two strokes per. second, by moving the stirrer up and down; when the materi- al begins to thicken, work into the melt portions of the material that have solidified on the walls of the tube.3 Read the temperature at I-minute intervals, using a magnifying device, and take the first three consecutive readings during which the tempera- ture remains constant as the initial setting-point.4 Apply corrections as necessary for the thermometer calibration and the emergent stem. Emergent-stem correction. The correction for the emergent stem in mercury-filled thermometers, to be t G A = rubber tube (to loop stirrer) B = drying-tube containing magnesium perchlorate or self-indicating silica-gel C = glass tap D = glass-wool plug E = ground-glass Joint F = pressure-regulating bottle containing mercury to a depth of 16 mm G = nitrogen supply from a cylinder, via reducing-valve 1 The electrically heated apparatus described by Toops, E. E. & Riddick J .A. (1951) Analyt. Chem., 23, 1106, is very suitable and may be used in place of the oil-bath. 2 It is preferable to use a short-range thermometer (99.5-130.5°C) such as is described in British Standard 593: 1954. Thermometers should have an official certificate of exa- mination and should be checked against a fixed reference point (e.g., the ice-point) every six months. 3 As the gamma-isomer of HCH undergoes slight sublimation, mobile crystals should always be present to initiate freezing. 4 In some instances, there may be a slight rise in the temperature of the material ; in such a case, record the highest steady temperature as the setting-point. In the event of superheating exceeding 0.25°C, results should be rejected and the determination repeated. -27- LIN DANE WHO/SIT/3.R3 added to the temperature reading, is calculated from the following formula: N x 0.00015 x (T - t) where N = number of degrees on the scale of the thermometer between the top of the inner boiling-tube and the level of the mercury T temperature reading on the thermometer in the melt t temperature of the stem at the mid-point of the exposed mercury thread. 2. 1. 3 Calculation The gamma-isomer content is calculated from the following formula : Mole per cent= antilog [2 - (0.00643 x T)] where T = 112.86 - setting-point of sample. 1% impurity depresses the setting-point by 0.7°C. 2 . 2 Solid Material Insoluble in Acetone Weigh accurately about 10 g of the sample into a clean, dry 250-ml flask, add 150 ml of anhydrous acetone, and warm under reflux until all soluble material has dissolved. Filter the solution through a tared Gooch or sintered-glass crucible of porosity No. 3, and wash well with more solvent. Dry at ll0°C for 30 minutes, cool, and weigh. 2.3 Water Content Determine the water content by the Karl Fischer electrometric titration method (see WHO/M/7, page 266), or, where practicable,1 by the Dean & Stark distillation method (see WHO/M/8, page 270). In the event of a dispute the Karl Fischer method shall govern. 1 At very low water contents, the Dean & Stark method is not reliable. -28- WHO/SIT/6.R3 TECHNICAL DIELDRIN TECHNICAL DIELDRIN * Specification WHO/SIT/6.RJ Approved 25 October 1965 1. SPECIFICATIONS 1.1 Material The material shall comprise essentially l,2,3,4,10,10-hexachloro-6,7- epoxy-l,4,4a,5 ,6, 7 ,8,8a-octahydro-endo-1,4-exo-5,8-dimethanonaphthalene (HEOD) and shall be in the form of white to tan granules, flakes, or powder, free from extraneous impurities or added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1.1 and with the following requirements : Minimum Maximum HEOD content (section 2.1), % by weight. . Acidity (Method WHO/M/3, page 250), % by weight, calculated as H2S04 • • • • • • • Solid material insoluble in xylene (section 2.2), % by weight ........... . Water content (section 2.3), % by weight 76.5 1. 3 Packing and Marking of Packages 0.3 0.5 0.3 The technical dieldrin shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : * In the ninth report of the WHO Expert Committee on Insecticides (unpublished), the decision was recorded that the following definitions should be used for the purposes of this specification : "'Dieldrin' is to refer to an insecticide containing 85% by weight of HEOD. The amount of dieldrin in any sample will therefore be calculated from the following formula: Dieldrin (% by weight) = HEOD (% by weight) x !~." " 'Technical dieldrin' is to refer to a product containing a minimum of 90% by weight of dieldrin, i.e., a minimum of 76.5% by weight of HEOD." 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. - 29- TECHNICAL DIELDRIN Manufacturer's name Technical dieldrin to Specification WHO/SIT/6.R3 Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : WHO/SIT/6.R3 .. Dieldrin is a toxic substance and may cause convulsions. It is poison- ous if swallowed. It may be absorbed through the skin or inhaled as dusts or mists. Avoid skin contact; wear protective gloves and clean protective clothing while using the material. Wash thoroughly with soap and water after using. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 HEOD Content 1 2 .1.1 Summary of method The sample is weighed and made up to a fixed volume with carbon disulfide. The infra-red spectrum of this solution is scanned in the wave- length region 10.0-13.0 µ. The absorbance of the solution is determined at 11.0 µ, 11.8 µ, and 12.35 µ, and the quantity of HEOD present is obtained by comparing the observed readings with a calibration graph relating absorption to various concentrations of HEOD. 2 .1. 2 Special apparatus 1. Infra-red spectrometer, capable ofrecording in the region 10.0-13.0 µ. The slit width and gain must be adjustable in order to give a satisfactory signal/noise ratio and an adequate resolution. In general, a signal/noise ratio of about 100: 1 is chosen. A sealed absorption-cell with sodium chloride windows and a path length of about 0.4 mm is required. 2. Hypodermic glass syringe, of 1.0 ml capacity, fitted with an 18-gauge (Stubbs), 5-cm, slip-on type needle. A Luer type is also suitable. 2 .1. 3 Special reagents HEOD standard solutions. Into three 10 ml volumetric flasks, weigh accurately 40 mg, 80 mg, and 120 mg of recrystallized HEOD (melting 1 The revised Stepanow (total organic chlorine) method (see Method WHO/M/16, page 288) may be used for routine purposes, but in the case of a dispute the method described here shall govern. -30- WHO/SIT/6.R3 TECHNICAL DIELDRIN point l 78°C).1 Dissolve in carbon disulfide, make up to the mark, and mix thoroughly. These solutions contain respectively 0.4 g, 0.8 g, and 1.2 g of HEOD per 100 ml.2 2 .1. 4 Preparation of calibration graph Fill the absorption cell with the most dilute of the standard solutions by means of the hypodermic syringe. Adjust the spectrometer to the optimum settings with respect to gain, slit width, response, speed, and drum drive. Make replicate scans of the solution over the wave-length region 10.0-13.0 µ. Fill the cell with the other two standard solutions in turn. Make replicate scans of each solution in the same wave-length region, using the same instrument settings as above. For each of the scans of the three different solutions, calculate the absorbance (£) at the band maxima as follows : (1) At 11.0 µ, E = log (;~) where P1 = transmission at 11.0 µ (peak maximum) P0 = transmission at 10.85 µ (peak minimum). (2) At 11.8 µ, E = log (;~) where P1 = transmission at 11.8 µ (peak maximum) P0 = transmission at 12.15 µ (peak minimum). (3) At 12.35 µ, E = log (;:) where P1 = transmission at 12.35 µ (peak maximum) P0 = transmission at 12.15 µ (peak minimum). For each of the three wave-lengths corresponding to the peak maxima, plot the absorbances observed for the three standard solutions against their 1 Samples of pure HEOD may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. 2 The concentrations of HEOD given are specific for a 0.4-mm cell and should be adjusted for different cell lengths in order to give a transmission of between 20% and 80% at the peak maxima. -31 - TECHNICAL DIELDRIN WHO/SIT/6.R3 HEOD content. An example of the calibration graph obtained is shown in Fig. 3. FIG. 3. SPECIMEN CALIBRATION GRAPH FOR DETERMINATION OF HEOD CONTENT 0.7 ,----------------------------~-·-:7-1 0.6 0.6 0.5 0.5 ~ 0.4 0.4 ~ ~ w u z < < "' "' °' °' SI 0.3 0 "' 0.3 ~ ' < < 0.2 ... 0.2 0.1 0.1 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 HEOD CONTENT ( g pe, 100 ml ) NOTE: Whilst the chosen characteristic maxima for HEOD occur at about 11.0 µ, 11.8 µ, and 12.35 µ, measurements of the absorption should be made at the actual peak maxima and not at exactly the wave-lengths mentioned. This applies also to the peak minima at 10.85 µ and 12.15 µ. 2 .1. 5 Analysis of sample Weigh accurately about 1 g of the sample into a 100-ml volumetric flask.1 Dissolve in carbon disulfide, make up to the mark, and mix thoroughly. Fill the absorption cell-the same one used in the calibration-with the above solution and make replicate scans in the region 10.0-13.0 µ, with the same instrument settings as used in the calibration (section 2.1.4). 1 The weight of sample given is specific for a 0.4-mm cell and should be adjusted for different cell lengths in order to give a transmission of between 20% and 80% at the peak maxima. -32 - WHO/SIT/6.R3 TECHNICAL DIELDRIN Calculate the absorbance at the l l .0-µ, 11.8-µ, and 12.35-µ peaks from the scans, as described in section 2.1.4. 2 .1. 6 Calculation For each peak, compute the mean absorbance and read from the calibra- tion graph the corresponding concentration of HEOD in grams per 100 ml of solution. Calculate the mean of the three concentrations.1 axlOO HEOD content(% w/w) = -- w where a = mean concentration (g/100 ml) of HEOD, as found from the calibration graph w = weight (g) of sample. Norn : Dieldrin being defined (see footnote on page 29) as containing 85% of HEOD (w/w), the dieldrin content may be obtained by multiplying the HEOD content by 100 85, i.e., by 1.175. 2 . 2 Solid Material Insoluble in Xylene Weigh accurately about 20 g of the sample and dissolve in sufficient xylene to make 100 ml of solution at 25°C. Allow the solution to stand for 1 hour at 25°C and filter through a tared Gooch or sintered-glass crucible. Wash any insoluble residue on the filter with two 10-ml portions of xylene. Dry the crucible and residue for 1 hour at 100°C, cool in a desiccator, and weigh. 2. 3 Water Content Determine the water content by the Karl Fischer electrometric titration method (see WHO/M/7, page 266) or, where practicable,2 by the Dean & Stark distillation method (see WHO/M/8, page 270). In the event of a dispute the Karl Fischer method shall govern. 1 In practice, the values obtained from the three different readings will be virtually identical. Although slight differences are to be expected owing to instrumental variations, the values should not differ from the mean by more than 5%, assuming that interfering substances have been effectively removed by the extraction procedure. If, in the calcula- tion, it is found that the results obtained from two wave-lengths agree within 5-10% of the mean but that the result obtained from the third wave-length is widely different, it can be assumed that interference is taking place at the third wave-length. In such an event, the result obtained from the third wave-length should be disregarded and the HEOD content calculated from the other two readings. 2 At very low water contents, the Dean & Stark method is not reliable. -33- PYRETHRUM WHO/SIT/7.R1 PYRETHRUM Specification WHO/SIT/7. Rl Approved 25 October 1965 1. SPECIFICATIONS 1.1 Material The material shall be composed of substances occurring naturally in commercial pyrethrum flowers and shall be free from extraneous impurities or added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1. 1 and with the following requirements : Content of "pyrethrins " 2 (section 2.1), % by weight ........... . Material insoluble in dichlorodifluoro- methane 3 (WHO/M/9, page 272), % by weight . . .......... . Minimum Maximum 19.0 1.5 1. 3 Packing and Marking of Packages The pyrethrum shall be packed in suitable, clean containers, as specified in the order. 4 All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Pyrethrum to Specification WHO/SIT/7. Rl " Pyrethrins ", . . . % w /w Batch or reference number, and date of test Net weight of contents. 1 A sampling procedure is described in Method WHO/M/l, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 Pyrethrin I and cinerin I are analysed together and called " pyrethrin I " ; similarly pyrethrin II and cinerin II are called " pyrethrin II ". Collectively these four esters are called the " pyrethrins ". a If the pyrethrum is for other than aerosol use this requirement may be omitted. ' Pyrethrum is sensitive to bright light and should therefore be kept in perfectly lacquered drums or dark-coloured bottles, the latter preferably stored in the dark. - 34- WHO/SIT /7 .R1 PYRETHRUM 2. METHODS FOR DETERMINATION OF CHEMICAL PROPERTIES 2 .1 " Pyrethrins " Content 2 .1.1 Summary of method 1 The " pyrethrins " are extracted from the sample with light petroleum and hydrolysed with alkali to chrysanthemummonocarboxylic and di- carboxylic acids. These acids are converted to their water-soluble barium salts, the aqueous solution filtered to remove insoluble materials and the acids liberated from the salts by treatment with sulfuric acid. Chrysanthemummonocarboxylic acid, which represents " pyrethrins I ", is extracted with light petroleum and reacted with Deniges reagent to form mercurous chloride (Hg2Cl2) which is then titrated with potassium iodate. Chrysanthemumdicarboxylic acid, which represents " pyrethrins II ", is extracted with ether from the water layer remaining from the mono- carboxylic acid extraction and is then determined by titration with standard alkali. 2. 1. 2 Special reagents Potassium iodate 0.01 M. Dissolve 2.14 g of pure potassium iodate, previously dried at 105°C, in distilled water and dilute to l litre. One millilitre of this solution is equivalent to 0.0057 g of pyrethrin I, without further standardization. Iodine monochloride solution. Dissolve 10.00 g of potassium iodide and 6.44 g of potassium iodate in 75 ml of distilled water in a glass-stoppered bottle. Add 75 ml of concentrated hydrochloric acid and 5 ml of chloro- form, and adjust to a faint iodine colour (in chloroform) by adding diluted potassium iodide or iodate solution. If much iodine is set free, use a stronger solution of potassium iodate than 0.01 M at first, making a final adjustment with the 0.01 M solution. Keep in a dark cupboard and re- adjust when necessary. Deniges reagent. Mix 5 g of yellow mercuric oxide with 40 ml of distilled water and, while stirring, add slowly 20 ml of concentrated sulfuric acid ; then add another 40-ml portion of distilled water and stir until solu- tion is complete. Test as follows for possible presence of mercurous mercury. To 10 ml of the reagent add a few drops of the iodine mono- chloride solution, 30 ml of concentrated hydrochloric acid and 20 ml of 1 This method is based on methods described in (a) Association of Agricultural Chemists (1965) Official methods of analysis of the association, lOth ed., Washington D.C., p. 501 and (b) Joint Committee of the Pharmaceutical Society and the Society for Analytical Chemistry on the Methods of Assay of Crude Drugs (1964) Analyst, 89, 689. -35- PYRETHRUM WHO/SIT /7 .R1 distilled water. Cool the flask, add 6 ml of chloroform (or carbon tetra- chloride), and titrate with the 0.01 M potassium iodate, shaking vigorously after each addition, until disappearance of the red colour from the chloro- form (or carbon tetrachloride) layer. Reject the reagent if the titre exceeds 0.2 ml of 0.01 M potassium iodate solution. 2.1.3 Procedure 2. l . 3. l Initial treatment of sample l. Ground flowers. Weigh accurately an amount of the sample con- taining 0.10-0.15 g of" pyrethrins" (about 10 g) and extract with light petroleum in a Soxhlet or other equivalent apparatus for 7 hours. When extraction is complete, evaporate to a volume of about 40 ml, stopper the flask, and place in a refrigerator at 0-5°C for at least 2 hours (preferably overnight). Filter the cold extract through a small cotton plug, placed in the stem of a glass funnel and previously wetted with cold light petroleum, and collect the filtrate in a 250-ml conical flask. Add 20 ml of chilled light petroleum to the extraction flask. With the aid of a rubber policeman dislodge the resinous material in the flask, swirl the contents without allowing the wash-liquid to warm up appreciably, and filter through the cotton. Repeat the operation twice with 10-ml portions of chilled light petroleum. Add several glass beads and evaporate on a water-bath, without attempt- ing to heat the residue long enough to remove the last traces of solvent. Add 15-20 ml of 0.5 N ethanolic sodium hydroxide to the residue, connect to a reflux condenser, and boil gently for 45 minutes. Transfer to a 600-ml beaker and add distilled water to bring the volume to 200 ml. Add a few glass beads, or preferably use a boiling rod, and boil until the volume is reduced to 150 ml. Transfer to a 250-ml volumetric flask and add l g of diatomaceous silica filtering aid and 10 ml of 10% barium chloride solution, without shaking. Make up to the mark with distilled water, mix thor- oughly, and filter off exactly 200 ml. Add l drop of phenolphthalein indi- cator solution, neutralize with 20% sulfuric acid, and add l ml in excess. (If it is necessary to leave the solution overnight at this point, it should be left in an alkaline condition.) 2. Pyrethrum extracts and concentrates.1 Weigh accurately an amount of the sample containing 0.10-0.15 g of" pyrethrins" into a 250-ml conical 1 If purified extracts are being analysed, test for the presence of oxidized " py- rethrins" by dissolving an amount of sample containing 0.10-0.15 g of" pyrethrins" in 50 ml of light petroleum. If the solution is clear or only slightly opalescent, omit the treatment with diatomaceous filtering aid given above, i.e., add 20 ml of 1 N ethanolic sodium hydroxide to the aliquot taken and proceed with the hydrolysis. - 36- WHO/SIT /7 .R1 PYRETHRUM flask, add 50 ml of light petroleum, mix, add 1 g of diatomaceous silica filtering aid, mix, stopper the flask and set aside in a refrigerator at 0-5°C for at least 2 hours (preferably overnight). Filter quantitatively through asbestos in a Gooch crucible into a 250-ml conical flask, washing with three 15-ml portions of cold light petroleum, and combine the filtrate and washings. Add several glass beads and evaporate on a water-bath, without attempt- ing to heat the residue long enough to remove the last traces of solvent. Add 20 ml (or more if necessary) of l N ethanolic sodium hydroxide, connect to a reflux condenser, and boil gently for 45 minutes. Transfer to a 600-ml beaker and add distilled water to make an aqueous layer of 200 ml. (If more than 20 ml of l N ethanolic sodium hydroxide has been used, add sufficient distilled water so that all the ethanol will be removed when the volume has been reduced to 150 ml.) Add a few glass beads, or preferably use a boiling rod, and boil until the volume of the aqueous layer is reduced to 150 ml, using an air stream if necessary to depress frothing. Transfer to a 500-ml separating funnel and draw off the aqueous layer into a 250-ml volumetric flask. Wash the oily layer with distilled water and add the washings to the aqueous layer. To the aqueous solution in the volumetric flask add l g of diatomaceous silica filtering aid and 10 ml (or more) of 10% barium chloride solution, without shaking. Make up to the mark with distilled water, mix thoroughly, and filter off 200 ml. Test the filtrate with 10% barium chloride solution to see if a sufficient quantity has been added to obtain a clear solution. Add 1 drop of phenolphthalein indicator solution, neutralize with 20% sulfuric acid and add l ml in excess. 2. l . 3. 2 Determination of" pyrethrin I" By means of a Buchner funnel, filter the solution obtained as described in section 2. l . 3. l through a 7-cm filter-paper that has been coated lightly with a suspension of diatomaceous silica filtering aid in distilled water and wash with three successive 15-ml portions of water. Transfer to a 500-ml separating funnel and extract with two 50-ml portions of light petroleum. Wash the extracts successively with two 10-ml portions of distilled water and filter the petroleum extract through a cotton plug into a clean 250-ml separating funnel. Wash the cotton plug with 5 ml of light petroleum. Reserve the aqueous phase from these extracts for the determination of "pyrethrin II" below. Extract the petroleum layer with 5 ml of 0.1 N sodium hydroxide, shaking vigorously. Draw off the aqueous layer into a 100-ml conical flask, wash the petroleum layer with a second 5-ml portion ofO.l N sodium hydroxide, and add the extract to the flask. Add 10.0 ml of the Deniges reagent and let stand for 60 minutes in the dark at 25°C ± ± 0.5°C (see NOTE, page 39). Add 20 ml of ethanol and precipitate the -37 - PYRETHRUM WHO/SIT/7.R1 mercurous chloride with 2 ml of saturated sodium chloride solution. Warm to about 60°C and set aside for several minutes, until the precipitated mercurous chloride has coagulated and settled. Decant the supernatant liquid through a 9-cm filter-paper in a small glass funnel (4.5 cm diameter) and drain carefully, retaining most of the precipitate in the flask. Wash the precipitate in the flask with 10 ml or more of hot anhydrous ethanol, decanting the ethanol through the same filter-paper as before. Wash the precipitate with three successive 10-ml portions of hot chloroform, decanting each washing through the same filter-paper, and transfer the filter-paper and any precipitate to the flask containing the bulk of the precipitate. Add 30 ml of concentrated hydrochloric acid and 20 ml of distilled water to the flask and cool. Add 6 ml of chloroform (or carbon tetrachloride) and I ml of the iodine monochloride and titrate with the 0.01 M potassium iodate, shaking vigorously after each addition, until disappearance of the red colour from the chloroform (or carbon tetrachloride) layer.1 Carry out a blank determination by repeating the whole procedure but omitting the " py- rethrins ". 2. l . 3. 3 Determination of" pyrethrin II" If necessary, filter the aqueous residue from the light petroleum extrac- tion (see section 2.1. 3. 2) through a Gooch crucible. Concentrate the filtrate to a volume of about 50 ml and transfer to a 500-ml separating funnel. Acidify with 10 ml of concentrated hydrochloric acid and saturate with sodium chloride.2 Extract with 50 ml of ether, draw off the aqueous layer into a second separating funnel, and extract again with 50 ml of ether. Run off the aqueous phase into a 100-ml conical flask and transfer the ether extract to the first separating funnel. Return the aqueous phase to the second separating funnel and extract in a similar manner with two 35-ml portions of ether, combining all ether extracts in the first separating funnel. Discard the aqueous phase and any aqueous liquid that separates from the combined ether extracts. Wash the ether extract with three successive 10-ml portions of saturated sodium chloride solution. Filter the ether extract through a cotton plug into a 500-ml conical flask and wash the cotton plug with 10 ml of fresh ether. Evaporate the ether on a water- bath, removing the last traces of vapour with a current of air. Dry the residue in an oven at I00°C for 10 minutes. Treat with 75 ml of boiling 1 KI03 reacts with mercurous Hg to form mercuric Hg and I ; further addition of KI03 in presence of HCl oxidizes I to ICl : 2H&Cl 2 + KI03 + 6HC1 = 4HgC12 + ICl + KCl + 3H20 Addition of ICl does not change the volume relationship between mercurous Hg and Kl03 solution and aids in determining the end-point in the titration of small quantities of Hg. 2 Care must be taken that the acidified aqueous layer is saturated with sodium chloride throughout the subsequent extractions. - 38- WHO/SIT/7.R1 PYRETHRUM water and filter the solution through a high-grade filter-paper, washing the flask and filter-paper with five successive 20-ml portions of boiling water or until the washings are neutral to litmus. Add 1 or 2 drops of phenol- phthalein indicator solution to the combined filtrate and titrate with 0.02 N sodium hydroxide. One millilitre of 0.02 N sodium hydroxide is equivalent to O .00372 g of" pyrethrin II". Carry out a blank determination by repeating the whole procedure on the aqueous liquid set aside in the blank determina- tion for " pyrethrin I " ( section 2. 1. 3. 2). 2 .1. 4 Calculation The pyrethrin contents are calculated as follows: 1. Content of" pyrethrin I" (% w/w) (a-b) x 0.7125 w where a = volume (ml) of 0.01 M potassium iodate required for titration of " pyrethrin I " b = volume (ml) of 0.01 M potassium iodate required for titration of the blank solution w = weight (g) of initial sample 2. Content of " pyrethrin II " (% w /w) (c-d) x 0.465 w where c = volume (ml) of 0.02 N sodium hydroxide required for titration of " pyrethrin II " d = volume (ml) of 0.02 N sodium hydroxide required for titration of the blank solution w = weight (g) of initial sample. Norn : Chrysanthemummonocarboxylic acid reacts with the Deniges reagent to form a series of colours beginning with red, which gradually changes to purple, then blue, and finally bluish-green. The colour reaction is very distinct with 5 mg of the acid, and quantities as low as 1 mg can usually be detected. Therefore, no "pyrethrin I" should be reported if the colour reaction is negative. - 39- TECHNICAL DIAZINON WHO/SIT/9.R2 TECHNICAL DIAZINON Specification WHO/ SIT/9. R2 Approved 25 October 1965 1. SPECIFICATIONS 1.1 Material The material shall comprise essentially O ,0-diethyl 0-(2-isopropyl-6- methyl-4-pyrimidinyl) phosphorothioate and shall be in the form of a dark liquid, free from extraneous impurities or added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1 . 1 and with the following requirements : Minimum Maximum 0 ,0-Diethyl 0-(2-isopropyl-6-methyl-4-pyrimi- dinyl) phosphorothioate content (section 2.1), % by weight . . . . . . . . . . . Acidity (WHO/M/3, page 250), % by weight, calculated as H2S04 ••••••••••• Solid material insoluble in acetone (section 2. 2), % by weight .......... . Water content (section 2.3), ~;,; by weight .. 85.0 1. 3 Packing and Marking of Packages 0.03 0.5 0.5 The technical diazinon shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical diazinon to Specification WHO /SIT /9. R2 Batch or reference number, and date of test Net weight of contents 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. -40- WHO/SIT/9.R2 TECHNICAL DIAZINON and the following minimum cautionary notice : " Diazinon is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed. It may be absorbed through the skin. Avoid skin contact; wear protective gloves, clean protective clothing and a respirator when handling the material. Wash thoroughly with soap and water after using. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. " If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respitation may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 0,0-Diethyl 0-(2-Isopropyl-6-methyl-4-pyrimidinyl) Phosphorothioate Content 2 .1.1 Summary of method The basic by-products of diazinon are absorbed in a column of diato· maceous silica filtering aid impregnated with 3N sulfuric acid. 0 ,0-Diethyl 0-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate is then deter- mined in the eluate by titration with perchloric acid. 2 .1. 2 Special apparatus 1. Chromatographic column 250 mm in length and 21-22 mm in internal diameter, fitted with glass or Teflon stopcock and a sintere~-glass disc and equipped to operate under pressure. 2. Steel tamping rod with perforated disc to fit loosely inside the chromatography column. 2 .1. 3 Special reagent Standardized 0.1 N perchloric acid in glacial acetic acid. Dilute a suitable quantity of perchloric acid, obtainable as a concentrated aqueous solution, to the required volume with glacial acetic acid and sufficient acetic anhydride to take up the entire water content of the perchloric acid. For example, to make up 5 litres of solution using 70% perchloric acid, the required quanti- ties would be : 70% perchloric acid . . . . Acetic anhydride . . . . . Glacial acetic acid, to make -41 - 73 g 124.l g 5 litres TECHNICAL DIAZINON WHO/SIT/9.R2 If the acetic acid used is not anhydrous, a correspondingly larger volume of acetic anhydride will be required, equivalent to the total water content of the other two reagents. Standardize the solution as follows: Dissolve 0.2 g of anhydrous sodium carbonate, accurately weighed, in 50 ml of glacial acetic acid and titrate this solution with the perchloric acid solution, using 1% o:-naphtholbenzein solution in benzene as indicator; 0.200 g of sodium carbonate corresponds to 37.73 ml of 0.1 N perchloric acid. 2 .1. 4 Preparation of the chromatographic column Weigh 250 g of diatomaceous silica filtering aid into the mixing bowl of a suitable mixer. This amount will provide enough material for 15 columns. Add slowly 150 ml of 3 N sulfuric acid while mixing. Continue mixing until the powder is homogeneous. Scrape the material adhering to the side of the bowl during the process. Add enough light petroleum to the diatomaceous silica filtering aid mixture to cover the paste with solvent, stirring by hand with a porcelain spatula during the addition. The resulting slurry is now ready for packing into the column. Pour into the dry column light petroleum to half of its height. With the help of the tamping rod, tamp a glass-wool plug into position above the fritted glass disc. Put the funnel on top of the column. Fill portions of the slurry into the column through the funnel and tamp each portion gently with the tamping rod until the column is two-thirds full. Stir the slurry with a glass rod to eliminate all air bubbles and to ensure homogen- eous packing. Adjust the pressure and open the stopcock. Press out excess of light petroleum, but retain enough solvent in the column so that the diatomaceous silica filtering aid is just covered at all times. Continue adding slurry and pressing until the height of the packed column is about 15 cm. Tamp with the rod to form an even surface and place a glass-wool plug of about 5 mm on top of the packed column. Properly packed columns allow the light petroleum to pass through dropwise. 2 .1. 5 Procedure Weigh accurately 0.5-0.7 g of the sample into a 25-ml flask and rinse it with 5-10 ml of light petroleum on to the column. Open the stopcock and let the level sink to about 1-3 mm above the packing. Rinse the flask twice with 5 ml and twice with 10 ml of light petroleum quantitatively. Pour each washing down the side of the column so as to wash the sample into the packing and allow it to penetrate to a level of 1-3 mm above the packing before adding the next portion. Add 30 ml of light petroleum. - 42 - WHO/SIT/9.R2 TECHNICAL DIAZINON Adjust the dropping funnel to the top of the column and elute with 200 ml of light petroleum. Collect the eluate in a 500-ml flask with ground-glass joint and distil off the solvent. Dissolve the residue in 50 ml of glacial acetic acid, connect to a reflux condenser and boil for 15 minutes. Cool, rinse with glacial acetic acid into a beaker, and titrate with 0.1 N perchloric acid in glacial acetic acid, 1 using a 1 % solution of a-naph- tholbenzein in benzene as indicator, until the solution becomes dark green. Alternatively, the titration may be done potentiometrically. Norn : The column can be used a second time if the first sample did not contain too large a quantity of by-products. Care has to be taken that the column does not get dry, but remains covered by light petroleum. 2 .1. 6 Calculation 0 ,0-Diethyl 0-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate content(% w/w) a x 3.044 w where a = volume (ml) of 0.1 N perchloric acid used w = weight (g) of sample. 2. 2 Solid Material Insoluble in Acetone Weigh accurately about 10 g of the sample into a clean, dry 250-ml flask, add 150 ml of anhydrous acetone, and warm under reflux until all soluble material has dissolved. Filter the solution through a tared Gooch or sintered-glass crucible of porosity No. 3, and wash well with more solvent. Dry at l l0°C for 30 minutes, cool, and weigh. 2.3 Water Content Determine the water content by the Karl Fischer electrometric titration method (see WHO/M/7, page 266) or, where practicable,2 by the Dean & Stark distillation method (see WHO/M/8, page 270). In the event of a dispute the Karl Fischer method shall govern. 1 Care must be taken that all titrations with perchloric acid are performed in abso- lutely anhydrous media. 2 At very low water contents, the Dean & Stark method is not reliable. - 43- TECHNICAL MALATHION WHO/SIT/10.R2 TECHNICAL MALATHION Specification WHO/ SIT/ JO. R2 Approved 25 October 1965 1. SPECIFICATIONS 1.1 Material The material shall comprise essentially O ,0-dimethyl S-(1,2-di-( ethoxy- carbonyl)ethyl] phosphorodithioate and shall be in the form of a clear, colourless to light amber liquid, free from extraneous impurities or added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1 . 1 and with the following requirements : 2 0 ,0-Dimethyl S-(1,2-di-( ethoxycarbonyl) ethyl] phosphorodithioate content (sec- tion 2.1), % by weight ...... . Acidity(WHO/M/3,page250), % byweight, calculated as H2S04 •••••.••• Solid material insoluble in acetone (section 2.2), % by weight ........ . Water content (section 2.3), % by weight Specific gravity at 25°C/25°C (section 2.4) Minimum 95.0 1.23 1. 3 Packing and Marking of Packages Maximum 0.5 0.5 0.1 The technical malathion shall be packed in suitable, clean containers, as specified in the order.3 All packages shall bear, durably and legibly marked on the container, the following : 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 If the material is required for use inside buildings, odour-free malathion should also be specified. 3 Malathion is likely to be decomposed under conditions of prolonged tropical storage. It is therefore recommended that this insecticide and all formulations based on it should be kept in a cool place and used as soon as possible after manufacture. -44- WHO/SIT/10.R2 TECHNICAL MALATHION Manufacturer's name Technical malathion to Specification WHO/SIT/10.R2 Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Malathion is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. " If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 0,0-Dimethyl S-(1,2-di(ethoxycarbonyl)ethyl) phosphorodithioate Content 2 .1.1 Summary of method The sample is quantitatively hydrolysed by ethanolic alkali to give the sodium salt of O ,0-dimethyl phosphorodithioic acid, which is then con- verted to a yellow-coloured cupric complex and immediately extracted into carbon tetrachloride. The intensity of the colour is proportional to the concentration of O ,0-dimethyl phosphorodithioic acid present and is measured colorimetrically at 420 mµ. The amount of 0,0-dimethyl S-[1,2- di( ethoxycarbonyl)ethyl] phosphorodithioate is then determined by com- parison with the absorbance at 420 mµ of a known sample of standard malathion that has been submitted to the same operations at the same time as the sample being analysed. Ferric reagent is added to oxidize materials that would reduce cupric ions to cuprous ions ; 0 ,0-dimethyl phosphoro- dithioic acid forms a colourless complex with cuprous ions, which is apparently more stable than the yellow-coloured cupric complex. 2 .1. 2 Special apparatus Spectrophotometer or photoelectric colorimeter with a blue filter (420 mµ). 2 .1. 3 Special reagents Acetonitrile. Boiling range 80-82°C. Pass through a column of silica gel and discard the first (yellow) portion of the eluate. Collect the colour- -45- TECHNICAL MALATHION WHO/SIT/10.R2 less eluate. A 10% aqueous solution of this should have a pH between 5 and 7. Ferric reagent. Dissolve 0.2 g of ferric chloride (FeC12 • 6H20) in distilled water, add 8 ml of concentrated hydrochloric acid, and dilute to 1 litre with distilled water. Cupric reagent. Dissolve 1.5 g of cupric sulfate (CuS04 • 5H20) in 100 ml of water. 2 .1. 4 Preparation of malathion standard solutions Standard solution A. Accurately weigh into a 5-ml or 10-ml beaker 1.00 g ± 0.02 g of malathion reference standard.1 Transfer quantitatively to a I-litre volumetric flask with anhydrous ethanol. Dilute with anhydrous ethanol to about 5 ml below the mark and allow to stand until a steady temperature is reached. Record the temperature, dilute to the mark with anhydrous ethanol and mix well. The solution is stable for about 2 weeks. Adjust to the recorded temperature before withdrawing aliquots. Standard solution B. Pipette a 15-ml aliquot of standard solution A into a 250-ml volumetric flask. Add 2.5 ml of acetonitrile and dilute with anhydrous ethanol to about 5 ml below the mark. Keep until the sample for analysis is ready for final dilution. At that time, dilute with anydrous ethanol to the mark and mix well. NOTE : Volume changes of alcohol solutions with temperature are appreciable. Bring each solution to volume just before proceeding to next step. 2 .1. 5 Preparation of sample Accurately weigh 0.30 g ± 0.02 g of the sample, transfer it quantitatively to a 50-ml volumetric flask and make up to the mark with acetonitrile. Immediately pipette a 25-ml aliquot into a 250-ml volumetric flask, dilute to the mark with anhydrous ethanol and mix well. Immediately pipette a 25-ml aliquot of the diluted solution into a 250-ml volumetric flask and again make up to volume with anhydrous ethanol. 2 .1. 6 Analysis of sample Determine the standard malathion and the sample separately but at the same time. Perform the entire analysis without interruption. 1 Samples of reference standard malathion may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. - 46 - WHO/SIT/10.R2 TECHNICAL MALATHION Pipette 25-ml aliquots of standard solution B and sample solutions into two 250-ml separating funnels. Add 2 ml ± 0.1 ml of0.5N sodium hydrox- ide to each and mix well by swirling gently for 5 to 10 seconds ( do not shake). Allow to stand for 120 seconds ± 10 seconds. Add 75 ml ± 1 ml of ferric reagent and mix well by swirling for 10 seconds. Allow to stand for 5 minutes. During this waiting period fill two 50-ml volumetric flasks to the mark with cyclohexane. Transfer the cyclohexane from each of the 50-ml flasks into one of the separating funnels and allow the flasks to drain for one minute. Add 2.0 ml ± 0.1 ml of cupric reagent and shake in the separating funnel for exactly one minute. NOTE: The copper-malathion complex is unstable in the aqueous phase. The cupric reagent should therefore be added from a fast-delivery Mohr pipette and shaking started without delay. A delay of only 15 seconds before extraction results in appreciable losses. Allow the phases to separate and discard the aqueous phase as soon as separation occurs. Allow a little of the solvent phase to rinse the stem of the separating funnel, then transfer the clear solvent phase to a small beaker and thence to the cell, or transfer directly to the cell. Manipulate phase separation of both standard and sample similarly. Immediately (within 5 minutes) determine the absorbance of the yellow cyclohexane solution at 420 mµ by means of the spectrophotometer (or photoelectric colorimeter) using cyclohexane as a reference. 2.1. 7 Calculation 0 ,0-dimethyl S-[1,2-di(ethoxycarbonyl)ethyl] phosphorodithioate content ( % w /w) A sample a X p X 0.3 = ·---- x ---- A standard b X 100 where Asample = absorbance of the sample at 420 mµ Astandard = absorbance of the standard at 420 mµ a = weight (g) of reference standard malathion taken to prepare standard solution b = weight (g) of sample p =purity(%) of reference standard malathion. 2. 2 Solid Material Insoluble in Acetone Weigh accurately about 10 g of the sample into a clean, dry 250-ml flask, add 150-ml of anhydrous acetone, and warm under reflux until all soluble material has dissolved. Filter the solution through a tared Gooch or sintered-glass crucible of porosity No. 3, and wash well with more solvent. Dry at l 10°C for 30 minutes, cool, and weigh. -47 - TECHNICAL MALATHION WHO/SIT/10.R2 2.3 Water Content Determine the water content by the Karl Fischer electrometric titration method (see WHO/M/7, page 266) or, where practicable,1 by the Dean & Stark distillation method (see WHO/M/8, page 270). In the event of a dispute the Karl Fischer method shall govern. 2.4 Specific Gravity Determine the specific gravity at 25°C/25°C by means of a pyknometer. 1 At very low water contents, the Dean & Stark method is not reliable. - 48- WHO/SIT/11.R2 TECHNICAL PARATHION TECHNICAL PARATHION l. SPECIFICATIONS 1. 1 Material Specification WHO/S/T/11. R2 Approved 25 October 1965 The material shall comprise essentially O, 0-diethyl 0-(p-nitrophenyl) phosphorothioate and shall be in the form of a dark liquid, free from extraneous impurities or added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l . l and with the following requirements : 0 ,0-Diethyl 0-(p-nitrophenyl) phosphorothio- ate content (section 2.1), % by weight . . Acidity (Method WHO/M/3, page 250), % by weight, calculated as H2S04 • • • • • • • Solid matter insoluble in acetone (section 2. 2), % by weight ........... . Water content (section 2.3), % by weight. Specific gravity at 25°C/25°C (section 2 .4) Minimum 90.0 l.25 1. 3 Packing and Marking of Packages Maximum 0.3 0.5 0.5 The technical parathion shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical parathion to Specification WHO/SIT/ll .R2 Batch or reference number, and date of test Net weight of contents 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. -49 - 4 TECHNICAL PARATHION WHO/SIT /11.R2 and the following minimum cautionary notice : "POISON [Skull-and-cross-bones insignia] " Parathion is an organophosphorus compound which inhibits cholin- esterase. It is a very toxic substance. Contact with the skin, inhalation of dust or spray, or swallowing may be fatal. Wear protective gloves, clean protective clothing, and a respirator of the organic-vapour type when handling this material. Bathe immediately after work. " Ensure that containers are stored under lock and key. Empty con- tainers must be disposed of in such a way as to prevent all possibility of accidental contact with them. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. " In case of contact, immediately remove contaminated clothing and wash the skin thoroughly with soap and water; for eyes, flush with water for 15 minutes. "If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and repeated doses may be necessary. Artificial respira- tion may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 0,0-Diethyl 0-(p-nitrophenyl) Phosphorothioate Content 2. 1.1 Summary of method Parathion is separated from p-nitrophenol, the most likely impurity, by mild alkaline extraction of an ether solution. The parathion in the ether layer is reduced by zinc and an acetic acid/hydrochloric acid mixture, and the amino groups formed are titrated with standard sodium nitrite solution. 2 .1. 2 Special reagents Acetic acid/ hydrochloric acid mixture. Mix 9 volumes of glacial acetic acid with 1 volume of concentrated hydrochloric acid. Sodium nitrite, 0.1 M, standardized against suljanilic acid. Dissolve 6.90 g of sodium nitrite in distilled water and make up to l litre with distilled water. Standardize this solution as follows : -50- WHO/SIT/11.R2 TECHNICAL PARATHION Weigh accurately 0.40-0.45 g of anhydrous sulfanilic acid (analytical grade or material, the purity of which has been checked by a nitrogen determination) into a 400-ml tall-form beaker. Add 80 ml of distilled water, 10 ml of concentrated hydrochloric acid, 30 ml of glacial acetic acid, and 5 g of sodium (or potassium) bromide. Cool the mixture to 0-10°C by the addition of clean, shaved ice and place under mechanical stirring. Titrate at 0-10°C with the 0.1 M sodium nitrite as rapidly as the spot test (section 2 .1. 3. 3) permits. Near the end-point, add the sodium nitrite in 4-drop portions. a x 5.774 Normality of 0.1 M sodium nitrite = b where a = weight (g) of sulfanilic acid used b = volume (ml) of 0.1 M sodium nitrite required 2.1.3 Procedure 2. 1 . 3 . 1 Separation of parathion from p-nitrophenol Weigh accurately from a weighing-pipette 0.6-0.9 g of the sample into 100 ml of ether contained in a 250-ml separating funnel. Extract the ether solution four times (or until the extract is colourless) with 20-ml portions of chilled 1 % (w/v) sodium carbonate solution, discarding the aqueous layers. 2. 1. 3. 2 Determination of parathion in ether layer Transfer the ether layer quantitatively to a 400-ml beaker, using small portions of ether. Add 35 ml of the acetic acid/hydrochloric acid mixture and 3 g of iron-free zinc dust.1 Cover the beaker with a watch-glass and heat gently on a steam-bath until most of the ether has evaporated and the solution is colourless. Add 10 ml of concentrated hydrochloric acid to complete the solution of the zinc dust. If undissolved zinc remains, filter the solution through a sintered-glass filter and subsequently wash with water. Cool, wash down the beaker and watch-glass with 100 ml of distilled water, and add 5 g of sodium (or potassium) bromide. Cool the mixture to O-l0°C by the addition of clean, shaved ice (about 100 g) and place under mechanical stirring. Titrate at O- l0°C with the standardized 0.1 M sodium nitrite as rapidly as the spot test (section 2. l. 3. 3) permits ; about 20-30 ml will be required. Near the end-point, add the sodium nitrite in 4-drop portions. 1 If less than 3 g of zinc dust are used or there is reason to doubt the purity of the zinc dust, this should be checked by analysis. -51 - TECHNICAL PARATHION WHO/SIT/11.R2 2. 1. 3. 3 Spot test Dip a glass rod into the solution to be tested and then touch the rod quickly to a piece of potassium iodide/starch paper. The end-point is reached when an intense blue-black colour appears immediately and can be obtained repeatedly during a I-minute period without further addition of sodium nitrite. 2 .1. 4 Calculation 0 ,0-Diethyl 0-(p-nitrophenyl) phosphorothioate content (% w /w) = c x f1!._ x 29 .13 x f where N= c = W= != w normality of the 0.1 M sodium nitrite (see section 2.1.2) volume (ml) of 0.1 M sodium nitrite required weight (g) of sample T A where T = quantity (ml) of 0.1 M sodium nitrite calculated for the nitro- group determination of l g of a mono-nitro reference sub- stance.1 A = quantity (ml) of 0.1 M sodium nitrite used in the nitro-group determination of 1 g of a recrystallized sample of the same substance, following the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2. 2 Solid Material Insoluble in Acetone Weigh accurately about 10 g of the sample into a clean, dry 250-ml flask, add 150 ml of anhydrous acetone, and warm under reflux until all soluble material has dissolved. Filter the solution through a tared Gooch or sintered-glass crucible of porosity No. 3, and wash well with more solvent. Dry at l l0°C for 30 minutes, cool, and weigh. 1 Since parathion is not usually available as a pure material, f is evaluated on the basis of another substance containing one nitro group. Suitable materials are : Melting-range m-nitrobenzoic acid. 140.5-14l.0°C p-nitrobenzoic acid. 239.6-240.l 0c - 52 - WHO/SIT /11 .R2 TECHNICAL PARATHION 2. 3 Water Content Determine the water content by the Karl Fischer electrometric titration method (see WHO/M/7, page 266) or, where practicable,1 by the Dean & Stark distillation method (see WHO/M/8, page 270). In the event of a dispute the Karl Fischer method shall govern. 2. 4 Specific Gravity Determine the specific gravity at 25°Cf25°C by means of a pyknometer. 1 At very low water contents, the Dean & Stark method is not reliable. -53- TECHNICAL TRICHLORFON WHO/SIT/13.R1 TECHNICAL TRICHLORFON Tentative Specification WHO /SIT/ 13. R I Approved 25 October 1965 1. SPECIFICATIONS 1. 1 Material The material shall comprise essentially dimethyl 1-hydroxy-2,2,2-tri- chloroethylphosphonate and shall be in the form of a white to pale yellow, crystalline powder, free from extraneous impurities or added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1. 1 and with the following requirements : Minimum Maximum Setting-point (section 2 .1) . . . . . . . . . 73 .5°C Dimethyl 1-hydroxy-2,2,2-trichloroethyl phos- phonate content (section 2.2), '.\ by weight 95.0 Acidity (section 2. 3), % by weight, calculated as H2S04 • • . • • • • . . • • . • • • 0.7 Solid material insoluble in acetone (section 2.4), % by weight . . . . . . . . . . . 0.5 Water content (section 2.5), % by weight .. 1.0 1. 3 Packing and Marking of Packages The technical trichlorfon shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical trichlorfon to Specification WHO/SIT /13. Rl Batch or reference number, and date of test Net weight of contents 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. -54 - WHO/SIT/13.R1 TECHNICAL TRICHLORFON and the following minimum cautionary notice : " Trichlorfon is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed or absorbed through the skin. Avoid skin contact: wear protective gloves and clean protective clothing while using the material. Wash thoroughly with soap and water after using. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. " If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Setting-Point Place in a 20-cm boiling-tube of 2.5 cm internal diameter a sufficient amount of the sample to give, when melted, a depth of liquid of approxi- mately 7.5 cm. Melt carefully by immersing the tube to a depth of 10 cm in an oil-bath, not allowing the temperature of the melt to exceed 80°C. Add 6 g of calcium sulfate previously dried at l80°C. Fit this boiling-tube with a cork collar and insert it to within 1.3 cm of the bottom of a 15-cm boiling-tube of approximately 4 cm diameter; then immerse the two tubes to a depth of 10 cm in a water-bath maintained at 55°C. Place in the inner boiling-tube a stirrer, consisting of a glass rod bent at one end in the form of a ring. A thermometer graduated in one- tenths of a degree is then clamped in a central position with its bulb 2.5 cm from the bottom of the tube. The temperature of the melt should be approximately 5°C higher than the anticipated setting-point at this stage. Stir at a rate of about two strokes per second, by moving the stirrer up and down, until the material begins to thicken, at which point stir vigorously to work into the melt material that has solidified on the walls of the tube. Stop stirring when the temperature ceases dropping and remains constant for some time.1 Record this temperature as the setting-point. Apply corrections as necessary for the thermometer calibration and the emergent stem. Emergent-stem correction. The correction for the emergent stem in mercury-filled thermometers, to be added to the temperature reading, is calculated from the following formula : N x 0.00015 x (T - t) 1 In some instances, there may be a slight rise in the temperature of the material; if this occurs, record the highest steady temperature as the setting-point. - 55 - TECHNICAL TRICHLORFON WHO/SIT/13.R1 where N = number of degrees on the scale of the thermometer between the top of the inner boiling-tube and the level of the mercury T temperature reading on the thermometer in the melt t temperature of the stem at the mid-point of the exposed mercury thread. 2. 2 Dimethyl 1-Hydroxy-2,2,2-trichloroethylphosphonate Content 2. 2 .1 Summary of method The sample is hydrolysed quantitatively in a mixture of methanol and ethanolamine to form chlorine ions. The dimethyl 1-hydroxy-2,2,2-tri- chloroethylphosphonate content is then measured by the difference between the total chlorine thus produced and the amount of inorganic (ionic) chlorine present in the sample before hydrolysis. 2.2.2 Procedure 2. 2. 2 .1 Determination of hydrolysable plus inorganic chlorine Weigh accurately about 1 g of the sample into a 250-ml conical flask and dissolve in 90 ml of anhydrous methanol. Add 10 ml of 2-aminoethanol of over 99% purity and keep for exactly I hour at 20°C ± 0.5°C. Cool in ice water and add 50 ml of chlorine-free 20% nitric acid. Keep at 20°C and titrate electrometrically with 0.1 N silver nitrate. 2. 2. 2. 2 Determination of inorganic chlorine Weigh accurately about 1 g of the sample into a 250-ml conical flask and dissolve in 100 ml of distilled water. Keep for 5 minutes at room temperature, acidify with 5 ml of chlorine-free 20% nitric acid, and titrate electrometrically with 0.1 N silver nitrate. 2. 2. 3 Calculation Dimethyl l-hydroxy-2,2,2-trichloroethylphosphonate content (% w /w) = (!!_ __ - !:_) x 2.575 x f W1 W2 where a b volume (ml) of 0.1 N silver nitrate equivalent to the hydro- lysable plus inorganic chlorine volume (ml) of 0.1 N silver nitrate equivalent to the inor- ganic chlorine -56 - WHO/SIT /13.R1 TECHNICAL TRICHLORFON weight (g) of sample used for the hydrolysable plus inor- ganic chlorine determination weight (g) of sample used for the inorganic chlorine deter- mination 13.77 f =-A where A value (%) for hydrolysable chlorine in recrystallized dimethyl l-hydroxy-2,2,2-trichloroethylphosphonate determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2.3 Acidity 2.3.1 Procedure Weigh exactly 10 g of the sample and dissolve in 100 ml of distilled water, with gentle warming if necessary. Titrate immediately at l0- l 5°C with 0.02 N sodium hydroxide, using methyl red as indicator. Carry out a blank determination on 100 ml of distilled water with 0.02 N sodium hydroxide. 2. 3. 2 Calculation Acidity, % by weight calculated as H2S04 = 0.0098 x (a-b) where a = volume (ml) of 0.02 N sodium hydroxide used for the sample b = volume (ml) of 0.02 N sodium hydroxide used for the blank. NOTE: The blank may take the form of a small titre with 0.02 N hydro- chloric acid, in which case acidity, % by weight calculated as H2S04 = 0.0098 x (a + c) where a = volume (ml) of 0.02 N sodium hydroxide used for the sample c = volume (ml) of 0.02 N hydrochloric acid used for the blank. Alternatively, the end-point may be determined electrometrically. 2. 4 Solid Material Insoluble in Acetone Weigh accurately about 10 g of the sample into a clean, dry 250-ml flask, add 150 ml of anhydrous acetone, and warm under reflux until all soluble material has dissolved. Filter the solution through a tared Gooch or sintered-glass crucible of porosity No. 3, and wash well with more solvent. Dry at l l0°C for 30 minutes, cool, and weigh. -57 - TECHNICAL TRICHLORFON WHO/SIT/13.R1 2. 5 Water Content Determine the water content by the Karl Fischer electrometric titra- tion method (see WHO/M/7, page 266) or, where practicable,1 by the Dean & Stark distillation method (see WHO/M/8, page 270). In the event of a dispute the Karl Fischer method shall govern. 1 At very low water contents, the Dean & Stark method is not reliable. - 58- WHO/SIT/14 TECHNICAL PARATHION-METHYL TECHNICAL PARATHION-METHYL Tentative Specification WHO/SIT/ 14 Approved 25 October 1965 1. SPECIFICATIONS 1.1 Material The material shall comprise essentially O ,0-dimethyl 0-(p-nitrophenyl) phosphorothioate and shall be in the form of a dark liquid, free from extraneous impurities and added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1. 1 and with the following requirements : Minimum Maximum 0 ,0-dimethyl 0-(p-nitrophenyl) phosphoro- thioate content (section 2.1), % by weight Acidity (Method WHO/M/3, page 250), % by weight, calculated as H2S04 • • • • • • Solid material insoluble in acetone (section 2. 2), % by weight . . . . . . . . . . Water content (section 2. 3), by weight . . Specific gravity at 25°C/25°C (section 2 .4) 80.0 1.24 1. 3 Packing and Marking of Packages 83.0 0.2 0.5 0.3 1.26 The technical parathion-methyl shall be packed in suitable, clean con- tainers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical parathion-methyl to Specification WHO /SIT/ 14 Batch or reference number, and date of test Net weight of contents 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. - 59- TECHNICAL PARATHION-METHYL and the following minimum cautionary notice : "POISON [Skull-and-cross-bones insignia] WHO/SIT/14 Parathion-methyl is an organophosphorus compound which inhibits cholinesterase. It is a very toxic substance. Contact with the skin, inhala- tion of dust or spray, or swallowing may be fatal. Wear protective gloves, clean protective clothing, and a respirator of the organic-vapour type when handling this material. Bathe immediately after work. " Ensure that containers are stored under lock and key. Empty con- tainers must be disposed of in such a way as to prevent all possibility of accidental contact with them. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. " In case of contact, immediately remove contaminated clothing and wash the skin thoroughly with soap and water; for eyes, flush with water for 15 minutes. "If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and repeated doses may be necessary. Artificial respira- tion may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 0,0-Dimethyl 0-(p-nitrophenyl) Phosphorothioate Content 2 .1.1 Summary of method Parathion-methyl is separated from p-nitrophenol, the most likely impurity, by mild alkaline extraction of an ether solution. The parathion- methyl in the ether layer is reduced by zinc and an acetic acid/hydrochloric acid mixture, and the amino groups formed are titrated with standard sodium nitrite solution. 2. 1. 2 Special reagents Acetic acid/hydrochloric acid mixture. Mix nine volumes of glacial acetic acid with one volume of concentrated hydrochloric acid. Sodium nitrite, 0.1 M, standardized against su!fanilic acid. Dissolve 6.90 g of sodium nitrite in distilled water and make up to one litre with distilled water. Standardize this solution as follows: Weigh accurately 0.40-0.45 g of anhydrous sulfanilic acid (analytical grade or material, the purity of which has been checked by a nitrogen - 60- WHO/SIT/14 TECHNICAL PARA THI ON-METHYL determination) into a 400-ml tall-form beaker. Add 80 ml of distilled water, 10 ml of concentrated hydrochloric acid, 30 ml of glacial acetic acid, and 5 g of sodium ( or potassium) bromide. Cool the mixture to O- l0°C by the addition of clean, shaved ice and place under mechanical stirring. Titrate at O- l0°C with the O. I M sodium nitrite as rapidly as the spot test (section 2 .1. 3. 3) permits. Near the end-point, add the sodium nitrite in four-drop portions. a x 1000 a x 5.774 Normality ofO.l M sodium nitrite= b x 173 _2 = b where a = weight (g) of sulfanilic acid used b = volume (ml) of 0.1 M sodium nitrite required. 2.1.3 Procedure 2. I . 3 . 1 Separation of parathion-methyl from p-nitrophenol Weigh accurately from a weighing-pipette 0.6-0.9 g of the sample into 100 ml of ether contained in a 250-ml separating funnel. Extract the ether solution four times (or until the extract is colourless) with 20-ml portions of chilled l % (w/v) sodium carbonate solution, discarding the aqueous layers. 2. l . 3. 2 Determination of parathion-methyl in ether layer Transfer the ether layer quantitatively to a 400-ml beaker, using small portions of ether. Add 35 ml of the acetic acid/hydrochloric acid mixture and 3 g of iron-free zinc dust. 1 Cover the beaker with a watch-glass and heat gently on a steam-bath until most of the ether has evaporated and the solution is colourless. Add 10 ml of concentrated hydrochloric acid to complete the solution of the zinc dust. If undissolved portions are present, filter through a sintered-glass crucible and subsequently wash with water. Cool, wash down the beaker and watch-glass with 100 ml of distilled water and add 5 g of sodium (or potassium) bromide. Cool the mixture to 0-10°C by the addition of clean, shaved ice (about 100 g) and place under mechan- ical stirring. Titrate at O- l0°C with the standardized O .1 M sodium nitrite as rapidly as the spot test (section 2. 1. 3. 3) permits ; about 20-30 ml will be required. Near the end-point, add the sodium nitrite in four-drop portions. 2. 1. 3. 3 Spot test Dip a glass rod into the solution to be tested and then touch the rod quickly on a piece of potassium iodide/starch paper. The end-point is 1 If less than 3 g of zinc dust are used or there is reason to doubt the purity of the zinc dust, this should be checked by analysis. - 61 - TECHNICAL PARATHION-METHYL WHO/SIT/14 reached when an intense blue-black colour appears immediately and can be obtained repeatedly during a one-minute period without further addition of sodium nitrite. 2. 1. 4 Calculation 0 ,0-Dimethyl 0-(p-nitrophenyl) phosphorothioate content (% w /w) c x N x 26.33 x f w where N = normality of the 0.1 M sodium nitrite (see section 2 .1. 2) c = volume (ml) of 0.1 M sodium nitrite required w = weight (g) of sample T f=-A where T = quantity (ml) of 0.1 M sodium nitrite calculated for the nitro- group determination of 1 g of a mono-nitro reference sub- stance.1 A = quantity (ml) of 0.1 M sodium nitrite used in the nitro-group determination of 1 g of a recrystallized sample of the same substance following the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its applica- tion by a given laboratory. Its value must lie within the range 0.98-1.02. 2. 2 Solid Material Insoluble in Acetone Weigh accurately about 10 g of the sample into a clean, dry 250-ml flask, add 150 ml of anhydrous acetone, and warm under reflux until all soluble material has dissolved. Filter the solution through a tared Gooch or sintered-glass crucible of porosity No. 3, and wash well with more solvent. Dry at l 10°C for 30 minutes, cool and weigh. 1 Since parathion-methyl is not usually available as a pure material, f is evaluated on the basis of another substance containing one nitro group. Suitable materials are : m-nitrobenzoic acid. p-nitrobenzoic acid . - 62 - Melting-range l40.5-l41.0°C 239.6-240.1 °C WHO/SIT/14 TECHNICAL PARATHION-METHYL 2.3 Water Content Determine the water content by the Karl Fischer electrometric titration method (see WHO/M/7, page 266) or, where practicable,1 by the Dean & Stark distillation method (see WHO/M/8, page 270). In the event of a dispute, the Karl Fischer method shall govern. 2. 4 Specific Gravity Determine the specific gravity at 25°C/25°C by means of a pyknometer. 1 At very low water contents, the Dean & Stark method is not reliable. -63- TECHNICAL FENTHION WHO/SIT/15 TECHNICAL FENTHION Tenta1ive Specification WHO/SIT/ 15 Approved 25 October 1965 1. SPECIFICATIONS 1. 1 Material The material shall comprise essentially 0,0-dimethyl 0-(4-methylthio- m-tolyl) phosphorothioate and shall be in the form of a dark brown liquid, free from extraneous impurities or added modifying agents. 1. 2 Chemical and Physical Requirements The material sampled from any part of the consignment 1 shall comply with the requirements of section 1. 1 and with the following requirements : 0 ,0-Dimethyl 0-( 4-methylthio-m-tolyl) phosphorothioate content (section 2.1), % by weight .......... . Acidity (Method WHO/M/3, page 250), % by weight calculated as H2S04 •• Solid material insoluble in acetone (sec- tion 2.2), % by weight ...... . Water content (section 2. 3), % by weight Minimum 90.0 1. 3 Packaging and Marking of Packages Maximum 0.3 0.5 0.5 The technical fenthion shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical fenthion to Specification WHO/SIT /15 Batch or reference number, and date of test Net weight of contents 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. - 64- WHO/SIT/15 TECHNICAL FENTHION and the following minimum cautionary notice : " Fenthion is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed or absorbed through the skin. Avoid skin contact; wear protective gloves, clean protective clothing and a respirator when handling the material. Wash thoroughly with soap and water after using. Keep the material out of reach of children and well away from foodstuffs, animal food and their containers. "If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed. " 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 0,0-Dimethyl 0-(4-methylthio-m-tolyl) Phosphorothioate Content 2 .1.1 Summary of method The sample is hydrolysed with a solution of potassium hydroxide in ethylene glycol monoethyl ether, and the resulting 4-methylthio-m-cresol, is coupled at pH 10.3-10.4 with diazo-3-nitraniline-4-sulfonic acid to form a red-violet azo dye, the absorbance of which is measured at 530 mµ. The free 4-methylthio-m-cresol, the most likely impurity, is determined by the same method on a sample that has not been hydrolysed. The difference between these values is then taken as the 0,0-dimethyl 0-(4-methylthio- m-tolyl) phosphorothioate content. 2 .1. 2 Special apparatus Spectrophotometer or photoelectric colorimeter capable of measuring absorbance at 530 mµ. 2 .1. 3 Special reagents 4-Methylthio-m-cresol, pure. This material should have a crystallizing point of 56°C + 0.5°C when tested by Method WHO/M/12, page 281. Ethylene glycol monoethyl ether (" ethyl glycol " or Cellosolve). Reflux 1 litre of pure " ethyl glycol " with 50 g of potassium hydroxide pellets for 1 hour, distil, and collect the fraction boiling at 130-140°C. 3-nitraniline-4-sulfonic acid. Dissolve lO g of the reagent in 1000 ml of hot distilled water. Add activated charcoal, filter, and cool the filtrate in ice. Filter off the crystalline acid, wash with water, and dry at 50°C. -65- TECHNICAL FENTHION WHO/SIT/15 Buffer solution. Dissolve 106 g of anhydrous sodium carbonate in distilled water and make up to 1000 ml-solution I. Dissolve 50 g of sodium bicarbonate in distilled water and make up to 1000 ml-solution II. Mix 350 ml of solution I with 130 ml of solution II. The pH of the mixture should be in the range 10.3-10.4 at 20°c. Diazo solution. Dissolve 0.35 g of 3-nitraniline-4-sulfonic acid, purified as described above, in 5 ml of l N sodium hydroxide, add about 350 ml of a mixture of ice and water, followed by 5 ml of concentrated hydro- chloric acid. Diazotize at 0.5°C with 0.1 N sodium nitrite (about 16.5 ml will be required). Determine the end-point of the diazotization by the spot test on starch-potassium iodide paper. Dilute the solution to 500 ml in a volumetric flask and use immediately. 2 .1. 4 Determination of free 4-methylthio-m-cresol 2.1.4. l Preparation of a standard curve Dissolve 0.400 g of pure 4-methylthio-m-cresol in 5 ml of l N sodium hydroxide and 10 ml of isopropanol and make up to the mark in a 1000-ml volumetric flask with distilled water. Transfer 50, 75, 100, 125, 150 and 175 ml of the thoroughly mixed solution into six 500-ml volumetric flasks and dilute to volume with distilled water. The stock solutions ( = S) thus obtained contain 20, 30, 40, 50, 60 and 70 mg of 4-methylthio-m-cresol in 500 ml. Transfer from each of these solutions 5 ml, corresponding to 0.2-0.7 mg of 4-methylthio-m-cresol, into six 50-ml volumetric flasks, add successively to each 10 ml of isopropanol, 20 ml of buffer solution and 10 ml of diazo solution, and make up to the mark with water. After mixing thoroughly, keep the volumetric flasks for two hours in a thermo- statically controlled bath at 20°C. Measure the absorbance in a 1-cm cell at 530 mµ in a suitable photometer, using the reagents as reference. Plot graphically the measured absorbances, corresponding to 0.2-0. 7 mg of 4-methylthio-m-cresol. The calibration curve depends on the photometer used and therefore has to be determined for each instrument individually. Because the standard solution of 4-methylthio-m-cresol deteriorates on standing, a fresh solution should be made each day. 2. l .4 . 2 Procedure Weigh accurately an amount of the sample contammg about 5 mg of this by-product. Rinse the sample with isopropanol into a 50-ml volu- metric flask and fill up to the mark. Shake and filter through a fast paper. Transfer 5 ml of the clear solution into a 50-ml volumetric flask, add successively 10 ml of isopropanol, 20 ml of buffer solution and 10 ml of diazo solution. Continue as described in section 2.1.4.1. Read from the standard curve the amount of 4-methylthio-m-cresol corresponding to the -66- WHO/SIT/15 TECHNICAL FENTHION absorbance. If the amount is not in the range 0.2-0.7 mg, repeat the deter- mination with a smaller or larger sample. 2. 1. 5 Determination of O, 0-dimethyl 0-( 4-methylthio-m-tolyl) phosphoro- thioate 2. l . 5. l Preparation of a standard curve Transfer 5 ml of each of the stock solutions S, the preparation of which is described in section 2. l . 4. l, into 50-ml volumetric flasks, add succes- sively 3 ml of isopropanol from a burette, 30 ml of buffer solution, and 7.5 ml of diazo solution, and fill up to the mark with buffer solution. Continue as described in section 2.1.4. l. Plot graphically the absorbance determined, corresponding to 0.2-0.7 mg of 4-methylthio-m-cresol. The calibration curve depends on the photometer used and therefore has to be determined for each instrument individually. 2.1.5.2 Procedure Weigh accurately about 0.08 g of the sample and rinse with 25 ml of ethylene glycol monoethyl ether into a 100-ml flask. Add about l .65 g of potassium hydroxide (approximately 7 pellets), connect to a reflux condenser, and boil for 7 hours. Cool, transfer to a 500-ml volumetric flask, dilute to the mark with water and mix thoroughly. Transfer 5 ml of this solution into a 50-ml volumetric flask, add successively 3 ml of isopropanol from a microburette, 30 ml of buffer solution, and 7.5 ml of diazo solution. Dilute to volume with buffer solution. Continue as described in section 2.1.4. l. Read from the standard curve the amount of 4-methylthio-m-cresol corresponding to the absorbance. 2 .1. 6 Calculation 0,0-Dimethyl 0-(4-methylthio-m-tolyl) phosphorothioate content (% w/w) [axlO b] = -- - - x l .805 W1 W2 where a = total amount (mg) of 4-methylthio-m-cresol found after hydrolysis in section 2. l. 5. 2. b = amount (mg) of free 4-methylthio-m-cresol found in section 2.1.4.2. w1 = weight (g) of sample used for the determination of the total amount of 4-methylthio-m-cresol in section 2. l . 5. 2. w2 = weight (g) of sample used for the determination of free 4-methylthio-m-cresol in section 2. 1 . 4. 2. -67- TECHNICAL FENTHION WHO/SIT/15 2. 2 Solid Material Insoluble in Acetone Weigh accurately about 10 g of the sample into a clean, dry 250-ml flask add 150 ml of anhydrous acetone, and warm under reflux until all soluble material has dissolved. Filter the solution through a tared Gooch or sintered-glass crucible of porosity No. 3 and wash well with more solvent. Dry at l I0°C for 30 minutes, cool, and weigh. 2.3 Water Content Determine the water content by the Karl Fischer electrometric titration method (see WHO/M/7, page 266) or, where practicable,1 by the Dean & Stark distillation method (see WHO/M/8, page 270). In the event of a dispute the Karl Fischer method shall govern. 1 At very low water contents, the Dean & Stark method is not reliable. -68- WHO/SIT/16 TECHNICAL DICHLORVOS TECHNICAL DICHLORVOS Tentative Specification WHO/ SIT/ 16 Approved 25 October 1965 1. SPECIFICATIONS 1.1 Material The material shall comprise essentially 2,2-dichlorovinyl dimethyl phosphate and shall be in the form of a pale amber-coloured liquid, free from extraneous impurities or added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment 1 shall comply with the requirements of section l. 1 and with the following requirements : 2,2-Dichlorovinyl dimethyl phosphate con- tent (section 2.1), % by weight . . . . Acidity (Method WHO/M/3, page 250), % by weight, calculated as H2S04 Solid material insoluble in acetone (sec- tion 2.3), % by weight ...... . Water content (section 2.4), % by weight . Minimum 93.0 1. 3 Packing and Marking of Packages Maximum 0.1 0.5 0.02 The technical dichlorvos shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical dichlorvos to Specification WHO/SIT /16 Batch or reference number, and date of test Net weight of contents 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. -69- TECHNICAL DICHLORVOS and the following minimum cautionary notice : "POISON [Skull-and-cross-bones insignia] WHO/SIT/16 " Dichlorvos is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed, inhaled or absorbed through the skin. Wear protective gloves, clean protective clothing, goggles and a respirator of the organic-vapour type when handling this material. Avoid prolonged exposure to fumes. Wash hands and exposed skin after handling and before eating, and bathe immediately after work. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. Ensure that containers are tightly sealed and stored and disposed of in such a way as to prevent accidental contact. " In case of contact, immediately remove contaminated clothing and wash the skin thoroughly with soap and water ; for eyes, flush with water for 15 minutes. "If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Infra-Red Method for 2,2-Dichlorovinyl Dimethyl Phosphate Content 1 2 .1.1 Summary of method The sample is diluted with chloroform and the infra-red absorbance measured at about 10.2 µ employing a reference-point technique. This net absorbance is used to obtain the concentration of dichlorvos from previously prepared calibration curve relating net absorbance to con- centration of dichlorvos. 2 .1. 2 Special apparatus I. Infra-red spectrophotometer, capable of recording in the region of 2-15 µ. The slit width and gain must be adjustable in order to give a satisfactory signal/noise ratio and an adequate resolution. In general, a signal/noise ratio of at least 100: I is chosen. A sealed absorption cell with sodium chloride windows, having a path length of about 0.2 mm is required. The path length of the cell need not be known accurately since the same cell is used for both the calibration and the sample measurements. 1 The iodometric method (section 2. 2) may be employed for routine purposes, but in the event of a dispute the infra-red method shall govern. - 70- WHO/SIT/16 TECHNICAL DICHLORVOS 2. Hypodermic syringe, glass, 1.0 ml capacity, fitted with an 18-gauge (Stubbs), 5-cm, slip-on type needle. A Luer type is suitable but not essential. 2. 1. 3 Preparation of calibration graph Into five 10-ml volumetric flasks, weigh, to the nearest 0.1 mg, 25, 75, 100, 150 and 200 mg, respectively, of reference-standard dichlorvos.1 When diluted to the mark with chloroform, these calibration solutions will contain concentrations of dichlorvos of the order of 2.5, 7.5, 10, 15 and 20 g/litre. FIG. 4. DICHLORVOS INFRARED ABSORPTION SPECTRUM - REFERENCE-POINT TECHNIQUE 100 .-----r-----r-----T----,----, 75 ~ lii 3: 8. 50 c 0 ~ '5 ~ 25 p 0 + 9.8 10.0 10.2 10.4 10.6 10.8 Wavelength ( ~) Fill the sealed liquid absorption cell with chloroform. Adjust the spectrophotometer to the optimum settings with respect to gain, slit width, response, chart speed and wave-length scanning speed, and obtain a scan of the chloroform and cell over the 9.9-10.6 µ wave-length range. 1 Samples of reference-standard dichlorvos may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. - 71 - TECHNICAL DICHLORVOS WHO/SIT/16 Without changing the instrument settings, fill the cell, in turn, with each of the calibration solutions starting with the most dilute. Scan each of these solutions over the 9.9-10.6 µ wave-length region. For each of the scans obtained draw perpendiculars to the zero radiation line through the absorption peak of the calibration solution at about 10.2 µ and the reference minima at about 10.0 µ and 10.4 µ. Measure the radiant power P0 and Pas shown in Fig. 4. The distances may be measured in any convenient units, provided the same units are used throughout the determination. Calculate the absorbance as the logarithm of the ratio of the incident power (P0) to the transmitted radiant power (P). Repeat the calculation of the absorbances of the calibration solutions using the reference minimum at about 10.4 µ. Subtract the absorbance of the cell plus chloroform from the absorbances of the cell plus calibration solutions. Make a plot of the net absorbances as ordinate against the corresponding concentrations of dichlorvos in g/litre as abscissa for each of the reference points used, i.e., the absorption minima at about 10.0 µ and 10.4 µ respectively. 2 .1. 4 Analysis of sample Weigh accurately into a 50-ml volumetric flask an amount of sample sufficient to give a I% w /v solution of dichlorvos and dilute to the mark with chloroform. Mix thoroughly and fill the calibrated liquid absorption cell with the sample solution. Using the same instrument settings that were used for the calibration, obtain a scan of the sample solution over the 9.9-10.6 µ region. Calculate the absorbances of the sample solution for the two reference minima as described in section 2. I . 3. 2 .1. 5 Calculation From the computed absorbances (section 2. 1.4) read the concentra- tions of 2,2-dichlorovinyl dimethyl phosphate from the calibration graph (section 2.1.3). 2,2-Dichlorovinyl dimethyl phosphate content (% w/w) a x v w x 10 where a = the mean value (g/litre) determined from the use of two refer- ence minima v = volume (ml) of sample solution w = weight (g) of sample - 72 - WHO/SIT/16 TECHNICAL DICHLORVOS 2. 2 lodometric Method for 2,2-Dichlorovinyl Dimethyl Phosphate Content 2. 2 .1 Summary of method The material is reacted with iodine (a) in the presence of sodium hydroxide and (b) in the presence of sodium carbonate. The difference between the two titrations is taken as a measure of the 2,2-dichlorovinyl dimethyl phosphate content. 2.2.2 Procedure Weigh accurately lg of sample, dissolve in water and make up to 100 ml. Pipette 10 ml of this solution into a flask and add 20.0 ml of O.l N iodine in potassium iodide followed by 20 ml of 2 N sodium hydroxide. Allow to stand for 5 minutes at 20-30°C, then acidify the solution with 20 ml of 5 N hydrochloric acid and titrate with O. l N sodium thiosulfate to the disappearance of colour, using starch indicator. The starch indicator should be added near the end of the titration when the solution has become a pale yellow, and the titration continued until the solution becomes colourless and remains colourless for two minutes. In a second flask pipette another 10.0 ml of the unknown solution and add 5.0 ml of 0. l N iodine in potassium iodide followed by 10 ml of 2 N sodium carbonate solution. Allow to stand for 5-10 minutes, acidify with 20 ml of 5 N hydrochloric acid and titrate as above with 0.1 N sodium thiosulfate. Norn : It is absolutely essential that the iodine be added before the sodium hydroxide or the sodium carbonate, since otherwise hydrolysis will occur and low values will be obtained. It is also advisable that the unknown solution should be freshly made up. 2. 2. 3 Calculation 2,2-Dichlorovinyl dimethyl phosphate content(% w/w) (a-b) x 11.05 IV where a = volume (ml) of O.l N iodine consumed in the presence of sodium hydroxide b = volume (ml) of O.l N iodine consumed in the presence of sodium carbonate IV = weight (g) of sample taken - 73- TECHNICAL DICHLORVOS WHO/SIT/16 2. 3 Solid Material Insoluble in Acetone Weigh accurately about 10 g of the sample into a clean, dry, 250-ml flask, add 150 ml of anhydrous acetone and warm under reflux until all soluble material has dissolved. Filter the solution through a tared Gooch or sintered-glass crucible of porosity No. 3 and wash well with more solvent. Dry at l 10°C for 30 minutes, cool and weigh. 2.4 Water Content The water content is determined by the Karl Fischer electrometric titration method described in Method WHO/M/7, page 266, with the following modifications : 2. 4. 1 Special apparatus 1. A 5-ml microburette calibrated in 0.02-ml divisions. 2. Reaction vessel similar to that described in WHO/M/7, page 266, but having a further inlet for a 5-ml microburette. 2. 4. 2 Special reagents 1. N-ethylpiperidine, catalyst. 2. Ethylene glycol monomethyl ether, anhydrous. Dry by distillation through a glass column, 1 ft (30 cm) long x l in (2.5 cm) internal diameter, packed with single-turn glass helices. The first 5% of distillate is rejected and the distilland is retained. Ethylene glycol monomethyl ether dried in this manner should contain not more than 0.02% by weight of water. 3. Dilute Fischer reagent. Reagent prepared as described in WHO/ M/7 is diluted with dried ethylene glycol monomethyl ether to give a solution of approximately 1 mg/ml water equivalent. 4. Dilute standard solution of water in methanol. Dry thoroughly in an oven a 100-ml graduated flask and allow to cool in a desiccator. Partially fill the flask with anhydrous methanol, add about 0.1 g of distilled water, accurately weighed, from a weighing pipette, make up to the mark with the methanol and mix thoroughly. This solution should be prepared immediately before use. 2.4.3 Standardization of the dilute Fischer reagent Remove moisture from the reaction vessel by the procedure detailed in WHO/M/7. Transfer 10 ml of anhydrous methanol to the reaction vessel and titrate with concentrated Fischer reagent (i.e., approximately - 74- WHO/SIT/16 TECHNICAL DICHLORVOS 3.5 mg/ml). Add l ml of N-ethylpiperidine and titrate with the dilute Fischer reagent (i.e., approximately 1 mg/ml). Ignore this titration. Add to the reaction vessel 5 ml of the dilute standard water solution and again titrate with the dilute Fischer reagent. 50 x w Water equivalent (mg/ml) of the dilute Fischer reagent = --- t where ir = weight (g) of distilled water used in preparing 100 ml of stand- ard solution of water in methanol t = volume (ml) of dilute Fischer reagent used for titration of the dilute standard solution of water in methanol. 2. 4. 4 Analysis of sample Transfer 10 ml of anhydrous methanol to the reaction vessel and titrate with concentrated Fischer reagent as detailed in WHO/M/7. Add 1 ml of N-ethylpiperidine and titrate with dilute Fischer reagent. Ignore this titration. Add to the reaction vessel 15 g of technical dichlorvos and again titrate with the dilute Fischer reagent. t x F Water content (% by weight) = IO wx where F = water equivalent (mg/ml) of the dilute Fischer reagent (sec- tion 2.4.3) = volume (ml) of dilute Fischer reagent used for titration w = weight (g) of sample. - 75 - DDT WATER-DISPERSIBLE POWDERS WHO/SIF/1.R3 DDT WATER-DISPERSIBLE POWDERS* Specification WHO/SIF/1.RJ Approved 25 October 1965 l. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of a homogeneous mixture of technical DDT in a filler and shall be in the form of a fine, free-flowing, white to cream-coloured powder, which wets out readily on stirring into water. The technical DDT used in the manufacture of the water-dispersible powder shall comply with the requirements of Specification WHO/SIT/l.R3. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l. l and with the following requirements. 1.2.1 DDT content (w/w basis) The content of DDT, determined by one of the methods described in section 2. l, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content ± 5% of (100 - nominal content) r The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Sieving after accelerated storage Not less than 98% of the powder (dry weight) after accelerated storage treatment ( section 2. 3) shall pass through a 7 4-µ sieve (BS 200 mesh ; 3 US Standard No. 200 4) when tested by the method described in WHO/M/4, page 253. * If a DDT water dispersible powder for shipment overseas is required, Specifica- tion WHO /SIF /26 should be used. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. i For a 50% water-dispersible powder, tolerance = ± 5% of 50% = == 2.5%. 3 British Standards Institution, British Standard 410: 1943. 4 American Society for Testing Materials, Standard E 11-39. - 76- WHO/SIF/1.R3 DDT WATER-DISPERSIBLE POWDERS 1. 2 . 3 Suspensibility 1 . 2. 3. 1 In distilled water without pretreatment When tested by the method described in section 2. 2, a minimum of 60% of the DDT (1.5% w/v) shall be in suspension 30 minutes after agitating a suspension containing 2.5% w/v of DDT, prepared in distilled water from the powder as received. 1 . 2. 3 . 2 In standard hard water after accelerated storage treatment When tested by the method described in section 2. 2, a minimum of 60% of the DDT (1.5% w/v) shall be in suspension 30 minutes after agitating a suspension containing 2.5% w/v DDT, prepared in standard hard water from powder subjected to the accelerated storage treatment described in section 2. 3. 1. 2.4 Acidity or alkalinity The acidity or alkalinity of the powder, determined by the method described in WHO/M/3, page 250, shall not be greater than 0.2%, calculated as H2S04, nor greater than 0.2%, calculated as NaOH. 1. 3 Packing and Marking of Packages The DDT water-dispersible powder shall be packed in suitable, clean drums, as specified in the order. The drums shall have a minimum capacity of two litres for every kilogram of DDT powder and shall contain an inner- liner or bag of polyethylene or equivalent, with a nominal thickness of 0.1 mm. The inner-liner or bag shall be hermetically sealed after filling. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name DDT water-dispersible powder to Specification WHO/SIF/l .R3 DDT, ... % (w/w) Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : "Keep well away from foodstuffs, animal feed and their containers." -77 - DDT WATER-DISPERSIBLE POWDERS 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 DDT Content WHO/SIF/1.R3 Of the two methods described below, only one need be performed. In the event of a dispute the method described in section 2 .1. 2 shall govern. 2.1.1 Total organic chlorine method (Stepanow method, revised) 2 . 1 . 1 . 1 Procedure Weigh accurately an amount of the sample containing about l g of DDT and extract it quantitatively with chlorine-free and thiophene-free benzene. If a Soxhlet apparatus is used, care must be taken to ensure that extraction is complete and that channelling has not occurred. Con- centrate the extract to such a volume that it can be transferred quanti- tatively to a 100-ml volumetric flask, make up to the mark with benzene, and mix thoroughly. Transfer a 10-ml aliquot to a 250-ml conical flask and add 25 ml of 99% isopropanol. Shake the flask and add 2.5 g of metallic sodium, in the form of ribbon or small pieces. Connect to a reflux condenser and boil gently for at least 1 hour, shaking the flask occasionally. Re- move the excess metallic sodium by cautiously adding 10 ml of 50% aqueous isopropanol through the condenser, at the rate of 1-2 drops per second. Boil for an additional 10 minutes and then add 60 ml of dis- tilled water. Cool, add 2 or 3 drops of phenolphthalein indicator solution, neutralize by adding 50% nitric acid dropwise, and then add 10 ml in excess. Cool, if necessary, to room temperature, add a slight excess of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam- bath for Y2 hour, with frequent stirring. Cool, filter through a fast paper, and wash thoroughly with distilled water. Add 5 ml of I 0% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the total chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. 2. 1 . 1 . 2 Calculation DDT content (% w/w) -78- a xf x 7.092 w WHO/SIF/1.R3 DDT WATER-DISPERSIBLE POWDERS where a = volume (ml) of 0.1 N silver nitrate used w = weight (g) of sample 50.01 f=-A where A = value found for chlorine in recrystallized DDT, when deter- mined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2 .1. 2 Hydrolysable chlorine method 2. 1. 2 . 1 Procedure Weigh accurately an amount of the sample containing about 0.5 g of DDT and extract it quantitatively with acetone. If a Soxhlet apparatus is used, care must be taken to ensure that extraction is complete and that channelling has not occurred. Concentrate the extract to a volume of about 50 ml and add 20 ml of l N ethanolic potassium hydroxide. Keep at 20-25°C for 15 minutes and add 50 ml of distilled water. Add 20 ml of 2 N nitric acid and exactly 25 ml of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam-bath for Y2 hour, with frequent stirring. Cool, filter the coagulated silver chloride through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the hydrolysable chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. 2. 1. 2. 2 Calculation a xf x 3.546 DDT content (% w/w) = IV where a = volume (ml) of 0.1 N silver nitrate used w = weight (g) of sample 10.00 f=-A -79- DDT WATER-DISPERSIBLE POWDERS WHO/SIF/1.Rt where A = value found (%) for hydrolysable chlorine in recrystallized DDT, determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2. 2 Suspensibility 2. 2. 1 Special apparatus 1 I. A 250-ml graduated cylinder with ground-in stopper and a distance of 20-21.5 cm between the bottom and the 250-ml calibration mark. 2. A glass tube, about 40 cm long and about 5 mm in internal diameter, pointed at one end to an opening of 2-3 mm, the other end being con- nected to a vacuum pump. 2. 2. 2 Special reagent Standard hard water. Dissolve 0.304 g of anhydrous calcium chloride and O .139 g of magnesium chloride hexahydrate in distilled water and make up to one litre. This provides water with a hardness of 342 parts per million, calculated as calcium carbonate. 2.2.3 Procedure Weigh accurately into a 100-ml beaker an amount of the sample, treated as described above, to form 250 ml of a suspension containing 2.5% w/v of DDT. Add a volume of water 2 at 30°C± l°C equal to at least twice the weight of the sample taken. Allow to stand for 30 seconds and then stir by hand for 30 seconds with a glass rod, 4-6 mm in diameter, at not more than four revolutions per second, making no deliberate attempt to break any lumps. Then immediately transfer the mixture quantitatively to the 250-ml graduated cylinder, using water 2 at 30°C ± 1 °C for rins- ing, and again avoiding mechanical disintegration of any lumps. Imme- diately add sufficient water 2 at 30°C ± I °C to bring the volume to the 250-ml mark. Stopper the cylinder and mix by inverting and righting it 30 times at a rate of one complete cycle every 2 seconds. This operation should be carried out as smoothly as possible, keeping the axis of rotation 1 A specially designed and fitted graduated cylinder which is suitable for use in this procedure is described in FAO Plant Protection Bulletin, 1962, vol. 10, No. 2, Method 10. 2 Use distilled water for testing the powder without pretreatment and standard hard water for testing the powder after accelerated storage. -80- WHO/SIF/1.R3 DDT WATER-DISPERSIBLE POWDERS fixed. The cylinder must be thermally insulated from the hands to main- tain the prescribed tt:mperature of the suspension. Allow the graduated cylinder to stand for 30 minutes in a water-bath at 30°C ± l °C, care being taken that the bath is free from vibrations. Norn : Should excessive flocculation occur during the test, accom- panied by the appearance of transparent liquid, the material is unsatis- factory. At the end of the settling period (30 minutes), insert the glass tube into the cylinder and, with a minimum of disturbance, withdraw during l0-15 seconds by means of the vacuum pump nine-tenths of the suspension, i.e., 225 ml. This is achieved by maintaining the tip of the glass tube just below the sinking top level of the suspension. Discard the suspension withdrawn. Determine the weight of DDT in the retained one-tenth of the suspen- sion, including the sediment, by one of the methods described in sec- tion 2. l. 2. 2. 4 Calculation From the value obtained in section 2.1 for the percentage content of DDT, calculate the weight of DDT in the initial sample taken for the suspensibility test. (b - a) x 111.l Suspensibility (%) = b where a= weight (g) of DDT in the retained one-tenth of the suspension b = weight (g) of DDT in the initial sample. 2. 3 Accelerated Storage Treatment Place 20 g of the sample in a 250-ml beaker having an internal diameter of 6-6.5 cm and level off without compacting. Place within the beaker, on top of the powder, a loose-fitting piston or disc so formed and weighted as to exert upon the powder an even pressure of 25 gf/cm2 • Keep the powder thus under pressure in an oven at 54°C ± 1 °C for 24 hours. Take the sample from the oven, remove the pressure assembly, and allow the powder to come naturally to room temperature in a closed container. After completion of the accelerated storage treatment, the sample should not be exposed to heat or to bright sunshine. Unnecessary exposure of the sample to high atmospheric humidity must also be avoided. Proceed with suspensibility test as described in section 1. 2. 3. 2. - 81 - 6 DDT WATER-DISPERSIBLE POWDERS FOR OVERSEAS SHIPMENT WHO/SIF/26 DDT WATER-DISPERSIBLE POWDERS FOR OVERSEAS SHIPMENT Specification WHO/SIF/26 Approved 25 October 1965 1. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of a homogeneous mixture of technical DDT and such inert diluents and other materials as are needed to meet the requirements of this specification. The product shall be in the form of a fine, free-flowing, white to cream-coloured powder which wets out readily on stirring into water. The technical DDT used in the manufacture of the water-dispersible powder shall comply with the requirements of Specification WHO/SIT/l.R3. 1. 2 Chemical and Physical Requirements The material sampled from any part of the consignment,1 shall comply with the requirements of section 1 . 1 and with the following requirements : 1.2.1 DDT content (w/w basis) The content of DDT, determined by one of the methods described in section 2. 1, shall not differ from the nominal content by more than the following amounts : Nominal content Tolerance permitted 50% and above ± 5% of (100 - nominal content) 2 The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Sieving after accelerated storage Not less than 98% of the powder (dry weight) after accelerated storage treatment (section 2.4) shall pass through a 74-µ sieve (BS 200 mesh; 3 US Standard No. 200 4) when tested by the method described in section 2.3. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. Samples for the Storage Life Test (section l. 2. 5) should be taken at the same time as samples are taken for tests for requirements in sections l.2.1, l.2.2, l.2.3 and l.2.4. 2 For a 75% water-dispersible powder, tolerance= ± 5%of(I00-75)% = ± l.25%. 3 British Standards Institution, British Standard 410 : 1943. 4 American Society for Testing Materials, Standard E 11-39. -82- WHO/SIF/26 DDT WATER-DISPERSIBLE POWDERS FOR OVERSEAS SHIPMENT 1. 2. 3 Suspensibility I . 2. 3. I In distilled water without pretreatment When tested by the method described in section 2. 2, a minimum of 70% of the DDT (l.75% w/v) shall be in suspension 30 minutes after agitating a suspension containing 2.5% w/v of DDT, prepared in distilled water from the powder as received. I . 2. 3. 2 In standard hard water after accelerated storage treatment When tested by the method described in section 2. 2, a minimum of 65% of the DDT (l.625% w/v) shall be in suspension 30 minutes after agitating a suspension containing 2.5% w/v of DDT, prepared in standard hard water from the powder subjected to the accelerated storage treatment described in section 2.4. 1. 2. 4 Acidity or alkalinity The acidity or alkalinity of the powder, determined by the method described in WHO/M/3, page 250, shall not be greaterthan0.2%, calculated as H2S04, nor greater than 0.2%, calculated as NaOH. 1. 2. 5 Storage life 1 Any sample 2 of powder taken from a consignment at the time of offer by the manufacturer and stored in moisture-proof containers at an ambient temperature between 21 °C and 38°C for a period of up to 12 months shall have a minimum of 50% (l.25% w/v) of the DDT in suspension 30 minutes after agitating a suspension containing 2.5% w/v of DDT prepared in standard hard water when tested by the method described in section 2. 2. 1 This requirement may be waived in certain circumstances, but it should be main- tained for all purchases of DDT powders intended for use in malaria eradication pro- grammes, in order to ensure that powders meeting all other requirements will maintain minimum suspensibility for a period of at least 12 months. It is recommended that before the end of the 12-month storage period, the purchaser or his agent should examine at his discretion a part or all of the batch samples held in storage for compliance with this requirement. The following procedure is suggested: (1) After at least nine months in storage, examine 20% of the batch samples in a given consignment by the visual suspensibility test (Method WHO/M/2, page 249) conducted in standard hard water without pretreatment. If any of the samples exhibit questionable suspensibility, retest them by the procedure described in section 2.2, using standard hard water. (2) If any of the samples so examined fail to pass the test, all the batch samples in the consignment should be examined as described in (1). 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. Samples should be taken at the same time as those for the tests described in sections 1 . 2. 1, 1.2.2, 1.2.3 and 1.2.4. - 83- DDT WATER-DISPERSIBLE POWDERS FOR OVERSEAS SHIPMENT WHO/SIF/26 1. 3 Packing and Marking of Packages The DDT water-dispersible powder shall be packed in suitable, clean drums or boxes 1 as specified in the order. The containers shall have a minimum capacity of two litres for every kilogram of powder and shall contain an inner-liner or bag of polyethylene or equivalent, with a nominal thickness of 0.1 mm. The inner-liner or bag shall be hermetically sealed after filling. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name DDT water-dispersible powder to Specification WHO /SIF /26 DDT, ... % (w/w) Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Keep well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 DDT Content Of the two methods described below, only one need be performed. In the event of a dispute the method described in section 2. 1. 2 shall govern. 2.1.1 Total organic chlorine method (Stepanow method, revised) 2.1.1.1 Procedure Weigh accurately an amount of the sample containing about 1 g of DDT and extract it quantitatively with chlorine-free and thiophene-free benzene. If a Soxhlet apparatus is used, care must be taken to ensure that extraction is complete and that channelling has not occurred. Con- centrate the extract to such a volume that it can be transferred quanti- tatively to a 100-ml volumetric flask, make up to the mark with benzene, and mix thoroughly. Transfer a 10-ml aliquot to a 250-ml conical flask and add 25 ml of 99% isopropanol. Shake the flask and add 2.5 g of metallic sodium, 1 Interim specifications for boxes of adequate strength to withstand overseas ship· ment may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. - 84- WHO/SIF/26 DDT WATER-DISPERSIBLE POWDERS FOR OVERSEAS SHIPMENT in the form of ribbon or small pieces. Connect to a reflux condenser and boil gently for at least 1 hour, shaking the flask occasionally. Remove the excess metallic sodium by cautiously adding 10 ml of 50% aqueous isopropanol through the condenser, at the rate of 1-2 drops per second. Boil for an additional 10 minutes, and then add 60 ml of distilled water. Cool, add 2 or 3 drops of phenolphthalein indicator solution, neutralize by adding 50% nitric acid dropwise, and then add 10 ml in excess. Cool, if necessary, to room temperature, add a slight excess of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digest- ing on a steam-bath for Y2 hour, with frequent stirring. Cool, filter through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the total chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. 2. 1 . 1 . 2 Calculation a x f x 7.092 DDT content(% w/w) = ·--·--- w where a = volume (ml) of 0.1 N silver nitrate used w = weight (g) of sample 50.0l f A where A = value(%) found for chlorine in recrystallized DDT, when deter- mined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2 .1. 2 Hydrolysable chlorine method 2.1.2.1 Procedure Weigh accurately an amount of the sample containing about 0.5 g of DDT and extract it quantitatively with acetone. If a Soxhlet apparatus is used, care must be taken to ensure that extraction is complete and that channelling has not occurred. Concentrate the extract to a volume of about 50 ml and add 20 ml of 1 N ethanolic potassium hydroxide. -85 - DDT WATER-DISPERSIBLE POWDERS FOR OVERSEAS SHIPMENT WHO/SIF/26 Keep at 20-25°C for 15 minutes and add 50 ml of distilled water. Add 20 ml of 2 N nitric acid and exactly 25 ml of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam-bath for '/2 hour, with frequent stirring. Cool, filter the coagulated silver chloride through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the hydrolysable chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. 2 . l . 2 . 2 Calculation a xf x 3.546 DDT content (% w/w) = ----- w where a = volume (ml) of 0.1 N silver nitrate used w = weight (g) of sample 10.00 f=-A where A = value (%) found for hydrolysable chlorine in recrystallized DDT, when determined by the above-described method an using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2. 2 Suspensibility 2. 2. 1 Special apparatus l. A 100-ml glass-stoppered graduated cylinder having the 100-ml mark situated 18.0 cm ± 1.5 cm from the bottom and 5.5 cm ± 0.5 cm from the top excluding the neck. 2. A 25-ml pipette fitted with a device to permit its insertion into the 100-ml cylinder so that it is held with the tip exactly at the 50-ml mark. This may be accomplished by fitting the pipette with a stopper having a centre bore for the stem and a shallow V-cut on one side as an air vent. - 86 - WHO/SIF/26 DDT WATER-DISPERSIBLE POWDERS FOR OVERSEAS SHIPMENT 3. A constant-temperature water-bath into which the 100-ml graduated cylinders can be immersed to the 100-ml mark and which can be maintained at 30°C ± l °C. The bath must be free from any vibration caused by stirring motors or other equipment. 2. 2. 2 Special reagent Standard hard water. Dissolve 0.304 g of anhydrous calcium chloride and O .139 g of magnesium chloride hexahydrate in distilled water and make up to one litre. This provides water with a hardness of 342 parts per mil- lion, calculated as calcium carbonate. 2.2.3 Procedure Weigh accurately into a 100-ml beaker an amount of the sample to form 100 ml of a suspension containing 2.5% w/v of DDT. Add 50 ml of water 1 at 30°C ± l °C, and allow to stand for 30 seconds. Stir the mixture with a glass rod by hand for 30 seconds and then transfer to the 100-ml graduated cylinder using additional water 1 for the transfer. Add sufficient water 1 at 30°C ± l °C to make 100 ml of suspension. Stopper the cylinder and mix by inverting and righting it 30 times at the rate of approximately one cycle every two seconds. This operation should be carried out as smoothly as possible, keeping the axis of rotation fixed. The cylinder must be thermally insulated from the hands to maintain the prescribed temperature of the suspension. Immerse the cylinder up to the 100-ml mark in the water-bath maintained at 30°C ± l °C. The prepara- tion of the suspension from the first addition of water to the placing of the cylinder in the constant-temperature bath should be a continuous operation and should be completed within three minutes. At the end of the 30-minute settling period, remove the cylinder from the water-bath, insert the specially fitted pipette so that the tip is exactly at the 50-ml mark and remove a 25-ml aliquot. (If this test is being per- formed on a sample after accelerated storage treatment [section 2.4], the remaining 75 ml of the suspension should be retained for the sieving test as described in section 2. 3). Transfer the 25-ml aliquot to a glass evaporat- ing dish of approximately 84 mm diameter x 45 mm depth and evaporate the water in a forced-draft oven at l00°C. Remove the dish from the oven as soon as the last traces of water have evaporated in order to avoid over- heating the sample. 1 Use distilled water for testing the powder without pretreatment (section l. 2. 3. l) and standard hard water for testing the powder after accelerated storage treatment (section l. 2. 3. 2) and after storage at 21-38°C for the Storage Life Test (section l. 2. 5). - 87 - DDT WATER-DISPERSIBLE POWDERS FOR OVERSEAS SHIPMENT WHO/SIF/26 The DDT content of the residue is determined by one of the methods described in section 2. 1 or by the method given in section 2. 2 .4. In the event of a dispute, the method described in section 2 .1. 2 shall govern. 2. 2. 4 DDT content of aliquot by gravimetric method The DDT is extracted quantitatively from the residue obtained in section 2. 2. 3 and estimated gravimetrically as follows : Add approximately 15 ml of benzene to the dried sample obtained in section 2. 2. 3 and allow to stand for about ten minutes to aid extraction. Prepare a filter by loosely packing the stem of a filter tube, 1 approximately 32 x 160 mm, with benzene-washed cotton wool. Wet the cotton wool well with benzene and filter the sample into a tared 50-ml beaker. Add a fresh 15-ml portion of benzene to the evaporating dish and scrub the bottom and sides with a rubber policeman to ensure complete extraction of the DDT. After the first extract has filtered into the beaker, pour the second portion through the filter tube ; after this has filtered through, rinse the tube with about 5 ml of benzene. Finally, wash the tip of the filter tube with a small amount of benzene from a wash-bottle. Evaporate the benzene in a 60°C water-bath under a gentle current of dry air. After the volume of benzene has evaporated to about 10 ml, add approximately 3 ml of 99% isopropanol and continue evaporating until about 5 ml of solution remains. Add a second 3-ml portion of 99% isopropanol and evaporate to dryness. Transfer the beaker to a forced-draft oven at 65°C and dry for 20 minutes. Weigh the residue and calculate as technical DDT (grams). 2. 2. 5 Calculation Calculate the suspensibility of the samples as follows : Suspensibility (%) = a x 160 where a = weight (g) of DDT found in the 25-ml aliquot. 2. 3 Sieving Test after Accelerated Storage Pour the 75 ml of suspension retained from the suspensibility test (see section 2. 2. 3) on to a 74-µ sieve and proceed with the sieving test as described in Method WHO/M/4, page 253. Jn this procedure it is assumed that all the particles in the 25-ml aliquot taken from the centre of the suspension would have passed through the 74-µ sieve. 1 Corning No. 9480 or equivalent. -88- WHO/SIF/26 DDT WATER-DISPERSIBLE POWDERS FOR OVERSEAS SHIPMENT 2. 4 Accelerated Storage Treatment Weigh 5 g of the powder into a 25-mm x 200-mm test tube. If the height of the powder in the tube exceeds 6.0 cm, gently tap the tube until the level is reduced to 6.0 cm. Immerse the tube to a depth of at least 9.0 cm in an oil bath maintained at 70°C ± O.l°C. The bath should be equipped with an electric stirrer and the tube must not be stoppered. Allow the sample to remain in the bath for two hours, then remove and cool to room temperature. -89- HCH WATER-DISPERSIBLE POWDERS WHO/SIF/2.R3 HCH WATER-DISPERSIBLE POWDERS Specification WHO/SIF/2.RJ Approved 25 October 1965 1. SPECIFICATIONS 1. 1 Description and Ingredients The material shall consist of a homogeneous mixture of technical HCH, refined HCH, or lindane in a filler and shall be in the form of a fine, free- flowing, white to cream-coloured powder, which wets out readily on stirring into water.1 The HCH used in the manufacture of the water-dispersible powder shall comply with the requirements of Specification WHO/SIT/ 2.R3 or WHO/SIT/3.R3. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,2 shall comply with the requirements of section 1. 1 and with the following requirements. 1. 2 .1 Gamma-isomer content (w/w basis) The content of gamma-isomer, determined by the method described in section 2. 1, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 3 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. 1 If substantial freedom from odour is required, this should be specified in the order. 2 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 3 For a 50% water-dispersible powder, tolerance= ± 5% of 50% = ± 2.5%; for a 75% water-dispersible powder, tolerance= ± 5% of (100- 75)% = ± l.25%. - 90- WHO/SIF/2.R3 HCH WATER-DISPERSIBLE POWDERS 1. 2. 2 Sieving after accelerated storage Not Jess than 98% of the powder (dry weight) after accelerated storage treatment (section 2. 3) shall pass through a 74-µ sieve (BS 200 mesh; 1 US Standard No. 200 2 when tested by the method described in WHO/ M/4, page 253. 1. 2. 3 Suspensibility 1 . 2. 3. 1 In distilled water without pretreatment When tested by the method described in section 2.2,3 a minimum of 50% of the gamma-isomer (0.25% w/v) shall be in suspension 30 minutes after agitating a suspension containing 0.5% w /v of gamma-isomer, prepared in distilled water from the powder as received. 1 . 2. 3 . 2 In standard hard water after accelerated storage treatment When tested by the method described in section 2.2,3 a minimum of 50% of the gamma-isomer (0.25% w/v) shall be in suspension 30 minutes after agitating a suspension containing 0.5% w/v of gamma-isomer, prepared in standard hard water from the powder subjected to accelerated storage treatment described in section 2. 3. 1. 2. 4 Acidity or alkalinity The acidity or alkalinity of the powder, determined by the method described in WHO/M/3, page 250, shall not be greater than 0.2%, cal- culated as H2S04, nor greater than 0.2%, calculated as NaOH. 1. 3 Packing and Marking of Packages The HCH water-dispersible powder shall be packed in suitable, clean drums, as specified in the order. The drums shall have a minimum capacity of two litres for every kilogram of powder and shall contain an inner-liner or bag of polyethylene or equivalent, with a nominal thickness of 0.1 mm. The inner-liner or bag shall be hermetically sealed after filling. All packages shall bear, durably and legibly marked on the container, the following : 1 British Standards Institution, British Standard 410: 1943. 2 American Society for Testing Materials, Standard E 11-39. 3 HCH water-dispersible powders with a higher gamma-isomer content are in some instances used in the field at a higher concentration than 0.5%. The test on suspensibility should then be carried out at this higher concentration, by agreement between the manu- facturer and purchaser. - 91 - HCH WATER-DISPERSIBLE POWDERS WHO/SIF/2.R3 Manufacturer's name HCH water-dispersible powder to Specification WHO /SIF /2. R3 Gamma-isomer, . . . % w /w Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : "Keep well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 Gamma-Isomer Content 1 Depending on the content of carrier (including wetting and dispersing agents) used in the manufacture of the powder, extract the sample in one of the following two ways : I. Water-dispersible powder containing up to 25% of carrier. Weigh accurately an amount of the sample containing 60-80 mg of gamma-isomer into a conical flask, add 15 ml of light petroleum,2 and heat under reflux for 3 minutes on a water-bath maintained at 60°C. Cool and decant on to the column.2 Treat the residue once more with 10 ml of light petro- leum and transfer the solution with the residue quantitatively to the column, rinsing the flask with a few millilitres of light petroleum. 2. Water-dispersible powder containing more than 25% of carrier. Weigh accurately an amount of the sample containing 60-80 mg of gamma-isomer into a conical flask, add 15 ml of light petroleum,2 and heat under reflux for 3 minutes on a water-bath maintained at 60°C. Cool and decant on to the column.2 Treat the residue with another 15-ml portion of light petroleum and transfer the solution quantitatively to the column. Repeat this operation three times, using successively 10 ml, 5 ml, and 5 ml of light petroleum, and transfer the solutions, and finally the residue, quanti- tatively to the column, rinsing the flask with a few millilitres of light petroleum. Proceed with the chromatographic separation and spectrophotometric determination as described in WHO/M/6, page 258. 1 The revised Stepanow (total organic chlorine) method and the hydrolysable chlorine method, described respectively in Methods WHO/M/16, page 288, and WHO/M/17, page 291, are not specific. However, this does not preclude a purchaser or user from applying these methods for routine purposes where the gamma-isomer content of the technical HCH used in the manufacture of the water-dispersible powder is known and when the use of the method has been agreed to between all parties concerned. 2 Light petroleum as specified in WHO/M/6, page 260, should be used. - 92 - WHO/SIF/2.R3 HCH WATER-DISPERSIBLE POWDERS Norn : If further removal of the dispersing or other auxiliary agents used in the manufacture of the water-dispersible powder is necessary to ensure good separation (see WHO/M/6, page 262), proceed as fol- lows: Weigh accurately an amount of the sample containing 60-80 mg of gamma-isomer, add 100 ml of pure methanol, and heat under reflux for 20 minutes. Filter through a crucible and rinse the residue with 50 ml of pure methanol. Combine the filtrates and distil off the solvent. Dissolve the residue in 30 ml of benzene and pass the solution through a column of 4 cm internal diameter containing a 5-cm layer of aluminium oxide. Elute the HCH from the column with 400 ml of ethyl acetate and distil off the solvent. Dissolve the residue in light petroleum and transfer the solu- tion quantitatively to the silica-gel column. 2. 2 Suspensibility 2. 2 .1 Special apparatus 1 1. A 250-ml graduated cylinder with ground-in stopper and a distance of 20-21.5 cm between the bottom and the 250-ml graduation. 2. A glass tube, about 40 cm long and about 5 mm in internal diameter, drawn out at one end to an opening of 2-3 mm, the other end being con- nected to a vacuum pump. 2. 2. 2 Special reagent Standard hard water. Dissolve 0.304 g of anhydrous calcium chloride and 0.139 g of magnesium chloride hexahydrate in distilled water and make up to one litre. This provides water with a hardness of 342 parts per million, calculated as calcium carbonate. 2.2.3 Procedure Weigh accurately into a 100-ml beaker an amount of the sample, treated as described above, to form 250 ml of a suspension containing 0.5% (w/v) of gamma-isomer. Add a volume of water2 at 30°C ± l°C equal to at least twice the weight of the sample taken. Allow to stand for 30 seconds and then stir by hand for 30 seconds with a glass rod, 4-6 mm in diameter, at not more than four revolutions per second, making no deliberate attempt to break any lumps. Then immediately transfer the mixture quantitatively to the 250-ml graduated cylinder, using water 2 at 30°C ± l °C for rinsing, and again avoiding mechanical disintegration 1 A specially designed and fitted graduated cylinder suitable for use in this procedure is described in FAO Plant Protection Bulletin, 1962, vol. 10, No. 2, Method 10. 2 Use distilled water for testing the powder without pretreatment and standard hard water for testing the powder after accelerated storage. - 93- HCH WATER-DISPERSIBLE POWDERS WHO/SIF/2.R3 of any lumps. Immediately add sufficient water1 at 30°C ± l °C to bring the volume to the 250-ml mark. Stopper the cylinder and mix by inverting and righting it 30 times at a rate of one complete cycle every 2 seconds. This operation should be carried out as smoothly as possible, keeping the axis of rotation fixed. The cylinder must be thermally insulated from the hands to maintain the prescribed temperature of the suspension. Allow the graduated cylinder to stand for 30 minutes in a water-bath at 30°C ± l °C, care being taken that the bath is free from vibrations. Norn : Should excessive flocculation occur during the test, accompanied by the appearance of transparent liquid, the material is unsatisfactory. At the end of the settling period (30 minutes), insert the glass tube into the cylinder and, with a minimum of disturbance, withdraw during 10-15 seconds by means of the vacuum pump nine-tenths of the suspension, i.e., 225 ml. This is achieved by maintaining the tip of the glass tube just below the sinking top level of the suspension. Discard the suspension withdrawn. Add to the retained one-tenth of the suspension, including the sediment, enough anhydrous sodium sulfate to absorb all the water present. Extract eight times with 25-ml portions of light petroleum. Determine whether extraction is complete by extracting once more with 5 ml of light petro- leum and distilling off the solvent ; there shall be no visible residue from this last extract. Combine all extracts and distil off the solvent to a volume of 20-30 ml. Transfer quantitatively to the chromatographic column and proceed as described in WHO/M/6, page 262. NOTE : It is important that the sample transferred to the chromatographic column should not contain more than 60-80 mg of gamma-isomer, in order to ensure satisfactory separation of the isomers. If the sample to be trans- ferred contains more than this amount, an aliquot should be taken to prevent overloading of the column. 2.2.4 Calculation From the value obtained for the percentage content of gamma-isomer in the test described in section 2.1 and WHO/M/6, page 258, calculate the weight of gamma-isomer in the initial sample taken for the suspensibility test. S .bT (%) (b - a) x 111.l uspens1 1 1ty O = b 1 Use distilled water for testing the powder without pretreatment and standard hard water for testing the powder after accelerated storage. - 94- WHO/SIF/2.R3 where a b HCH WATER-DISPERSIBLE POWDERS weight (g) of gamma-isomer in the retained one-tenth of the suspension weight (g) of gamma-isomer in the initial sample. 2. 3 Accelerated Storage Treatment Place 20 g of the sample in a 250-ml beaker having an internal diameter of 6-6.5 cm and level off without compacting. Place within the beaker, on top of the powder, a loose-fitting piston or disc so formed and weighted as to exert upon the powder an even pressure of 25 gf/cm 2• Keep the powder thus under pressure in an oven at 54°C ± 1 °C for 24 hours. Take the sample from the oven, remove the pressure assembly, and allow the powder to come naturally to room temperature in a closed container. After completion of the accelerated storage treatment, the sample should not be exposed to heat or to bright sunshine. Unnecessary exposure of the sample to high atmospheric humidity must also be avoided. Proceed with the suspensibility test as described in section l. 2. 3. 2. - 95- DIELDRIN WATER-DISPERSIBLE POWDERS WHO/SIF/3.R3 DIELDRIN WATER-DISPERSIBLE POWDERS* Specification WHO/SIF/3.RJ Approved 25 October 1965 l. SPECIFICATIONS 1. 1 Description and Ingredients The material shall consist of a homogeneous mixture of technical dieldrin in a filler and shall be in the form of a fine, free-flowing, white to cream-coloured powder, which wets out readily on stirring into water. The technical dieldrin used in the manufacture of the water-dispersible powder shall comply with the requirements of Specification WHO/SIT/ 6.R3. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1.1 and with the following requirements. 1.2.1 Dieldrin content (w/w basis) The content of dieldrin, determined by the method described in sec- tion 2. 1, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 2 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. • In the ninth report of the WHO Expert Committee on Insecticides (unpublished), the decision was recorded that the following definitions should be used for the purposes of this specification : "'Dieldrin' is to refer to an insecticide containing 85% by weight of HEOD. The amount of dieldrin in any sample will therefore be calculated from the following formula : Dieldrin (% by weight) = HEOD (% by weight) x ~;o. " " 'Technical dieldrin' is to refer to a product containing a minimum of 90% by weight of dieldrin, i.e., a minimum of 76.5% by weight of HEOD." 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 For a 50% water-dispersible powder, tolerance = ± 5% of 50% = ± 2.5%; for a 75% water-dispersible powder, tolerance = ± 5% of (100- 75)% = ± l.25%, -96- WHO/SIF/3.R3 DIELDRIN WATER-DISPERSIBLE POWDERS 1. 2. 2 Sieving after accelerated storage Not less than 98% of the powder (dry weight) after accelerated storage treatment (section 2.3) shall pass through a 74-µ sieve (BS 200 mesh; 1 US Standard No. 200 2) when tested by the method described in WHO/ M/4, page 253. 1. 2. 3 Suspensibility l. 2. 3. l In distilled water without pretreatment When tested by the method described in section 2. 2, a minimum of 80% of the dieldrin (0.500% w/v) shall be in suspension 30 minutes after agitating a suspension containing 0.625% w/v of dieldrin, prepared in distilled water from the powder as received. l. 2. 3. 2 In standard hard water. after accelerated storage treatment When tested by the method described in section 2. 2, a minimum of 60% of the dieldrin (0.375% w/v) shall be in suspension 30 minutes after agitating a suspension containing 0.625% w/v of dieldrin, prepared in standard hard water from the powder subjected to the accelerated storage treatment described in section 2. 3. 1. 2. 4 Acidity or alkalinity The acidity or alkalinity of the powder, determined by the method described in WHO/M/3, page 250, shall not be greater than 0.2%, calcu- lated as H2S04, nor greater than 0.5%, calculated as NaOH. 1.2.5 Water content The water content of the powder shall not be more than 2.0% when determined by the method given in WHO/M/8, page 270. 1. 3 Packing and Marking of Packages The dieldrin water-dispersible powder shall be packed in suitable, clean drums, as specified in the order. The drums shall have a minimum capacity of 2 litres for every kilogram of powder and shall contain an inner-liner or bag of polyethylene or equivalent, with a nominal thickness of 0.1 mm. The inner-liner or bag shall be hermetically sealed after filling. All packages shall bear, durably and legibly marked on the container, the following : 1 British Standards Institution, British Standard 410: 1943. 2 American Society for Testing Materials, Standard E 11-39. -97- 7 DIELDRIN WATER-DISPERSIBLE POWDERS WHO/SIF/3.R3 Manufacturer's name Dieldrin water-dispersible powder to Specification WHO/SIF /3. R3 Dieldrin, ... % w/w Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Dieldrin is a toxic substance and may cause convulsions. It is poison- ous if swallowed. It may be absorbed through the skin or inhaled as dusts or mists. Avoid skin contact; wear protective gloves and clean protective clothing while using the material. Wash thoroughly with soap and water after using. Keep the material out of reach of children and well away from foodstuffs, animal feed, and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Dieldrin Content 1 2 .1.1 Summary of method The sample is extracted with hexane and the extract is evaporated to dryness and weighed. The residue is dissolved in carbon disulfide and the solution is made up to a fixed volume with carbon disulfide. The infra- red spectrum of this solution is scanned in the wave-length region 10.0- 13.0 µ. The absorbance of the solution is determined at 11.0 µ, 11.8 µ, and 12.35 µ, and the quantity of HEOD present is obtained by comparing the observed readings with a calibration graph relating absorption to various concentrations of HEOD. 2. 1. 2 Special apparatus l. Chromatographic column, constructed as shown in Fig. 5 and charged with 3 g of activated charcoal. 2. Infra-red spectrometer, capable of recording in the region 10.0-13.0 µ. The slit width and gain must be adjustable in order to give a satisfactory signal/noise ratio and an adequate resolution. In general, a signal/noise ratio of atout 10::> : l is chosrn. A sealed absorption-cell with sodium chloride windows and a path length of about 0.4 mm is required. 3. Hypodermic glass syringe, of 1.0-ml capacity, fitted with an 18-gauge (Stubbs), 5-cm, slip-on type needle. A Luer type is also suitable. 1 The revised Stepanow (total organic chlorine) method (see Method WHO/M/16, page 288) may be used for routine purposes, but in the event of a dispute the method described here shall govern. - 98- WHO/SIF/3.R3 DIELDRIN WATER-DISPERSIBLE POWDERS FIG. 5. CHROMATOGRAPHIC COLUMN FOR EXTRACTION OF DIELDRIN 100 mm (approx.) 260 mm (approx.) ---a -b •ttO "))9 A = air pressure C = sintered-glass plate of medium porosity B = activated charcoal D = wide-mouthed conical flask or beaker a = outside diameter 60 mm b = Inside diameter 20 mm 2 .1. 3 Special reagents Reproduced by kind permission of the Shell International Chemical Compan.v, Ltd, London. 1. Hexane. A commercial C6 petroleum fraction having a boiling-range of 62-68°C and a paraffin content of at least 98%, and leaving on evapora- tion a residue of less than 0.0016% (w/w). - 99- DIELDRIN WATER-DISPERSIBLE POWDERS WHO/SIF/3.R3 2. Extraction solvent. Commercial hexane (as defined above) containing 5% (v/v) of acetone. 3. HEOD standard solutions. Into three 10-ml volumetric flasks, weigh accurately 40 mg, 80 mg, and 120 mg of recrystallized HEOD (melting point l 78°C).1 Dissolve in carbon disulfide, make up to the mark, and mix thoroughly. These solutions contain respectively 0.4 g, 0.8 g, and 1.2 g of HEOD per 100 ml.2 2. 1. 4 Preparation of calibration graph Fill the absorption cell with the most dilute of the standard solutions by means of the hypodermic syringe. Adjust the spectrometer to the optimum settings with respect to gain, slit width, response, speed, and drum drive. Make replicate scans of the solution over the wave-length region 10.0-13.0 µ. Fill the cell with the other two standard solutions in turn. Make replicate scans of each solution in the same wave-length region, using the same instrument settings as above. For each of the scans of the three different solutions. calculate the absorbance (E) at the band maxima as follows : (l) Atll.0µ,E=log(;:) transmission at 11.0 µ (peak maximum) transmission at 10.85 µ (peak minimum). (2) At 11.8 µ, E =log(;:) transmission at 11.8 µ (peak maximum) transmission at 12.15 µ (peak minimum). ( p \ (3) At 12.35 µ, E = log P;) where P1 = transmission at 12.35 µ (peak maximum) P0 = transmission at 12.15 µ (peak minimum). For each of the three wave-lengths corresponding to the peak maxima, plot the absorbances observed for the three standard solutions against their 1 Samples of pure HEOD may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. 2 The concentrations of HEOD given are specific for a 0.4-mm cell and should be adjusted for different cell lengths in order to give a transmission of between 20% and 80% at the peak maxima. -100 - WHO/SIF/3.R3 DIELDRIN WATER-DISPERSIBLE POWDERS HEOD content. An example of the calibration graph obtained is shown in Fig. 3 (see page 32). NOTE: Whilst the chosen characteristic maxima for HEOD occur at about 11.0 µ, 11.8 µ, and 12.35 µ, measurements of the absorbance should be made at the actual peak maxima and not at exactly the wave-lengths mentioned. This applies also to the peak minima at 10.85 µ and 12.15 µ. 2.1.5 Procedure 2. l . 5. l Extraction of dieldrin Mix the sample thoroughly and weigh accurately an amount containing about l g of dieldrin. Transfer quantitatively to the chromatographic column, tamping or vibrating the material slightly to settle the contents of the tube. Place a 600-ml beaker under the tip of the column. Working in a well-ventilated hood, add extraction solvent to the top of the column. Allow 300 ml of the solvent to percolate through into the beaker. Replace the 600-ml beaker by a 100-ml beaker and collect further 50-ml portions of the solvent, changing the 100-ml receiver after each 50-ml portion has been collected. Whilst the percolation is in progress, evaporate the solvent from the 600-ml and 100-ml beakers, e.g., by placing them in the mouth of a fume chamber the window of which has been pulled down to the level of the top of the receivers. Avoid too rapid evaporation, as this may cause condensation of moisture on the walls of the beaker. Discontinue the percolation when it is apparent from the residue in the 100-ml receivers that no further dieldrin is being extracted. With the help of the solvent, transfer the residues from all the receivers to a weighed 100-ml wide-mouthed conical flask. Ensure that no crystalline deposit remains on or inside the tip of the chromatographic column by washing with additional solvent into the weighed flask. Evaporate the solution to dryness in a current of air. Break up the residue by means of a weighed glass rod, leaving the rod in the flask. Place the flask and contents for 15 minutes in an oven maintained at 75°C ± 2°C and atmospheric pres- sure. Remove from the oven, cool in a desiccator, weigh, and calculate the weight of the residue. NOTE : With a correctly prepared sample that has not undergone any degradation through storage, the weight of the final residue is a good preliminary indication of the dieldrin content of the sample. Should the weight be less than the nominal dieldrin content, it may be concluded that the sample has been incorrectly prepared and/or has undergone some degradation-although slight degradation would not seriously affect the weight of the residue. Should the weight of the final residue be higher than the nominal dieldrin content, it may be concluded that the sample - 101 - DIELDRIN WATER-DISPERSIBLE POWDERS WHO/SIF/3.R3 has been incorrectly prepared and/or contains other extractable material that may well interfere with the infra-red determination. 2. l . 5. 2 Analysis of sample Dissolve the residue in the flask (obtained by the procedure described in section 2. l . 5. l) in a small volume of carbon disulfide, transfer the solution quantitatively to a 100-ml graduated flask, and make up to the mark with carbon disulfide. Fill the absorption cell-the same one used in the calibration-with the above solution and make replicate scans in the region 10.0-13.0 µ, with the same instrument settings as used in the calibration (section 2.1.4). Calculate the absorbance at the l l .0-µ, 11.8-µ, and 12.35-µ peaks from the scans, as described in section 2.1.4. 2. 1. 6 Calculation For each peak, compute the mean absorbance and read from the calibra- tion graph the corresponding concentration of HEOD in grams per 100 ml of solution. Calculate the mean of the three concentrations.1 axb HEOD content(% w/w) = -- w where a mean concentration (g/100 ml) of HEOD, as found from the calibration graph b = volume (ml) of carbon disulfide solution containing the residue w = weight (g) of initial sample. Since dieldrin is defined (see footnote on page 96) as containing 85% of HEOD (w/w), the dieldrin content is obtained by multiplying the HEOD 100 content by 85, i.e., by l.175. Therefore a x b x 1.175 Dieldrin content (% w/w) = w 1 In practice, the values obtained from the three different readings will be virtually identical. Although slight differences are to be expected owing to instrumental variations, the values should not differ from the mean by more than 5%, assuming that interfering substances have been effectively removed by the extraction procedure. If, in the calcula- tion, it is found that the results obtained from two wave-lengths agree within 5-10% of the mean but that the result obtained from the third wave-length is widely different, it can be assumed that interference is taking place at the third wave-length. In such an event, the result obtained from the third wave-length should be disregarded and the HEOD content calculated from the other two readings. - 102 - WHO/SIF/3.R3 DIELDRIN WATER-DISPERSIBLE POWDERS 2. 2 Suspensibility 2. 2 .1 Special apparatus 1 1. A 250-ml graduated cylinder with ground-in stopper and a distance of 20-21.5 cm between the bottom and the 250-ml graduation. 2. A glass tube, about 40 cm long and about 5 mm in internal dia- meter, drawn out at one end to an opening of 2-3 mm, the other end being connected to a vacuum pump. 2. 2. 2 Special reagent Standard hard water. Dissolve 0.304 g of anhydrous calcium chloride and O .139 g of magnesium chloride hexahydrate in distilled water and make up to one litre. This provides water with a hardness of 342 parts per million, calculated as calcium carbonate. Hexane-a commercial C6 petroleum fraction having a boiling-range of 62-68°C and a paraffin content of at least 98%, and leaving on evaporation a residue of less than 0.0016% (w/w). 2.2.3 Procedure Weigh accurately into a 100-ml beaker an amount of the sample, treated as described above, to form 250 ml of a suspension containing 0.625% (w/v) of dieldrin. Add a volume of water 2 at 30°C ± l°C equal to at least twice the weight of the sample taken. Allow to stand for 30 seconds and then stir by hand for 30 seconds with a glass rod, 4-6 mm in diameter, at not more than four revolutions per second, making no deliberate attempt to break any lumps. Then immediately transfer the mixture quantitatively to the 250-ml graduated cylinder, using water 2 at 30°C ± 1 °C for rinsing, and again avoiding mechanical disintegration of any lumps. Immediately add sufficient water 2 at 30°C ± l °C to bring the volume to the 250-ml mark. Stopper the cylinder and mix by inverting and righting it 30 times at a rate of one complete cycle every 2 seconds. This operation should be carried out as smoothly as possible, keeping the axis of rotation fixed. The cylinder must be thermally insulated from the hands to maintain the prescribed temperature of the suspension. Allow the graduated cylinder to stand for 30 minutes in a water-bath at 30°C ± l °C, care being taken that the bath is free from vibrations. 1 A specially designed and fitted graduated cylinder suitable for use in this procedure is described in FAO Plant Protection Bulletin, 1962, vol. 10, No. 2, Method 10. 2 Use distilled water for testing the powder without pretreatment and standard hard water for testing the powder after accelerated storage. - 103 - DIELDRIN WATER-DISPERSIBLE POWDERS WHO/SIF/3.R3 NoTE : Should excessive flocculation occur during the test, accompanied by the appearance of transparent liquid, the material is unsatisfactory. At the end of the settling period (30 minutes), insert the glass tube into the cylinder and, with a minimum of disturbance, withdraw during I0-15 seconds by means of the vacuum pump nine-tenths of the suspension, i.e., 225 ml. This is achieved by maintaining the tip of the glass tube just below the sinking top level of the suspension. Discard the suspension withdrawn. Filter the retained one-tenth of the suspension, including the sediment, through a Buchner funnel or a sintered-glass crucible of medium porosity, using distilled water to wash out the 250-ml graduated cylinder. If solids or turbidity appear in the filtrate, re-filter through the same filter until the filtrate is perfectly clear. Discard the filtrate. Place the funnel or crucible (with its contents) in a vacuum desiccator over phosphorus pentoxide and allow the filter cake to dry overnight or for at least 8 hours. Place the funnel or crucible on a clean, dry filter flask. Break up the filter cake by means of a glass rod and extract the residue in the funnel or crucible with successive 10-ml portions of hexane until extraction is complete; in practice, a total volume of 100 ml of hexane is likely to be sufficient. The first 10-ml portion of hexane should be used to wash the glass rod. Each IO-ml portion should be pipetted into the funnel or crucible and allowed to remain there for about 15 seconds before the solvent (containing any dissolved solids) is drawn through the filter by gentle suction. Transfer the combined extracts quantitatively to a beaker, previously weighed to the nearest 0.1 g, and evaporate the solution to dryness in a current of air. Place the beaker and contents for 15 minutes in the atmospheric oven maintained at 75°C ± 2°C. Allow the beaker to cool to room temperature in a desiccator, weigh to the nearest 0.1 g, and calculate the approximate weight of the residue. Dissolve the residue in the beaker in a small volume of carbon disulfide, transfer the solution quantitatively to a graduated flask, and make up to volume with the same solvent. The volume of the flask should be such that the solution, when made up to the mark, will contain about l g of the residue per l 00 ml. Determine the HEOD content of the above solution by the infra-red spectrophotometric method (section 2.1). , 2. 2. 4 Calculation From the HEOD content obtained in section 2.2.3, calculate the weight of dieldrin that was present in the retained one-tenth of the sus- pension. From the value obtained in section 2. l. 6 for the percentage - 104 - WHO/SIF/3.R3 DIELDRIN WATER-DISPERSIBLE POWDERS content of dieldrin, calculate also the weight of dieldrin in the initial sample taken for the suspensibility test. where a b Suspensibility (%) = (b a) x 111.l h weight (g) of dieldrin in the retained one-tenth of the suspension weight (g) of dieldrin in the initial sample. 2. 3 Accelerated Storage Treatment Weigh 5 g of the powder into a 25-mm x 200-mm test tube. If the height of the powder in the tube exceeds 6.0 cm, gently tap the tube until the level is reduced to 6.0 cm. Immerse the tube to a depth of at least 9.0 cm in an oil bath maintained at 90°C ± O.l°C. The bath should be equipped with an electric stirrer and the tubes must not be stoppered. Allow the sample to remain in the bath for two hours, then remove and cool to room temperature. After completion of the accelerated storage treatment, the sample should not be exposed to heat or to bright sunshine. Unnecessary exposure of the sample to high atmospheric humidity must also be avoided. -105 - DIAZINON WATER-DISPERSIBLE POWDERS WHO/SIF/9.R2 DIAZINON WATER-DISPERSIBLE POWDERS Specification WHO/SIF/9. R2 Approved 25 October /965 l. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of a homogeneous mixture of technical diazinon in a filler and shall be in the form of a fine, free-flowing, yellow to light brown powder, which wets out readily on stirring into water. The technical diazinon used in the manufacture of the water-dispersible powder shall comply with the requirements of Specification WHO/SIT/ 9.R2. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l. l and with the following requirements. 1.2.1 Diazinon content (w/w basis) The content of O ,0-diethyl 0-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate, determined by the method described in section 2. I, shall not differ from the nominal content by more than the following amounts: Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 2 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. 1. 2 . 2 Sieving after accelerated storage Not less than 98% of the powder (dry weight) after accelerated storage treatment (section 2.3) shall pass through a 74-µ sieve (BS 200 mesh; 3 US Standard No. 200 4) when tested by the method described in WHO/ M/4, page 253. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 For a 50% water-dispersible powder, tolerance = ± 5% of 50% = ± 2.5%; for a 75% water-dispersible powder, tolerance = ± 5% of (100 - 75)~~ = ± 1.25%. 3 British Standards Institution, British Standard 410: 1943. 4 American Society for Testing Materials, Standard E 11-39. - 106 - WHO/SIF/9.R2 DIAZINON WATER-DISPERSIBLE POWDERS 1. 2. 3 Suspeosibility l . 2. 3. l In distilled water without pretreatment When tested by the method described in section 2. 2, a minimum of 50% of the diazinon (0.5% w/v) shall be in suspension 30 minutes after agitating a suspension containing 1.0% w /v of diazinon, prepared in dis- tilled water from the powder as received. I . 2. 3. 2 In standard hard water after accelerated storage treatment When tested by the method described in section 2. 2, a minimum of 50% of the diazinon (0.5% w/v) shall be in suspension 30 minutes after agitating a suspension containing 1.0% w /v of diazinon, prepared in stand- ard hard water from powder subjected to the accelerated storage treatment described in section 2. 3. 1. 2. 4 Acidity The acidity of the powder, determined by the method described in WHO/M/3 (see page 250), shall not be greater than 0.3%, calculated as H2S04 • 1. 3 Packing and Marking of Packages The diazinon water-dispersible powder shall be packed in suitable, clean drums, as specified in the order. The drums shall contain an inner- liner or bag of polyethylene or equivalent, with a nominal thickness of 0.1 mm. The inner-liner or bag shall be hermetically sealed after filling. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Diazinon water-dispersible powder to Specification WHO/SIF /9. R2 Diazinon, ... % w/w Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Diazinon is an organophosphorus compound which inhibits cholines- terase. It is poisonous if swallowed. It may be absorbed through the skin. Avoid skin contact; wear protective gloves, clean protective clothing and a respirator when handling the material. Wash thoroughly with soap and water after using. Keep the material out of reach of children and well away from foodstuffs, animal feed, and their containers. - 107 - DIAZINON WATER-DISPERSIBLE POWDERS WHO/SIF/9.R2 "If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Diazinon Content 2 .1.1 Summary of method The sample is extracted with ethyl ether and re-extracted with light petroleum. The basic by-products of diazinon are absorbed in a column of diatomaceous silica filtering aid impregnated with 3 N sulfuric acid. 0 ,0-Diethyl 0-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate is determined in the eluate by titration with perchloric acid. 2. 1. 2 Special apparatus 1. Chromatographic column, 250 mm in length and 21-22 mm in internal diameter, equipped with glass or Teflon stopcock and a sintered- glass disc and equipped to operate under pressure. 2. Steel tamping rod with perforated disc to fit loosely inside the chromatographic column. 2. 1. 3 Special reagent Standardized 0.1 N perchloric acid in glacial acetic acid. Dilute a suitable quantity of perchloric acid, obtainable as a concentrated aqueous solution, to the required volume with glacial acetic acid and sufficient acetic anhydride to take up the entire water content of the perchloric acid. For example, to make up 5 litres of solution using 70% perchloric acid, the required quantities would be : 70% perchloric acid . . . . Acetic anhydride . . . . . Glacial acetic acid, to make 73 g 124.1 g 5 litres If the acetic acid is not anhydrous, a correspondingly larger volume of acetic anhydride will be required, equivalent to the total water content of the other two reagents. Standardize the solution as follows : Dissolve 0.2 g of anhydrous sodium carbonate, accurately weighed, in 50 ml of glacial acetic acid and - 108 - WHO/SIF/9.R2 DIAZINON WATER-DISPERSIBLE POWDERS titrate this solution with the perchloric acid solution, using 1 % a-naphthol- benzein solution in benzene as indicator; 0.200 g of sodium carbonate corresponds to 37.73 ml of 0.1 N perchloric acid. 2 .1. 4 Preparation of the chromatographic column Weigh 250 g of diatomaceous silica filtering aid into the mixing bowl of a suitable mixer. This amount will provide enough material for 15 columns. Add slowly 150 ml of 3 N sulfuric acid while mixing. Continue mixing until the powder is homogeneous. Scrape the material adhering to the side of the bowl during the process. Add enough light petroleum to the diatomaceous silica filtering aid mixture to cover the paste with solvent, stirring by hand with a porcelain spatula. The resulting slurry is now ready for packing into the column. Pour into the dry column light petroleum to half of its height. Tamp a glass-wool plug with the help of the tamping rod into position above the sintered-glass disc. Put the funnel on top of the column. Fill portions of the slurry into the column through the funnel and tamp each portion gently with the tamping rod until the column is two-thirds full. Stir the slurry with a glass rod to eliminate all air bubbles and to ensure homo- geneous packing. Adjust the pressure and open the stopcock. Press out excess of light petroleum, but retain enough solvent in the column so that the diatomaceous silica filtering aid is just covered at all times. Continue adding slurry and pressing until the height of the packing is about 15 cm. Tamp with the tamping rod to form an even surface and place a glass- wool plug of about 5 mm on top of the packed column. Properly packed columns allow the light petroleum to pass through dropwise. 2.1.5 Procedure Weigh accurately an amount of the sample containing 0.6-0.8 g of diazinon into a 200-ml conical flask, add 70 ml of ethyl ether and agitate for 5 minutes. Filter through a paper placed in a sintered-glass funnel. Rinse the flask and funnel with about 150 ml of ethyl ether. Distil off the solvent, eliminating the last traces by an air-stream. Dissolve the residue in 10-15 ml of light petroleum and pour on to the column. Open the stopcock and let the level sink to about 1-3 mm above the diatomaceous silica filtering aid. Rinse the flask quantitatively, twice with 5 ml and twice with 10 ml of light petroleum. Pour each washing down the side of the column so as to wash the sample into the diatomaceous silica filtering aid and allow the solution to penetrate to a level of 1-3 mm above the packing before adding the next portion. Add 30 ml of light petroleum. Adjust the dropping funnel to the top of the column and elute with 200 ml -109 - DIAZINON WATER-DISPERSIBLE POWDERS WHO/SIF/9.R2 of light petroleum. Collect the eluate in a 500-ml conical flask and distil off the solvent. Dissolve the residue in 50 ml of glacial acetic acid, connect to a reflux condenser, and boil for 15 minutes. Cool, rinse with glacial acetic acid into a beaker and titrate potentio- metrically with 0.1 N perchloric acid in glacial acetic acid.1 Alternatively, the titration can be done visually using a 1 % cx-naphthol- benzein solution in benzene as indicator, the end-point being reached when the solution becomes dark green. NOTE : The column can be used a second time if the first sample did not contain too large a quantity of by-products. Care has to be taken that the column does not get dry, but remains covered by light petroleum. 2 .1. 6 Calculation 0 ,0-Diethyl 0-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate content (% w /w) a x 3.044 w where a = volume (ml) of 0.1 N perchloric acid used w = weight (g) of sample. 2. 2 Suspensibility 2. 2. 1 Special apparatus 2 1. A 250-ml graduated cylinder with ground-in stopper and a distance of 20-21.5 cm between the bottom and the 250-ml graduation. 2. A glass tube, about 40 cm long and about 5 mm in internal diameter, drawn out at one end to an opening of 2-3 mm, the other end being con- nected to a vacuum pump. 2. 2. 2 Special reagent Standard hard water. Dissolve 0.304 g of anhydrous calcium chloride and 0.139 g of magnesium chloride hexahydrate in distilled water and make up to one litre. This provides water with a hardness of 342 parts per million, calculated as calcium carbonate. 1 Care must be taken that all titrations with perchloric acid are performed in absolu- tely anhydrous media. 2 A specially designed and fitted graduated cylinder suitable for use in this procedure is described in FAO Plant Protection Bulletin, 1962, vol. 10, No. 2, Method JO. - 110 - WHO/SIF/9.R2 DIAZINON WATER-DISPERSIBLE POWDERS 2.2.3 Procedure Weigh accurately into a 100-ml beaker an amount of the sample, treated as described above, to form 250 ml of a suspension containing 1 % (w /v) of diazinon. Add a volume of water 1 at 30°C ± 1 °C equal to at least twice the weight of the sample taken. Allow to stand for 30 seconds and then stir by hand for 30 seconds with a glass rod, 4-6 mm in diameter, at not more than four revolutions per second, making no deliberate attempt to break any lumps. Then immediately transfer the mixture quantitatively to the 250-ml graduated cylinder, using water 1 at 30°C ± 1 °C for rinsing, and again avoiding mechanical disintegration of any lumps. Immediately add sufficient water 1 at 30°C ± 1 °C to bring the volume to the 250-ml mark. Stopper the cylinder and mix by inverting and righting it 30 times at a rate of one complete cycle every 2 seconds. This operation should be carried out as smoothly as possible, keeping the axis of rotation fixed. The cylinder must be thermally insulated from the hands to maintain the prescribed temperature of the suspension. Allow the graduated cylinder to stand for 30 minutes in a water-bath at 30°C ± 1 °C, care being taken that the bath is free from vibrations. NOTE: Should excessive flocculation occur during the test, accompanied by the appearance of transparent liquid, the material is unsatisfactory. At the end of the settling period (30 minutes), insert the glass tube into the cylinder and, with a minimum of disturbance, withdraw during 10-15 seconds by means of the vacuum pump nine-tenths of the suspension, i.e., 225 ml. This is achieved by maintaining the tip of the glass tube just below the sinking top level of the suspension. Discard the suspen- sion withdrawn. Transfer the retained one-tenth of the suspension, including the sedi- ment, to a 250-ml separating funnel. Rinse the cylinder twice with 50-ml portions of light petroleum (boiling range 40-60°C), adding both rinsings to the contents of the separating funnel. Shake and drain the aqueous layer into a second 250-ml separating funnel. If there is an emulsion between the two phases, leave it in the first separating funnel. Add to the second separating funnel 100 ml of light petroleum and 10 ml of saturated sodium chloride solution and shake again. Let the phases separate while occasionally swirling, then discard the aqueous layer. Wash the two light petroleum extracts separately and successively twice with 50-ml portions of distilled water to which 10 ml of saturated sodium chloride solution have been added, using the same solutions for both extracts. 1 Use distilled water for testing the powder without pretreatment and standard hard water for testing the powder after accelerated storage. - 111 - DIAZINON WATER-DISPERSIBLE POWDERS WHO/SIF/9.R2 Remove all water droplets from the two light petroleum extracts and filter them through a cotton plug in the order in which the extracts were made. Rinse the cotton with light petroleum. Distil off the solvent, dis- solve the residue in 20 ml of glacial acetic acid, and titrate with the stand- ardized 0.1 N perchloric acid in glacial acetic acid,1 using 1 % o:-naphthol- benzein solution in benzene as indicator, the end-point being reached when the solution becomes dark green. 2. 2. 4 Calculation From the volume of 0.1 N perchloric acid used, calculate the weight of diazinon in the retained one-tenth of the suspension by the formula given in section 2. l. 6. From the value obtained in the same section for the percentage content of diazinon, calculate also the weight of diazinon in the initial sample taken for the suspensibility test. where a b S 'bT (%) (b - a) x lll.l uspens1 1 1ty O = b weight (g) of diazinon in the retained one-tenth of the sus- pension weight (g) of diazinon in the initial sample. 2. 3 Accelerated Storage Treatment Place 20 g of the sample in a 100-ml wide-mouthed bottle fitted with a vinyl-plastic-lined screw-cap. Place in a forced-draught oven main- tained at 70°C ± 1 °C for 2 hours. Take the sample from the oven and allow to cool to room temperature before removing the cap. After comple- tion of the accelerated storage treatment, the sample should not be exposed to heat or to bright sunshine. Unnecessary exposure of the sample to high atmospheric humidity must also be avoided. 1 Care must be taken that all titrations with perchloric acid are performed in absolutely anhydrous media. -112 - WHO/SIF/10.R2 MALATHION WATER-DISPERSIBLE POWDERS MALATHION WATER-DISPERSIBLE POWDERS l. SPECIFICATIONS Specification WHO/SIF/JO.R2 Approved 25 October 1965 1.1 Description and Ingredients The material shall consist of a homogeneous mixture of technical mala- thion in a filler and shall be in the form of a fine, free-flowing, yellow to brown powder, which wets out readily on stirring into water. The technical malathion used in the manufacture of the water-dispersible powder shall comply with the requirements of Specification WHO/SIT/l0.R2. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1.1 and with the following requirements.2 1.2.1 Malathion content (w/w basis) The content of O ,0-dimethyl S-[l ,2-di-(ethoxycarbonyl)ethyl] phos- phorodithioate, determined by the method described in section 2 .1, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content l 3 ± 5% of (100 - nominal content) f The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Sieving after accelerated storage Not less than 98% of the powder (dry weight) after accelerated storage treatment (section 2.4) shall pass through a 74-µ sieve (BS 200 mesh; 4 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 If the material is required for use inside buildings, odour-free malathion should also be specified. 3 For a 50% water-dispersible powder, tolerance= ± 5% of 50% = ± 2.5%; for a 75% water-dispersible powder, tolerance= ± 5% of (100 - 75) % = ± 1.25%. ' British Standards Institution, British Standard 410: 1943. - 113 - MALATHION WATER-DISPERSIBLE POWDERS WHO/SIF/10.R2 US Standard No. 200 1) when tested by the method described in WHO/ M/4 (see page 253). 1.2.3 Suspensibility 1 . 2. 3 . 1 In distilled water without pretreatment When tested by the method described in section 2. 2, a minimum of 60% of the malathion (l.5% w/v) shall be in suspension 30 minutes after agitating a suspension containing 2.5% w/v of malathion, prepared in distilled water from the powder as received. 1 . 2. 3 . 2 In standard hard water cifter accelerated storage treatment When tested by the method described in section 2. 2, a minimum of 50% of the malathion (l.25% w/v) shall be in suspension 30 minutes after agitating a suspension containing 2.5% w/v of malathion, prepared in stand- ard hard water from powder subjected to the accelerated storage treatment described in section 2 .4. 1. 2. 4 Acidity The acidity of the powder, determined by the method described in WHO/M/3 (see page 250), shall not be greater than 0.5% calculated as H2S04 . 1. 3 Packing and Marking of Packages The malathion water-dispersible powder shall be packed in suitable, clean drums, as specified in the order. The drums shall contain an inner- liner or bag of polyethylene or equivalent with a nominal thickness of 0.1 mm. The inner-liner or bag shall be hermetically sealed after filling. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Malathion water-dispersible powder to Specification WHO/SIF/IO.R2 Malathion, ... % w /w Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Malathion is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed. Keep the material out of reach of children and well away from foodstuffs, animal feed, and their containers. 1 American Society for Testing Materials, Standard E 11-39. - 114 - WHO/SIF/10.R2 MALATHION WATER-DISPERSIBLE POWDERS " If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Malathion Content 2 .1.1 Special apparatus Spectrophotometer or photoelectric colorimeter with a blue filter (420 mµ). 2 .1. 2 Special reagents Acetonitrile, boiling range 80-82°C. Pass through a column of silica gel and discard the first yellow portion of the eluate. Collect the colour- less eluate. A 10% aqueous solution of this should have a pH between 5 and 7. Ferric reagent. Dissolve 0.2 g of ferric chloride (FeCl2 • 6H20) in dis- tilled water, add 8 ml of concentrated hydrochloric acid, and dilute to l litre with distilled water. Cupric reagent. Dissolve 1.5 g of cupric sulfate (CuS04 • 5H20) in 100 ml of water. 2. 1. 3 Preparation of malathion standard solutions Standard solution A. Accurately weigh into a 5-ml or 10-ml beaker 1.00 g ± 0.02 g of malathion reference standard.1 Transfer quantitatively to a I-litre volumetric flask with anhydrous ethanol. Dilute with anhydrous ethanol to about 5 ml below the mark and allow to stand until a steady temperature is reached. Record the temperature, dilute to the mark with anhydrous ethanol and mix well. The solution is stable for about 2 weeks. Adjust to recorded temperature before withdrawing aliquots. Standard solution B. Pipette a 15-ml aliquot of standard solution A into a 250-ml volumetric flask. Add 2.5 ml of acetonitrile and dilute with anhydrous ethanol to about 5 ml below the mark. Keep until the sample for analysis is ready for final dilution. At that time, dilute with anhydrous ethanol to the mark and mix well. 1 Samples of reference standard malathion may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. - 115 - MALATHION WATER-DISPERSIBLE POWDERS WHO/SIF/10.R2 NOTE: Volume changes of alcohol solutions with temperature are appreciable. Bring each solution to volume just before proceeding to next step. 2 .1. 4 Preparation of sample Accurately weigh a sample containing 0.30 g ± 0.02 g of malathion and transfer it quantitatively to a 100-ml bottle fitted with a screw cap having a polyethylene liner. Pipette 50 ml of acetonitrile into the bottle. Cap the bottle tightly and shake vigorously for 2 to 3 minutes. Allow the solids to settle for 3 to 5 minutes. If necessary, centrifuge for 2 to 3 minutes at 1500 to 3000 rev/min. Immediately pipette a 25-ml aliquot of the super- natant liquid into a 250-ml volumetric flask, dilute with anhydrous ethanol to the mark and mix well. Immediately pipette a 25-ml aliquot of the diluted solution into a 250-ml volumetric flask and again make up to volume with anhydrous ethanol. 2 .1. 5 Analysis of sample Determine the standard malathion and the sample separately but at the same time. Perform the entire analysis without interruption. Pipette 25-ml aliquots of standard solution B and sample solutions into two 250-ml separating funnels. Add 2 ml ± 0.1 ml of 0.5 N sodium hydroxide to each and mix well by swirling gently for 5 to 10 seconds (do not shake). Allow to stand for 120 seconds ± lO seconds. Add 75 ml ± l ml of ferric reagent and mix well by swirling for lO seconds. Allow to stand for 5 minutes. During this waiting period fill two 50-ml volumetric flasks to the mark with cyclohexane. Transfer the cyclohexane from these flasks to the two separating funnels and allow the flasks to drain for one minute. Add 2.0 ml ± 0.1 ml of cupric reagent and shake in the separating funnel for exactly one minute. NoTE : The copper-malathion complex is unstable in the aqueous phase. The cupric reagent should therefore be added from a fast-delivery Mohr pipette and shaking started without delay. A delay of only 15 seconds before extraction results in appreciable losses. Allow the phases to separate and discard the aqueous phase as soon as separation occurs. Allow a little of the solvent phase to rinse the stem of the separating funnel, then transfer the clear solvent phase to a small beaker and thence to the cell, or transfer directly to the cell. Manipulate phase separation of both standard and sample similarly. Immediately (within 5 minutes) determine the absorbance of the yellow cyclohexane solution at 420 mµ by means of the spectrophotometer ( or photoelectric colorimeter) using cyclohexane reference. - 116 - WHO/SIF/10.R2 MALATHION WATER-DISPERSIBLE POWDERS 2. 1. 6 Calculation 0 ,0-dimethyl S-[l ,2-di(ethoxycarbonyl)ethyl] phosphorodithioate content (% w /w) Asamp1c a x p x 0.3 = x Astandard b X 100 where Asampte = absorbance of the sample at 420 mµ Asiandard = absorbance of the standard at 420 mµ a = weight (g) of reference standard malathion taken to pre- pare standard solution b = weight (g) of sample p = purity (%) of reference standard malathion. 2. 2 Suspensibility 2 . 2. 1 Special apparatus l. A 100-ml glass-stoppered graduated cylinder having the 100-ml mark situated 18.0 cm ± l.5 cm from the bottom and 5.5 cm ± 0.5 cm from the top, excluding the neck. 2. A 25-ml pipette fitted with a device to permit its insertion into the 100-ml cylinder so that it is held with the tip exactly at the 50-ml mark. This may be accomplished by fitting the pipette with a stopper having a centre bore for the stem and a shallow V-cut on one side as an air vent. 3. A constant-temperature water-bath into which the 100-ml graduated cylinders can be immersed to the 100-ml mark and which can be main- tained at 30°C ± 1 °C. The bath must be free from any vibration caused by stirring motors or other equipment. 2. 2. 2 Special reagent Standard hard water. Dissolve 0.304 g of anhydrous calcium chloride and 0.139 g of magnesium chloride hexahydrate in distilled water and make up to one litre. This provides water with a hardness of 342 parts per million, calculated as calcium carbonate. 2.2.3 Procedure Weigh accurately into a 100-ml beaker an amount of the sample to form 100 ml ofa suspension containing 2.5% (w/v) of malathion. Add 50 ml of water 1 at 30°C ± 1 °C. Stir the mixture with a glass rod by hand for 1 Use distilled water for testing the powder without pretreatment and standard hard water for testing the powder after accelerated storage. -117 - MALATHION WATER-DISPERSIBLE POWDERS WH0/$1F/10.R2 30 seconds, making no deliberate attempt to break up any lumps and then immediately transfer the sample quantitatively to the 100-ml cylinder using additional water 1 for the transfer. Add sufficient water 1 at 30°C ± l °C to make 100 ml of suspension. Stopper the cylinder and mix by inverting and righting it 30 times at the rate of approximately one cycle every two seconds. This operation should be carried out as smoothly as possible keeping the axis of rotation fixed. The cylinder must be thermally insulated from the hands to maintain the prescribed temperature of the suspension. Immerse the cylinder up to the 100-ml mark in the water-bath maintained at 30°C ± 1 °C. The preparation of the suspension from the first addition of water to the placing of the cylinder in the constant-temperature bath should be a continuous operation and should be completed within three minutes. Allow the cylinder to stand for 30 minutes in the water-bath at 30°C ± 1 °C. During this period care should be taken that the bath and cylinder are free from vibrations. Norn : Should excessive flocculation occur during the test, accompanied by the appearance of transparent liquid, the material is unsatisfactory. At the end of the 30-minute settling period, remove the cylinder from the water-bath, insert the specially fitted pipette so that the tip is exactly at the 50-ml mark and remove a 25-ml aliquot. (If this test is being per- formed on a sample after accelerated storage treatment [section 2.4], the remaining 75 ml of the suspension should be retained for the sieving test as described in section 2. 3.) Transfer the 25-ml aliquot to a 100-ml beaker and add 2 g of potassium bromide. Stir to dissolve the potassium bromide and allow to stand for about 5 minutes to coagulate the powder. Place a 5.5-cm glass-fibre filter- paper 2 in a 5 .5-cm Buchner funnel, insert the funnel in a 250-ml suction flask and wet the paper with water. Transfer the coagulated 25-ml aliquot to the paper and wash with six 20-ml portions of water. Transfer the funnel to a dry 250-ml suction flask and extract the malathion with six 20-ml portions of acetone. Evaporate the acetone in the flask in a steam- bath to a volume of approximately 10 ml. Transfer the residue quantitatively to a tared 50-ml beaker, using additional acetone. Evaporate the acetone at 60°C in a stream of dry air. Add two 5-ml portions of isopropanol during the evaporation to remove traces of water. Dry the sample in an oven at 55°C for 20 minutes and weigh as malathion. 1 Use distilled water for testing the powder without pretreatment and standard hard water for testing the powder after accelerated storage. 2 Reeve Angel No. 934, AHO, or equivalent. -118 - WHO/SIF/10.R2 MALATHION WATER-DISPERSIBLE POWDERS 2. 2. 4 Calculation Suspensibility (%) = a x 160 where a = weight (g) of malathion found in the 25-ml aliquot (section 2.2.3). 2. 3 Sieving Test after Accelerated Storage Pour the 75-ml of suspension retained from the suspensibility test (see section 2.2.3) on to a 74-µ sieve and proceed with the sieving test as de- scribed in Method WHO/M/4, page 253. In this procedure it is assumed that all the particles in the 25-ml aliquot taken from the centre of the suspension would have passed through the 74-µ sieve. 2. 4 Accelerated Storage Treatment Weigh 20 g of the powder into a 100-ml wide-mouthed bottle fitted with a vinyl-plastic-lined screw-cap. Place in a forced-draught oven maintained at 90°C ± 2°C. Allow the sample to remain in the oven for 20 hours and then cool to room temperature before removing the cap. After completion of the accelerated storage treatment, the sample should not be exposed to heat or to bright sunshine. Unnecessary exposure to high atmospheric humidity must also be avoided. - 119 - DOT EMULSION CONCENTRATES WHO/SIF/4.R3 DDT EMULSION CONCENTRATES* Specification WHO/SIF/4.RJ Approved 25 October 1965 1. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of technical DDT dissolved in a suitable solvent, with an emulsifying agent or agents added, and shall be in the form of a stable liquid, free from extraneous impurities. The technical DDT used in the manufacture of the concentrate shall comply with the requirements of Specification WHO /SIT /1 . R3. 1. 2 Chemical and Physical Reqnirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1.1 and with the following requirements. 1.2.1 DDT content (w/w basis) The content of DDT, determined by one of the methods described in section 2. I, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 2 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. * The term" emulsion concentrate" is used here to mean a single-phase liquid system containing an insecticide together with one or more surface-active agents having the property of forming an emulsion on dilution with water. Such a system is more correctly termed an " emulsifiable concentrate ", but " emulsion concentrate " has been given preference on account of established usage. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 For a 50% emulsion concentrate, tolerance = ± 5% of 50% = ± 2.5%; for a 75% emulsion concentrate, tolerance = ± 5% of (100 - 75)% = ± 1.25%. -120 - WHO/SIF/4.R3 DDT EMULSION CONCENTRATES 1.2.2 Cold test No separation of solid or oily material shall occur when the concentrate is tested as described in section 2. 2. 1. 2. 3 Flash-point The flash-point of the concentrate shall not be lower than 22.8°C when determined by one of the methods referred to in section 2. 3. 1. 2. 4 Stability of the emulsion l . 2 .4. l In distilled water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in distilled water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/ M/13 (see page 284). l . 2. 4. 2 In standard hard water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in standard hard water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/ M/13 (see page 284). 1. 2. 5 Heat stability The concentrate, after treatment as described in section 2. 4, shall comply with the requirements of sections 1 . 2. l, l . 2. 2, 1. 2. 3, and l. 2. 4 of this specification. 1. 2. 6 Acidity or alkalinity The acidity or alkalinity of the concentrate, determined by the method described in WHO/M/3, page 250, shall not be greater than 0.05%, calculated as H2S04 , nor greater than 0.05%, calculated as NaOH. 1.2.7 Staining and odour The staining of surfaces produced by the diluted emulsion, prepared as described in WHO/M/13, page 284, and applied at the recommended rate, shall not be greater than that produced by equal volumes of (1) standard hard water and (2) xylene when these liquids are sprayed separately at the same rate of application. The DDT deposit left after the application shall be ignored in assessing the staining, but the deposit must be free from - 121 - DDT EMULSION CONCENTRATES WHO/SIF/4.R3 objectionable odour after standing for 24 hours in a still atmosphere at room temperature. 1.3 Packing and Marking of Packages The DDT emulsion concentrate shall be packed in suitable, clean con- tainers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name DDT emulsion concentrate to Specification WHO/SIF/4.R3 DDT, ... % w/w Batch or reference number, and date of test Net weight of contents Instructions for dilution and the following minimum cautionary notice : "Keep well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 DDT Content Of the two methods described below, only one need be performed. In the event of a dispute the method described in section 2. 1 . 2 shall govern. 2 .1.1 Total organic chlorine method (Stepanow method, revised) 2. l . 1 . l Determination of total chlorine Weigh accurately an amount of the sample containing about 0.1 g of DDT and transfer to a 250-ml conical flask. Add 25 ml of 99% iso- propanol, shake the flask, and add 2.5 g of metallic sodium, in the form of ribbon or small pieces. Connect to a reflux condenser and boil gently for at least 2 hours, shaking the flask occasionally. Remove the excess metallic sodium by cautiously adding 10 ml of 50% aqueous isopropanol through the condenser, at the rate of 1-2 drops per second. Boil for an additional 10 minutes, and then add 60 ml of distilled water. Cool, add 2 or 3 drops of phenolphthalein indicator solution, neutralize by adding 50% nitric acid dropwise, and then add 10 ml in excess. Cool, if necessary, to room temperature, add a slight excess of 0.1 N silver nitrate, -122 - WHO/SIF/4.R3 DDT EMULSION CONCENTRATES and coagulate the precipitated silver chloride by digesting on a steam- bath for Y2 hour, with frequent stirring. Cool, filter through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the total chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. 2. I . 1 . 2 Determination of inorganic chlorine Weigh accurately about 1 g of the sample and transfer with 100 ml of distilled water to a 250-ml conical flask. Acidify with 50% nitric acid and proceed as described in section 2. l . l . l. 2. l . l . 3 Calculation DDT content (% w/w) = { --- - -- xf x 0.7092 'a b) \ W1 W2 where a = volume (ml) of 0.1 N silver nitrate equivalent to the total chlorine b = volume (ml) of 0.1 N silver nitrate equivalent to the inorganic chlorine w1 = weight (g) of sample used for the total chlorine determination w2 = weight (g) of sample used for the inorganic chlorine determi- nation 50.0l f =-A where A = value found for chlorine in recrystallized DDT, determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2 .1. 2 Hydrolysable chlorine method 2. I . 2. 1 Determination of hydrolysable plus inorganic chlorine Weigh accurately an amount of the sample containing about 0.5 g of DDT and transfer to a 250-ml conical flask. Add 50 ml of acetone -123 - DDT EMULSION CONCENTRATES WHO/SIF/4.R3 and 20 ml of 1 N ethanolic potassium hydroxide. Keep at 20-25°C for 15 minutes and add 50 ml of distilled water. Add 20 ml of 2 N nitric acid and exactly 25 ml of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam-bath for Y1 hour, with frequent stirring. Cool, filter the coagulated silver chloride through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with O .1 N potassium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the hydrolysable plus inorganic chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. 2. 1 . 2 . 2 Determination of inorganic chlorine Weigh accurately about 1 g of the sample and transfer with 100 ml of distilled water to a 250-ml conical flask. Acidify with 50% nitric acid, add exactly 25 ml of 0.1 N silver nitrate, and proceed as described in section 2. 1 . 2. 1. 2. l . 2. 3 Calculation ( a b · DDT content (% w/w) = --- - --) x f x 3.546 WI W2 where a = volume (ml) of 0.1 N silver nitrate equivalent to the hydrolys- able plus inorganic chlorine b = volume (ml) of 0.1 N silver nitrate equivalent to the inorganic chlorine w1 = weight (g) of sample used for the hydrolysable plus inorganic chlorine determination w2 = weight (g) of sample used for the inorganic chlorine determi- nation 10.00 I=-A where A = value found for hydrolysable chlorine in recrystallized DDT, determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its - 124 - WHO/SIF/4.R3 DDT EMULSION CONCENTRATES application by a given laboratory. Its value must lie within the range 0.98-1.02. 2.2 Cold Test Cool SO ml of the sample to 0°C and add a small seeding-crystal of DDT having an edge of approximately 1 mm. Stir gently with a thermometer at intervals for I hour, maintaining the temperature at 0°C. 2.3 Flash-Point Determine the flash-point by the TAG Closed Tester method, WHO/ M/10, page 276, the Cleveland Open Tester method, WHO/M/11, page 279, or any other equivalent standard method. 2. 4 Heat Stability Keep 50 ml of the sample for three days at a temperature of 50°C ± l °C in a glass container sealed to avoid loss of volatile solvent, and then cool to room temperature. -125 - HCH EMULSION CONCENTRATES WHO/SIF/5.R3 HCH EMULSION CONCENTRATES * 1. SPECIFICATIONS Specification WHO/SIF/5.RJ Approved 25 October 1965 1.1 Description and Ingredients The material shall consist of technical HCH, refined HCH, or lindane, dissolved in a suitable solvent, with an emulsifying agent or agents added, and shall be in the form of a stable liquid, free from extraneous impurities. The HCH used in the manufacture of the concentrate shall comply with the requirements of Specification WHO /SIT /2. R3 or WHO /SIT /3. R3. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1.1 and with the following requirements.2 1.2.1 Gamma-isomer content (w/w basis) The content of gamma-isomer, determined by the method described in section 2. 1, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 3 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. * The term " emulsion concentrate " is used here to mean a single-phase liquid system containing an insecticide together with one or more surface-active agents having the property of forming an emulsion on dilution with water. Such a system is more correctly termed an " emulsifiable concentrate", but " emulsion concentrate" has been given preference on account of established usage. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 If substantial freedom from odour is required, this should be specified in the order. 3 For a 50% emulsion concentrate, tolerance = ± 5% of 50?{ = ± 2 .5~~; for a 75% emulsion concentrate, tolerance = ± 5% of (100- 75)% = ± 1.25%. -126 - WHO/SIF/5.R3 HCH EMULSION CONCENTRATES 1.2.2 Cold test No separation of solid or oily material shall occur when the concentrate is tested as described in section 2.2. 1. 2. 3 Flash-point The flash-point of the concentrate shall not be lower than 22.8°C when determined by one of the methods referred to in section 2. 3. 1. 2. 4 Stability of the emulsion l . 2. 4. l In distilled water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in distilled water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). l. 2. 4. 2 In standard hard water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in standard hard water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/ M/13 (see page 284). 1. 2. 5 Heat stability The concentrate, after treatment as described in section 2. 4, shall comply with the requirements of sections 1.2. l, 1.2.2, 1.2.3, and 1.2.4 of this specification. 1. 2. 6 Acidity or alkalinity The acidity or alkalinity of the concentrate, determined by the method described in WHO/M/3, page 250, shall not be greater than 0.05%, cal- culated as H2S04 , nor greater than 0.05%, calculated as NaOH. 1. 2. 7 Staining and odour The staining of surfaces produced by the diluted emulsion, prepared as described in WHO/M/13, page 284, and applied at the recommended rate, shall not be greater than that produced by equal volumes of (1) standard hard water and (2) xylene when these liquids are sprayed separately at the -127 - HCH EMULSION CONCENTRATES WHO/SIF/5.R3 same rate of application. The HCH deposit left after the application shall be ignored in assessing the staining, but the deposit must be free from objectionable odour after standing for 24 hours in a still atmosphere at room temperature. 1. 3 Packing and Marking of Packages The HCH emulsion concentrate shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name HCH emulsion concentrate to Specification WHO/SIF /5. R3 Gamma-isomer, ... % w/w Batch or reference number, and date of test Net weight of contents Instructions for dilution and the following minimum cautionary notice : "Keep well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 Gamma-Isomer Content 1 Weigh accurately an amount of the sample containing 60-80 mg of gamma-isomer into a conical flask, distil off the volatile solvent under reduced pressure on a water-bath and dissolve the residue in light petrol- eum.2 Proceed with the chromatographic separation and spectrophoto- metric determination as described in WHO/M/6, page 258. Norn : If removal of the emulsifying agent is necessary to ensure good separation (see WHO/M/6, page 262), proceed as follows: After distilling 1 The revised Stepanow (total organic chlorine) method and the hydrolysablechlorine method, described respectively in Methods WHO/M/16 and WHO/M/17, pages 288 and 291, are not specific. However, this does not preclude a purchaser or user from applying these methods for routine purposes where the gamma-isomer content of the technical HCH used in the manufacture of the emulsion concentrate is known and when the use of the methods has been agreed to by all parties concerned. 2 Light petroleum as specified in WHO/M/6, page 260, should be used. - 128 - WHO/SIF/5.R3 HCH EMULSION CONCENTRATES off the volatile solvent, dissolve the residue in 30 ml of benzene and pass the solution through a column 4 cm in internal diameter containing a 5-cm layer of aluminium oxide. Elute the HCH from the column with 400 ml of ethyl acetate and distil off the solvent. Dissolve the residue in light petro- leum and transfer the solution quantitatively to the silica-gel column. 2.2 Cold Test Cool 50 ml of the sample to 0°C. Stir gently with a thermometer at intervals for I hour, maintaining the temperature at 0°C. 2.3 Flash-Point Determine the flash-point by the TAG Closed Tester method (see WHO/M/10, page 276), the Cleveland Open Tester method (see WHO/ M/l l, page 279), or any other equivalent standard method. 2. 4 Heat Stability Keep 50 ml of the sample for three days at a temperature of 50°C ± l °C in a glass container sealed to avoid loss of volatile solvent, and then cool to room temperature. -129 - 9 DIELDRIN EMULSION CONCENTRATES WHO/SIF/6.R3 DIELDRIN EMULSION CONCENTRATES* Specification WHO/SIF/6 .R3 Approved 25 October 1965 l. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of technical dieldrin1 dissolved in a suitable solvent, with an emulsifying agent or agents added, and shall be in the form of a stable liquid, free from extraneous impurities. The technical dieldrin used in the manufacture of the concentrate shall comply with the require- ments of Specification WHO/SIT/6.R3. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,2 shall comply with the requirements of section I. I and with the following requirements. 1.2.1 Dieldrin content (w/w basis) The content of dieldrin, determined by the method described in section 2. I, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } s ± 5% of (100 - nominal content) * The term" emulsion concentrate" is used here to mean a single-phase liquid system containing an insecticide together with one or more surface-active agents having the property of forming an emulsion on dilution with water. Such a system is more correctly termed an " emulsifiable concentrate ", but " emulsion concentrate " has been given preference on account of established usage. 1 ln the ninth report of the WHO Expert Committee on Insecticides (unpublished), the decision was recorded that the foHowing definitions should be used for the purposes of this specification : "'Dieldrin' is to refer to an insecticide containing 85% by weight of HEOD. The amount of dieldrin in any sample will therefore be calculated from the following formula : Dieldrin (% by weight) = HEOD (% by weight) x 18~." " 'Technical dieldrin' is to refer to a product containing a minimum of 90% by weight of dieldrin, i.e., a minimum of 76.5% by weight of HEOD." 2 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 3 For a 50% emulsion concentrate, tolerance= ± 5% of 50% = ± 2.5%; for a 75% emulsion concentrate, tolerance = ± 5% of (100-- 75) % = ± l.25%. - 130 - WHO/SIF/6.R3 DIELDRIN EMULSION CONCENTRATES The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Cold test No separation of solid or oily material shall occur when the concentrate is tested as described in section 2. 2. 1. 2. 3 Flash-point The flash-point of the concentrate shall not be lower than 22. 8°C when determined by one of the methods referred to in section 2. 3. 1. 2. 4 Stability of the emulsion l . 2. 4. l In distilled water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in distilled water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). 1 . 2. 4. 2 In standard hard water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in standard hard water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/ M/13 (see page 284). 1. 2. 5 Heat stability The concentrate, after treatment as described in section 2.4, shall comply with the requirements of sections 1. 2 .1, 1. 2. 2, l. 2. 3, and 1. 2. 4 of this specification. 1. 2. 6 Acidity or alkalinity The acidity or alkalinity of the concentrate, determined by the method described in WHO/M/3, page 250, shall not be greater than 0.05%, calculated as H2S04 , nor greater than 0.05%, calculated as NaOH. 1. 2. 7 Staining and odour The staining of surfaces produced by the diluted emulsion, prepared as described in Method WHO/M/13, page 284, and applied at the recom- mended rate, shall not be greater than that produced by equal volumes of (1) standard hard water and (2) xylene when these liquids are sprayed separately at the same rate of application. The dieldrin deposit left after - 131 - DIELDRIN EMULSION CONCENTRATES WHO/SIF/6.R3 the application shall be ignored in assessing the staining, but the deposit must be free from objectionable odour after standing for 24 hours in a still atmosphere at room temperature. 1. 3 Packing and Marking of Packages The dieldrin emulsion concentrate shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Dieldrin emulsion concentrate to Specification WHO/SIF/6.R3 Dieldrin, ... % w /w Batch or reference number, and date of test Net weight of contents Instructions for dilution and the following minimum cautionary notice : " Dieldrin is a toxic substance and may cause convulsions. It is poison- ous if swallowed. It may be absorbed through the skin or inhaled as dusts or mists. Avoid skin contact; wear protective gloves and clean protective clothing while using the material. Wash thoroughly with soap and water after using. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Dieldrin Content 1 2 .1.1 Summary of method The sample is introduced into a chromatographic column packed with activated charcoal, which is then eluted with hexane and the extract evapo- rated to dryness and weighed.2 The residue is dissolved in carbon disulfide 1 The revised Stepanow (total organic chlorine) method (see Method WHO/M/16, page 288) may be used for routine purposes, but in the event of a dispute the method described here shall govern. 2 The extraction procedure described in this method is not applicable to concentrates containing a solvent that has a boiling-range extending beyond about 200°C. With such concentrates, difficulty may be encountered in evaporating the solvent completely without, at the same time, losing some of the dieldrin by volatilization ; the residual solvent may interfere with the infra-red determination. In such instances, the analyst should be provided with a formulation sample from which the dieldrin has been omitted, so that the amount of " background interference " (if any) can be determined. - 132 - WHO/SIF/6.R3 DIELDRIN EMULSION CONCENTRATES and the solution made up to a fixed volume with carbon disulfide. The infra-red spectrum of this solution is scanned in the wave-length region 10.0-13.0 µ. The absorbance of the solution is determined at 11.0 µ, 11.8 µ, and 12.35 µ, and the quantity of HEOD present is obtained by comparing the observed readings with a calibration graph relating absorption to various concentrations of HEOD. 2. 1. 2 Special apparatus l. Chromatographic column, constructed as shown in Fig. 5, page 99, and charged with 3 g of activated charcoal. 2. Infra-red spectrometer, capable of recording in the region 10.0-13.0 µ. The slit width and gain must be adjustable in order to give a satisfactory signal/noise ratio and an adequate resolution. In general, a signal/noise ratio of about 100 : 1 is chosen. A sealed absorption cell with sodium chloride windows and a path length of about 0.4 mm is required. 3. Hypodermic glass syringe, of 1.0-ml capacity, fitted with an 18-gauge (Stubbs), 5-cm, slip-on type needle. A Luer type is also suitable. 2 .1. 3 Special reagents 1. Hexane - a commercial C6 petroleum fraction having a boiling-range of 62-68°C and a paraffin content of at least 98%, and leaving on evapora- tion a residue of less than 0.0016% (w/w). 2. Extraction solvent. Commercial hexane (as defined above) containing 5% (v/v) of acetone. 3. HEOD standard solutions. Into three 10-ml volumetric flasks, weigh accurately 40 mg, 80 mg, and 120 mg of recrystallized HEOD (melting point l 78°C).1 Dissolve in carbon disulfide, make up to the mark, and mix thoroughly. These solutions contain respectively 0.4 g, 0.8 g, and 1.2 g of HEOD per 100 ml.2 2 .1. 4 Preparation of calibration graph Fill the absorption cell with the most dilute of the standard solutions by means of the hypodermic syringe. Adjust the spectrometer to the optimum settings with respect to gain, slit width, response, speed, and drum drive. Make replicate scans of the solution over the wave-length region 10.0-13.0 µ, 1 Samples of pure HEOD may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. 2 The concentrations of HEOD given are specific for a 0.4-mm cell and should be adjusted for different cell lengths in order to give a transmission of between 20% and 80% at the peak maxima. - 133 - DIELDRIN EMULSION CONCENTRATES WHO/SIF/6.R3 Fill the cell with the other two standard solutions in turn. Make replicate scans of each solution in the same wave-length region, using the same instrument settings as above. For each of the scans of the three different solutions, calculate the absorbance (E) at the band maxima as follows : (1) At 11.0 µ, E = log(~:) transmission at 11.0 µ (peak maximum) transmission at 10.85 µ (peak minimum). (2) At 11.8 µ, E = log (~:) transmission at 11.8 µ (peak maximum) transmission at 12.15 µ (peak minimum). ( p' (3) At 12.35 µ, E = log p:) transmission at 12.35 µ (peak maximum) transmission at 12.15 µ (peak minimum). For each of the three wave-lengths corresponding to the peak maxima, plot the absorbances observed for the three standard solutions against their HEOD content. An example of the calibration graph obtained is shown in Fig. 3 (see page 32). Norn : Whilst the chosen characteristic maxima for HEOD occur at about 11.0 µ, 11.8 µ, and 12.35 µ, measurements of the absorbance should be made at the actual peak maxima and not at exactly the wave-lengths mentioned. This applies also to the peak minima at 10.85 µ and 12.15 µ. 2.1.5 Procedure 2. 1 . 5. 1 Extraction of dieldrin Mix the sample thoroughly and weigh accurately an amount containing about 1 g of dieldrin. Transfer quantitatively to the chromatographic column by means of a jet of extraction solvent applied from a wash bottle. Place a 600-ml beaker under the tip of the column. Working in a well ventilated hood, add extraction solvent to the top of the column. Allow 300 ml of the solvent to percolate through into the beaker. Replace the - 134 - WHO/SIF/6.R3 DIELDRIN EMULSION CONCENTRATES 600-ml beaker by a 100-ml beaker and collect further 50-ml portions of the solvent, changing the 100-ml beaker after each 50-ml portion has been col- lected. Whilst the percolation is in progress, evaporate the solvent from the 600-ml and 100-ml beakers, e.g., by placing them in the mouth of a fume chamber the window of which has been pulled down to the level of the top of the beakers. Avoid too rapid evaporation, as this may cause condensation of moisture on the walls of the beaker. Discontinue the percolation when it is apparent from the residue in the 100-ml beakers that no further dieldrin is being extracted. With the help of the solvent, transfer the residues from all the beakers to a weighed 100-ml beaker. Ensure that no crystalline deposit remains on or inside the tip of the chromatographic column by washing with additional solvent into the weighed beaker. Evaporate the solution to dryness in a current of air. Break up the residue by means of a weighed glass rod, leaving the rod in the beaker. Place the beaker and contents for 15 minutes in an oven main- tained at 75°C ± 2°C and atmospheric pressure. Remove from the oven, cool in a desiccator, weigh and calculate the weight of the residue. NOTE : With a correctly prepared sample that has not undergone any degradation through storage, the weight of the final residue is a good preliminary indication of the dieldrin content of the sample. Should the weight be less than the nominal dieldrin content, it may be concluded that the sample has been incorrectly prepared and/or has undergone some degra- dation-although slight degradation would not seriously affect the weight of the residue. Should the weight of the final residue be higher than the nominal dieldrin content, it may be concluded that the sample has been incorrectly prepared and/or contains other extractable material that may well interfere with the infrared determination. 2. 1. 5. 2 Analysis of sample Dissolve the residue in the beaker (obtained by the procedure described in section 2. 1. 5 .1) in a small volume of carbon disulfide, transfer the solution quantitatively to a 100-ml volumetric flask, and make up to the mark with carbon disulfide.1 Fill the absorption cell-the same one used in the calibration-with the above solution and make replicate scans in the region 10.0-13.0 µ, with the same instrument settings as used in the calibration (section 2.1.4). Calculate the absorbance at the 11.0-µ, 11.8-µ, and 12.35-µ peaks from the scans, as described in section 2. 1.4. 1 A solution containing approximately 1% of dieldrin (w/v) provides optimum operating conditions with a 0.4-mm cell, giving a transmission of between 20% and 80% at the peak maxima. - 135 - DIELDRIN EMULSION CONCENTRATES WHO/SIF/6.R3 2. 1. 6 Calculation For each peak, compute the mean absorbance and read from the cali- bration graph the corresponding concentration of HEOD in grams per 100 ml of solution. Calculate the mean of the three concentrations.1 axb HEOD content(% w/w) = -- w where a = mean concentration (g per 100 ml) of HEOD, as found from the calibration graph b = volume (ml) of carbon disulfide solution containing the residue w = weight (g) of initial sample. Since dieldrin is defined (see footnote on page 130) as containing 85% of HEOD (w/w), the dieldrin content is obtained by multiplying the HEOD 100 content by 85, i.e., by l.175. Therefore, axbx l.175 dieldrin content (% w /w) = ----- w 2. 2 Cold Test Cool 50 ml of the sample to 0°C and add a small seeding-crystal of dieldrin having an edge of about 1 mm. Stir gently with a thermometer at intervals for 1 hour, maintaining the temperature at 0°C. 2.3 Flash-Point Determine the flash-point by the TAG Closed Tester method (see WHO/M/10, page 276, the Cleveland Open Tester method (see WHO/M/11, page 279), or any other equivalent standard method. 2.4 Heat Stability Keep 50 ml of the sample for three days at a temperature of 50°C ± l °C in a glass container sealed to avoid loss of volatile solvent, and then cool to room temperature. 1 In practice, the values obtained from the three different readings will be virtually identical. Although slight differences are to be expected owing to instrumental variations, the values should not differ from the mean by more than 5%, assuming that interfering substances have been effectively removed by the extraction procedure. If, in the calcula- tion, it is found that the results obtained from two wave-lengths agree within 5-10% of the mean but that the result obtained from the third wave-length is widely different, it can be assumed that interference is taking place at the third wave-length. In such an event, the result obtained from the third wave-length should be disregarded and the HEOD content calculated from the other two readings. -136 - WHO/SIF/13.R2 DIAZINON EMULSION CONCENTRATES DIAZINON EMULSION CONCENTRATES* 1. SPECIFICATIONS Specification WHO/SIF/13.Rl Approved 25 October 1965 1.1 Description and Ingredients The material shall consist of technical diazinon dissolved in a suitable solvent, with an emulsifying agent or agents added, and shall be in the form of a stable liquid, free from extraneous impurities. The technical diazinon used in the manufacture of the concentrate shall comply with the require- ments of Specification WHO/SIT/9.R2. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1.1 and with the following requirements. 1. 2 .1 Diazinon content ( w /w basis) The content of 0,0-diethyl 0-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate, determined by the method described in section 2. 1, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 2 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. 1.2.2 Cold test No separation of solid or oily material shall occur when the concen- trate is tested as described in section 2. 2. * The term " emulsion concentrate " is used here to mean a single-phase liquid system containing an insecticide together with one or more surface-active agents having the property of forming an emulsion on dilution with water. Such a system is more correctly termed an " emulsifiable concentrate ", but " emulsion concentrate " has been given preference on account of established usage. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this is only a guide and does not preclude the purchaser from sampling in any way considered desirable. 2 For a 50% emulsion concentrate, tolerance = ± 5% of 50% = ± 2.5%; for a 75% emulsion concentrate, tolerance = ± 5% of (100 - 75)% = ± 1.25%. - 137 - DIAZINON EMULSION CONCENTRATES WHO/SIF/13.R2 1. 2. 3 Flash-point The flash-point of the concentrate shall not be lower than 22.8°C when determined by one of the methods referred to in section 2. 3. 1. 2. 4 Stability of the emulsion l . 2. 4. l In distilled water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in distilled water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). I . 2. 4. 2 In standard hard water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in standard hard water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). 1. 2. 5 Heat stability The concentrate, after treatment as described in section 2.4, shall comply with the requirements of sections 1.2.l, 1.2.2, 1.2.3, and 1.2.4 of this specification. 1. 2. 6 Acidity The acidity of the concentrate, determined by the method described in WHO/M/3 (see page 250), shall not be greater than 0.05% calculated as H2S04 • 1. 3 Packing and Marking of Packages The diazinon emulsion concentrate shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following: Manufacturer's name Diazinon emulsion concentrate to Specification WHO/SIF/l3.R2 Diazinon, ... % w /w Batch or reference number, and date of test Net weight of contents Instructions for dilution -138 - WHO/SIF/13.R2 DIAZINON EMULSION CONCENTRATES and the following minimum cautionary notice : " Diazinon is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed. It may be absorbed through the skin. Avoid skin contact; wear protective gloves, clean protective clothing and a respirator when handling the material. Wash thoroughly with soap and water after using. Keep the material out of reach of children and well away from foodstuffs, animal feed, and their containers." ·· If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Diazinon Content 2 .1. 1 Summary of method The sample is mixed with diethyl ether and passed through a silica gel column to remove the emulsifier. The basic by-products are then removed by passing the sample in light petroleum through a column containing diatomaceous silica filtering aid impregnated with sulfuric acid. The 0,0- diethyl 0-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate content of the eluate is then determined by titration with standard perchloric acid. 2 . 1. 2 Special apparatus 1. Two chromatographic columns each 250 mm in length and 21-22 mm in internal diameter, fitted with glass or Teflon stopcocks and sintered- glass discs and equipped to operate under pressure. 2. Steel tamping rod with perforated disc to fit loosely inside the chromatographic column. 2 .1. 3 Special reagents Standardized O. I N perch/oric acid in glacial acetic acid. Dilute a suitable quantity of perchloric acid, obtainable as a concentrated aqueous solution, to the required volume with glacial acetic acid and sufficient acetic anhy- dride to take up the entire water content of the perchloric acid. For example to make up take up 5 litres of solution using 70% perchloric acid, the required quantities would be: 70% perchloric acid Acetic anhydride . Glacial acetic acid, to make -139 - 73 g 124.1 g 5 litres DIAZINON EMULSION CONCENTRATES WHO/SIF/13.R2 If the acetic acid is not anhydrous, a correspondingly larger volume of acetic anhydride will be required, equivalent to the total water content of the other two reagents. Standardize the solution as follows: Dissolve 0.2 g of anhydrous sodium carbonate, accurately weighed, in 50 ml of glacial acetic and titrate this solution with the perchloric acid solution, using 1% a-naphthol- benzein solution in benzene as indicator; 0.200 g of sodium carbonate corresponds to 37.73 ml of0.1 N perchloric acid. 2 .1. 4 Preparation of the silica-gel column Fill one of the chromatographic columns to two-thirds of its height with diethyl ether and add slowly 30 ml of silica gel while stirring with a glass rod. Drain the ethyl ether until its level is about 2 mm above the silica gel. 2 .1. 5 Preparation of the diatomaceous silica filtering aid column Weigh 250 g of diatomaceous silica filtering aid into the mixing bowl of a suitable mixer. This amount will provide enough material for 15 columns. Add slowly 150 ml of 3N sulfuric acid while mixing. Continue mixing until the powder is homogeneous. Scrape the material adhering to the side of the bowl during the process. Add enough light petroleum to the diatomaceous silica filtering aid mixture to cover the paste with solvent, stirring by hand with a porcelain spatula. The resulting slurry is now ready for packing into the column. Pour into the dry column light petroleum to half of its height. Tamp a glass-wool plug with the help of the tamping rod into position above the fritted glass disc. Put the funnel on top of the column. Fill portions of the slurry into the column through the funnel and tamp each portion gently with the tamping rod until the column is two-thirds full. Stir the slurry with a glass rod to eliminate all air bubbles and to ensure homogeneous packing. Adjust the pressure and open the stopcock. Press out excess of light petroleum, but retain enough solvent in the column so that the diatoma- ceous silica filtering aid is just covered at all times. Continue adding slurry and pressing until the height of the packing is about 15 cm. Tamp with the tamping rod to form an even surface and place a glass- wool plug of about 5 mm on top of the packed column. Properly packed columns allow the light petroleum to pass through dropwise. 2.1.6 Procedure Weigh accurately an amount of the sample containing about 0.4 to 0.6 g of diazinon into a 25-ml conical flask and rinse with 5-10 ml of diethyl ether on to the silica-gel column (section 2 .1 .4). Open the stopcock and - 140 - WHO/SIF/13.R2 DIAZINON EMULSION CONCENTRATES let the level sink to 1-3 mm above the surface of the silica gel. Add 30 ml of diethyl ether. Adjust the dropping funnel to the top of the column and elute with 200 ml of ether. Collect the eluate in a 500-ml flask and distil off the solvent. Rinse this residue with l 0-15 ml of light petroleum on to the column packed with diatomaceous silica filtering aid ( section 2. l . 5). Open the stopcock and let the level sink to about 1-3 mm above the level of the packing. Rinse the flask twice with 5 ml and twice with 10 ml of light petroleum quantitatively. Pour each washing down the side of the column so as to wash the sample into the packing and allow to penetrate to a level of 1-3 mm above the packing before adding the next portion. Add 30 ml of light petroleum. Adjust the dropping funnel to the top of the column and elute with 200 ml of light petroleum. Collect the eluate in a 500-ml conical flask and distil off the solvent. Dissolve the residue in 50 ml of glacial acetic acid, connect to a reflux condenser and boil for 15 minutes. Cool, rinse with glacial acetic acid into a beaker and titrate potentio- metrically with 0.1 N perchloric acid in glacial acetic acid.1 Alternatively the titration can be done visually using a l % a-naphthol- benzein solution in benzene as indicator, the end-point being reached when the solution becomes dark green. NOTE : The column can be used a second time if the first sample did not contain too large a quantity of by-products. Care has to be taken that the column does not get dry, but remains covered by light petroleum. 2.1. 7 Calculation 0 ,0-Diethyl 0-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate content (%) a x 3.044 w where a = volume (ml) of 0.1 N perchloric acid used w = weight (g) of sample. 2.2 Cold Test Cool 50 ml of the sample to 0°C. Stir gently with a thermometer at intervals for 1 hour, maintaining the temperature at 0°C. 1 Care must be taken that all titrations with perchloric acid are performed in abso- lutely anhydrous media. -141 - DIAZINON EMULSION CONCENTRATES WHO/SIF/13.R2 2.3 Flash-Point Determine the flash-point by the TAG Closed Tester method (see WHO/M/10, page 276), the Cleveland Open Tester method (see WHO/ M/11, page 279), or any other equivalent standard method. 2 . 4 Heat Stability Keep 50 ml of the sample for three days at a temperature of 50°C ± 1 °C in a glass container sealed to avoid loss of volatile solvent, and then cool to room temperature. - 142 - WHO/SIF/14.R2 MALATHION EMULSION CONCENTRATES MALATHION EMULSION CONCENTRATES* 1. SPECIFICATIONS Specification WHO/SIF/14.R2 Approved 25 October 1965 1.1 Description and Ingredients The material shall consist of technical malathion dissolved in a suitable solvent, with an emulsifying agent or agents added, and shall be in the form of a stable liquid, free from extraneous impurities. The technical malathion used in the manufacture of the concentrate shall comply with the requirements of Specification WHO/SIT/10.R2. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1 . l and with the following requirements.2 1.2.1 Malathion content (w/w basis) The content of 0,0-dimethyl S-[l,2-di-(ethoxycarbonyl)ethyl] phos- phorodithioate, determined by the method described in section 2. l, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 3 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. * The term " emulsion concentrate " is used here to mean a single-phase liquid system containing an insecticide together with one or more surface-active agents having the property of forming an emulsion on dilution with water. Such a system is more correctly termed an " emulsifiable concentrate ", but " emulsion concentrate " has been given preference on account of established usage. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 If the material is required for use inside buildings, odour-free malathion should also be specified. 3 For a 50% emulsion concentrate, tolerance= ± 5% of 50% = ± 2.5%; for a 75% emulsion concentrate, tolerance = ± 5% of (100 - 75) % = ± 1.25%. - 143 - MALATHION EMULSION CONCENTRATES WHO/SIF/14.R2 1. 2. 2 Cold test No separation of solid or oily material shall occur when the concentrate is tested as described in section 2. 2. 1. 2. 3 Flash-point The flash-point of the concentrate shall not be lower than 22.8°C when determined by one of the methods referred to in section 2. 3. 1. 2. 4 Stability of the emulsion 1 . 2 .4. 1 In distilled water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in distilled water with 5 ml of con- centrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). 1 . 2 .4. 2 In standard hard water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in standard hard water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/ M/13 (see page 284). 1. 2. 5 Heat stability The concentrate, after treatment as described in section 2. 4, shall comply with the requirements of sections 1.2.1, 1.2.2, 1.2.3, and 1.2.4 of this specification. 1. 2. 6 Acidity The acidity of the concentrate, determined by the method described in WHO/M/3 (see page 250), shall not be greater than 0.5%, calculated as H2S04 • 1. 3 Packing and Marking of Packages The malathion emulsion concentrate shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : -144 - WHO/SIF/14.R2 MALATHION EMULSION CONCENTRATES Manufacturer's name Malathion emulsion concentrate to Specification WHO/SIF/14. R2 Malathion, ... % w /w Batch or reference number, and date of test Net weight of contents Instructions for dilution and the following minimum cautionary notice : " Malathion is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Malathion Content 2 .1. 1 Special apparatus Spectrophotometer or photoelectric colorimeter with a blue filter (420 mµ). 2. 1. 2 Special reagents Acetonitrile- boiling range 80-82°C. Pass through a column of silica gel and discard the first (yellow) portion of the eluate. Collect the colourless eluate. A 10% aqueous solution of this should have a pH between 5 and 7. Ferric reagent. Dissolve 0.2 g of ferric chloride (FeC13 • 6H20) in dis- tilled water, add 8 ml of concentrated hydrochloric acid, and dilute to l litre with distilled water. Cupric reagent. Dissolve 1.5 g of cupric sulfate (CuS04 • 5H20) in 100 ml of water. 2. 1. 3 Preparation of malathion standard solutions Standard solution A. Accurately weigh into a 5-ml or 10-ml beaker 1.00 g ± 0.02 g of malathion reference standard.1 Transfer quantitatively to a I-litre volumetric flask with anhydrous ethanol. Dilute with anhydrous 1 Samples of reference standard malathion may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. -145 - 10 MALATHION EMULSION CONCENTRATES WHO/SIF/14.R2 ethanol to about 5 ml below the mark and allow to stand until a steady temperature is reached. Record the temperature, dilute to the mark with anhydrous ethanol and mix well. The solution is stable for about 2 weeks. Adjust to the recorded temperature before withdrawing aliquots. Standard solution B. Pipette a 15-ml aliquot of standard solution A into a 250-ml volumetric flask. Add 2.5 ml of acetonitrile and dilute with anhydrous ethanol to about 5 ml below the mark. Keep until the sample for analysis is ready for final dilution. At that time, dilute with anhydrous ethanol to the mark and mix well. NOTE : Volume changes of alcohol solutions with temperature are appreciable. Bring each solution to volume just before proceeding to next step. 2 .1. 4 Preparation of sample Accurately weigh a sample containing 1.00 g + 0.02 g of malathion into a 5-ml or 10-ml beaker. Transfer quantitatively to a I-litre volumetric flask with anhydrous ethanol, dilute to the mark with additional anhydrous ethanol and mix well. Immediately pipette a 15-ml aliquot into a 250-ml volumetric flask, add 2.5 ml of acetonitrile, dilute to the mark with anhyd- rous ethanol and mix well. 2 .1. 5 Analysis of sample Determine the standard malathion and the sample separately but at the same time. Perform the entire analysis without interruption. Pipette 25-ml aliquots of standard solution B and sample solutions into two 250-ml separating funnels. Add 2 ml ± 0.1 ml of 0.5 N sodium hydroxide to each and mix well by swirling gently for 5 to 10 seconds (do not shake). Allow to stand for 120 seconds ± 10 seconds. Add 75 ml ± l ml of ferric reagent and mix well by swirling for 10 seconds. Allow to stand for 5 minutes. During this waiting period fill two 50-ml volumetric flasks to the mark with cyclohexane. Transfer the cyclohexane from each of these flasks into one of the separating funnels and allow the flasks to drain for one minute. Add 2.0 ml ± O.l ml of cupric reagent and shake in the separating funnel for exactly one minute. NOTE : The copper-malathion complex is unstable in the aqueous phase. The cupric reagent should therefore be added from a fast delivery Mohr pipette and shaking started without delay. A delay of only 15 seconds before extraction results in appreciable losses. Allow the phases to separate and discard the aqueous phase as soon as separation occurs. Allow a little of the solvent phase to rinse the stem of -146 - WHO/SIF/14.R2 MALATHION EMULSION CONCENTRATES the separating funnel, then transfer the clear solvent phase to a small beaker and thence to the cell, or transfer directly to the cell. Manipulate phase separation of both standard and sample similarly. Immediately (within 5 minutes) determine the absorbance of the yellow cyclohexane solution at 420 mµ by means of the spectrophotometer ( or photoelectric colorimeter) using cyclohexane reference. 2 .1. 6 Calculation 0 ,0-Dimethyl S-[l ,2-di(ethoxycarbonyl)ethyl] phosphorodithioate content (% w /w) Asample a X p X 0.3 A x bxlOO standard where Asample = absorbance of the sample at 420 mµ Asiandard = absorbance of the standard at 420 mµ a = weight (g) of reference standard malathion taken to pre- pare standard solution b = weight (g) of sample p = purity (%) of reference standard malathion. 2.2 Cold Test Cool 50 ml of the sample to 0°C. Stir gently with a thermometer at intervals for 1 hour, maintaining the temperature at 0°C. 2.3 Flash-Point Determine the flash-point by the TAG Closed Tester method (see WHO/M/10, page 276), the Cleveland Open Tester method (see WHO/ M/ll, page 279), or any other equivalent standard method. 2 .4 Heat Stability Keep 50 ml of the sample for three days at a temperature of 50°C ± 1 °C in a glass container sealed to avoid loss of volatile solvent, and then cool to room temperature. -147 - PARATHION EMULSION CONCENTRATES WHO/SIF/15.R2 PARATHION EMULSION CONCENTRATES* Specification WHO/SIF/15.R2 Approved 25 October 1965 l. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of technical parathion dissolved in a suitable solvent, with an emulsifying agent or agents added, and shall be in the form of a stable liquid, free from extraneous impurities. The technical parathion used in the manufacture of the concentrate shall comply with the requirements of Specification WHO/SIT/11. R2. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment, 1 shall comply with the requirements of section l. 1 and with the following requirements. 1. 2 .1 Parathion content ( w /w basis) The content of O ,0-diethyl 0-(p-nitrophenyl) phosphorothioate deter- mined by the method described in section 2. 1, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 2 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. * The term " emulsion concentrate " is used here to mean a single-phase liquid system containing an insecticide together with one or more surface-active agents having the property of forming an emulsion on dilution with water. Such a system is more correctly termed an " emulsifiable concentrate ", but " emulsion concentrate " has been given preference on account of established usage. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 For a 50% emulsion concentrate, tolerance = ± 5% of 50% = ± 2.5% ; for a 75% emulsion concentrate, tolerance = ± 5% of (100 - 75)% = ± 1.25%. -148 - WHO/SIF/15.R2 PARATHION EMULSION CONCENTRATES 1. 2. 2 Cold test No separation of solid or oily material shall occur when the concentrate is tested as described in section 2. 2. 1. 2. 3 Flash-point The flash-point of the concentrate shall not be lower than 22.8°C when determined by one of the methods referred to in section 2 . 3. 1. 2 . 4 Stability of the emulsion l . 2. 4. 1 In distilled water Any separation, including creaming at the top and sedimentation at the bottom, of l 00 ml of emulsion prepared in distilled water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). l . 2. 4. 2 In standard hard water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in standard hard water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/ M/13 (see page 284). 1. 2. 5 Heat stability The concentrate, after treatment as described in section 2. 4, shall comply with the requirements of sections 1.2.1, 1.2.2, 1.2.3, and 1.2.4 of this specification. 1. 2. 6 Acidity The acidity of the concentrate, determined by the method described in WHO/M/3 (see page 250), shall not be greater than 0.1 % calculated as H2S04 • 1. 3 Packing and Marking of Packages The parathion emulsion concentrate shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : -149- PARATHION EMULSION CONCENTRATES WHO/SIF/15.R2 Manufacturer's name Parathion emulsion concentrate to Specification WHO /SIF /15. R2 Parathion, ... % w/w Batch or reference number, and date of test Net weight of contents Instructions for dilution and the following minimum cautionary notice : "POISON [Skull-and-cross-bones insignia] " Parathion is an organophosphorus compound which inhibits cholin- esterase. It is a very toxic substance. Contact with the skin, inhalation of dust or spray, or swallowing may be fatal. Wear protective gloves, clean protective clothing, and a respirator of the organic-vapour type when handling this material. Bathe immediately after work. " Ensure that containers are stored under lock and key. Empty con- tainers must be disposed of in such a way as to prevent all possibility of accidental contact with them. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. " In case of contact, immediately remove contaminated clothing and wash the skin thoroughly with soap and water ; for eyes, flush with water for 15 minutes. "If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and repeated doses may be necessary. Artificial respira- tion may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Parathion Content 2 .1.1 Summary of method The emulsifying agent and p-nitrophenol, the most likely impurity, are removed by treating the sample with light petroleum and aqueous ethanol, followed by extraction with sodium bicarbonate solution. The 0,0-diethyl 0-(p-nitrophenyl) phosphorothioate in the light-petroleum layer is reduced by zinc and an acetic acid/hydrochloric acid mixture, and the amino groups formed are titrated with standard sodium nitrite solution. - 150 - WHO/SIF/15.R2 PARATHION EMULSION CONCENTRATES 2 .1. 2 Special reagents Acetic acid/hydrochloric acid mixture. Mix 9 volumes of glacial acetic acid with 1 volume of concentrated hydrochloric acid. Sodium nitrite, 0.1 M, standardized against sulfanilic acid. Dissolve 6.90 g of sodium nitrite in distilled water and make up to 1 litre with distilled water. Standardize this solution as follows : Weigh accurately 0.40-0.45 g of anhydrous sulfanilic acid (analytical grade or material, the purity of which has been checked by a nitrogen determination) into a 400-ml tall-form beaker. Add 80 ml of distilled water, 10 ml of concentrated hydrochloric acid, 30 ml of glacial acetic acid, and 5 g of sodium (or potassium) bromide. Cool the mixture to 0-10°C by the addition of clean, shaved ice and place under mechanical stirring. Titrate at 0-10°C with the 0.1 M sodium nitrite as rapidly as the spot test (section 2.1.3.3) permits. Near the end-point, add the sodium nitrite in 4-drop portions. where a b a x 5.774 Normality of 0.1 M sodium nitrite = -- ·--b weight (g) of sulfanilic acid used volume (ml) of 0.1 M sodium nitrite required. 2.1.3 Procedure 2. 1 . 3. 1 Separation and extraction Weigh accurately an amount of the sample containing about 1 g of 0 ,0-diethyl 0-(p-nitrophenyl) phosphorothioate and rinse with 100 ml of 50% aqueous ethanol and 100 ml of light petroleum (boiling-range 40-60°C) into a 250-ml separating funnel. Add 10 ml of saturated sodium chloride solution and shake during 30 seconds. Drain the aqueous-ethanolic layer into a second separating funnel and extract with 100 ml of light petroleum. Repeat this operation with a third 250-ml separating funnel and discard the aqueous-ethanolic layer. Wash the three light-petroleum extracts separately and successively with the following reagents, using the same reagent for the successive treatments of the three extracts : (a) twice with 30-ml portions of 50% aqueous ethanol; (b) twice with 30-ml portions of chilled 10% sodium bicarbonate solution ; and (c) finally with 50 ml of distilled water to which 10 ml of saturated sodium chloride solution have been added. - 151 - PARATHION EMULSION CONCENTRATES WHO/SIF/15.R2 2.1.3.2 Determination of 0,0-diethyl 0-(p-nitrophenyl) phosphorothioate in light-petroleum layer Combine the three light-petroleum extracts in a 400-ml tall-form beaker and evaporate the solvent. Add 35 ml of acetic acid/hydrochloric acid mixture and 3 g of iron-free zinc dust.1 Cover the beaker with a watch-glass and heat on a steam-bath for at least 15 minutes, or until the solution is colourless. Add 10 ml of concentrated hydrochloric acid and continue the heating until the zinc is completely dissolved. If undissolved zinc remains, filter the solution through a sintered-glass filter and then wash with water. Cool, and then add 50 ml of distilled water and 5 g of sodium (or potassium) bromide. Cool to O-l0°C by the addition of clean, shaved ice and place under mechanical stirring. Titrate at O-l0°C with the stand- ardized 0.1 M sodium nitrite as rapidly as the spot test (section 2. I. 3. 3) permits. Near the end-point, add the sodium nitrite in 4-drop portions. 2.1.3.3 Spot test Dip a glass rod into the solution to be tested and then touch the rod quickly to a piece of potassium iodide/starch paper. The end-point is reached when an intense blue-black colour appears within 15-20 seconds and can be obtained repeatedly during a I-minute period without further addition of sodium nitrite. 2 .1. 4 Calculation 0 ,0-Diethyl 0-(p-nitrophenyl) phosphorothioate content (% w /w) c x N x 29 .13 x f w where N = c = W= normality of the O.l M sodium nitrite (see section 2.1.2) volume (ml) of 0.1 M sodium nitrite required weight (g) of sample != . T correction factor A where T = quantity (ml) of 0.1 M sodium nitrite calculated for the nitro- group determination of l g of a mono-nitro reference substance.2 1 If less than 3 g of zinc dust are used or there is reason to doubt the purity of the zinc dust, this should be checked by analysis. 2 Since 0,0-diethyl 0-(p-nitrophenyl) phosphorothioate is not usually available as a pure material, f is evaluated on the basis of another substance containing one nitro group. Suitable materials are : m-nitrobenzoic acid. p-nitrobenzoic acid . - 152 - Melting-range 140.5-14l.0°C 239.6-240.1 °C WHO/SIF/15.R2 PARATHION EMULSION CONCENTRATES A = quantity (ml) of 0.1 M sodium nitrite used in the nitro-group determination of l g of a recrystallized sample of the same substance, following the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2. 2 Cold Test Cool 50 ml of the sample to 0°C. Stir gently with a thermometer at intervals for 1 hour, maintaining the temperature at 0°C. 2. 3 Flash-Point Determine the flash-point by the TAG Closed Tester method (see WHO/M/10, page 276), the Cleveland Open Tester method (see WHO/ M/ll, page 279), or any other equivalent standard method. 2. 4 Heat Stability Keep 50 ml of the sample for three days at a temperature of 50°C ± 1 °C in a glass container sealed to avoid loss of volatile solvent, and then cool to room temperature. -153 - TRICHLORFON EMULSION CONCENTRATES WHO/SIF/20.R1 TRICHLORFON EMULSION CONCENTRATES* Tentative Specification WHO/SIF/20.Rl Approved 25 October 1965 1. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of technical trichlorfon dissolved in a suitable solvent, with an emulsifying agent or agents added, and shall be in the form of a stable liquid, free from extraneous impurities. The technical trichlorfon used in the manufacture of the concentrate shall comply with the requirements of Specification WHO/SIT /13. Rl. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1. 1 and with the following requirements. 1.2.1 Trichlorfon content (w/w basis) The content of dimethyl 1-hydroxy-2,2,2-trichloroethylphosphonate de- termined by the method described in section 2. 1, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 2 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. * The term " emulsion concentrate " is used here to mean a single-phase liquid system containing an insecticide together with one or more surface-active agents having the property of forming an emulsion on dilution with water. Such a system is more correctly termed an " emulsifiable concentrate ", but " emulsion concentrate " has been given preference on account of established usage. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 For a 50% emulsion concentrate, tolerance = ± 5% of 50% = ± 2.5%; for a 75% emulsion concentrate, tolerance = ± 5% of (100 - 75)% = ± 1.25%. - 154 - WHO/SIF/20.R1 TRICHLORFON EMULSION CONCENTRATES 1. 2. 2 Cold test No separation of solid or oily material shall occur when the con- centrate is tested as described in section 2. 2. 1. 2. 3 Flash-point The flash-point of the concentrate shall not be lower than 22.8°C when determined by one of the methods referred to in section 2. 3. 1. 2. 4 Stability of the emulsion 1 . 2. 4. 1 In distilled water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in distilled water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). 1 . 2. 4. 2 In standard hard water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in standard hard water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). 1. 2. 5 Heat stability The concentrate, after treatment as described in section 2.4, shall comply with the requirements of sections 1 . 2. 1, 1 . 2. 2, 1 . 2. 3, and 1 . 2. 4 of this specification. 1. 2. 6 Acidity The acidity of the concentrate, determined by the method described in section 2. 5 shall not be greater than 1.0%, calculated as H2SO 4• 1. 3 Packing and Marking of Packages The trichlorfon emulsion concentrate shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Trichlorfon emulsion concentrate to Specification WHO /SIF /20 - 155 - TRICHLORFON EMULSION CONCENTRATES Trichlorfon, ... % w /w Batch or reference number, and date of test Net weight of contents Instructions for dilution and the following minimum cautionary notice : WHO/SIF/20.R1 " Trichlorfon is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed or absorbed through the skin. Avoid skin contact ; wear protective gloves and clean protective clothing while using the material. Wash thoroughly with soap and water after using. " Keep the material out of reach of children and well away from food- stuffs, animal feed and their containers. "If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Dimethyl 1-hydroxy-2,2,2-trichloroethylphosphonate Content 2.1.1 Procedure 2. l. l. l Determination of hydrolysable plus inorganic chlorine Weigh accurately an amount of the sample containing about l g of dimethyl l-hydroxy-2,2,2-trichloroethylphosphonate into a 250-ml conical flask. Add 90 ml of anhydrous methanol and 10 ml of pure 2-aminoethanol of over 99% purity, mix well, and keep for exactly 1 hour at 20°C ± 0.5°C. Cool in ice water and add 50 ml of chlorine-free 20% nitric acid. Keep at 20°C and titrate electrometrically with 0.1 N silver nitrate. 2. l . 1 . 2 Determination of inorganic chlorine Weigh accurately about l g of the sample and transfer with 100 ml of distilled water to a 250-ml conical flask. Acidify with 5 ml of chlorine- free 20% nitric acid and titrate electrometrically with 0.1 N silver nitrate. 2.1.2 Calculation Dimethyl 1-hydroxy-2,2,2-trichloroethylphosphonate content (% w /w) = (!!_ - !!____) x 2.575 x f W1 W2 -156 - WHO/SIF/20.R1 where a b TRICHLORFON EMULSION CONCENTRATES volume (ml) of 0.1 N silver nitrate equivalent to the hydro- lysable plus inorganic chlorine volume (ml) of 0.1 N silver nitrate equivalent to the inor- ganic chlorine w1 = weight (g) of sample used for the hydrolysable plus inor- ganic chlorine determination w2 = weight (g) of the sample used for the inorganic chlorine de- termination f where A . ~ 13.77 correct10n 1actor -- A value found for hydrolysable chlorine in recrystallized di- methyl l-hydroxy-2,2,2-trichloroethylphosphonate determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2.2 Cold Test Cool 50 ml of the sample to 0°C. Stir gently with a thermometer at intervals for 1 hour, maintaining the temperature at 0°C. 2.3 Flash-Point Determine the flash-point by the TAG Closed Tester method (see WHO/M/10, page 276), the Cleveland Open Tester method (see WHO/ M/11, page 279), or any other equivalent standard method. 2. 4 Heat Stability Keep 50 ml of the sample for three days at a temperature of 50°C ± 1 °C in a glass container sealed to avoid loss of volatile solvent, and then cool to room temperature. 2.5 Acidity 2.5.1 Procedure Weigh a sample containing approximately 10 g of trichlorfon and dis- solve it in 100 ml of distilled water, with gentle warming if necessary. Titrate immediately at 10-15°C with 0.02 N sodium hydroxide, using -157 - TRICHLORFON EMULSION CONCENTRATES WHO/SIF/20.R, methyl red as indicator. Carry out a blank determination on 100 ml of distilled water with 0.02 N sodium hydroxide. 2.5.2 Calculation Acidity % by weight calculated as H2S04 = 0.0098 x (a-b) where a = volume (ml) of 0.02 N sodium hydroxide used for the sample b = volume (ml) of 0.02 N sodium hydroxide used for the blank. NOTE: The blank may take the form of a small titre with 0.02 N hydro- chloric acid, in which case acidity % by weight calculated as H2S04 = 0.0098 x (a+ c) where a = volume (ml) of 0.02 N sodium hydroxide used for the sample c = volume (ml) of 0.02 N hydrochloric acid used for the blank. Alternatively, the end-point may be determined electrometrically. -158 - WHO/SIF/27 PARATHION-METHYL EMULSION CONCENTRATES PARATHION-METHYL EMULSION CONCENTRATES* Tentative Specification WHO/SIF/27 Approved 25 October 1965 l. SPECIFICATIONS 1. 1 Description and Ingredients The material shall consist of technical parathion-methyl dissolved in a suitable solvent, with an emulsifying agent or agents added, and shall be in the form of a stable liquid, free from extraneous impurities. The technical parathion-methyl used in the manufacture of the concentrate shall comply with the requirements of Specification WHO/SIT/14. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l . l and with the following requirements. 1.2.1 Parathion-methyl content (w/w basis) The content of O ,0-dimethyl 0-(p-nitrophenyl) phosphorothioate, determined by the method described in section 2. l , shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 2 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. * The term " emulsion concentrate " is used here to mean a single-phase liquid system containing an insecticide together with one or more surface-active agents having the property of forming an emulsion on dilution with water. Such a system is more correctly termed an " emulsifiable concentrate ", but " emulsion concentrate " has been given preference on account of established usage. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 For a 50% emulsion concentrate, the tolerance = ± 5% of 50% = ± 2.5% ; for a 75% emulsion concentrate, the tolerance = ± 5% of (100 - 75)% = ± 1.25%. -159 - PARA THI ON-METHYL EMULSION CONCENTRATES WHO/SIF/27 1. 2. 2 Cold test No separation of solid or oily material shall occur when the concentrate is tested as described in section 2.2. 1. 2. 3 Flash-point The flash-point of the concentrate shall not be lower than 22.8°C when determined by one of the methods referred to in section 2. 3. 1. 2. 4 Stability of the emulsion l . 2. 4. 1 In distilled water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in distilled water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). 1 . 2. 4. 2 In standard hard water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in standard hard water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/ M/13 (see page 284). 1. 2. 5 Heat stability The concentrate, after treatment as described in section 2. 4, shall comply with the requirements of sections 1 . 2. 1, 1 . 2. 2, 1 . 2. 3 and 1 . 2 .4 of this specification. 1. 2. 6 Acidity The acidity of the concentrate, determined by the method described in WHO/M/3, page 250, shall not be greater than 0.1 %, calculated as H2S04• 1. 3 Packing and Marking of Packages The parathion-methyl emulsion concentrate shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Parathion-methyl emulsion concentrate to Specification WHO/ SIF/27 -160 - WHO/SIF/27 PARATHION-METHYL EMULSION CONCENTRATES Parathion-methyl, ... % w /w Batch or reference number, and date of test Net weight of contents Instructions for dilution and the following minimum cautionary notice : "POISON [Skull-and-cross-bones insignia] " Parathion-methyl is an organophosphorus compound which inhibits cholinesterase. It is a very toxic substance. Contact with the skin, inhala- tion of dust or spray, or swallowing may be fatal. Wear protective gloves, clean protective clothing, and a respirator of the organic-vapour type when handling this material. Bathe immediately after work. " Ensure that containers are stored under lock and key. Empty con- tainers must be disposed of in such a way as to prevent all possibility of accidental contact with them. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. "In case of contact, immediately remove contaminated clothing and wash the skin thoroughly with soap and water ; for eyes, flush with water for 15 minutes. "If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and repeated doses may be necessary. Artificial respira- tion may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSlCAL PROPERTIES 2.1 O,O-Dimethyl-0-(p-nitrophenyl) Phosphorothioate Content 2 .1.1 Summary of method The emulsifying agent and 4-nitrophenol, the most likely impurity, are removed by treating the sample with light petroleum and aqueous ethanol, followed by extraction with sodium bicarbonate solution. The O,O-dimethyl-0-(p-nitrophenyl) phosphorothioate in the light-petroleum layer is reduced by zinc and an acetic acid/hydrochloric acid mixture, and the amino groups formed are titrated with standard sodium nitrite solu- tion. -161 - LI PARATHION-METHYL EMULSION CONCENTRATES WHO/SIF/27 2 .1. 2 Special reagents Acetic acid/hydrochloric acid mixture. Mix nine volumes of glacial acetic acid with one volume of concentrated hydrochloric acid. Sodium nitrite, 0.1 M, standardized against sulfanilic acid. Dissolve 6.90 g of sodium nitrite in distilled water and make up to one litre with distilled water. Standardize this solution as follows : Weigh accurately 0.40-0.45 g of anhydrous sulfanilic acid (analytical grade or material, the purity of which has been checked by a nitrogen determination) into a 400-ml tall-form beaker. Add 80 ml of distilled water, 10 ml of concentrated hydrochloric acid, 30 ml of glacial acetic acid, and 5 g of sodium (or potassium) bromide. Cool the mixture to O-l0°C by the addition of clean, shaved ice and place under mechanical stirring. Titrate at O-l0°C with the O.l M sodium nitrite as rapidly as the spot test (section 2. l . 3. 3) permits. Near the end-point, add the sodium nitrite in 4-drop portions. N l. f O l M d" . . a x 5.774 orma 1ty o . so mm mtnte = --b-- where a = weight (g) of sulfanilic acid used b = volume (ml) of O.l M sodium nitrite required. 2.1.3 Procedure 2. l . 3. l Extraction Weigh accurately an amount of the sample containing about l g of parathion-methyl and rinse with 100 ml of 50% aqueous ethanol and 100 ml of light petroleum (boiling-range 40-60°C) into a 250-ml separating funnel. Add lO ml of saturated sodium chloride solution and shake during 30 seconds. Drain the aqueous-ethanolic layer into a second separating funnel and extract with 100 ml of light petroleum. Repeat this operation with a third 250-ml separating funnel and discard the aqueous-ethanolic layer. Wash the three light-petroleum extracts separately and successively with the following reagents, using the same reagent for the successive treatments of the three extracts : (a) twice with 30-ml portions of 50% aqueous ethanol; (b) twice with 30-ml portions of chilled l0% sodium bicarbonate solu- tion; and ( c) finally with 50 ml of distilled water to which lO ml of saturated sodium chloride solution have been added. - 162 - WHO/SIF/27 PARATHION-METHYL EMULSION CONCENTRATES 2. 1 . 3 . 2 Determination of O, 0-dimethyl 0-( p-nitrophenyl) phosphorothioate in light-petroleum layer Combine the three light-petroleum extracts in a 400-ml tall-form beaker and evaporate the solvent. Add 35 ml of acetic acid/hydrochloric acid mixture and 3 g of iron-free zinc dust.1 Cover the beaker with a watch-glass and heat on a steam-bath for at least 15 minutes, or until the solution is colourless. Add 10 ml of concentrated hydrochloric acid and continue the heating until the zinc is completely dissolved. If undissolved portions are present, filter through a sintered-glass filter and subsequently wash with water. Cool, and then add 50 ml of distilled water and 5 g of sodium ( or potassium) bromide. Cool to 0-10°C by the addition of clean, shaved ice and place under mechanical stirring. Titrate at 0-10°C with the standardized 0.1 M sodium nitrite as rapidly as the spot test (section 2.1.3.3) permits. Near the end-point, add the sodium nitrite in 4-drop portions. 2.1. 3. 3 Spot test Dip a glass rod into the solution to be tested and then touch the rod quickly on to a piece of potassium iodide/starch paper. The end-point is reached when an intense blue-black colour appears within 15-20 seconds and can be obtained repeatedly during a 1-minute period without further addition of sodium nitrite. 2 .1. 4 Calculation 0,0-dimethyl 0-(p-nitrophenyl) phosphorothioate content(% w/w) = c x N x 26.33 x f w where N = normality of the 0.1 M sodium nitrite (see section 2 .1. 2) c = volume (ml) of 0.1 M sodium nitrite required w = weight (g) of sample f . r. T = correction 1actor -A T = volume (ml) of 0.1 M sodium nitrite calculated for the nitro- group determination of 1 g of a mono-nitro reference ma- terial 2 1 If less than 3 g of zinc dust are used or there is reason to doubt the purity of the zinc dust, this should be checked by analysis. 2 Since parathion-methyl is not usually available as a pure material, f is evaluated on the basis of another substance containing one nitro group. Suitable materials are : Melting-range m-nitrobenzoic acid. . . 140.5-14l.0°C p-nitrobenzoic acid . . . . . . . . . . . . 239.6-240.1 °c -163 - PARATHION-METHYL EMULSION CONCENTRATES WHO/SIF/27 A = volume (ml) of 0.1 M sodium nitrite used in the nitro-group determination of l g of the same mono-nitro reference material following the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its appli- cation by a given laboratory. Its value must lie within the range 0.98-1.02. 2.2 Cold Test Cool 50 ml of the sample to 5°C. Stir gently with a thermometer at intervals for one hour, maintaining the temperature at 5°C. 2. 3 Flash-point Determine the flash-point by the TAG Closed Tester method (see WHO/M/10, page 276), the Cleveland Open Tester method (see WHO/ M/11, page 279), or any other equivalent standard method. 2 .4 Heat Stability Keep 50 ml of the sample for three days at a temperature of 50°C ± l °C in a glass container sealed to avoid loss of volatile solvent, and then cool to room temperature. -164 - WHO/SIF/28 FENTHION EMULSION CONCENTRATES FENTHION EMULSION CONCENTRATES * Tentative Specification WHO/SIF/28 Approved 25 October 1965 l. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of technical fenthion dissolved in a suitable solvent, with an emulsifying agent or agents added, and shall be in the form of a stable liquid, free from extraneous impurities. The technical fenthion used in the manufacture of the concentrate shall comply with the requirements of Specification WHO/SIT/15. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l . l and with the following requirements. 1.2.1 Fenthion content (w/w basis) The content of O ,0-dimethyl 0-( 4-methylthio-m-tolyl) phosphoro- thioate, determined by the method described in section 2. l, shall not differ from the nominal content by more than the following amounts : Nominal content Up to 50% Above 50% Tolerance permitted ± 5% of the nominal content } 2 ± 5% of (100 - nominal content) The average content of all samples taken shall not be lower than the nominal content. * The term " emulsion concentrate " is used here to mean a single-phase liquid system containing an insecticide together with one or more surface-active agents having the property of forming an emulsion on dilution with water. Such a system is more correctly termed an " emulsifiable concentrate ", but " emulsion concentrate " has been given preference on account of established usage. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 For a 50% emulsion concentrate, the tolerance = ± 5% of 50% = ± 2.5% ; for a 75% emulsion concentrate, the tolerance= ± 5% of (100 - 75)% = ± 1.25%. -165 - FENTHION EMULSION CONCENTRATES WHO/SIF/28 1. 2. 2 Cold test No separation of solid or oily material shall occur when the concentrate is tested as described in section 2. 2. 1. 2. 3 Flash-point The flash-point of the concentrate shall not be lower than 22.8°C when determined by one of the methods referred to in section 2. 3. 1. 2. 4 Stability of the emulsion l . 2. 4. l In distilled water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in distilled water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). l . 2. 4. 2 In standard hard water Any separation, including creaming at the top and sedimentation at the bottom, of 100 ml of emulsion prepared in standard hard water with 5 ml of concentrate, shall not exceed 2 ml when tested as described in WHO/M/13 (see page 284). 1. 2. 5 Heat stability The concentrate, after treatment as described in section 2. 4, shall comply with the requirements of sections l . 2. l, l . 2. 2, 1. 2. 3, and 1. 2 .4 of this specification. 1. 2. 6 Acidity or alkalinity The acidity or alkalinity of the concentrate, determined by the method described in WHO/M/3, page 250, shall not be greater than 0.3% calculated as H2S04 • 1. 3 Packing and Marking of Packages The fenthion emulsion concentrate shall be packed in suitable clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following -166 - WHO/SIF/28 FENTHION EMULSION CONCENTRATES Manufacturer's name Fenthion emulsion concentrate to specification WHO /SIF /28 Fenthion ... % w /w Batch or reference number, and date of test Net weight of contents Instructions for dilution and the following minimum cautionary notice : " Fenthion is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed or absorbed through the skin. Avoid skin contact ; wear protective gloves, clean protective clothing and a res- pirator when handling the material. Wash thoroughly with soap and water after using. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. " If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 0,0-Dimethyl 0-(4-methylthio-m-tolyl) Phosphorothioate Content 2 .1.1 Summary of method The active ingredient is hydrolysed with a solution of potassium hydrox- ide in ethylene glycol monoethyl ether, and the resulting 4-methylthio-m- cresol is coupled at pH l0.3-l0.4 with diazo-3-nitraniline-4-sulfonic acid to form a red-violet azo dye, the absorption of which is measured at 530 mµ. The free 4-methylthio-m-cresol, the most likely impurity of fenthion ori- ginally presented in the sample, is determined by the same method and the quantity present deducted from the amount found after hydrolysis. 2 .1. 2 Special apparatus Spectrophotometer or photoelectric colorimeter capable of measuring absorbance at 530 mµ. 2.1. 3 Special reagents 4-Methylthio-m-creso/, pure. This material should have a crystallizing point of 56° ± 0.5°C when tested by WHO/M/12, page 281. -167 - FENTHION EMULSION CONCENTRATES WHO/SIF/28 b = amount (mg) of free 4-methylthio-m-cresol found in section 2.1.4.2 w1 = weight (g) of sample used for the determination of the total amount of 4-methylthio-m-cresol in section 2. I. 5. 2 w2 = weight (g) of sample used for the determination of free 4-methylthio-m-cresol in section 2. 1 .4. 2. 2. 2 Cold test Cool 50 ml of the sample to 0°C. Stir gently with a thermometer at intervals for one hour, maintaining the temperature at 0°C. 2 . 3 Flash-point Determine the flash-point by the TAG Closed Tester method (see WHO/M/10, page 276), the Cleveland Open Tester method (see WHO/ M/11, page 279), or any other equivalent standard method. 2. 4 Heat Stability Keep 50 ml of the sample for three days at a temperature of 50°C ± 1 °C in a glass container sealed to avoid loss of volatile solvent, and then cool to room temperature. - 170 - WHO/SIF/16.R2 DDT DUSTING POWDERS DDT DUSTING-POWDERS 1. SPECIFICATIONS Specification WHO/SIF/16.R2 Approved 25 October 1965 1.1 Description and Ingredients The material shall consist of a homogeneous mixture of technical DDT in a filler and shall be in the form of a fine, free-flowing, white to cream or grey powder, free from extraneous impurities or added modifying agents. The technical DDT used in the manufacture of the powder shall comply with the requirements of Specification WHO/SIT/1.R3. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1 . 1 and with the following requirements. 1.2.1 DDT content (w/w basis) The content of DDT, determined by one of the methods descril::ed in section 2 .1, shall not differ from the nominal DDT content by more than ± 10%. The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Sieving after accelerated storage Not less than 98% of the powder (dry weight), after accelerated storage treatment as described in section 2.2, shall pass through a 150-µ sieve (BS 100 mesh; 2 US Standard No. 100 3) when tested by the method described in WHO/M/4, page 253. For powders intended for personal use, the residue remaining on the sieve shall be free from grittiness. 1. 2. 3 Dustability after accelerated storage After accelerated storage treatment as ·described in section 2. 2, the powder shall issue freely without clogging or bridging, when tested in a hand dusting apparatus conforming to specification WHO/EQP/4.22.4 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 British Standards Institution, British Standard 410: 1943. 3 American Society for Testing Materials, Standard E 11-39. 4 World Health Organization (1964) Equipment for vector control, Geneva, p. 112. -171 - DDT DUSTING-POWDERS WHO/SIF/16.R2 1. 2 .4 Acidity or alkalinity The acidity or alkalinity of the powder, determined by the method described in WHO/M/3, page 250, shall not be greater than 0.1 %, calculated as H2S04 , nor greater than 0.2%, calculated as NaOH. 1. 3 Packing and Marking of Packages The DDT dusting-powder shall be packed in suitable, clean, air-tight drums, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name DDT dusting-powder to Specification WHO/SIF/l6.R2 DDT, ... %w/w Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Keep well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 DDT Content Of the two methods described below, only one need be performed. In the event of a dispute the method described in section 2. l. 2 shall govern. 2.1.1 Total organic chlorine method (Stepanow method, revised) 2.1.1.l Procedure Weigh accurately an amount of the sample containing about 1 g of DDT and extract it quantitatively with chlorine-free and thiophene-free benzene. If a Soxhlet apparatus is used, care must be taken to ensure that extraction is complete and that channelling has not occurred. Concentrate the extract to such a volume that it can be transferred quantitatively to a 50-ml volumetric flask, and make up to the mark with benzene. Transfer a 5-ml aliquot to a 250-ml conical flask and add 25 ml of 99% isopropanol. Shake the flask and add 2. 5 g of metallic sodium, in the form of ribbon or small pieces. Connect to a reflux condenser - 172 - WHO/SIF/16.R2 DDT DUSTING POWDERS and boil gently for at least 1 hour, shaking the flask occasionally. Remove the excess metallic sodium by cautiously adding 10 ml of 50% aqueous isopropanol through the condenser, at the rate of 1-2 drops per second. Boil for an additional 10 minutes, and then add 60 ml of distilled water. Cool, add 2 or 3 drops of phenolphthalein indicator solution, neutralize by adding 50% nitric acid dropwise, and then add 10 ml in excess. Cool, if necessary, to room temperature, add a slight excess of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam-bath for Yz hour, with frequent stirring. Cool, filter through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the total chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. 2 . 1 . 1 . 2 Calculation DDT content (% w/w) a xf x 7.092 w where a = volume (ml) of 0.1 N silver nitrate used w = weight (g) of sample 50.01 f =-A where A = value (%) found for total chlorine in recrystallized DDT, when determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2 .1. 2 Hydrolysable chlorine method 2.1.2.1 Procedure Weigh accurately an amount of the sample containing about 0.5 g of DDT and extract it quantitatively with acetone. If a Soxhlet appa- ratus is used, care must be taken to ensure that extraction is complete and that channelling has not occurred. Concentrate the extract to a vol- -173 - DDT DUSTING-POWDERS WHO/SIF/16.R2 ume of about 50 ml and add 20 ml of 1 N ethanolic potassium hydrox- ide. Keep at 20-25°C for 15 minutes and add 50 ml of distilled water. Add 20 ml of 2 N nitric acid, exactly 25 ml of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam-bath for Y2 hour, with frequent stirring. Cool, filter the coagulated silver chloride through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate. Alternatively, the end-point may be determined electrometrically. 2 . 1 . 2 . 2 Calculation DDT content (% w/w) axfx3.546 w where a = volume (ml) of 0.1 N silver nitrate used w = weight (g) of sample 10.00 I - --X- where A = value found for hydrolysable chlorine in crystallized DDT, when determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2. 2 Accelerated Storage Treatment A sample of the powder is placed in a beaker and levelled off without compacting. For the sieve test, section 1.2.2, 20 g of sample are placed in a 250-ml beaker of 6-6.5 cm internal diameter and for the dustability test, section 1.2.3, 250 g of sample are placed in a beaker of 12-13 cm internal diameter. Place within the beaker, on top of the powder, a loose-fitting piston or disc so formed and weighted as to exert upon the powder an even pressure of 25 gf/cm2• Keep the powder thus under pressure in an oven at 54°C ± 1 °C for 24 hours. Take the sample from the oven, remove the pressure assembly, and allow the powder to come to room temperature. -174 - WHO/SIF/17.R2 HCH DUSTING-POWDERS HCH DUSTING-POWDERS Specification WHO/SIF/17.R2 Approved 25 October 1965 1. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of a homogeneous mixture of technical HCH, refined HCH, or lindane in a filler and shall be in the form of a fine, free- flowing, white to cream or grey powder, free from extraneous impurities or added modifying agents. The HCH used in the manufacture of the powder shall comply with the requirements of Specification WHO /SIT /2. R3 or WHO/SIT/3. R3. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1.1 and with the following requirements.2 1. 2 .1 Gamma-isomer content (w /w basis) The content of gamma-isomer, determined by the method described in section 2. 1, shall not differ from the nominal content by more than ± 10%. The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Sieving after accelerated storage Not less than 98% of the powder (dry weight), after accelerated storage treatment as described in section 2.2, shall pass through a 150-µ sieve (BS 100 mesh; 3 US Standard No. 100 4) when tested by the method des- cribed in WHO/M/4, page 253. For powders intended for personal use, the residue remaining on the sieve shall be free from grittiness. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 If substantial freedom from odour is required, this should be specified in the order. 3 British Standards Institution, British Standard 410: 1943. 4 American Society for Testing Materials, Standard E 11-39. -175 - MALATHION DUSTING-POWDERS WHO/SIF/22.R1 MALATHION DUSTING-POWDERS Tentative Specification WHO/SIF/22.RI Approved 25 October 1965 l. SPECIFICATIONS 1. 1 Description and Ingredients The material shall consist of a homogeneous mixture of technical malathion in a filler and shall be in the form of a fine, free-flowing, yellow to brown powder, free from extraneous impurities or added modifying agents. The technical malathion used in the manufacture of the powder shall comply with the requirements of Specification WHO/SIT/l0.R2. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1.1 and with the following requirements.2 1.2.1 Malathion content (w/w basis) The content of O ,0-dimethyl S-[1,2-di-(ethoxycarbonyl)ethyl] phos- phorodithioate, determined by the method described in section 2. 1, shall not differ from the nominal content by more than -10% or + 25%. The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Sieving after accelerated storage Not less than 98% of the powder (dry weight), after accelerated storage treatment as described in section 2. 2, shall pass through a 150-µ sieve (BS 100 mesh; 3 US Standard No. 100 4) when tested by the method described in WHO/M/4, page 253. For powders intended for personal use, the residue remaining on the sieve shall be free from grittiness. 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 If the material is required for use inside buildings, odour-free malathion should also be specified. 3 British Standards Institution, British Standard 410: 1943. 4 American Society for Testing Materials, Standard E ll-39. -178 - WHO/SIF/22.R1 MALATHION DUSTING-POWDERS 1. 2. 3 Dustability after accelerated storage After accelerated storage treatment as described in section 2. 2, the pow- der shall issue freely without clogging or bridging, when tested in a hand dusting apparatus conforming to specification WHO /EQP /4. R2.1 1. 2. 4 Acidity or alkalinity The acidity or alkalinity of the powder, determined by the method described in WHO/M/3, page 250, shall not be greater than 0.1 %, cal- culated as H2S04, nor greater than 0.2%, calculated as NaOH. 1. 3 Packing and Marking of Packages The malathion dusting-powder shall be packed in suitable, clean, air-tight drums, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Malathion dusting-powder to Specification WHO /SIF /22. Rl Malathion, ... % w/w Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Malathion is an organophosphorus compound which inhibits cholin- esterase. It is poisonous if swallowed. Keep the material out of reach of children and well away from foodstuffs, animal feed and their containers. " If poisoning occurs, call a physician. Atropine and pralidoxime are specific antidotes and artificial respiration may be needed." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Malathion Content 2. 1.1 Special apparatus Spectrophotometer or photoelectric colorimeter with a blue filter (420 mµ). 1 World Health Organization (1964) Equipment for vector control, Geneva, p. 112. -179 - MALATHION DUSTING-POWDERS WHO/SIF/22.R1 2 .1. 2 Special reagents Acetonitrile. Boiling range 80-82°C. Pass through a column of silica gel and discard the first (yellow) portion of the eluate. Collect the colour- less eluate. A 10% aqueous solution of this should have a pH between 5 and 7. Ferric reagent. Dissolve 0.2 g offerric chloride (FeC13 • 6H20) in distilled water, add 8 ml of concentrated hydrochloric acid, and dilute to l litre with distilled water. Cupric reagent. Dissolve 1.5 g of cupric sulfate (CuS04 • 5H20) in 100 ml of distilled water. 2 .1. 3 Preparation of malathion standard solutions Standard solution A. Accurately weigh into a 5-ml or 10-ml beaker 1.00 g ± 0.02 g of malathion reference standard.1 Transfer quantitatively to a I-litre volumetric flask with anhydrous ethanol. Dilute with anhydrous ethanol to about 5 ml below the mark and allow to stand until a steady temperature is reached. Record the temperature and dilute to the mark with anhydrous ethanol and mix well. The solution is stable for about 2 weeks. Adjust to the recorded temperature before withdrawing aliquots. Standard solution B. Pipette a 15-ml aliquot of standard solution A into a 250-ml volumetric flask. Add 2. 5 ml of acetonitrile and dilute with anhydrous ethanol to about 5 ml below the mark. Keep until the sample for analysis is ready for final dilution. At that time, dilute with anhydrous ethanol to the mark and mix well. Norn : Volume charges of alcohol solutions with temperature are appreciable. Bring each solution to volume just before proceeding to next step. 2 .1. 4 Preparation of sample Accurately weigh a sample containing 0.30 g ± 0.02 g of malathion and transfer it quantitatively to a 100-ml bottle fitted with a screw cap having a polyethylene liner. Pipette 50 ml of acetonitrile into the bottle. Cap the bottle tightly and shake vigorously for 2 to 3 minutes. Allow the solids to settle for 3 to 5 minutes. If necessary, centrifuge for 2 to 3 minutes at 1500 to 3000 rev/min. Immediately pipette a 25-ml aliquot of the super- natant liquid into a 250-ml volumetric flask, dilute with anhydrous ethanol 1 Samples of reference standard malathion may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. - 180 - WHO/SIF/22.R1 MALATHION DUSTING-POWDERS to the mark and mix well. Immediately pipette a 25-ml aliquot of the diluted solution into a 250-ml volumetric flask and again make up to volume with anhydrous ethanol. 2 .1. 5 Analysis of sample Determine the standard malathion and the sample separately but at the same time. Perform the entire analysis without interruption. Pipette 25-ml aliquots of standard solution B and sample solutions into two 250-ml separating funnels. Add 2 ml ± 0.1 ml of 0.5 N sodium hydroxide to each and mix well by swirling gently for 5 to 10 seconds (do not shake). Allow to stand for 120 seconds ± 10 seconds. Add 75 ml ± 1 ml of ferric reagent and mix well by swirling for 10 seconds. Allow to stand 5 minutes. During this waiting period fill two 50-ml volumetric flasks to the mark with cyclohexane. Transfer the cyclohexane from each of these flasks into one of the separating funnels and allow the flasks to drain for one minute. Add 20 ml ± 0.1 ml of cupric reagent and shake in the separating funnel for exactly one minute. Norn: The copper-malathion complex is unstable in the aqueous phase. The cupric reagent should therefore be added from a fast-delivery Mohr pipette and shaking started without delay. A delay of only 15 seconds before extraction results in appreciable losses. Allow the phases to separate and discard the aqueous phase as soon as separation occurs. Allow a little of the solvent phase to rinse the stem of the separating funnel, then transfer the clear solvent phase to a small beaker and thence to the cell, or transfer directly to the cell. Manipulate phase separation of both standard and sample similarly. Immediately (within 5 minutes) determine the absorbance of the yellow cyclohexane solu- tion at 420 mµ by means of the spectrophotometer ( or photoelectric colori- meter) using cyclohexane as a reference. 2.1. 6 Calculation 0 ,0-dimethyl S-[1,2-di(ethoxycarbonyl)ethyl] phosphorodithioate content (% w/w) A,ampte a x p x 0.3 = x A standard b X 100 where Asampte = absorbance of the sample at 420 mµ A standard = absorbance of the standard at 420 mµ a = weight (g) of reference standard malathion taken to pre- pare standard solution b = weight (g) of sample p = purity (%) of reference standard malathion. - 181 - MALATHION DUSTING-POWDERS WHO/SIF/22.R1 2. 2 Accelerated Storage Treatment For the sieve test, section l. 2. 2, place 20 g of the sample in a 100-ml wide-mouthed bottle fitted with a vinyl-plastic-lined screw cap. For the dustability test, section 1.2.3, place 250 g of the sample in a 1000-ml wide- mouthed bottle fitted with a vinyl-plastic-lined screw cap. Place the bottles in a forced-draught oven maintained at 70° ± 1 °C for 2 hours. Take the samples from the oven and allow them to cool to room temperature before removing the cap. After completion of the accelerated storage treatment, the samples should not be exposed to heat or to bright sunshine. Unneces- sary exposure of the samples to high atmospheric humidity must also be avoided. -182 - WHO/SIF/23 LARVICIDAL OILS WITHOUT INSECTICIDE LARVICIDAL OILS WITHOUT INSECTICIDE Tentative Specification WHO/SIF/23 Approved 26 November 1958 l. SPECIFICATIONS 1. 1 Material The material shall consist of a mineral oil in the form of a homogeneous mobile liquid, free from dirt, water, and other extraneous impurities. It may, if so specified, have additives incorporated to improve its physical performance. At the rates ordinarily used, it must not be toxic to fish, domestic animals, man, or plant life. Any additives used in the manufacture of the larvicidal oil shall comply with the requirements of the current approved specifications, where such specincations exist. 1. 2 Chemical, Physical, and Biological Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l . l and with the following requirements : Specific gravity at 30°Cf30°C (section 2. l). Distillation ( section 2. 2) : volume distilling at 200°C ............. . Flash-point (section 2.3) ....... . Kinematic viscosity at 2l.1°C (section 2.4) Spreading pressure (section 2. 5): Grade l '' 2 ....... . 3 ....... . Stability of film ( section 2. 6) Material soluble in water and oil layers (section 2. 7), % by volume Toxicity to mosquito larvae (WHO/M/18, page 294): Anopheles stephensi kill at 25°C Aedes aegypti kill at 25°C . . . Minimum Maximum 0.940 5% 65.6°C 10 centistokes 46 dynes/cm 25 " 18 " 2 hours 90% 75% 2.5 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. - 183 - LARVICIDAL OILS WITHOUT INSECTICIDE WHO/SIF/23 1. 3 Packing and Marking of Packages The larvicidal oil shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Larvicidal oil to Specification WHO /SIF /23 Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : "Keep well away from foodstuffs, animal feed and their containers." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Specific Gravity Determine the specific gravity at 30°C/30°C by means of a specific- gravity bottle or a pyknometer. 2. 2 Distillation Determine the volume of the sample that distils at 200°C when using the appropriate method described in the Institute of Petroleum Standard IP 123/58,1 the American Society for Testing Materials Standard D 158-54,2 or any other equivalent standard method. 2.3 Flash-Point Determine the flash-point by the TAG Closed Tester method (see WHO/M/10, page 276), the Cleveland Open Tester method (see WHO/ M/11, page 279), or any other equivalent standard method. 2. 4 Kinematic Viscosity Determine the kinematic viscosity at 21. l °C by one of the relevant methods described in BS 188: 1957,3 the American Society for Testing 1 Institute of Petroleum, London (1953) Standard methods for testing petroleum and its products, 13th ed., London. 2 American Society for Testing Materials (1958) Book of ASTM Standards, Phila- delphia. 3 British Standards Institution, British Standard 188 : 1957 (Determination of the viscosity of liquids in c.g.s. units). -184 - WHO/SIF/23 LARVICIDAL OILS WITHOUT INSECTICIDE Materials Standard D 445-53T,1 or by any other equivalent standard method. 2. 5 Spreading Pressure 2. 5 .1 Standard solutions Solution 1 (spreading pressure 46 dynes/cm). A 10%(w/v) solution of oleyl alcohol in medicinal paraffin (BP 2 or equivalent grade). Solution 2 (spreading pressure 25 dynes/cm). A 1% (w/v) solution of oleyl alcohol in medicinal paraffin (BP 2 or equivalent grade). Solution 3 (spreading pressure 18 dynes/cm). A 1% (v/v) solution of terpineol in medicinal paraffin (BP 2 or equivalent grade). 2.5.2 Procedure Thoroughly clean a large glass funnel, not less than 20 cm in diameter, with chromic acid solution and remove all traces of acid by thorough washing with distilled water. Fix the funnel in a vertical position in a retort stand over a sink or receptacle to collect the water that overflows. Connect the stem to a water supply by means of rubber tubing. Turn on the water and allow it to overflow in order to give a clean surface for testing. Turn off the water and tilt the funnel slightly to bring the water level about 3 mm below the rim of the funnel. Take a clean glass rod in each hand, dip one rod into the sample and the other into the standard solution corresponding to the grade of the larvicidal oil, and lower a drop from each of the rods simultaneously on to the water surface. Observe the spreading of the oils on the water surface. It is often easier to see the films if they are viewed from the level of the water surface. The funnel must be cleaned between tests by allowing the water to overflow, or, if necessary, by cleaning with chromic acid solution, to ensure that the water surface used is not contaminated. 2. 5. 3 Interpretation of results If the sample occupies more than half the surface, its spreading pressure is greater than that of the standard. If both the sample and the standard solution occupy about equal areas, the spreading pressures are approxi- mately equal. In these two instances the sample is acceptable from the point of view of spreading pressure. If the standard solution occupies 1 American Society for Testing Materials (1958) Book of ASTM Standards, Part 7, Philadelphia. 2 British Pharmacopoeia. -185 - LARVICIDAL OILS WITHOUT INSECTICIDE WHO/SIF/23 more than half the surface, the spreading pressure of the sample is lower than that of the standard. In this instance the sample is unsatisfactory and is not acceptable. Only initial observations should be recorded. The standard solution may subsequently occupy a smaller area than when first allowed to spread on the surface, on account of the solubility of the spreading agent in water. The test should be repeated, especially when " borderline " oils are under examination. 2. 6 Stability of Film 2. 6. 1 Special apparatus Test-bowl of china or enamelled iron, 30 cm ± 1 cm in diameter. 2.6.2 Procedure Thoroughly clean the test-bowl with light petroleum and then with chromic acid solution. Rinse thoroughly, first with hot distilled water, then with acetone, and finally dry. Thereafter, do not touch the inside of the bowl. Fill the bowl almost completely with distilled water. Pipette 0.8-1.0 ml of the sample gently on to the surface of the water so that a complete film is formed extending to the edge of the bowl. The film must remain uniform and unbroken for at least 2 hours. 2. 7 Material Soluble in Water and Oil Layers 2. 7 .1 Special apparatus Graduated cylinder, 100-ml capacity, with 0.2-ml graduations, fitted with a ground-glass stopper. 2.7.2 Procedure Measure accurately 50 ml of the sample and 50 ml of distilled water into the cylinder at room temperature. Shake the mixture vigorously for 10 minutes so that thorough mixing of the two layers occurs. Then leave the cylinder undisturbed for 24 hours at room temperature. Express any reduction in volume of either layer as a percentage of the total volume of sample taken. -186 - WHO/SIF/24 LARVICIDAL OILS WITH INSECTICIDE LARVICIDAL OILS WITH ADDED INSECTICIDE Tentative Specification WHO/SIF/24 Approved 26 November 1958 l. SPECIFICATIONS 1.1 Material The material shall consist essentially of a solution of a specified insec- ticide 1 in a mineral oil, in the form of a homogeneous mobile liquid, free from dirt, water, and other extraneous impurities. It may, if so specified, have additives incorporated to improve its physical performance. At the rates ordinarily used, it must not be toxic to fish, domestic animals, man, or plant life. The technical insecticide and any additives used in the manu- facture of the larvicidal oil shall comply with the requirements of the current approved specifications, where such specifications exist. 1. 2 Chemical, Physical, and Biological Requirements The material, sampled from any part of the consignment,2 shall comply with the requirements of section l . l and with the following requirements : Specific gravity at 30°C/30°C (section 2 .1). Distillation ( section 2. 2) : volume distilling at 200°C ............. . Flash-point (section 2. 3) . . . . . . . . Kinematic viscosity at 21.l °C (section 2 .4). Spreading pressure (section 2.5): Grade l '' 2 ....... . 3 ....... . Stability of film (section 2. 6) Minimum Maximum 0.940 5% 65.6°C 10 centistokes 46 dynes/cm 25 " 18 2 hours 1 The nature and content of the insecticide shall be agreed between the purchaser and manufacturer at the time of placing the order. 2 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. -187 - LARVICIDAL OILS WITH INSECTICIDE Material soluble in water and oil layers (section 2. 7), % by volume ..... Toxicity to mosquito larvae (WHO/M/18, page 294): Anopheles stephensi kill at 25°C Aedes aegypti kill at 25°C . . . Minimum 100% 100% WHO/SIF/24 Maximum 2.5 Insecticide content (w/w basis). The content of insecticide, determined as indicated in section 2. 8, shall not differ from the nominal content by more than ± 5%. The average content of all samples taken shall not be lower than the nominal content. 1. 3 Packing and Marking of Packages The larvicidal oil shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Larvicidal oil to Specification WHO /SIF /24 Insecticide added : ... , . . . % w /w Batch or reference number, and date of test Net weight of contents and a cautionary notice appropriate to the insecticide added.1 METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Specific Gravity Determine the specific gravity at 30°C/30°C by means of a specific- gravity bottle or a pyknometer. 2. 2 Distillation Determine the volume of the sample that distils at 200°C when using the appropriate method described in the Institute of Petroleum Standard 1 For the wording of the notice, see the specification for the corresponding technical product. -188 - WHO/SIF/24 LARVICIDAL OILS WITH INSECTICIDE IP 123/58,1 the American Society for Testing Materials Standard D 158-54,2 or any other equivalent standard method. 2. 3 Flash-Point Determine the flash-point by the TAG Closed Tester method (see WHO/M/10, page 276), the Cleveland Open Tester method (see WHO/M/ 11, page 279), or any other equivalent standard method. 2. 4 Kinematic Viscosity Determine the kinematic viscosity at 21.1 °C by one of the relevant methods described in BS 188: 1957,3 the American Society for Testing Materials Standard D 445-53T,4 or by any other equivalent standard method. 2. 5 Spreading Pressure 2. 5. 1 Standard solutions Solution I (spreading pressure 46 dynes/cm). A 10% (w/v) solution of oleyl alcohol in medicinal paraffin (BP 5 or equivalent grade). Solution 2 (spreading pressure 25 dynes/cm). A 1% (w/v) solution of oleyl alcohol in medicinal paraffin (BP 6 or equivalent grade). Solution 3 (spreading pressure 18 dynes/cm). A 1% (v/v) solution of terpineol in medicinal paraffin (BP 5 or equivalent grade). 2.5.2 Procedure Thoroughly clean a large glass funnel, not less than 20 cm in diameter, with chromic acid solution and remove all traces of acid by thorough washing with distilled water. Fix the funnel in a vertical position in a retort stand over a sink or receptacle to collect the water that overflows. Connect the stem to a water supply by means of rubber tubing. Turn on the water and allow it to overflow in order to give a clean surface for testing. 1 Institute of Petroleum, London (1953) Standard methods for testing petroleum and its products, 13th ed., London. 2 American Society for Testing Materials (1958) Book of ASTM Standards, Phila- delphia. 3 British Standards Institution, British Standard 188 : 1957 (Determination of the viscosity of liquids in c .g .s. units). 4 American Society for Testing Materials (1958) Book of ASTM Standards, Part 7, Philadelphia. 5 British Pharmacopoeia. -189 - LARVICIDAL OILS WITH INSECTICIDE WHO/SIF/24 Turn off the water and tilt the funnel slightly to bring the water level about 3 mm below the rim of the funnel. Take a clean glass rod in each hand, dip one rod into the sample and the other into the standard solution corresponding to the grade of the larvicidal oil, and lower a drop from each of the rods simultaneously on to the water surface. Observe the spreading of the oils on the water surface. It is often easier to see the films if they are viewed from the level of the water surface. The funnel must be cleaned between tests by allowing the water to overflow, or, if necessary, by cleaning with chromic acid solution, to ensure that the water surface used is not contaminated. 2. 5. 3 Interpretation of results If the sample occupies more than half the surface, its spreading pressure is greater than that of the standard. If both the sample and the standard solution occupy about equal areas, the spreading pressures are approxi- mately equal. In these two instances the sample is acceptable from the point of view of spreading pressure. If the standard solution occupies more than half the surface, the spreading pressure of the sample is lower than that of the standard. In this instance the sample is unsatisfactory and is not acceptable. Only initial observations should be recorded. The standard solution may subsequently occupy a smaller area than when first allowed to spread on the surface, on account of the solubility of the spreading agent in water. The test should be repeated, especially when " borderline " oils are under examination. 2. 6 Stability of Film 2. 6 .1 Special apparatus Test-bowl of china or enamelled iron, 30 cm ± 1 cm in diameter. 2.6.2 Procedure Thoroughly clean the test-bowl with light petroleum and then with chromic acid solution. Rinse thoroughly, first with hot distilled water, then with acetone, and finally dry. Thereafter, do not touch the inside of the bowl. Fill the bowl almost completely with distilled water. Pipette 0.8-l.O ml of the sample gently on to the surface of the water so that a complete film is formed extending to the edge of the bowl. The film must remain uniform and unbroken for at least 2 hours. -190 - WHO/SIF/24 LARVICIDAL OILS WITH INSECTICIDE 2. 7 Material Soluble in Water and Oil Layers 2. 7 .1 Special apparatus Graduated cylinder, 100-ml capacity, with 0.2-ml graduations, fitted with ground-glass stopper. 2.7.2 Procedure Measure accurately 50 ml of the sample and 50 ml of distilled water into the cylinder at room temperature. Shake the mixture vigorously for 10 minutes so that thorough mixing of the two layers occurs. Then leave the cylinder undisturbed for 24 hours at room temperature. Express any reduction in volume of either layer as a percentage of the total volume of sample taken. 2. 8 Insecticide Content Determine the content of the added insecticide by the method included in the relevant specification. The method shall be agreed between the purchaser and manufacturer at the time of placing the order. -191 -

Part II RODENTICIDES 13

WHO/SRT/3.R1 TECHNICAL COUMACHLOR TECHNICAL COUMACHLOR 1. SPECIFICATIONS 1. 1 Material Specification WHO/SRT/3.Rl Approved 25 October 1965 The material shall comprise essentially 3-(a-acetonyl-4-chlorobenzyl)- 4-hydroxycoumarin, together with other related compounds normally resulting from the manufacturing process, and shall be in the form of a free-flowing, brownish powder, free from any lumps, extraneous impurities, added modifying agents or odours that may limit its suitability for baiting rodents or impair its effectiveness. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1 . 1 and with the following requirements : Minimum Maximum Total organic chlorine content (section 2.1), % by weight . . . . . . . . . . . . 9.8 10.8 Loss on drying (section 2.2), % by weight . 2.0 Melting-point (WHO/M/5, page 255) . . . . 103°C 1. 3 Packing and Marking of Packages The technical coumachlor shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical coumachlor to Specification WHO/SRT/3.RI Batch or reference number, and date of test Net weight of contents 1 A sampling procedure is described in Method WHO/Mll, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. -195 - TECHNICAL COUMACHLOR WHO/SRT/3.R1 and the following minimum cautionary notice : " Keep this material and baits containing it well away from children, domestic animals, foodstuffs, empty foodstuff containers, and animal feed. " In cases of poisoning call a physician. The specific antidote is vita- min K1• Blood transfusions may be necessary." 2. METHODS FOR DETERMINATION OF CHEMICAL PROPERTIES 2.1 Total Organic Chlorine Content (Stepanow Method, Revised) 2.1.1 Procedure 2. I . l . l Determination of total chlorine Weigh accurately about 0.5 g of the sample, transfer to a 250-ml conical flask, and dissolve in 25 ml of 99% isopropanol. Add 2.5 g of metallic sodium, in the form of ribbon or small pieces. Connect to a reflux condenser and boil gently for at least I hour, shaking the flask occasionally. Remove the excess metallic sodium by cautiously adding 10 ml of 50% aqueous isopropanol through the condenser, at the rate of l-2 drops per second. Boil for an additional lO minutes, and then add 60 ml of distilled water. Cool, add 2 or 3 drops of phenolphthalein indicator solution, neutralize by adding 50% nitric acid dropwise, and then add 10 ml in excess. Cool, if necessary, to room temperature, add a slight excess of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam-bath for Yz hour, with frequent stirring. Cool, filter through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with O.l N potas- sium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the total chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. 2. l . l . 2 Determination of inorganic chlorine Weigh accurately about 1 g of the sample and transfer with 100 ml of distilled water to a 250-ml conical flask. Acidify with 50% nitric acid and proceed as described in section 2. I . I . I. -196 - WHO/SRT/3.R1 TECHNICAL COUMACHLOR 2 .1. 2 Calculation Total organic chlorine content(% w/w) = (!!_ - !!__) x 0.3546 x f W1 W2 where a = volume (ml) of 0.1 N silver nitrate equivalent to the total chlorine b = volume (ml) ofO.l N silver nitrate equivalent to the inorganic chlorine w1 = weight (g) of sample used for the total chlorine determination w2 = weight (g) of sample used for the inorganic chlorine determina- tion 10.34 f =-A where A = value (%) found for total chlorine in recrystallized coumachlor, when determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its appli- cation by a given laboratory. Its value must lie within the range 0.98-1.02. 2. 2 Loss on Drying Weigh accurately about 0.5 g of the sample into a dried, tared dish. Dry for 16 hours at 100°C, cool in a desiccator, and weigh. Calculate the percentage loss in weight. - 197 - TECHNICAL WARFARIN WHO/SRT/6.R1 TECHNICAL WARFARIN Specification WHV/SRT/6.Rl Approved 25 October 1965 1. SPECIFICATIONS 1. 1 Material The material shall comprise essentially 3-(a-acetonylbenzyl)-4-hydroxy- coumarin and shall be in the form of a free-flowing, white or light-tan, crystalline powder, free from any lumps, extraneous impurities, added modifying agents or odours that may limit its suitability for baiting rodents or impair its effectiveness. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section I . 1 and with the following requirements : Minimum 3-( a-acetonylbenzyl)-4-hydroxycoumarin content (section 2.1), % by weight 98.0 Loss on drying (section 2.2), % by weight Melting-point (Method WHO/M/5, page 255) 159°C 1. 3 Packing and Marking of Packages Maximum 1.0 164°C The technical warfarin shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical warfarin to Specification WHO/SRT/6. Rl Batch or reference number, and date of test Net weight of contents 1 A sampling procedure is described in Method WHO/M/11, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. -198 - WHO/SRT/6.R1 TECHNICAL WARFARIN and the following minimum cautionary notice : " Keep this material and baits containing it well away from children, domestic animals, foodstuffs, empty foodstuff containers, and animal feed. " In cases of poisoning, call a physician. The specific antidote is vita- min K1 • Blood transfusions may be necessary." 2. METHODS FOR DETERMINATION OF CHEMICAL PROPERTIES 2. 1 3-( cx-Acetonylbenzyl)-4-hydroxycoumarin Content 2 .1. 1 Special apparatus Beckman spectrophotometer, model DU, or equivalent instrument, with 1-cm quartz cells. 2.1.2 Procedure Weigh accurately about 50 mg of the sample into a 100-ml volumetric flask, add 0.1 N sodium hydroxide to effect solution, and make up to the mark with 0.1 N sodium hydroxide. Dilute 2 ml of this solution to 100 ml with 0.1 N sodium hydroxide. With the spectrophotometer set at maximum sensitivity, determine the absorbance of the final solution at 308 mµ, using 0.1 N sodium hydroxide as reference. 2. 1. 3 Calculation 3-( cx-acetonylbenzyl)-4-hydroxycoumarin content (%) E x 15.42 x 104 Mx w where E = absorbance of the final solution at 308 mµ w = weight (g) of sample M = molar extinction coefficient determined with pure 3-( cx-acetonyl- benzyl)-4-hydroxycoumarin. 2. 2 Loss on Drying Weigh accurately about 0.5 g of the sample into a dried, tared dish. Dry for 16 hours at 100°C, cool in a desiccator, and weigh. Calculate the percentage loss in weight. -199 - TECHNICAL PINDONE WHO/SRT/9.R1 TECHNICAL PINDONE Tentative Specification WHO/SRT/9.Rl Approved 25 October 1965 1. SPECIFICATIONS 1. 1 Material The material shall comprise essentially 2-pivaloyl-1,3-indanedione and shall be in the form of a free-flowing, yellow to brownish powder, free from any lumps, extraneous impurities, added modifying agents or odours that may limit its suitability for baiting rodents or impair its effectiveness. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l. l and with the following requirements : Minimum Maximum Setting-point ( section 2. l) l07°C 2-Pivaloyl-l ,3-indanedione content (section 2.2), % by weight 98.0 Sieving (section 2.3): % by weight passing through a 150-µ sieve (BS 100 mesh; 2 US Standard No. 100 3) 95.0 Moisture content ( section 2. 4), % by weight l.O 1. 3 Packing and Marking of Packages The technical pindone shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 British Standards Institution, British Standard 410: 1943. 3 American Society for Testing Materials, Standard E 11-39. -200 - WHO/SRT/9.R1 Manufacturer's name Technical pindone to Specification WHO/SRT/9. Rl Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : TECHNICAL PINDONE "Keep this material and baits containing it well away from children, domestic animals, foodstuffs, empty foodstuff containers, and animal feed. " In cases of poisoning, call a physician. The specific antidote is vita- min K1• Blood transfusions may be necessary." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Setting-Point Place in a 20-cm boiling-tube of 2.5 cm internal diameter a sufficient amount of the sample to give, when melted, a depth of liquid of approxi- mately 7 .5 cm. Melt carefully by immersing the tube to a depth of 10 cm in an oil-bath at approximately 131 °C. Fit this boiling-tube with a cork collar and insert it to within 1.3 cm of the bottom of a 15-cm boiling-tube of approximately 4 cm diameter; then immerse the two tubes to a depth of 10 cm in an oil-bath main- tained at 96°C. Place in the inner boiling-tube a stirrer, consisting of a glass rod bent at one end in the form of a ring. A thermometer graduated in one-tenths of a degree is then clamped in a central position with its bulb 2.5 cm from the bottom of the tube. The temperature of the melt should be approximately l0°C higher than the anticipated setting-point at this stage. Stir at a rate of about two strokes per second, by moving the stirrer up and down, until the material begins to thicken, at which point stir vigorously to work into the melt material that has solidified on the walls of the tube. Stop stirring when the temperature ceases dropping and remains constant for some time.1 Record this temperature as the setting-point. Apply corrections as necessary for the thermometer calibration and the emergent stem. Emergent-stem correction. The correction for the emergent stem in mercury-filled thermometers, to be added to the temperature reading, is calculated from the following formula : 1 In some instances, there may be a slight rise in the temperature of the material ; if this occurs, record the highest steady temperature as the setting-point. - 201 - TECHNICAL PINDONE WHO/SRT/9.R1 N x 0.00015 x (T - t) where N = number of degrees on the scale of the thermometer between the top of the inner boiling-tube and the level of the mercury T temperature reading on the thermometer in the melt t temperature of the stem at the mid-point of the exposed mercury thread. 2.2 2-Pivaloyl-1,3-indanedione Content 2. 2. 1 Special apparatus Beckman spectrophotometer, model DU, or equivalent instrument, with 1-cm quartz cells. 2.2.2 Procedure Weigh accurately about 0.1 g of the sample and transfer quantitatively to a 200-ml volumetric flask with anhydrous ethanol 1 made alkaline with sodium hydroxide.2 After the material has completely dissolved, make up to the mark with anhydrous ethanol. Dilute exactly 5 ml of this solu- tion to 500 ml with the same alkaline anhydrous ethanol. With the spectrophotometer set at maximum sensitivity, determine the absorbance of the final solution at 281-283 mµ, using anhydrous ethanol as reference. 2. 2. 3 Calculation 2-Pivaloyl-1,3-indanedione content (% w /w) E x 46.05 x 104 Mx w where E w absorbance of the final solution at 281-283 mµ weight (g) of sample M = molar extinction coefficient determined with pure 2-pivaloyl- 1,3-indanedione. 1 Anhydrous ethanol denatured with about 5% of anhydrous methanol-e.g., anhydrous ethanol SD2B (USA) or absolute industrial methylated spirit 74° overproof (United Kingdom)-may be used. 2 To each litre of anhydrous ethanol is added 1.0 ml of a stock solution prepared by dissolving one pellet (0.13 g) of sodium hydroxide in 50 ml of anhydrous ethanol. - 202 - WHO/SRT/9.R1 TECHNICAL PINDONE 2.3 Sieving Test Place 200 g of the sample on a clean, dry 150-µ sieve (BS 100 mesh; 1 US Standard No. 100 2) and screen in a Ro-Tap Shaker or equivalent machine 3 for 10 minutes. Stop the machine, break up any soft lumps by brushing lightly over the sieve, and restart the machine. Completion of screening is determined by shaking the sieve by hand over a clean, glazed paper and noting if any appreciable amount passes through. Weigh the residue remaining on the sieve. Calculate the portion passing through and express it as a percentage of the weight of sample taken. 2. 4 Moisture Content Weigh accurately about 2 g of the sample into a dry, tared dish (nickel, platinum, or aluminium) and dry for 10 hours under atmospheric pressure at 100-102°C; then cool in a desiccator and weigh. Dry again for 1 hour, or until the change in weight is not greater than 2 mg. Report the loss in weight as moisture content. 1 British Standards Institution, British Standard 410: 1943. 2 American Society for Testing Materials, Standard E 11-39. 3 The Ro-Tap Testing Sieve Shaker, supplied by the W. S. Tyler Company, Cleveland, Ohio, USA, reproduces the circular and tapping motion given testing sieves in hand- sieving, but with a uniform, mechanical action. One model is manufactured with ge1r to operate either with 60-cycle, 1750-rev/min motors, or with SO-cycle, 1450-rev/min motors, and still have the same standard speed of operation. The unit operates the sieves with a horizontal circular motion of 285 rev/min, together with a tapping action at the rate of approximately 150 taps per minute. The tapping action is very important for ensuring comparable results. This is especially true in the sieving of materials such as insecticides. The tapping motion is imparted to the top of the sieves and is transmitted through the entire nest. - 203 - COUMACHLOR CONCENTRATES WHO/SRF/1.R1 COUMACHLOR CONCENTRATES Specification WHO/SRF/1.Rl Approved 25 October 1965 1. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of technical coumachlor compounded with cornstarch or other suitable diluent, and with added colouring matter as required,1 and shall be in the form of a uniformly blended, free-flowing powder, free from lumps and extraneous impurities that may limit its suitability for baiting rodents or impair its effectiveness. The technical coumachlor used in the manufacture of the concentrate shall comply with the requirements of Specification WHO/SRT /3. Rl. 1. 2 Chemical, Physical, and Biological Requirements The material, sampled from any part of the consignment,2 shall comply with the requirements of section 1.1 and with the following requirements. 1.2.1 Coumachlor content (w/w basis) The content of 3-(o:-acetonyl-4-chlorobenzyl)-4-hydroxycoumarin, deter- mined by the method described in section 2. 1, shall not differ from the nominal content by more than ± 5%. The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Sieving Not less than 95% of the concentrate (dry weight) shall pass through a 150-µ sieve (BS 100 mesh ; 3 US Standard No. 100 4) when tested by the method described in section 2. 2. 1 The colouring matter added shall be consistent with the laws of the country in which the concentrate is to be used. A sufficient quantity must be added to ensure that the concentrate and baits prepared from it are clearly recognizable. The colouring matters described as Direct Blue l (sodium salt of dimethoxydiphenyldiazo-bis-(8- amino-1-naphthol) 5,7-disulfonic acid) and Direct Blue 14 (sodium salt of ditolyldiazo- bis-(8-amino-l-naphthol) 3,6-disulfonic acid) in the Colour lndex (2nd ed., 1956, Part I, Vol. 2, Bradford, England & Lowell, Mass.) have been found to be satisfactory. 2 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 3 British Standards Institution, British Standard 410: 1943. 4 American Society for Testing Materials, Standard E 11-39. - 204- WHO/SRF/1.R1 COUMACHLOR CONCENTRATES 1. 2. 3 Toxicity to rats When tested by the method described in WHO/M/14, page 285, the concentrate shall have an effectiveness of at least 90% of that of the standard material.1 1. 3 Packing and Marking of Packages The coumachlor concentrate shall be packed in suitable, clean con- tainers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Coumachlor concentrate to Specification WHO/SRF/1.Rl Coumachlor, ... % (w/w) Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Keep this material and baits containing it well away from children, domestic animals, foodstuffs, empty foodstuff containers, and animal feed. " In cases of poisoning, call a physician. The specific antidote is vita- min K1• Blood transfusions may be necessary." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 3-( cx-Acetonyl-4-chlorobenzyl)-4-hydroxycoumarin Content (Stepanow Method, Revised) 2.1.1 Procedure Weigh accurately an amount of the sample containing about 0.2 g of coumachlor, transfer to a 250-ml conical flask, and add 150 ml of acetone. Heat on a water-bath, while stirring. Decant through a Gooch crucible and repeat the extraction with acetone until the solvent is no longer coloured. Distil off the acetone from the combined acetone extracts. 1 Samples of standard material may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. - 205 - COUMACHLOR CONCENTRATES WHO/SRF/1.R1 Dissolve the residue in 25 ml of 99% isopropanol and add 2.5 g of metallic sodium, in the form of ribbon or small pieces. Connect to a reflux condenser and boil gently for at least 1 hour, shaking the flask occasionally. Remove excess metallic sodium by cautiously adding 10 ml of 50% aqueous isopropanol through the condenser, at the rate of 1-2 drops per second. Boil for an additional 10 minutes, and then add 60 ml of distilled water. Cool, add 2 or 3 drops of phenolphthalein indicator solution, neutralize by adding 50% nitric acid dropwise, and then add 10 ml in excess. Cool, if necessary, to room temperature, add a slight excess of 0.02 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam-bath for Y2 hour, with frequent stirring. Cool, filter through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.02 N potassium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the chlorine originally present in the sample. One millilitre of 0.02 N silver nitrate is equivalent to 0.000709 g of chlorine. Alternatively, the end-point may be determined electrometrically. 2 .1. 2 Calculation 3-( a-acetonyl-4-chlorobenzyl)-4-hydroxycoumarin content (% w /w) a x f x 0.6856 w where a = volume (ml) of 0.02 N silver nitrate equivalent to the total chlorine w = weight (g) of sample 10.34 f A where A = value(%) found for total chlorine in recrystallized coumachlor, when determined by the above-described method and using the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. - 206 - WHO/SRF/1.R1 COUMACHLOR CONCENTRATES 2.2 Sieving Test Place 200 g of the sample on a clean, dry 150-µ sieve (BS 100 mesh; 1 US Standard No. 100 2) and screen in a Ro-Tap Shaker 3 or equivalent machine for 10 minutes. Stop the machine, break up any soft lumps by brushing lightly over the sieve, and restart the machine. Completion of screening is determined by shaking the sieve by hand over a clean, glazed paper and noting if any appreciable amount passes through. Weigh the residue remaining on the sieve. Calculate the portion passing through and express it as a percentage of the weight of sample taken. 1 .British Standards Institution, British Standard 410: 1943. 2 American Society for Testing Materials, Standard E 11-39. 3 The Ro-Tap Testing Sieve Shaker, supplied by the W. S. Tyler Company, Cleve- land, Ohio, USA, reproduces the circular and tapping motion given testing sieves in handsieving, but with a uniform, mechanical action. One model is manufactured with · gear to operate either with 60-cycle, 1750-rev/min motors, or with 50-cycle, 1450-rev/min motors, and still have the same standard speed of operation. The unit operates the sieves with a horizontal circular motion of 285 rev/min, together with a tapping action at the rate of approximately 150 taps per minute. The tapping action is very important for ensuring comparable results. This is especially true in the sieving of materials such as insecticides. The tapping motion is imparted to the top of the sieves and is transmitted through the entire nest. - 207 - WARFARIN CONCENTRATES WHO/SRF/2.R1 WARFARIN CONCENTRATES Specification WHO/ SRF/ 2. RJ Approved 25 October 1965 1. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of technical warfarin compounded with cornstarch or other suitable diluent, and with added colouring matter as required,1 and shall be in the form of a uniformly blended, free-flowing powder, free from lumps and extraneous impurities that may limit its suitability for baiting rodents or impair its effectiveness. The technical warfarin used in the manufacture of the concentrate shall comply with the requirements of Specification WHO/SRT/6.Rl. 1. 2 Chemical, Physical, and Biological Requirements The material, sampled from any part of the consignment,2 shall comply with the requirements of section 1. 1 and with the following requirements. 1.2.1 Warfarin content (w/w basis) The content of 3-(cx-acetonylbenzyl)-4-hydroxycoumarin, determined by the method described in section 2. 1, shall not differ from the nominal content by more than ± 5%. The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Sieving Not less than 95% of the concentrate (dry weight) shall pass through a 150-µ sieve (BS 100 mesh; 3 US Standard No. 100 4) when tested by the method described in section 2.2. 1 The colouring matter added shall be consistent with the laws of the country in which the concentrate is to be used. A sufficient quantity must be added to ensure that the concentrate and baits prepared from it are clearly recognizable. The colouring matters described as Direct Blue 1 (sodium salt of dimethoxydiphenyldiazo-bis-(8- amino-1-naphthol) 5,7-disulfonic acid) and Direct Blue 14 (sodium salt of ditolyldiazo- bis-(8-amino-1-naphthol) 3,6-disulfonic acid) in the Colour Index (2nd ed., 1956, Part I, Vol. 2, Bradford, England & Lowell, Mass.) have been found to be satisfactory. 2 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 3 British Standards Institution, British Standard 410: 1943. 4 American Society for Testing Materials, Standard E 11-39. - 208- WHO/SRF/2.R1 WARF ARIN CONCENTRATES 1. 2. 3 Toxicity to rats When tested by the method described in WHO/M/14, page 285, the concentrate shall have an effectiveness of at least 90% of that of the standard material.1 1. 3 Packing and Marking of Packages The warfarin concentrate shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Warfarin concentrate to Specification WHO/SRF/2.Rl Warfarin, ... % (w/w) Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Keep this material and baits containing it well away from children, domestic animals, foodstuffs, empty foodstuff containers, and animal feed. " In cases of poisoning, call a physician. The specific antidote is vita- min K1• Blood transfusions may be necessary." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 3-(a:-Acetooylbenzyl)-4-hydroxycoumarin Content 2. 1.1 Special apparatus Beckman spectrophotometer, model DU, or equivalent instrument, with I-cm quartz cells. 2 .1. 2 Special reagent Reference solution. Pipette 2 ml of ethylene dichloride into a glass- stoppered cylinder, add 10 ml of 1 % sodium hydroxide solution with a pipette, and shake by hand for 1 minute. Decant the sodium hydroxide layer into a centrifuge tube and centrifuge until clear. 1 Samples of standard material may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. - 209 - 14 WARFARIN CONCENTRATES WHO/SRF/2.R1 2.1.3 Procedure Weigh accurately about 0.6 g of the sample into a 125-ml glass-stoppered flask, add 50 ml of ethylene dichloride with a pipette, and shake on a shaking-machine for at least 10 minutes. Transfer to a centrifuge tube, stopper, and centrifuge at high speed for 5 minutes, or until clear. Pipette 2 ml of the ethylene dichloride extract into a glass-stoppered cylinder, add 10 ml of 1% sodium hydroxide solution with a pipette, and shake by hand for l minute. Decant the sodium hydroxide layer into a centrifuge tube and centrifuge until clear. Pipette a sufficient amount (about 3 ml) of the alkali layer into a 1-cm quartz cell and, with the spectrophotometer set at maximum sensitivity, determine its absorbance at 308 mµ against the reference solution. 2 .1. 4 Calculation 3-( cx-acetonylbenzyl)-4-hydroxycoumarin content (% w /w) E x 7.71 x 10a Mx w where E = absorbance of the final solution at 308 mµ w = weight (g) of sample M = molar extinction coefficient determined with pure 3-( cx-acetonyl- benzyl)-4-hydroxycoumarin. 2.2 Sieving Test Place 200 g of the sample on a clean, dry 150-µ sieve (BS 100 mesh; 1 US Standard No. 100 2) and screen in a Ro-Tap Shaker 3 or equivalent machine for 10 minutes. Stop the machine, break up any soft lumps by brushing lightly over the sieve, and restart the machine. Completion of screening is determined by shaking the sieve by hand over a clean, glazed paper and noting if any appreciable amount passes through. Weigh the residue remaining on the sieve. Calculate the portion passing through and express it as a percentage of the weight of sample taken. 1 British Standards Institution, British Standard 410: 1943. 2 American Society for Testing Materials, Standard E 11-39. 3 The Ro-Tap Testing Sieve Shaker, supplied by the W. S. Tyler Company, Cleve- land, Ohio, USA, reproduces the circular and tapping motion given testing sieves in handsieving, but with a uniform, mechanical action. One model is manufactured with gear to operate either with 60-cycle, 1750-rev/min motors, or with 50-cycle, 1450-rev/min motors, and still have the same standard speed of operation. The unit operates the sieves with a horizontal circular motion of 285 rev /min, together with a tapping action at the rate of approximately 150 taps per minute. The tapping action is very important for ensuring comparable results. This is especially true in the sieving of materials such as insecticides. The tapping motion is imparted to the top of the sieves and is transmitted through the entire nest. - 210 - WHO/SRF/4 PINDONE CONCENTRATES PINDONE CONCENTRATES Tentative Specification WHO/SRF/4 Approved 25 October 1965 l. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of technical pindone compounded with corn- starch or other suitable diluent, and with added colouring matter as required,1 and shall be in the form of a uniformly blended, free-flowing powder, free from lumps and extraneous impurities that may limit its suitability for baiting rodents or impair its effectivenes. The technical pindone used in the manufacture of the concentrate shall comply with the requirements of Specification WHO/SRT/9.Rl. 1. 2 Chemical, Physical, and Biological Requirements The material, sampled from any part of the consignment,2 shall comply with the requirements of section 1. 1 and with the following requirements. 1. 2 .1 Pindone content ( w /w basis) The content of 2-pivaloyl-l,3-indanedione determined by the method described in section 2. l, shall not differ from the nominal content by more than ± 5%. The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Sieving Not less than 95% of the concentrate (dry weight) shall pass through a 150-µ sieve (BS 100 mesh; 3 US Standard No. 100 4) when tested by the method described in section 2.2. 1 The colouring matter added shall be consistent with the laws of the country in which the concentrate is to be used. A sufficient quantity must be added to ensure that the concentrate and baits prepared from it are clearly recognizable. The colouring matters described as Direct Blue I (sodium salt of dimethoxy-diphenyl-diazo-bis-(8- amino-l-naphthol) 5,7-disulfonic acid) and Direct Blue 14 (sodium salt of ditolyl-diazo- bis-(8-amino-l-naphthol) 3,6-disulfonic acid) in the Colour Index (2nd ed., 1956, Part I, Vol. 2, Bradford, England & Lowell, Mass.) have been found to be satisfactory. 2 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 3 British Standards Institution, British Standard 410: 1943. 4 American Society for Testing Materials, Standard E 11-39. - 211 - PINDONE CONCENTRATES WHO/SRF/4 1. 2. 3 Toxicity to rats When tested by the method described in WHO/M/14, page 285, the concentrate shall have an effectiveness of at least 90% of that of the standard material.1 1. 3 Packing and Marking of Packages The pindone concentrate shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Pindone concentrate to Specification WHO/SRF/4.Rl Pindone, ... % (w/w) Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : '' Keep this material and baits containing it well away from children, domestic animals, foodstuffs, empty foodstuff containers, and animal feed. " In cases of poisoning, call a physician. The specific antidote is vita- min K1. Blood transfusions may be necessary." 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 2-Pivaloyl-1,3-indanedione Content 2 .1.1 Special apparatus Beckman spectrophotometer, model DU, or equivalent instrument, with 1-cm quartz cells. 2 .1. 2 Special reagent Reference solution. Weigh accurately 0.100 g 2 of the diluent used in the manufacture of the concentrate into a 100-ml beaker or conical flask 1 Samples of standard material may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. 2 The weights of diluent and sample given here are convenient for concentrates containing 0.5% of pindone. They should be suitably varied for concentrates of different pindone content. - 212 - WHO/SRF/4 PINDONE CONCENTRATES and add 50 ml of ethanol.1 Heat on a steam-bath for 10 minutes, with constant stirring or swirling. Filter through a filter paper (Whatman No. 44 or equivalent) into a 100-ml volumetric flask, wash the filter paper twice with 20-ml portions of warm ethanol, combine the filtrate and washings, and make up to 100 ml with ethanol. 2.1.3 Procedure Weigh accurately about 2.5 g 2 of the sample into a 250-ml beaker or conical flask and add 40 ml of ethanol.1 Heat on a steam-bath for 10 minutes, with constant stirring or swirling. Decant the alcohol solution through a filter paper (Whatman No. 44 or equivalent) into a 250-ml volumetric flask. Repeat the extraction four times, using 40-ml portions of ethanol. Wash the filter paper thoroughly with two 30-ml portions of warm ethanol and add the washings to the combined filtrates. Cool the flask and contents and adjust the volume to 250 ml. Pipette 10 ml of this solution into a 100-ml volumetric flask, make up to the mark with ethanol, and mix thoroughly. With the spectrophoto- meter set at maximum sensitivity, determine the absorbance of the final solution at 281-283 mµ against the reference solution. 2 .1. 4 Calculation 2-Pivaloyl-1,3-indanedione content (% w /w) Ex 57.56 x 10s M x w where E = absorbance of the final solution at 281-283 mµ w = weight (g) of sample M = molar extinction coefficient determined with pure 2-pivaloyl- 1,3-indanedione. 2.2 Sieving Test Place 200 g of the sample on a clean, dry 150-µ sieve (BS 100 mesh; 3 US Standard No. 100 4) and screen in a Ro-Tap Shaker or equivalent 1 Throughout this procedure 95% ethanol should be used. 2 The weights of diluent and sample given here are convenient for concentrates containing 0.5% of pindone. They should be suitably varied for concentrates of different pindone content. 3 British Standards Institution, British Standard 410: 1943. 4 American Society for Testing Materials, Standard E 11-39. - 213 - PIN DONE CONCENTRATES WHO/SRF/4 machine 1 for 10 minutes. Stop the machine, break up any soft lumps by brushing lightly over the sieve, and restart the machine. Completion of screening is determined by shaking the sieve by hand over a clean, glazed paper and noting if any appreciable amount passes through. Weigh the residue remaining on the sieve. Calculate the portion passing through and express it as a percentage of the weight of sample taken. 1 The Ro-Tap Testing Sieve Shaker, supplied by the W. S. Tyler Company, Cleve- land, Ohio, USA, reproduces the circular and tapping motion given testing sieves in handsieving, but with a uniform, mechanical action. One model is manufactured with gear to operate either with 60-cycle, 1750-rev /min motors, or with 50-cycle, 1450-rev /min motors, and still have the same standard speed of operation. The unit operates the sieves with a horizontal circular motion of 285 rev/min, together with a tapping action at the rate of approximately 150 taps per minute. The tapping action is very important for ensuring comparable results. This is especially true in the sieving of materials such as insecticides. The tapping motion is imparted to the top of the sieves and is transmitted through the entire nest. - 214 - Part III MOLLUSCICIDES

WHO/SMT /1.R1 TECHNICAL COPPER SULFATE TECHNICAL COPPER SULFATE 1. SPECIFICATIONS 1.1 Material Specification WHO/SMT/1.Rl Approved 25 October 1965 The material shall comprise essentially copper sulfate pentahydrate and shall be in the form of blue or bluish-green crystals, free from visible impurities and hard-caking properties. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section 1 . 1 and with the following requirements : Copper sulfate pentahydrate content (sec- tion 2.1), % by weight ..... . Acidity (section 2.2), % by weight, cal- culated as H2S04 • • Water-insoluble material (section 2. 3), % by weight ....... . Minimum 98.0 1. 3 Packing and Marking of Packages Maximum 0.1 0.2 The technical copper sulfate shall be packed in suitable, clean con- tainers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical copper sulfate to Specification WHO/SMT/1. Rl Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Keep well away from foodstuffs, animal feed and their containers." 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. - 217 - TECHNICAL COPPER SULFATE 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Copper Sulfate Pentahydrate Content 2 .1.1 Special reagent WHO/SMT/1.R1 Sodium thiosulfate solution, 0.1 N to 0.2 N. This solution has to be standardized immediately before the determination, as described in sec- tion 2.1.2.l. 2.1.2 Procedure 2. l . 2. l Standardization of sodium thiosulfate solution Weigh accurately about 0.5 g of electrolytic copper foil into a 600-ml flask, add 5 ml of concentrated nitric acid and 5 ml of distilled water, and evaporate nearly to dryness. Cool, add 100 ml of distilled water, boil, and cool. Add a slight excess of saturated sodium carbonate solution and then dilute acetic acid until the mixture is slightly acid. Add 10 ml of saturated sodium fluoride solution, followed by 5 g of potassium iodide. Titrate the liberated iodine with the sodium thiosulfate solution until the yellow colour has nearly disappeared, add a small quantity of freshly pre- pared soluble-starch solution, and continue the titration until the blue colour disappears. Strength of sodium thiosulfate solution in terms of copper sulfate pentahydrate (g/ml) a x 3.928 b where a weight (g) of copper taken b = volume (ml) of sodium thiosulfate solution required. 2. 1 . 2. 2 Analysis of sample Weigh accurately about 2 g of the sample, coarsely crushed if necessary, by difference from a stoppered weighing-bottle, and dissolve in 100 ml of distilled water. Add 3 drops of concentrated nitric acid, boil, and cool. Add a slight excess of saturated sodium carbonate solution and then dilute acetic acid until the mixture is slightly acid. Add 10 ml of saturated sodium fluoride solution, followed by 5 g of potassium iodide. Titrate the liberated iodine with the standardized sodium thiosulfate solution until the yellow colour has nearly disappeared, add a small quantity of freshly prepared soluble-starch solution, and continue the titration until the blue colour disappears. - 218 - WHO/SMT/1.R1 TECHNICAL COPPER SULFATE 2 .1. 3 Calculation cxSxlOO Copper sulfate pentahydrate content (% w /w) = ----- w where S = strength of sodium thiosulfate solution (see section 2.1.2.1) c = volume (ml) of sodium thiosulfate solution required w = weight (g) of sample. NOTE : The electrolytic method may be used in laboratories equipped for this work. 2.2 Acidity Weigh exactly 4 g of the sample, dissolve in 100 ml of distilled water, and add 4 drops of methyl orange indicator solution (1% w/v) to obtain a purplish colour. Divide into two equal portions. Add 0.4 ml of 0.1 N sodium hydroxide to one of the portions, keeping the other portion for comparison of the colour. The prescribed limit shall be taken as not having been exceeded if the purplish colour of the portion under test disappears on the addition of the sodium hydroxide. 2.3 Water-Insoluble Material Weigh accurately about 25 g of the sample into a beaker, add 150 ml of distilled water and 2 drops of concentrated sulfuric acid, and heat to dissolve. Filter while hot through a tared Gooch crucible, and wash thoroughly with hot distilled water. Dry to constant weight in an oven at ll0°C, cool, and weigh. - 219 - TECHNICAL PENT ACHLOROPHENOL WHO/SMT/2.R1 TECHNICAL PENTACHLOROPHENOL Specification WHO/SMT/2.RI Approved 25 October 1965 l. SPECIFICATIONS 1.1 Material The material shall comprise essentially pentachlorophenol and shall be in the form of free-flowing, dark greyish flakes, with a phenolic odour and free from dust, extraneous impurities, or added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l . l and with the following requirements : Minimum Maximum Pentachlorophenol content (section 2.1), % by weight . . . . . . . . . . . 96.0 Crystallizing-point (WHO/M/12, page 281) l 74°C Alkali-insoluble material (section 2.2), % by weight . . . . . . . . 1.0 1. 3 Packing and Marking of Packages The technical pentachlorophenol shall be packed in suitable, clean con- tainers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical pentachlorophenol to Specification WHO/SMT/2.Rl Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " A void inhalation of fumes and do not allow the material to come into contact with the skin and mucous membranes ; wash after any accidental contamination. Keep well away from foodstuffs, animal feed and their containers." 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. - 220 - WHO/SMT/2.R1 TECHNICAL PENT ACHLOROPHENOL 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2 .1 Pentachlorophenol Content 2.1.1 Procedure Weigh accurately about 5 g of the sample into a 250-ml conical flask. Add 100 ml of neutral absolute ethanol and swirl the contents of the flask, warming on a steam-bath if necessary, until solution is as complete as possible. Cool, add 0.5 ml of phenolphthalein indicator solution, and titrate with 1 N sodium hydroxide until a definite pink colour is obtained. If the alcoholic solution, before titration, is rather highly coloured, a solution of 5 g of the sample in 100 ml of absolute ethanol, contained in a second flask, may be used for comparative purposes to help in detecting the colour change. 2 .1. 2 Calculation a x 26.64 Pentachlorophenol content (% w /w) = ---- w where a = volume (ml) of 1 N sodium hydroxide required w = weight (g) of sample. 2. 2 Alkali-Insoluble Material Weigh accurately about 5 g of the sample into a 250-ml beaker and add 50 ml of 5% sodium hydroxide solution. Boil for 5 minutes or until as much as possible of the sample has dissolved. Make up to 200 ml with distilled water, heat to boiling, filter while hot through a dry, tared Gooch crucible, and wash until free from alkali. Dry in an oven at 120°C for 1 hour, cool, and weigh. - 221 - TECHNICAL 2',5-DICHLOR0-4'-NITRO- SALICYLANILIDE ETHANOLAMINE SALT WHO/SMT/4 TECHNICAL 2' ,5-DICHLOR0-4'-NITROSALICYLANILIDE ETHANOLAMINE SALT Tentative Specification WHO/SMT/4 Approved 25 October 1965 1. SPECIFICATIONS 1.1 Material The material shall comprise essentially the ethanolamine salt of 2' ,5- dichloro-4' -nitrosalicylanilide and shall be in the form of a yellow crystal- line product free from extraneous impurities and added modifying agents. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l . l and with the following requirements : Minimum Maximum Melting point of the ethanolamine salt of 2' ,5-dichloro-4' -nitrosalicylanilide (WHO/M/5, page 255) . . . . . . Content of 2' ,5-dichloro-4' -nitrosalicyl- anilide ethanolamine salt ( section 2. 1), % by weight .......... . Water content (section 2.2), % by weight 191°C 94.0 1. 3 Packing and Marking of Packages 1.0 The technical 2' ,5-dichloro-4' -nitrosalicylanilide ethanolamine salt shall be packed in suitable clean drums, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical 2' ,5-dichloro-4'-nitrosalicylanilide ethanolamine salt Specification to WHO/SMT/4 Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : "Keep well away from foodstuffs, animal feed and their containers." 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. - 222 - WHO/SMT/4 TECHNICAL 2' ,5-DICHLOR0-4' -NITRO- SALICYLANILIDE ETHANOLAMINE SALT 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 Content of 2',5-Dichloro-4'-nitrosalicylanilide Ethanolamine Salt 2.1.1 Summary of method The active ingredient is determined by reduction of the nitro-group with titanous chloride solution. 2.1. 2 Special apparatus 1. Carbon dioxide source : carbon dioxide bomb with reducing or needle valve. In order to remove traces of oxygen, the gas current is passed through a bubbler containing 15% titanous chloride solution. 2. Titration apparatus: two 5-litre storage bottles for 0.1 N titanous chloride and ferric chloride solutions, closed with rubber stoppers, and two 50-ml burettes with side-arms. The single parts of the apparatus are joined with glass tubes fitted with rubber connexions and the entire apparatus is connected to the carbon dioxide source. 2 .1. 3 Special reagents Titanous chloride solution, 0.1 N. Mix 500 ml of hydrochloric acid (sp. gr. 1.16-1.18) and 550 g of 15% titanous chloride solution and make up to 5 litres with distilled water in a volumetric flask. Transfer to the storage bottle, mix well and preserve the solution in an atmosphere of carbon dioxide. As a further precaution against oxidation add about 75 ml of zinc amalgam. Zinc amalgam. Mix about 30 g of pure zinc granules, 1000 g of mercury and some dilute sulfuric acid and heat on a steam bath. Cool, wash well with distilled water in a separating funnel and separate from undissolved zinc. Standard ferric chloride solution, 0.05 N. Dissolve 19.9625 g of ferric oxide reagent grade in 200 ml of hydrochloric acid (sp. gr. 1.124) by heating to gentle boiling. Allow to cool, transfer the clear solution to a 5-litre volumetric flask, and dilute to volume with distilled water saturated with carbon dioxide. Transfer to the storage bottle, mix well, and preserve the solution in an atmosphere of carbon dioxide. The normality of the ferric chloride solution is checked as follows : Pipette 100 ml of the ferric chloride solution into a 250-ml conical flask, buffer the hydrochloric acid by dropwise addition of dilute sodium - 223 - TECHNICAL 2',5-DICHLOR0-4'-NITRO- SALICYLANILIDE ETHANOLAMINE SALT WHO/SMT/4 hydroxide, the final pH being at least 1, and displace the air in the flask by passing in carbon dioxide. Add 10 g of potassium iodide, bring into solution and allow the closed flask to stand in the dark for 20 minutes. Then titrate the free iodine with 0.1 N sodium thiosulfate solution 1 using starch indicator. It should be noted that after refilling the flask with carbon dioxide, the solution must remain colourless for several minutes. 2.1.4 Procedure 2. 1 . 4. 1 Standardization of titanous chloride solution Mix 25 ml of pyridine, 50 ml of dimethylformamide and 25 ml of glacial acetic acid in a 500-ml wide-necked conical flask and pass a stream of carbon dioxide through the solution. Keep the stream of carbon dioxide flowing throughout the entire titration. When all the air has been displaced by carbon dioxide (5 min), add from a burette 50 ml of 0.1 N titanous chloride solution, mix well and allow the mixture to stand for 5-10 minutes at room temperature. Acidify with 50 ml of hydrochloric acid (sp. gr. 1.15-1.18), add 10 ml of 10% potassium thiocyanate solution and titrate, without cooling, with the standard ferric chloride solution until the colour changes to red. 2. 1 . 4. 2 Analysis of sample Weigh accurately 5.00 g of the sample, dissolve it in about 200 ml of pyridine by heating on a water bath, cool to 20°C, transfer to a 250-ml volumetric flask and dilute to the mark with pyridine. Pipette 10 ml of the solution into a 500-ml wide-necked conical flask, add 15 ml of pyridine, 50 ml of dimethylformamide and 25 ml of glacial acetic acid and pass a stream of carbon dioxide through the solution. Keep the stream of carbon dioxide flowing throughout the entire titration. When all the air has been displaced by carbon dioxide (5 min), add from a burette 50 ml of 0.1 N titanous chloride solution and proceed with the titration as described in section 2. 1. 4. 1. 2. 1. 5 Calculation 2' ,5-dichloro-4'-nitrosalicylanilide ethanolamine salt content (% w /w) (a - b) x 6.47 x N w where a = volume (ml) of 0.05 N ferric chloride required for 50 ml of 0.1 N titanous chloride 1 50 ml of 0.1 N sodium thiosulfate solution should be used. - 224 - WHO/SMT/4 TECHNICAL 2' ,5-DICHLOR0-4'-NITRO- SALICYLANILIDE ETHANOLAMINE SALT b = volume (ml) of 0.05 N ferric chloride required for back- titration of excess titanous chloride in analysis of sample N = normality of the 0.05 N ferric chloride (see section 2 .1. 3) w = weight (g) of sample. 2. 2 Water Content Determine the water content by the Karl Fischer electrometric titration method (see WHO/M/7, page 266). - 225 - 15 TECHNICAL N-TRIPHENYLMETHYLMORPHOLINE WHO/SMT/5 TECHNICAL N-TRIPHENYLMETHYLMORPHOLINE Tentative Specification WHO/SMT/5 Approved 25 October 1965 l. SPECIFICATIONS 1. 1 Material The material shall comprise essentially N-triphenylmethylmorpholine and shall be in the form of an off-white to yellow powder. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l . l and with the following requirements : N-Triphenylmethylmorpholine content ( sec- tion 2.1), % by weight ..... Acidity (WHO/M/3, page 250), % by weight, calculated as H2S04 . • • • • Water content (section 2.2), % by weight . Tar impurities (section 2.3), % by weight . Volatile matter (section 2.4), % by weight . Minimum 90.0 1. 3 Packing and Marking of Packages Maximum 0.1 0.1 2.0 10.0 The technical N-triphenylmethylmorpholine shall be packed in suitable, clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical N-triphenylmethylmorpholine to Specification WHO/SMT/5 Batch or reference number and date of test Net weight of contents and the following minimum cautionary notice : "Keep well away from foodstuffs, animal feed and their containers." 1 A sampling procedure is described in Method WHO/M/11, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. - 226 - WHO/SMT/5 TECHNICAL N-TRI PHENYLMETHYLMORPHOLINE 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 N-Triphenylmethylmorpholine Content 2 .1.1 Summary of method The sample is weighed and chromatographed on alumina using diethyl ether as the eluting solvent. After evaporation of the solvent the N-tri- phenylmethylmorpholine content is determined by titration with perchloric acid. 2 .1. 2 Special apparatus I. Chromatographic column (18 mm x 500 mm) packed with 50 g of 100/200-mesh dry alumina (dried for at least 5 hours at 105°C) and fitted with glass stopcock and reservoir (approximately 500 ml). 2. 10-ml burette graduated in 0.02-ml divisions. 2 .1. 3 Special reagents 1. Indicator solution. Dissolve 0.5 g of crystal violet in 100 ml of glacial acetic acid. 2. Perchloric acid in glacial acetic acid. To 900 ml of glacial acetic acid add slowly with stirring 8.5 ml of perchloric acid (72%) followed by 30 ml of acetic anhydride. Allow the mixture to stand for 24 hours before use. 2. 1. 4 Standardization of perchloric acid in glacial acetic acid Weigh out accurately about 0.25 g of pure N-triphenylmethylmorpholine 1 and transfer quantitatively to a 100-ml conical flask with 10 ml of chloro- form. Add 50 ml of glacial acetic acid, 3 drops of indicator solution and titrate with perchloric acid in glacial acetic acid until the colour changes from blue to green. Carry out a blank determination on the reagents by titrating a mixture of 50 ml of glacial acetic acid and 10 ml of chloroform in the presence of 3 drops of indicator solution and a small amount (50 mg) of triphenyl carbinol. NOTES 1. The addition of triphenylmethanol in the blank determination is desirable in order to reproduce the same colour changes as those obtained 1 Samples of standard N-triphenylmethylmorpholine may be obtained, on request, from the World Health Organization, Avenue Appia, 12ll Geneva, Switzerland. - 227 - TECHNICAL N-TRIPHENYLMETHYLMORPHOLINE WHO/SMT/5 on titrating N-triphenylmethylmorpholine, since both these compounds produce the yellow triphenylmethane carbonium ion in acid solution. If pure triphenylmethanol is not available, the blank determination may be carried out with very little loss in accuracy by adding only crystal violet. 2. Alternatively, the end-point may be determined potentiometrically. 3. If pure N-triphenylmethylmorpholine is not available, the perchloric acid may be standardized against pure potassium hydrogen phthalate: Weigh accurately about 0.5 g of potassium hydrogen phthalate, dissolve in 10 ml of chloroform and add 50 ml of glacial acetic acid. Titrate the solution with the prepared perchloric acid in glacial acetic acid, using 3 drops of crystal violet indicator. The end-point is shown by the first change from violet to green. (The crystalline precipitate of potassium perchlorate which forms during the titrations has no adverse effect on the accuracy of the method.) Calculate the normality assuming that l litre of l N perchloric acid in glacial acetic acid is equivalent to l g mol of potas- sium hydrogen phthalate. 2.1.5 Procedure Weigh accurately about 0.25 g of the dried N-triphenylmethylmorpholine (section 2.4) and add approximately 5 ml of diethyl ether. Allow to stand for 5 minutes to permit the material to dissolve and then transfer the solu- tion or suspension quantitatively to the alumina column, using the minimum quantity of ether from a wash bottle.1 Place a tared 250-ml beaker under the column and continue eluting with small quantities of ether to wash the toxicant thoroughly into the alumina column. When solvent emerges from the column add two further 100-ml portions of diethyl ether and allow to elute into the beaker. Evaporate the solvent carefully in a forced draught, either at room temperature or (preferably) on an electrically- heated water bath kept at 30-40°C. When dry, reweigh the beaker to obtain an approximate value for the content of the sample. Transfer the extract to a 100-ml conical flask with 10 ml of chloroform, add 50 ml of glacial acetic acid, 3 drops of indicator solution and titrate with standard per- chloric acid in glacial acetic acid until the colour changes from blue to green. Whenever possible a blank determination should be carried out along- side the samples, including, if necessary, the chromatographic stage. In any case a reagent blank (i.e., 10 ml of chloroform plus 50 ml of glacial acetic acid) should always be carried out for the titration stage. Alternatively the end-point may be determined potentiometrically. 1 If the technical material is known to be free from basic impurities (e.g., morpholine) the chromatographic stage may be omitted. - 228- WHO/SMT/5 TECHNICAL N-TRIPHENYLMETHYLMORPHOLINE 2.1. 6 Calculation N-Triphenylmethylmorpholine content (% w/w) = 0.3295 x (T - B) x N x lOO w where T = volume (ml) of perchloric acid used in titration B = volume (ml) of perchloric acid used in blank determination N = normality of perchloric acid W = weight (g) of sample taken. 2.2 Water Content The water content is determined, on the undried sample, by the Karl Fischer electrometric titration method described in WHO/M/7, page 266, with the following modifications : 2. 2. 1 Special apparatus l. A 10-ml microburette graduated in 0.02-ml divisions. 2. Reaction vessel similar to that described in WHO/M/7, page 266, but having a further inlet for a 10-ml microburette. 2. 2. 2 Special reagents Titration solvent. Anhydrous methanol and chloroform in 1 : 4 ratio. N-ethylpiperidine. Ethylene glycol monomethyl ether, anhydrous. Dry by distillation through a glass column 30 cm long and 2.5 cm in internal diameter, packed with single-turn glass helices. The first 5% by volume of distillate is rejected and the distilland is retained. Ethylene glycol monomethyl ether dried in this manner should contain not more than 0.02% by weight of water. Dilute Fischer reagent. Reagent prepared as described in WHO/M/7 is diluted with dried ethylene glycol monomethyl ether to give a solution of approximately 1 mg/ml water equivalent. Dilute standard solution of water in methanol. Dry thoroughly in an oven a 100-ml volumetric flask and then allow it to cool in a desiccator. Partially fill the flask with anhydrous methanol, add about 0.1 g of distilled water, accurately weighed, from a weighing pipette, make up to the mark with the methanol and mix thoroughly. This solution should be prepared immediately before use. - 229 - TECHNICAL N-TRIPHENYLMETHYLMORPHOLINE WHO/SMT/5 2. 2. 3 Standardization of the dilute Fischer reagent Remove moisture from the reaction vessel by the procedure detailed in WHO /M/7. Transfer 10 ml of titration solvent to the reaction vessel and titrate with concentrated Fischer reagent (i.e., approximately 3.5 mg/ml). Add l ml of N-ethylpiperidine and titrate with the dilute Fischer reagent (i.e., approximately l mg/ml). Ignore this titration. Add to the reaction vessel 5 ml of the dilute standard water solution and again titrate with the dilute Fischer reagent. Water equivalent of the dilute Fischer reagent (mg/ml) 50 x w where w = weight (g) of distilled water used in preparing 100 ml of stand- ard solution of water in methanol t = volume (ml) of dilute Fischer reagent used for titration of the dilute standard solution of water in methanol. 2. 2. 4 Analysis of sample Transfer 25 ml of titration solvent to the reaction vessel and titrate with concentrated Fischer reagent as described in WHO/M/7. Add l ml of N-ethylpiperidine and titrate with dilute Fischer reagent. Ignore this titration. Add to the reaction vessel 5 g of technical N-triphenylmethyl- morpholine, allow to dissolve, and again titrate with the dilute Fischer reagent. Water content (% by weight) = t x F w x 10 where F = water equivalent (mg/ml) of the dilute Fischer reagent (sec- tion 2.2.3) t = volume (ml) of dilute Fischer reagent used for titration w = weight (g) of sample. 2.3 Tar Impurities A 1-g sample of the dried material (section 2.4) is shaken with petroleum spirit (b.p. 62-68°C) for 5 minutes. The solution is filtered through a sintered glass crucible (porosity 4), the residue washed with petroleum spirit, dried at 75°C for 30 minutes, cooled and weighed. - 230 - WHO/SMT/5 TECHNICAL N-TRI PHENYLMETHYLMORPHOLINE 2. 4 Volatile Matter A representative 5-g sample of the material " as received " is kept under continuously maintained vacuum (about 2 Torr) for a period of two hours. The residue is weighed and the process repeated to constant weight. - 231 - I 2' ,5-DICHLOR0-4' -NITROSALICYLAN Ill DE ETHANOLAMINE SALT, WATER-DISPERSIBLE POWDER WHO/SMF/1 2',5-DICHLOR0-4'-NITROSALICYLANILIDE ETHANOLAMINE SALT, WATER-DISPERSIBLE POWDER Tentative Specification WHO/SMF/1 Approved 25 October 1965 l. SPECIFICATIONS 1.1 Description and Ingredients The material shall consist of a homogeneous mixture of the ethanol- amine salt of 2' ,5-dichloro-4' -nitrosalicylanilide in a filler and shall be in the form of a yellow-coloured powder. The ingredients used in the manu- facture of the powder shall comply with the requirements of specification WHO/SMT/4, page 222. 1. 2 Chemical and Physical Requirements The material, sampled from any part of the consignment,1 shall comply with the requirements of section l . l and with the following requirements. 1.2.1 Content of 2',5-dichloro-4'-nitrosalicylanilide ethanolamine salt The content of 2' ,5-dichloro-4' -nitrosalicylanilide ethanolamine salt determined by the method described in section 2. l, shall not differ from the nominal content by more than the following amounts : Nominal content Tolerance permitted above 50% ± 5% of (100-nominal content) 2 The average content of all samples taken shall not be lower than the nominal content. 1. 2. 2 Sieving after accelerated storage Not less than 96% of the powder (dry weight) after accelerated storage ( section 2. 3) shall pass through a 7 4-µ sieve (BS 200 mesh ; 3 US Stand- 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. 2 For a 70% water-dispersible powder, tolerance= ± 5% of(l00-70)% = ± l.5%. 3 British Standards Institution, British Standard 410: 1943. -232 - WHO/SMF/1 2'5-DICHLOR0-4' -NITROSALICYLANILI DE ETHANOLAMINE SALT, WATER-DISPERSIBLE POWDER ard No. 200 1) when tested by the method described in WHO/M/4, page 253. 1. 2. 3 Suspensibility 1 . 2. 3. 1 In distilled water without pretreatment When tested by the method described in section 2.2, a minimum of 50% of the 2' ,5-dichloro-4'-nitrosalicylanilide ethanolamine salt (0.07% w /v) shall be in suspension 30 minutes after agitating a suspension con- taining O .14 % ( w /v) of the 2' ,5-dichloro-4' -nitrosalicylanilide ethanolamine salt, prepared in distilled water from the powder as received. 1 . 2. 3. 2 In standard hard water after accelerated storage treatment When tested by the method described in section 2. 2, a minimum of 50% of the 2' ,5-dichloro-4'-nitrosalicylanilide ethanolamine salt (0.07% w/v) shall be in suspension 30 minutes after agitating a suspension contain- ing 0.14% (w/v) of the 2',5-dichloro-4'-nitrosalicylanilide ethanolamine salt, prepared in standard hard water from the powder subjected to acceler- ated storage treatment, as described in section 2. 3. 1. 3 Packing and Marking of Packages The 2' ,5-dichloro-4' -nitrosalicylanilide ethanolamine salt, water-dis- persible powder shall be packed in suitable, clean drums, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name 2' ,5-Dichloro-4'-nitrosalicylanilide ethanolamine salt, water-dispersible powder to Specification WHO/SMF/1 2',5-Dichloro-4'-nitrosalicylanilide ethanolamine salt, ... % (w/w) Batch or reference number, and date of test Net weight of contents and the following minimum cautionary notice : " Keep well away from foodstuffs, animal feed and their containers." 1 American Society for Testing Materials, Standard E 11-39. - 233- 2',5-DICHLOR0-4'-NITROSALICYLANILIDE ETHANOLAMINE SALT, WATER-DISPERSIBLE POWDER 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES WHO/SMF/1 2.1 Content of 2',5-Dichloro-4'-Nitrosalicylanilide Ethanolamine Salt 2 .1.1 Summary of method The active ingredient is determined by reduction of the nitro-group with titanous chloride solution. 2 .1. 2 Special apparatus 1. Carbon dioxide source : carbon dioxide bomb with reducing or needle valve. In order to remove traces of oxygen, the gas current is passed through a bubbler containing 15% titanous chloride solution. 2. Titration apparatus : two 5-litre storage bottles for 0.1 N titanous chloride and ferric chloride solutions, closed with rubber stoppers, and two 50-ml burettes with side-arms. The single parts of the apparatus are joined with glass tubes fitted with rubber connexions and the entire apparatus is connected to the carbon dioxide source. 2 .1. 3 Special reagents Titanous chloride solution, 0.1 N. Mix 500 ml of hydrochloric acid (sp. gr. 1.16-1.18) and 550 g of 15% titanous chloride solution and make up to 5 litres with distilled water in a volumetric flask. Transfer to the storage bottle, mix well and preserve the solution in an atmosphere of carbon dioxide. As a further precaution against oxidation add about 75 ml of zinc amalgam. Zinc amalgam. Mix about 30 g of pure zinc granules, 1000 g of mercury and some dilute sulfuric acid and heat on a steam bath. Cool, wash well with distilled water in a separating funnel and separate from undissolved zinc. Standard ferric chloride solution, 0.05 N. Dissolve 19.9625 g of ferric oxide, reagent grade, in 200 ml of hydrochloric acid (sp. gr. 1.124) by heating to gentle boiling. Allow to cool, transfer the clear solution to a 5-litre volumetric flask and dilute to volume with distilled water saturated with carbon dioxide. Transfer to the storage bottle, mix well and preserve the solution in an atmosphere of carbon dioxide. The normality of the ferric chloride solution is checked as follows : Pipette 100 ml of the ferric chloride solution into a 250-ml conical flask, buffer the hydrochloric acid by dropwise addition of dilute sodium hydroxide, the final pH being at least 1, and displace the air in the flask - 234 - WHO/SMF/1 2' ,5-DICHLOR0-4'-NITROSALICYLANILIDE ETHANOLAMINE SALT, WATER-DISPERSIBLE POWDER by passing in carbon dioxide. Add lO g of potassium iodide, bring into solution, and allow the closed flask to stand in the dark for 20 minutes. Then titrate the free iodine with 0.1 N sodium thiosulfate solution 1 using starch indicator. It should be noted that after refilling the flask with carbon dioxide, the solution must remain colourless for several minutes. 2.1.4 Procedure 2. 1 . 4. 1 Standardization of titanous chloride solution Mix 25 ml of pyridine, 50 ml of dimethylformamide and 25 ml of glacial acetic acid in a 500-ml wide-necked conical flask and pass a stream of carbon dioxide through the solution. Keep the stream of carbon dioxide flowing throughout the entire titration. When all the air has been displaced by carbon dioxide (5 min), add from a burette 50 ml of 0.1 N titanous chloride solution, mix well and allow the mixture to stand for 5-10 minutes at room temperature. Acidify with 50 ml of hydrochloric acid (sp. gr. 1.15-1.18), add lO ml of 10% potassium thiocyanate solution and titrate, without cooling, with the standard ferric chloride solution until the colour changes to red. 2. l.4.2 Analysis of sample Weigh accurately 5.00 g of the sample, dissolve it in about 200 ml of pyridine by heating on a water bath, cool to 20°C, transfer to a 250-ml volumetric flask and dilute to the mark with pyridine. Pipette lO ml of the solution into a 500-ml wide-necked conical flask, add 15 ml of pyridine, 50 ml of dimethylformamide and 25 ml of glacial acetic acid and pass a stream of carbon dioxide through the solution. Keep the stream of carbon dioxide flowing throughout the entire titration. When all the air has been displaced by carbon dioxide (5 min), add from a burette 50 ml of O.l N titanous chloride solution and proceed with the titration as described in section 2. 1 .4. I. 2. 1. 5 Calculation 2' ,5-dichloro-4' -nitrosalicylanilide ethanolamine salt content (% w /w) (a - b) x 6.47 x N w where a = volume (ml) of 0.05 N ferric chloride required for 50 ml of 0.1 N titanous chloride 1 50 ml of 0.1 N sodium thiosulfate solution should be used. - 235 - 2',5-DICHLOR0-4'-NITROSALICYLANILIDE ETHANOLAMINE SALT, WATER-DISPERSIBLE POWDER WHO/SMF/1 b = volume (ml) of0.05 N ferric chloride required for back-titration of excess titanous chloride in analysis of sample N = normality of 0.05 N ferric chloride solution (section 2. l. 3) w = weight (g) of sample. 2. 2 Suspensibility 2. 2.1 Special apparatus 1 l. A 250-ml graduated cylinder with ground-in stopper and a distance of 20-21.5 cm between the bottom and the 250-ml graduation. 2. A glass tube, about 40 cm long and about 5 mm in internal diameter, drawn out at one end to an opening of 2-3 mm, the other end being con- nected to a vacuum pump. 2. 2. 2 Special reagent Standard hard water. Dissolve 0.304 g of anhydrous calcium chloride and O .139 g of magnesium chloride hexahydrate in distilled water and make up to one litre. This provides water with a hardness of 342 parts per million, calculated as calcium carbonate. 2.2.3 Procedure Weigh accurately into a 100-ml beaker an amount of the sample, treated as described above, to form 250 ml of a suspension containing 0.14% of 2',5-dichloro-4'-nitrosalicylanilide ethanolamine salt (w/v). Add a volume of water 2 at 30°C ± l °C equal to at least twice the weight of the sample taken. Allow to stand for 30 seconds and then stir by hand for 30 seconds with a glass rod, 4-6 mm in diameter, at not more than four revolutions per second, making no deliberate attempt to break any lumps. Then immediately transfer the mixture quantitatively to the 250-ml graduated cylinder using water 2 at 30°C ± l °C for rinsing, and again avoiding mechanical disintegration of any lumps. Immediately add sufficient water 2 at 30°C ± l °C to bring the volume to the 250-ml mark. Stopper the cylin- der and mix by inverting and righting it 30 times at a rate of one complete cycle every 2 seconds. This operation should be carried out as smoothly as possible, keeping the axis of rotation fixed. The cylinder must be ther- mally insulated from the hands to maintain the prescribed temperature 1 A specially designed and fitted graduated cylinder suitable for use in this procedure is described in FAO Plant Protection Bulletin, 1962, vol. 10, No. 2, Method 10. 2 Use distilled water for testing the powder without pretreatment and standard hard water for testing the powder after accelerated storage. - 236- WHO/SMF/1 2',5-DICHLOR0-4'-NITROSALICYLANILIDE ETHANOLAMINE SALT, WATER-DISPERSIBLE POWDER of the suspension. Allow the graduated cylinder to stand for 30 minutes in a water-bath at 30°C ± l °C, care being taken that the bath is free from vibrations. NOTE : Should excessive flocculation occur during the test, accompanied by the appearance of transparent liquid, the material is unsatisfactory. At the end of the settling period (30 minutes), insert the glass tube into the cylinder and, with a minimum of disturbance, withdraw during 10-15 seconds by means of the vacuum pump nine-tenths of the suspension, i.e., 225 ml. This is achieved by maintaining the tip of the glass tube just below the sinking top level of the suspension. Discard the suspension withdrawn. Transfer the retained one-tenth of the suspension, including the sedi- ment, with 25 ml of pyridine and 50 ml of dimethylformamide into a 500-ml wide-necked conical flask. Heat on the water bath to complete solution (if necessary add more dimethylformamide). Cool to room temperature, add 25 ml of acetic acid and pass a stream of carbon dioxide through the solution. When all the air has been displaced by carbon dioxide (5 min) proceed with the titration as described in section 2. l. 4. 2. 2. 4 Calculation Calculate the weight of 2' ,5-dichloro-4' -nitrosalicylanilide ethanolamine salt in the retained one-tenth of the suspension by the formula given in section 2. l. 5. From the value obtained in the same section for the percentage con- tent of active ingredient, calculate also the weight of 2' ,5-dichloro-4' -nitro- salicylanilide ethanolamine salt in the initial sample taken for the suspensi- bility test. Suspensibility (%) (b - a) x 111.l b where a = weight (g) of 2' ,5-dichloro-4'-nitrosalicylanilide ethanolamine salt in the retained one-tenth of the suspension b = weight (g) of 2' ,5-dichloro-4'-nitrosalicylanilide ethanolamine salt in the initial sample. 2. 3 Accelerated Storage Test Place 20 g of the sample in a 250-ml beaker having an internal diameter of 6-6.5 cm and level off without compacting. Place within the beaker, on top of the powder, a loose-fitting piston or disc so formed and weighted - 237 - 2' ,5-DICHLOR0-4' -NITROSALICYLANILI DE ETHANOLAMINE SALT, WATER-DISPERSIBLE POWDER WHO/SMF/1 as to exert upon the powder an even pressure of 25 gf/cm2• Keep the powder thus under pressure in an oven at 54°C ± 1 °C for 24 hours. Take the sample from the oven, remove the pressure assembly, and allow the powder to come naturally to room temperature in a closed container. After comple- tion of the accelerated storage test, the sample should not be exposed to heat or to bright sunshine. Unnecessary exposure of the sample to high atmospheric humidity must also be avoided. Continue with the suspensibility test as described in section 1 . 2. 3. 2. - 238 - Part IV REPELLENTS

WHO/SRpT/1 TECHNICAL DEET TECHNICAL DEET Tentative Specification WHO/SRpT/1 Approved 25 October 1965 l. SPECIFICATIONS 1.1 Material The material shall comprise essentially N,N-diethyl-m-toluamide and shall be in the form of a clear, nearly odourless liquid. 1. 2 Chemical and Physical Requirements The material sampled from any part of the consignment,1 shall comply with the requirements of section 1. 1 and with the following requirements : Content of meta-isomer of N,N-diethyl- toluamide (section 2.1), % by weight. Acidity (WHO/M/3, page 250), % by weight, calculated as H2S04 • • • • • Water content (WHO/M/7, page 266) .. Specific gravity at 25°Cj25°C (section 2. 2) Refractive index n/50c ( section 2. 3) . Colour (section 2.4), Hazen number Minimum 95.0 0.992 1.520 1. 3 Packing and Marking of Packages Maximum 0.03 0.3 0.999 1.524 100 The technical N,N-diethyl-m-toluamide shall be packed in suitable, clean containers as specified in the order. All packages shall bear, durably and legibly marked on the container, the following : Manufacturer's name Technical N,N-diethyl-m-toluamide to Specification WHO/SRpt/1 Batch or reference number, and date of test Net weight of content~ 1 A sampling procedure is described in Method WHO/M/1, page 247. However, this does not preclude the purchaser from sampling in any way considered desirable. - 241 - 16 TECHNICAL DEET 2. METHODS FOR DETERMINATION OF CHEMICAL AND PHYSICAL PROPERTIES 2.1 N,N-Diethyl-m-toluamide Content 2 .1.1 Summary of method WHO/SRpT/1 The sample is dissolved in carbon disulfide and the difference in absorb- ance at 14.18 µ and at 14.48 µ is determined. The quantity of meta-isomer is obtained from this value by means of a calibration curve prepared by the use of a reference standard. 2.1. 2 Special apparatus l. Double-beam infrared spectrophotometer, Perkins-Elmer model 21 or equivalent. 2. Two equivalent infrared absorption cells, with sodium chloride windows and a path length of approximately 0.4 mm. 2. 1. 3 Preparation of calibration curve Weigh accurately into separate volumetric flasks sufficient amounts of the reference standard 1 to give approximately 20, 40, 60, and 80 mg/ml when dissolved in carbon disulfide. Fill the reference cell with carbon disulfide and the sample cell with each of the standard solutions in turn, and record the spectra. The spectrum may be scanned rapidly, except for the region 12-15 µ, where a normal speed should be used. Carry out a carbon disulfide blank to correct for any inequality in the paired cells and to see if a cell correction is required. Measure the absorbance at 14.18 µ and at 14.48 µ and calculate the difference between these values, AA, for each of the solutions. Plot the values of AA against the concentration (mg/ml) of the meta-isomer. If a cell correction is required, the value of AA is determined from the formula: AA = (A14.1s - A14,4s)rer. - (A14.ts - A14.4s)b1ank where ref. = determination with reference standard and blank = determination on CS2 blank. 1 Reference standard N,N-diethyl-m-toluamide with b.p. lll°C at 1 Torr and refractive index nd25 1.5206 is available, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerland. - 242 - WHO/SRpT/1 TECHNICAL DEET 2.1.4 Procedure Weigh accurately about 0.5 g of sample, transfer quantitatively to a 10-ml volumetric flask and make up to the mark with carbon disulfide. Measure the infrared absorption at 14.18 µ and 14.48 µ using the same conditions as described in section 2 .1. 3. Determine the concentration of meta-isomer by comparing this value with the calibration curve. A standard sample should be run each day to check the calibration of the instrument. 2. 1. 5 Calculation a x 100 N,N-diethyl-m-toluamide content (% w /w) = b where a = concentration (mg/ml) of meta-isomer found from calibration curve b = concentration (mg/ml) of sample taken. 2. 2 Specific Gravity The specific gravity shall be determined by a pycnometer or equivalent method accurate to the third decimal place. 2. 3 Refractive Index The refractive index shall be determined by any method accurate to the third decimal place. 2.4 Colour 2. 4. 1 Preparation of Hazen colour standard solutions Dissolve 1.245 g of potassium chloroplatinate (K2PtC16) and 1 g of crystallized cobaltous chloride (CoC12 • 6H20) in 100 ml of concentrated hydrochloric acid and dilute with distilled water to make one litre. This standard solution has a Hazen colour number of 500. Solutions having Hazen colour numbers of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, and 100 are made by diluting in standard tall- form 500-ml Nessler tubes 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 and 10 ml of the standard solution with distilled water to make 50 ml. 2.4.2 Procedure Place 50 ml of technical N,N-diethyl-m-toluamide in a matched Nessler colour-comparison tube and match with comparison standards (section 2.4.1). Use a suitable colorimeter provided with a white light. - 243 -

Part V METHODS

WHO/M/1 SAMPLING PROCEDURES SAMPLING PROCEDURES Method WHO/M/1 Approved 25 October 1965 1. SAMPLING BY THE PuRCHASER 1.1 Sampling of Solids (other than Water-Dispersible Powders) The purchaser or his agent should collect composite samples to be tested for complete compliance with the requirements of the specification. A composite sample should represent not more than 20 OOO kg of the material and should be prepared by thoroughly mixing 200-g samples taken from not less than 10 individual batches representing not more than 2000 kg each. The product may be sampled with either a slotted tube sampler or a scoop ; the tube sampler is preferable. The 200-g samples should be combined and thoroughly mixed. The mixture should be repeatedly quartered and mixed until a composite sample of 100-150 g is obtained. The quartering may be done on an oil cloth or with a riffle sample divider. The composite sample should be placed in a sealed container with appropriate identification and sent to the laboratory for analysis. 1. 2 Sampling of Water-Dispersible Powders As a general rule, a composite sample reflects the overall quality of the individual samples taken to make up the composite ; however, in the case of water-dispersible powders it has been observed that the suspensibility of a composite sample may be satisfactory even though it includes several samples of low suspensibility. For this reason the following sampling procedure should be used for water-dispersible powders : The purchaser or his agent should collect a 500-g sample of powder from each batch or grind offered by the manufacturer, each sample representing not more than 5000 kg of product. The samples should be taken at random from filled containers in which the product is to be shipped. Samples may be taken with a scoop or a slotted tube sampler, but the latter is preferable. Each batch sample should be thoroughly mixed and 100-150 g transferred to a sealed container with appropriate identification and sent to the labora- tory for analysis. In the case of DDT water-dispersible powders for over- seas shipment, furnished under Specification WHO/SIF /26, an additional portion of 100-150 g should be transferred to a sealed container with appro- priate identification and stored under the conditions specified in section l . 2. 5 of the Specification (page 83) for future testing. - 247- SAMPLING PROCEDURES WHO/M/1 All batch samples should be sieve tested and also tested for suspensibility in hard water after accelerated storage pretreatment, as directed by the appropriate specification. However, the test for active ingredient content, the suspensibility test in distilled water without pretreatment, and the tests for acidity or alkalinity need be performed on only one sample from each 25 OOO-kg lot representing consecutive production. If any sample from a given 25 OOO-kg lot fails to meet any of these requirements, then all samples from that lot should be tested for compliance with the requirement( s) that the initial sample failed to meet. 1. 3 Sampling of Liquids In the case of liquids, samples may be taken from bulk storage or shipping containers. In either case, the purchaser should take a minimum of one sample from each batch. If the size of the batch exceeds 10 OOO litres, at least one sample should be taken from each 10 OOO litres. Samples may be taken by means of a liquid sieve sampler or similar device that permits the operator to collect a sample from any desired level of the tank or drum. One-half litre of each sample should be transferred to a sealed container with appropriate identification and sent to the labora- tory for analysis. 2. SAMPLING BY THE MANUFACTURER The manufacturer should take a production control sample from each batch of material produced for the purchaser. The size of a batch repre- sented by a given sample should not exceed the limits defined in section 1. The manufacturer should conduct whatever tests are necessary on each batch sample to insure that all requirements of the specification are met. In no case should the manufacturer offer to the purchaser as part of a consignment any batch that does not meet the requirements of the specification. The manufacturer should follow the same procedures for collection of batch samples as those given in section 1. All batch samples should be retained until final acceptance of the order by the purchaser. Records of tests made on the manufacturer's batch samples should be maintained for at least one year and should be made available to the purchaser on request. - 248 - WHO/M/2 VISUAL SUSPENSIBILITY VISUAL SUSPENSIBILITY TEST FOR 75% DDT WATER-DISPERSIBLE POWDERS * Method WHO/M/2 Approved 25 October 1965 When the manufacturer or the purchaser, or both, require a rapid suspensibility test for use with 75% DDT water-dispersible powders, the following should be used : Weigh 3.3 g of the powder into a 100-ml beaker. Add 50 ml of water 1 at 30°C, allow to stand for 30 seconds and then stir the mixture with a glass rod by hand for another 30 seconds. Transfer this mixture to a 100-ml glass-stoppered graduated cylinder having the 100-ml mark situated 18.0 cm ± 1.5 cm from the bottom and 5.5 cm± 0.5 cm from the top excluding the neck. Add water 1 at 30°C to bring to the 100-ml mark. Stopper the cylinder and mix by inverting and righting it 30 times at the rate of approxi- mately one cycle every two seconds. This operation should be carried out as smoothly as possible, keeping the axis of rotation fixed. The cylinder must be thermally insulated from the hands to maintain the prescribed temperature of the suspension. Allow to stand for 15 minutes and then observe the volume of sediment in the bottom of the cylinder.2 * This is designed to provide a rapid test of the suspensibility of a powder whatever its history, i.e., newly manufactured, as received by the purchaser, after accelerated storage treatment, after ambient storage, or at the time of use in the field. 1 The test may be conducted in distilled water or standard hard water. 2 If the sediment is more than 4 ml, the sample should be subjected to the normal suspensibility tests. - 249 - ACIDITY AND ALKALINITY WHO/M/3 DETERMINATION OF ACIDITY AND ALKALINITY Method WHO/M/3 Approved 25 October 1965 Determine the acidity or alkalinity by titration using methyl red as the indicator where possible. In those cases where this is impractical because the sample is so highly coloured that the change in colour of the indicator is masked, because the indicator is adsorbed, or because the active ingre- dient is not sufficiently soluble in a l : 3 mixture of acetone and water, the end-point can be determined electrometrically in solution in a 10 : l mixture of acetone and water. l. METHYL RED INDICATOR DETERMINATION 1.1 Procedure 1.1. 1 Technical products Weigh exactly 10 g of the sample and dissolve in 25 ml of acetone, with gentle warming if necessary. Add 75 ml of distilled water and titrate immediately with 0.02 N sodium hydroxide, using methyl red as indicator. Carry out a blank determination on 25 ml of acetone and 75 ml of distilled water with 0.02 N sodium hydroxide. 1. 1. 2 Water-dispersible powders and dusting powders Weigh exactly 10 g of the sample, disperse in 25 ml of acetone, and warm to effect solution of the active ingredient. Add 75 ml of distilled water, filter, and titrate immediately using methyl red as indicator. Depend- ing upon the reaction of the material use either 0.02 N sodium hydroxide or 0.02 N hydrochloric acid. Carry out a blank determination on 25 ml of acetone and 75 ml of distilled water. 1. 1. 3 Emulsion concentrates Weigh exactly 10 g of the sample, dilute with 100 ml of distilled water and titrate immediately using methyl red as indicator. Depending upon the reaction of the material use either 0.02 N sodium hydroxide or 0.02 N hydrochloric acid. Carry out a blank determination on 100 ml of distilled water. - 250 - WHO/M/3 ACIDITY AND ALKALINITY 1. 2 Calculation 1. 2. 1 Acidity Acidity calculated as H2S04 is found as follows : % weight as H2S04 = 0.0098 x (a - b) where a = volume (ml) of 0.02 N sodium hydroxide used for the sample b = volume (ml) of 0.02 N sodium hydroxide used for the blank. NOTE : The blank may take the form of a small titre with 0.02 N hydro- chloric acid, in which case: % weight as H2S04 = 0.0098 x (a+ c) where c = volume (ml) of 0.02 N hydrochloric acid used for the blank. 1. 2. 2 Alkalinity Alkalinity calculated as NaOH is found as follows : % weight as NaOH = 0.008 x (d + e) where d = volume (ml) of 0.02 N hydrochloric acid used for the sample e = volume (ml) of 0.02 N sodium hydroxide used for the blank. NoTE : The blank may take the form of a small titre with 0.02 N hydro- chloric acid, in which case alkalinity as NaOH (%) = 0.008 x (d - f) where f = volume (ml) of 0.02 N hydrochloric acid used for the blank. 2. ELECTROMETRIC DETERMINATION 2. 1 Special Reagent Buffer solution. Mix 100 ml of 2 N acetic acid and 100 ml of l N sodium hydroxide and make up to 1000 ml with demineralized water. 2. 2 Procedure 2. 2 .1 Apparent pH of acetone/buffer-solution mixture Determine the apparent pH at 20°C of a mixture of 50 ml of distilled acetone and 5 ml of buffer solution with the electrodes and pH meter to be used in the titration. - 251 - ACIDITY AND ALKALINITY WHO/M/3 2.2.2 Technical products In the titration vessel of the pH meter dissolve 10 g of the technical product to be tested in 50 ml of distilled acetone.1 Add 5 ml of distilled water and titrate electrometrically with 0.02 N sodium hydroxide to the apparent pH of the acetone/buffer-solution mixture at 20°C. 2. 2. 3 Water-dispersible powders and dusting powders Transfer 10 g of the sample to a 200-ml conical flask. Add 75 ml of distilled acetone and stir for 5 minutes. Filter through a sintered glass crucible into a 250-ml suction flask. Rinse the conical flask and the crucible with four portions of 5 ml of acetone each. Transfer the combined acetone extracts to the titration vessel, rinsing the suction flask with 5 ml of acetone. Add 10 ml of water and titrate electrometrically with 0.02 N sodium hydroxide or with a 0.02 N hydrochloric acid to the apparent pH of the acetone/buffer-solution mixture at 20°c. 2. 2. 4 Emulsion concentrates In the titration vessel of the pH meter dissolve 10 g of the sample in 50 ml of acetone, add 5 ml of distilled water and titrate electrometrically with 0.02 N sodium hydroxide or with 0.02 N hydrochloric acid to the apparent pH of the acetone/buffer-solution mixture at 20°C. 2. 3 Calculation Acidity calculated as % by weight H2S04 is found as follows: = 0.0098 x a where a = volume (ml) of 0.02 N sodium hydroxide used for the titration of the sample to the apparent pH found in section 2. 2. 1. Alkalinity calculated as % by weight NaOH is found as follows : = 0.008 x b where b = volume (ml) of 0.02 N hydrochloric acid used for the titration of the sample to the apparent pH found in section 2. 2. l. 1 Some technical products such as dieldrin require more solvent to prevent pre- cipitation during titration. In such cases 100 ml of distilled acetone and 10 ml of distilled water should be used. - 252 - WHO/M/4 SIEVING TEST SIEVING TEST AFTER ACCELERATED STORAGE TREATMENT Method WHO/M/4 Approved 25 October 1965 Samples for this sieving test should be subjected to the accelerated storage treatment required for the material being tested and described in the appropriate specification. After completion of the accelerated storage treatment, the sample should not have been exposed to heat or to bright sunshine. 1. Water-Dispersible Powders Prepare a 74-µ sieve (BS 200 mesh; 1 US Standard No. 200 2) of about 20 cm diameter by freeing it from any film, grease, or other water-repellent material. Weigh accurately 10 g of the sample,3 after it has been subjected to the appropriate accelerated storage treatment, into a 250-ml beaker and add 100 ml of tap water. Allow to stand for 30 seconds, then stir with a glass rod by hand for 30 seconds at not more than four revolutions per second, making no deliberate attempt to break any lumps. Transfer the slurry immediately to the sieve, rinsing with tap water. Wash the material on the sieve with an oscillating, moderately vigorous spray of tap water, using a rubber hose of 10 mm internal diameter delivering 4 to 5 litres of water per minute. Continue the washing for 10 minutes, directing the water from the circumference of the sieve towards the centre and keeping the end of the hose not more than 5 cm from the surface of the sieve. Transfer the residue to a tared Gooch crucible, dry, and weigh. From this weight and the weight of the original sample taken, calculate the percentage residue on the sieve. 2. Dusting-Powders Weigh accurately 20 g of the sample which has been subjected to the appropriate accelerated storage treatment. Transfer this sample to a clean, dry 150-µ sieve (BS 100 mesh; 1 US Standard No. 100 2) and screen 1 British Standards Institution, British Standard 410: 1943. 2 American Society for Testing Materials, Standard E 11-39. 3 When this test is applied to DDT water-dispersible powders for overseas shipment (WHO/SIF/26) and to malathion water-dispersible powders (WHO/SIF/10.R2), the suspension remaining in the cylinder after the suspensibility test should be used. In these two cases the weight of the sample will be less than 10 g and is equal to the weight taken for the suspensibility test. - 253- SIEVING TEST WHO/M/4 in a Ro-Tap Shaker 1 or equivalent machine for 10 minutes. Stop the machine, break up any soft lumps by brushing lightly over the sieve, restart the machine and screen for an additional 5 minutes. Completion of the screening is made by shaking the sieve by hand for 4 minutes. Weigh the residue remaining on the sieve. Calculate the portion passing through and express it as a percentage of the weight of the sample taken. 1 The Ro-Tap Testing Sieve Shaker supplied by the W. S. Tyler Company, Cleve- land, Ohio, USA, reproduces the circular and tapping motion given testing sieves in hand-sieving, but with a uniform, mechanical action. One model is manufactured with gear to operate either with 60-cycle, 1750-rev/min motors, or with 50-cycle, 1450- rev/min motors, and still have the same standard speed of operation. The unit operates the sieves with a horizontal circular motion of 285 rev/min, together with a tapping action at the rate of approximately 150 taps per minute. The tapping action is very important to ensure comparable results. This is especially true in the sieving of materials such as insecticides. The tapping motion is imparted to the top of the sieves and is transmitted through the entire nest. - 254 - WHO/M/5 MEL TING-POINT AND MIXED MEL TING-POINT DETERMINATION OF MELTING-POINT AND MIXED MELTING-POINT* Method WHO/M/5 Approved 25 October 1965 1. Special apparatus 1. A capillary tube of borosilicate glass closed at one end and having the following dimensions; thickness of the well, about 0.10-0.15 mm; length, suitable for the apparatus used; internal diameter, 0.9-1.1 mm. 2. Accurately standardized thermometers covering the range - l0°C to 360°C, the one-degree graduations being not less that 0.8 mm apart. These thermometers should be of the mercury-in-glass, solid-stem type, with cylindrical bulbs, and made of approved thermometric glass. They may be of the 76-mm or 100-mm partial-immersion type or calibrated for total immersion. If thermometers of the latter type are used they should be surrounded by a glass tube. 3. A glass heating vessel of a suitable construction and capacity fitted with a suitable stirring device, capable of rapidly mixing the liquid. Certain liquid silicones are suitable for use in the heating vessel. 4. A magnifying-glass for observation of the capillary tube. 2. Procedure Spread a small quantity of the finely-powdered substance in a thin layer and dry it in a vacuum desiccator over silica gel or phosphorus pentoxide for 24 hours or according to the requirements given in the specification. Transfer a quantity of the dried powder to a dry capillary tube and pack the powder by tapping the tube on a hard surface so as to form a tightly packed column about 3 mm in height. Introduce the capillary tube into the heated bath at a temperature 5°C below the expected lower limit of the melting-range, the rise of temperature being regulated beforehand to about 1 °C per minute, unless otherwise stated. The capillary tube should be fitted in the bath in such a way that its closed end is at the level of the middle of the bulb of the standard thermometer. * This method is based on the work of Bervenmark, et al. (1963) Bull. Wld Hlth Org., 28, 175-178 and is equivalent to the method given in Specifications for the Quality Control of Pharmaceutical Substances-Second Edition of the International Pharmacopoeia, 1967, World Health Organization, Geneva, Appendix 6 (in press). Any other apparatus or method capable of equal accuracy may be used but it should be checked for accuracy with the melting point reference substances. - 255 - MEL TING-POINT AND MIXED MEL TING-POINT WHO/M/5 Unless otherwise directed, readings are taken of the temperature at which the substance is observed to collapse or form droplets at the wall of the tube and of the temperature at which it is completely melted as indicated by the disappearance of the solid phase. To the temperature readings add the correction for deviation of the standard thermometer, and the emergent-stem correction, which is obtained as follows: Before starting the determination of the melting-range, the auxiliary thermometer is attached so that the bulb touches the standard thermometer at a point midway between the graduation for the expected melting-tempera- ture and the surface of the heating material. When the substance has melted the temperature is read on the auxiliary thermometer. The correc- tion to be added to the temperature reading of the standard thermometer is calculated from the following formula : N x 0.00015 x (T - t) where T is the temperature reading of the standard thermometer ; t is the temperature reading of the auxiliary thermometer ; N is the number of degrees of the scale of the standard thermometer between the surface of the heating material and the level of the mercury. The accuracy of the apparatus or method used should be checked by means of the international melting-point reference substances.1 The set of international melting point reference substances includes the following materials: Reference substance Azobenzene Vanillin .. Benzil ... Acetanilide . Phenacetin . Benzanilide . Sulfanilimide Acetaminosalol Sulfapyridine Dicyandiamide Saccharin .. Caffeine ... Phenolphthalein . Melting temperature 69°C 83°C 96°C ll6°C 136°C 165°C l66°C 192°C l93°C 210°C 229°C 237°C 263°C 1 The substances are available in a polystrene plastic package together with a leaflet on " Directions for use". Orders for the substances should be sent to : WHO Interna- tional Reference Centre for Chemical Reference Substances, Apotekens Centrallabora- torium, Box 333, Solna 3, Sweden. - 256- WHO/M/5 MEL TING-POINT AND MIXED MEL TING-POINT 3. Definitions The melting-range of a substance is the range between the corrected temperature at which the substance begins to collapse or form droplets at the wall of a capillary tube and the corrected temperature at which it is completely melted as shown by the disappearance of the solid phase. The melting-temperature of a substance is the corrected temperature at which it is completely melted as shown by the disappearance of the solid phase. - 257 - 17 GAMMA-ISOMER CONTENT: INFRA-RED METHOD INFRA-RED SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF GAMMA-ISOMER CONTENT OF HCH WHO/M/6 Method WHO/M/6 Approved 25 October 1965 1. Special apparatus 1.1 Chromatographic column and attachments (see Fig. 6) The column consists of a hard glass tube 80-90 cm long ; the upper portion, over a length of about 10 cm has an internal diameter of 35-45 mm, whereas the remainder of the column has an internal diameter of 17-20 mm. The lower end is fitted with a sintered glass plate of porosity No. 2 and is connected to a siphon of 10-ml capacity by means of a ground-glass joint. The upper end of the column is fitted with a ground-glass spherical joint for connexion to the solvent reservoir. This reservoir consists of a cylinder of about 700-ml capacity, fitted at the lower end with a tap, a re-entrant joint and ground-glass spherical joint. At the top, the reservoir is connected to a filling funnel by a further tap. There are two side-arms attached to the top of the reservoir, both fitted with. taps ; one leads to the atmosphere, the other is connected by a T-joint to the pressure system and by a further tap to the re-entrant joint at the bottom of the reservoir. All taps in the system are spring loaded so that the apparatus can be operated under increased pressure. Since the operating pressure of the column is approximately 0.6-0.8 kgf/cm2 above atmospheric-Le., an absolute pressure of about 1.6-1.8 kgf/cm2-as a safety precaution, the whole apparatus is surrounded by a wire-gauze jacket or operated behind a safety screen. The reservoir is connected to a cylinder of compressed nitrogen fitted with the appropriate needle valves, gauges, safety valve set at 2.5 kgf/cm2 (absolute pressure), and release valve. 1 . 2 Flasks and container 50-ml round-bottomed flasks with a mouth about 25 mm in diameter. A wooden case with 42 compartments (arranged in 6 rows of 7) makes a convenient container for the flasks. 1 . 3 Device for removal of solvent A Y-type vacuum-pipe, equipped on both sides with 4 stop-cocks to which the flasks, provided with loose-fitting cork stoppers, are connected - 258- WHO/M/6 GAMMA-ISOMER CONTENT: INFRA-RED METHOD FIG. 6. CHROMATOGRAPHIC COLUMN FOR SEPARATING ISOMERS OF HCH c E u .,, "' 35· 45 mm 5-6 cm E u 0 °' 0 co F G H / / - 259 - GAMMA-ISOMER CONTENT: INFRA-RED METHOD WHO/M/6 for evaporation of the solvent from 8 flasks simultaneously. Ground-in connexions have proved to be less satisfactory. The loose-fitting cork stoppers permit an air stream to pass through the flasks, thus ensuring constant distillation. It has been found that if a water pump is used the pressure does not fall below 20 Torr with a bath temperature of 60°C ; under these conditions there is no loss of the gamma-isomer. 1 . 4 Infra-red spectrophotometer Any of the commercially available instruments equipped with a sodium- chloride or potassium-bromide prism may be used. 2. Special reagents 2. l . Silica-gel The silica-gel should be of chromatographic grade. Since the success of this method depends mainly on the absorptive characteristics of the silica-gel, its suitability should be checked as follows : Run a test with the silica-gel in the apparatus described in section 1, according to the method described in section 3, using a mixture of 50 mg of pure gamma- isomer 1 and 200 mg of pure alpha-isomer .1 The isomers should be clearly separated, with one or two flasks containing pure solvent between the two fractions. The weight of the isomers should lie within the range 96-104% of the above-mentioned quantities. 2.2 Mobile solvent The mobile solvent should consist of a freshly distilled, colourless light petroleum, miscible in any proportion with ethanol and ether, insoluble in distilled water, and complying with the following additional require- ments: 1. Specific gravity. The specific gravity should lie within the range 0.620- 0.640 at 20°c. 2. Distillation. The material should distil as follows: No fraction should distil below 40°C A maximum of 20% should distil up to 45°C A maximum of 80% should distil up to 55°C A minimum of 95% should distil up to 60°C. 1 Samples of alpha- and gamma-isomers of hexachlorocyclohexane are obtainable, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzer- land. - 260 - WHO/M/6 GAMMA-ISOMER CONTENT: INFRA-RED METHOD 3. Neutrality. Shake 10 ml of the light petroleum with 10 ml of distilled water. Separate the aqueous layer and add 1 drop of phenolphthalein indicator. The solution should remain colourless, but change to red on addition of 1 drop of 0.02 N sodium hydroxide. 4. Residue. Pour 50 ml of the light petroleum into a clean porcelain dish and evaporate on a water-bath. Dry the residue at 105°C for 4 hours and weigh. The residue should not exceed 2.5 mg. 5. Sulfur compounds. Mix 5 ml of the light petroleum and 10 ml of ethan- olic ammonia silver solution. Keep the mixture, protected from light, at 50°C for 5 minutes. No change of colour to brown should occur. 6. Aromatic hydrocarbons. Shake 5 ml of the light petroleum with 12 ml of a colourless mixture of 4 parts of concentrated nitric acid and 1 part of concentrated sulfuric acid for 5 minutes. Dilute with distilled water and cool to 0°C. No crystals should be deposited and no odour ofnitro- benzene produced. If the light petroleum is not satisfactory in this respect, it must be purified by running it through an activated-charcoal column according to the following procedure : Fill a chromatographic column having an internal diameter of about 25 mm, to a height of about 100 cm, with a granular activated charcoal ground to pass through a 300-µ sieve (BS 52 mesh; 1 US Standard No. 50 2). Allow the light petroleum to percolate through the column at a rate of about 500 ml per hour until 2 litres of percolate have been collected. Pass this partially purified light petroleum through a similar column charged with fresh charcoal and collect the purified light petroleum. (The second column may be used for the first treatment of a further 2 litres of light petroleum.) This treatment will satisfactorily remove the aromatic hydrocarbons from the light petroleum usually available. If grades with a high content of aromatic compounds are encountered, it may be necessary either to reduce the volume of light petroleum treated, or to pass it through a third column of activated charcoal. Pure gamma-isomer. Recrystallize a sample of commercially available gamma-isomer of benzene hexachloride successively from solutions in : (a) its own weight of acetone, (b) ten times its weight of the mobile solvent, and (c) four times its weight of iso-octane. Dry at 60°C for at least4 hours. Check the purity of the material thus obtained by determining its melting- point and mixed melting-point with standard gamma-isomer, using the 1 British Standards Institution, British Standard 410: 1943. 2 American Society for Testing Materials, Standard E ll-39. - 261 - GAMMA-ISOMER CONTENT: INFRA-RED METHOD WHO/M/6 method described in Method WHO/M/5, page 255. The melting-point should be at least 112°C, and the mixed melting-point should not be lower than the melting-point of the standard material. 3. Procedure 3. 1 Preparation of column Weigh 38-42 g of the silica-gel, previously dried at 110°C, transfer to a mortar, and add 17% of distilled water (calculated on the weight of silica- gel) by means of a burette. Mix thoroughly, without grinding the silica-gel, add 150 ml of the mobile solvent while stirring, and transfer the suspension thus obtained to the column. Eliminate any air bubbles from the column by tapping. Connect the column to the pressure line and apply a pressure of 0.6-0.8 kgf/cm2 above atmospheric, in order to compress the silica-gel suspension to a height of 35-45 cm, care being taken to ensure that there is always mobile solvent above the silica-gel. 3 . 2 Chromatographic separation Pour the solution of the sample into the column and add mobile solvent to a height of 16-19 cm above the silica-gel. Adjust the flow of the solvent in such a way that this height is maintained during the whole operation. Adjust the pressure to 0.6-0.8 kgf/cm2 above atmospheric, to ensure that the solution passes through the column at a rate of about 5 ml per minute. The various fractions appear at the bottom of the column in the follow- ing sequence : (1) heptachlorocyclohexane, (2) alpha-isomer, (3) gamma- + epsilon-isomers, (4) beta-isomer, (5) delta-isomer. The alpha-isomer starts to appear after approximately 150 ml of the eluate have passed the overflow. The gamma- and epsilon-isomers may be expected approxi- mately between the 250th and the 450th millilitre of the eluate. However, this has to be checked by a test, owing to possible variations due to small differences in the apparatus and reagents. Drain the eluate in successive 10-ml portions into the 50-ml round- bottomed flasks and distil off the solvent from the flasks. Avoid vigorous ebullition of the solutions, particularly in the final stages, by releasing the vacuum when necessary, otherwise the characteristic, crystalline form of the isomers may be impaired. Identify the gamma- + epsilon-isomer fraction by the shape of the crystals in the residue (see Fig. 7). Dissolve the residues in the mobile solvent, or in analytical grade acetone, rinse the flasks with the same solvent, and combine the solutions and rinsings in one flask. Distil off the solvent on a water-bath maintained at 60°C. Norn: If there is no clear separation of the fractions-i.e., if a flask of eluate consisting of pure solvent is not obtained between the fractions- the last flask containing alpha-isomer and the first flask containing beta- - 262 - WHO/M/6 GAMMA-ISOMER CONTENT: INFRA-RED METHOD FIG. 7. CRYSTALLINE APPEARANCE OF HCH RESIDUES AFTER CHROMATOGRAPHIC SEPARATION alpha-isomer fraction beta-isomer fraction gamma ( + epsi lon)-isomer fraction delta-isomer fraction Magnification (all four photographs) x 30 - 263 - GAMMA-ISOMER CONTENT: INFRA-RED METHOD WHO/M/6 isomer must be added to the gamma-+ epsilon-isomer fraction.1 This procedure does not involve an error, since the correct gamma-isomer content in the fraction is determined subsequently by infra-red spectro- photometry. 3. 3 Spectrophotometric determination of gamma-isomer Dissolve the residues of the gamma- + epsilon-isomer fraction in a small amount of carbon disulfide and transfer to a graduated flask. Rinse the collecting flasks twice with carbon disulfide, transfer the rinsings to the graduated flask, and make up to the mark with the same solvent. The capacity of the flask should be such that the final solution contains 60-80 mg of gamma-isomer per lO ml. Prepare a standard solution containing 70-80 mg of pure gamma- isomer per 10 ml of carbon disulfide. Fill one of the spectrophotometer cells with the sample solution and bring it into the sample beam. Fill the second cell with carbon disulfide and bring it into the reference beam. Determine the spectrum of absorption at a wave-length of 13.6-15.0 µ. Subsequently, fill the first cell with the standard solution and determine the spectrum of absorption in the same range. 3.4 Calculation For each of the recorded curves, calculate the absorbance at 13.80 µ as the basis point, and at 14.56 µ as the absorption maximum. Correct the absorbance at 14.56 µ as follows : corrected absorbance at 14.56 µ recorded absorbance at 14.56 µ recorded absorbance at 13.80 µ. From the corrected absorbances, calculate the gamma-isomer content from the following formula : . CxExvxlO Gamma-isomer content(% w/w) = ---8---- Et x w 1 In some instances, it may be impossible to obtain good separation between the alpha-isomer and gamma- + epsilon-isomer fractions, or between the gamma- + epsilon- isomer and beta-isomer fractions, owing to the wetting, emulsifying, or other auxiliary agents used in the manufacture of the product. In these circumstances, these agents should be removed by passage of the sample through a column containing aluminium oxide, as described in the relevant specifications. - 264 - WHO/M/6 GAMMA-ISOMER CONTENT: INFRA-RED METHOD where C = concentration (mg per 10 ml) of standard solution E. = corrected absorbance of the sample solution at 14.56 µ Et = corrected absorbance of the standard solution at 14.56 µ v volume (ml) of sample solution w weight (mg) of initial sample taken for chromatographic separation. - 265 - WATER CONTENT: KARL FISCHER METHOD WHO/M/7 KARL FISCHER ELECTROMETRIC TITRATION METHOD FOR DETERMINATION OF WATER CONTENT* 1. Special apparatus Method WHO/M/7 Approved 25 October 1965 The apparatus required, a suitable arrangement of which is shown in Fig. 8, comprises the following items : 1. Reaction vessel, working capacity not less than 60 ml. The wide neck terminates in a suitable air-tight detachable junction, fitted to accom- modate a burette jet ( extended to suitable length) and tubes for entry and exit of nitrogen, the inlet tube extending nearly to the bottom of the vessel. The nitrogen supply is passed through a drying-train consisting of a guard- tube, filled with freshly activated silica-gel or other efficient desiccant, and a flask containing Fischer reagent. Two platinum-wire electrodes extending nearly to the bottom of the reaction vessel are either fused in through the wall of the vessel or inserted through the seal if preferred. A side arm, closed by a stopper or a vaccine cap, provides for insertion of the sample. 2. Burette with pressure-filling device and automatic zero, 25-ml capacity, fitted with a guard-tube. The reagent reservoir forming part of the pressure-filling device is protected by a trap and both the trap and the guard-tube are filled with freshly activated silica-gel or other efficient desiccant. 3. Electrical circuit incorporating a dry cell, or other source of l.5-volt direct current, a potential divider to provide a working potential of about 300 millivolts, a galvanometer with a sensivity such that full-scale deflection is obtained when a current of not more than 100 microamperes is passed, and a switch. The galvanometer may be in series with the reaction vessel or in a shunt position. In the former case the galvanometer will give a " null" reading at the end-point, and in the latter case a positive reading. 4. One-mark bulb pipettes, 5-ml, 10-ml, and 20-ml capacity, with a cotton-wool plug in the upper part, above the calibration mark. * Reproduced-with slight modifications-by kind permission of the British Stand- ards Institution from British Standard 2511 : 1954 (Determination of water by the Karl Fischer method). - 266- WHO/M/7 WATER CONTENT: KARL FISCHER METHOD FIG. 8. APPARATUS FOR DETERMINATION OF WATER CONTENT A H A = 25-ml class B burette G = trap filled with freshly activated silica-gel B = reagent reservoir or other desiccant C = reaction vessel H = rubber blowing-ball D = guard-tube filled with freshly activated I = tap silica-gel or other desiccant J = galvanometer, 0-100 microamperes E = nitrogen supply K = dry cell, 1.5-volt direct current F = flask containing Fischer reagent L = switch M = 7000-ohm resistance ~ The actual resistances will depend to some extent N = 2000-ohm resistance / on the size and distance apart of the electrodes. 2. Special reagents Methanol, anhydrous. If not already anhydrous, dry by distillation over magnesium turnings and a little iodine, or by fractionation through a 1.5-m rectifying column, and distil into a receiver protected from atmos- pheric moisture by a guard-tube. Methanol dried in this manner will contain not more than 0.03% of water. Standard solution of water in methanol. Dry thoroughly in an oven a 100-ml graduated flask and allow to cool in a desiccator. Partially fill the flask with the anhydrous methanol, add about 0.5 g of distilled water, accurately weighed, from a weighing pipette, make up to the mark with the methanol, and mix thoroughly by shaking. This solution should be pre- pared immediately before use. Pyridine. Distil the pyridine, collecting the fraction boiling between 1 l4°C and 1 l6°C at 760 Torr, the receiver being protected from atmospheric moisture by a guard-tube. - 267 - WATER CONTENT: KARL FISCHER METHOD WHO/M/7 Nitrogen, dry, supplied from a cylinder under pressure. Fischer reagent. This reagent should have a water equivalent of between 3.0 mg/ml and 4.0 mg/ml and may be prepared as follows: Measure 800 ml of the anhydrous methanol into a dry 1500-ml glass- stoppered flask and add 160 g of pyridine. Exchange the glass stopper for a two-hole rubber stopper fitted with an inlet tube reaching nearly to the bottom of the flask and a short outlet tube protected by a guard-tube. Weigh the flask with its fittings and contents and cool in ice-water. Connect the inlet tube to a glass siphon containing sulfur dioxide, refrigeration grade, and allow the gas to pass into the mixture, with continuous agitation, until the total weight of the flask and its contents is increased by about 40 g. Then remove the rubber stopper and delivery tubes and add 90 g of iodine, resublimed. Replace the glass stopper, shake to dissolve the iodine, and allow the mixture to stand for 24 hours. Finally, transfer to the reagent reservoir. The water equivalent of the Fischer reagent thus prepared will be about 3.5 mg/ml. 3. Procedure 3 . 1 Standardization of the Fischer reagent Transfer to the reaction vessel, through the side arm by means of a pipette, 10 ml of the anhydrous methanol. Turn on a slow stream of nitrogen, sufficient to produce adequate agitation, close the electrical circuit, and titrate with the Fischer reagent until the galvanometer records a large deflection and remains above the half-scale reading for at least 30 seconds. Ignore the volumes or the reagents used up to this stage. Run out most of the reaction mixture, leaving just enough to cover the drain tap. (This operation is intended to remove any moisture in the reaction vessel.) Transfer to the reaction vessel a further 10 ml of the methanol and titrate as above with the Fischer reagent, noting the amount used. Then add to the reaction vessel 10 ml of the standard solution of water in methanol and again titrate with the Fischer reagent. 100 x w Water equivalent of the Fischer reagent (mg/ml) = --- f2 - f1 where w weight (g) of distilled water used in preparing 100 ml of standard solution of water in methanol volume (ml) of Fischer reagent used for titration of the methanol alone volume (ml) of Fischer reagent used for titration of the standard solution of water in methanol. - 268 - WHO/M/7 WATER CONTENT: KARL FISCHER METHOD 3. 2 Analysis of sample 3.2.1 Liquid samples. Add to the reaction vessel 10 ml of the anhydrous methanol.1 Turn on a slow stream of nitrogen, sufficient to produce adequate agitation, switch on the current, and titrate with the Fischer reagent as described in section 3. 1. Ignore this titration. Immediately add an amount of the sample containing between 0.06 and 0.08 g of water (sufficient to give a titration of about 20 ml of the reagent) and titrate with the Fischer reagent. 3. 2. 2 Solid samples. Prepare a solution of the sample in the anhydrous methanol,1 taking all possible precautions to avoid contamination with atmospheric moisture. By means of a pipette, transfer an aliquot of this solution containing between 0.06 and 0.08 g of water (sufficient to give a titration of about 20 ml of the reagent) through the side arm into the dry reaction vessel. Titrate this solution with the Fischer reagent as described in section 3. 1. Carry out a blank titration on a volume of the anhydrous methanol equal to that present in the aliquot of the sample solution. 4. Calculation 4. 1 Liquid samples where F s a v Fxa Water content (% w/w) = v x S x 10 water equivalent of the Fischer reagent (mg/ml) (section 3. 1) specific gravity of the sample at room temperature 2 volume (ml) of Fischer reagent used volume (ml) of sample. 4.2 Solid samples F x (b1 - b2) Water content (% by weight)= w x 10 where F water equivalent of the Fischer reagent (mg/ml) (section 3 .1) b1 volume (ml) of Fischer reagent used for titration of the aliquot b2 = volume (ml) of Fischer reagent used for the blank titration w = weight (g) of sample in the aliquot. 1 When the sample is not soluble in methanol, another suitable solvent should be used, e.g., pyridine, dioxan, or a mixture of benzene and methanol. 2 For the purpose of this calculation, the specific gravity of the sample at 15.6°C 2o·c, or 25°C may be used. - 269 - WATER CONTENT: DEAN & ST ARK METHOD DEAN & STARK DISTILLATION MEIBOD FOR DETERMINATION OF WATER CONTENT WHO/M/8 Method WHO/M/8 Approved 25 October 1965 1. Special apparatus 1 The Dean & Stark distillation apparatus consists of the following components : 1. A 500-ml hard glass distillation flask provided with a ground-glass socket. 2. A water-cooled glass reflux condenser, the outside diameter of the inner tube being 16-17 mm, and that of the jacket 23-25 mm. The lower end of the condenser is provided with a ground-glass cone. The tip of the condenser is ground off at an angle of approximately 30° from the vertical axis. 3. A receiver (or trap) made of hard glass and consisting essentially of an upper chamber, provided with a side-arm leading to the distillation flask, and a cylindrical graduated portion, the lower end of which is sealed. The opening of the upper chamber is provided with a ground-glass socket fitting the cone of the condenser. The lower end of the side-arm is provided with a ground-glass cone fitting the socket of the distillation flask. The graduated portion has a capacity of 2 ml when filled to the highest gradua- tion mark. The scale covers the range 0-2 ml, with graduation marks at 0.05-ml intervals. The error at any indicated capacity should not exceed 0.02 ml. 2. Entraining solvent One of the following solvents may be used : (a) petroleum naphtha, with a boiling-range of 90-210°C ; (b) n-heptane, boiling-point around 98°C; or (c) toluene, boiling-point around l l0°C. Shake the solvent with a small quantity of distilled water and distil off; use the distillate. 3. Procedure Clean the entire apparatus with chromic acid solution to minimize the adherence of water droplets to the sides of the condenser and receiver. Rinse thoroughly with distilled water and dry completely before using. 1 Adapted from British Standard 756: 1952 (Dean and Stark apparatus). - 270- WHO/M/8 WATER CONTENT: DEAN & ST ARK METHOD Place an appropriate amount of the sample,1 accurately weighed, into the distillation flask. Add sufficient of the entraining solvent to cover the sample (100-200 ml),2 swirl to mix, and add an anti-bumping agent to ensure steady boiling. Assemble the apparatus and fill the receiver with the entraining solvent by pouring the solvent through the condenser until it begins to overflow into the distillation flask. Insert a loose cotton plug in the top of the condenser to prevent condensation of atmospheric moisture within the tube. In order that the refluxing may be under control, wrap the distillation flask and the tube leading to the receiver with asbestos cloth. Heat the flask at such a rate that about 100 drops are dis- tilled per minute. When the greater part of the water has been distilled over, increase the distillation rate to about 200 drops per minute and continue heating until no more water is collected. Purge the condenser occasionally during the distillation with 5-ml portions of the entraining solvent, in order to wash down any moisture adhering to the walls of the condenser. The water in the trap may be made to separate from the solvent by means of a copper wire, one end of which is twisted into a helix. The copper wire is moved up and down in the condenser and receiver occasionally, thus causing the water to settle at the bottom of the trap. Reflux until the water level in the receiver remains unchanged for 30 minutes, and then shut off the source of heat. Flush the condenser with the entraining sol- vent, making use of the copper wire to discharge any moisture droplets. Allow to stand for about 15 minutes and then read the volume of water. 4. Calculation 100 xv Water content(% w/w) = --- w where v = volume (ml) of water collected w = weight (g) of sample. NOTE : The result of a second determination of water content of the sample should not vary by more than ± 0.05% from that of the first deter- mination. 1 The amount of sample taken should be such that the distillation flask is not more than one-third full ; it should not contain more than I.8 ml of water. 2 The sample should be completely covered by the entraining solvent while the distillation is in progress. - 271 - MATERIAL INSOLUBLE IN DICHLORODIFLUOROMETHANE DETERMINATION OF MATERIAL INSOLUBLE IN DICHLORODIFLUOROMETHANE WHO/M/9 Method WHO/M/9 Approved 25 October 1965 1. Special apparatus 1 The apparatus (Fig. 9) is constructed from a strong glass bottle with a special pressure-cap and needle-valve. The pressure-cap (C), which is held tightly on to the bottle by an adjustable frame provided with a tightening- screw (E), is constructed from 32-mm round brass stock by machining on a turning-lathe to the same shape as the glass stopper furnished with the bottle. The lower end of the cap is first drilled to a depth of 32 mm with a 5-mm drill, and then at right angles on the side for the outlet to the valve. The lower end of the cap is then drilled to a depth of 5 mm with an 8-mm drill. This opening is threaded on the lathe for a distance of 10 mm at 36 threads per inch, to fit the filter-screen unit (B), of the type commonly used on oil-burner tips. The side outlet is drilled to a depth of 9.5 mm with an 8-mm drill and threaded with a 3-mm pipe-tap. A depression is made in the centre of the top of the cap, to hold a 9.5-mm ball-bearing (D). The frame (A) for the bottle is constructed from two 9.5-mm steel rods threaded on both ends. The upper end of each rod is threaded for a distance of 5 cm, to permit adjustment of the cross-bar for irregularly shaped bottles. The cross-bar is made from 13-mm x 32-mm brass stock and drilled with two 9.5-mm holes, 5 cm apart. The pressure-screw hole is then made with an 8-mm drill and threaded with a 9.5-mm tap. The end of the pressure-screw is recessed with a 9.5-mm drill, to hold the ball- bearing. The base for the frame (J) is made from 100-mm round brass stock. After a 13-mm section has been cut from the brass stock, a depres- sion 5 mm deep and 70 mm in diameter is made in the centre, to hold the bottom of the bottle in place. A 70-mm disc is cut from a 3-mm rubber stock, to serve as a cushion between the bottom of the bottle and the brass base. Two 8-mm holes are then drilled diametrically opposite in the base and threaded with a 9.5-mm tap. The steel rods are heated to a cherry red and bent in the shape shown in the figure. The correct adjustment of 1 Fulton, R. A. (1949) An improved apparatus for observing aerosol solutions and determining Freon-insoluble matter in pyrethrum extract (US Department of Agriculture, Bureau of Entomology and Plant Quarantine, Entomological Technique Series, No. ET-273). - 272 - WHO/M/9 MATERIAL INSOLUBLE IN DICHLORODIFLUOROMETHANE the height of the cross-bar is then made by placing the bottle in the frame and turning the lock-nuts to obtain sufficient clearance for removing the bottle. The ball-bearing is used to obtain uniform pressure on the neoprene washer in the top of the bottle. The safety shield (F) is made by 18 FIG. 9. APPARATUS FOR DETERMINATION OF MATERIAL INSOLUBLE IN DICHLORODIFLUOROMETHANE . • . • I ' II A ' ( I I ' I ' I ~- ---··-·: - . = .·:::. I = VERTICAL SECTION OF PRESSURE-BOTTLE AND FRAME II = DETAIL OF NEEDLE-VALVE ASSEMBLY A = bottle frame B = filter-screen unit C = pressure-cap D = ball-bearing J = frame base E = tightening-screw F = safety shield G = needle-valve H = notch Reproduced by kind permission of the Bureau of Entomology and Plant Quarantine, Agricultural Research Administration, US Department of Agri- culture. - 273 - MATERIAL INSOLUBLE IN DICHLORODIFLUOROMETHANE WHO/M/9 cutting a 180-mm length from 95-mm transparent plastic tubing. By cutting a notch (H) in the tubing, to a depth of 38 mm, it is possible to adjust the needle-valve (G) and still have the shield extend above the level of the bottle for maximum protection. When the apparatus is being used, the filter-screen unit (B), filled with lamb's wool, is attached to the pressure-cap (C) as shown. Before use, wash the pressure-cap and the filter-screen unit with acetone and chloro- form. Clean the bottle with a l : 9 mixture of ethanol and sulfuric acid, and later rinse several times with distilled water. Dry all parts in an oven at l05°C for l hour and then cool in a desiccator. Suitable bottles are those used for carbonated beverages and those used by the pharmaceutical trade in some countries for citrate of magnesia. Since any glass under continuous strain will fatigue, it is suggested that, as a precautionary measure, bottles allowed to stand for any period be kept behind a safety-glass screen. 2. Procedure Weigh accurately about 6 g of the sample, by difference from a weigh- ing-bottle, into the pressure-bottle of the apparatus. Place the bottle inside the frame of the pressure-holding unit. Assemble the filter and the pressure- cap and place in the top of the bottle. Place a 9.5-mm ball-bearing between the top of the pressure-cap and the retaining screw, and tighten the screw. Place a transparent safety shield over the unit. Evacuate the bottle to a vacuum of at least 635 Torr. Connect by means of a refrigeration hose to a source of dichlorodifluoromethane (refrigeration grade) and weigh 294 g into the bottle.1 Place the assembled unit in a rack so as to allow the bottle to rest at an angle of 30°. At 10-minute intervals over a period of 2 hours, rotate the unit in the rack through approximately 45°. Allow the unit to remain in the rack overnight. Remove the dichlorodifluoromethane by holding the valve down and releasing the liquid slowly. When all the liquid has been discharged, add 150 g of dichlorodifluoromethane to the bottle, rinse the contents by shaking, and release again. Remove the filter from the pressure-cap and place the filter in a 10-ml beaker. Add 5-7 ml of chloroform and allow to stand. Add 15 ml of chloroform to the bottle and rotate while holding it in a horizontal position. Transfer the contents into a weighed 125-ml conical flask. Wash the bottle four times with 15-ml portions of chloroform and add each portion 1 To achieve this, after opening the tank valve to fill the hose with the liquefied gas. weigh the bottle and place on the balance pan additional weights equivalent to 294 g ; then open the needle-valve on the bottle and allow the gas to flow from the reservoir until the necessary amount has been added. - 274 - WHO/M/9 MATERIAL INSOLUBLE IN DICHLORODIFLUOROMETHANE to the weighed flask. Transfer the chloroform from the 10-ml beaker to the flask and wash the beaker and filter twice with 5-ml portions of chloroform. Remove the chloroform from the flask by heating slowly on a steam- bath. Place the flask in an oven at l05°C for l hour ; then transfer to a desiccator and allow to cool for 2 hours. Weigh the flask and express the amount of insoluble material as a percentage of the weight of sample taken. -275- 18• FLASH-POINT: TAG CLOSED TESTER METHOD WHO/M/10 TAG CLOSED TESTER METHOD FOR DETERMINATION OF FLASH-POINT 1. Special apparatus Method WHO /M/10 Approved 25 October 1965 TAG Closed Tester (see Fig. 10). The apparatus should be surrounded on three sides with an enclosure to keep away draughts. Tests made in a laboratory hood or near ventilators will give unreliable results. 2. Procedure 2. 1 Preparation of apparatus The test should be made in a dim light so as to see the flash plainly. FIG. 10. TAG CLOSED TESTER Reproduced by kind permission of lhe Fisher Scientifi c Company . Set the tester firm and level , put the water-bath thermometer in place, and fill the water-bath with water at such a temperature that, when testing is started, the tempera- ture of the water-bath will be at least 11 °C below the probable flash- point. Put the testing cup in place in the water-bath and measure into it 50 ml of the sample. Remove any air bubbles from the surface of the liquid , using a piece of clean, dry paper. The temperature of the sample must be at least 11 °C below its probable flash-point when test- ing is started. If gas is available, put on the cup cover with the flash-point thermo- meter in place and the gas hose at- tached , light the test flame on the cover, and adjust it to the size of the small white bead on the cover by means of the regulating-valve on the gas-hose connexion. If no gas is available , insert a wick of cotton cord through the - 276 - WHO/M/10 FLASH-POINT: TAG CLOSED TESTER METHOD burner tip. Put a small quantity of cotton waste in the oil chamber and fill with signal, sperm, or lard oil after closing the valve. Replace the filling-cap on the oil chamber, but do not screw in tightly, as a small air- hole should be left when oil is used. 2. 2 Performance of test Adjust the rate of heating so that the temperature of the sample rises at the rate of l °C ± O.l °C per minute. Record the barometric pressure, which, in the absence of a laboratory instrument, may be obtained from the nearest meteorological station. Record the temperature of the sample at the start. When the temperature of the sample reaches about 5°C below the probable flash-point, turn the knob so as to introduce the test flame into the cup and turn it promptly back again. Do not let it snap back. The time consumed in turning the knob down and back should be about one second. On making the first introduction of the test flame, record the time as well as the temperature of the sample. Repeat the introduction of the test flame at every 0.6°C rise in tem- perature of the sample, until there is a flash within the cup. Do not mistake for the flash an enlargement of the test flame or halo around it when it is introduced into the cup, or a slight flickering of the flame ; the true flash consumes the gas in the top of the cup and causes a very slight explosion. Record the flash-point and the time at which it is reached. If the rise in temperature of the sample, from the first introduction of the test flame to the time at which the flash-point is reached, was faster than· l.l°C or slower than 0.9°C per minute, the test should be repeated and the rate of heating adjusted. Do not turn off the test flame with the small regulating-valve. Leave it adjusted to give the proper size of flame. Turn off the heat, lift up the cup cover and wipe the thermometer bulb. Lift out the testing cup, empty it, and carefully wipe it. Throw away all samples when once used in making the test. Pour cold water into the water-bath, allowing it to overflow into the receptacle, until the temperature of the water in the bath is lowered to 8°C below the flash-point obtained in the preliminary test. Place the testing cup back in the water-bath and measure into it a fresh 50-ml portion of the sample. Remove any air bubbles from the surface of the liquid, put on the cover with its thermometer, record the tempera- ture of the sample and water, and proceed to repeat the test as described above. Introduce the test flame for the first time at a temperature of 5.5°C below the flash-point obtained in the preliminary test. - 277 - FLASH-POINT: TAG CLOSED TESTER METHOD WHO/M/10 3. Interpretation of results If two (or more) determinations agree within 0.6°C, then the average of these results, corrected if necessary for barometric pressure, shall be consi- dered the flash-point. If two determinations do not check within 0.6°C, a third determination should be made and, if the maximum variation of the three tests is not greater than 1.1 °C, then their average, corrected if necessary for baro- metric pressure, shall be considered the flash-point. A correction for barometric pressure should be made only in case of dispute or when the barometric reading deviates by more than 13 Torr from the standard pressure of 760 Torr ; the flash-point figure should then be corrected according to the following table. FLASH·POINT CORRECTION FOR BAROMETRIC PRESSURE Barometer reading Correction Barometer reading Correction (Torr) (•C) (Torr) (•C) 700 + 2.2 745 + 0.5 705 + 2.0 750 + 0.4 710 + 1.8 755 + 0.2 715 + 1.6 760 0 720 + 1.4 765 -0.2 725 + 1.3 770 -0.4 730 + 1.1 775 -0.5 735 + 0.9 780 -0.7 740 + 0.7 785 -0.9 - 278- WHO/M/11 FLASH-POINT: CLEVELAND OPEN TESTER METHOD CLEVELAND OPEN TESTER METHOD FOR DETERMINATION OF FLASH-POINT 1. Special apparatus Cleveland Open Tester (see Fig. 11). Method WHO /M /ll Approved 25 October 1965 FIG. 11. CLEVELAND OPEN TESTER I Reproduced by kind permission of the Fisher Scientific Company. - 279 - FLASH-POINT: CLEVELAND OPEN TESTER METHOD WHO/M/11 2. Procedure 2. 1 Preparation of apparatus The test should be made in a room or compartment free from draughts and darkened sufficiently so that the flash may be seen readily. The operator should avoid breathing over the surface of the test liquid. Suspend the thermometer in a vertical position. The bottom of the bulb should be 6 mm from the bottom of the cup and above a point half-way between the centre and the back of the cup. Fill the cup with the sample in such a manner that the top of the meniscus is exactly at the filling-line at room temperature. Remove any air bubbles from the surface of the liquid, using a piece of clean, dry paper, and check that there is no liquid above the filling-line or on the outside of the appara- tus. Use a test flame the same size as the reference bead (approximately 4 mm in diameter). 2. 2 Performance of test Heat the sample at a rate not exceeding l 7°C per minute, until the temperature reached is approximately 56°C below the probable flash-point. Thereafter, decrease the rate of heating; for at least the last 28°C before the probable flash-point is reached, the rate should be not less than 5°C and not more than 6°C per minute. Apply the test flame as the temperature on the thermometer reaches each successive 3°C mark. The time for the passage of the test flame across the cup should be approximately one second. Record as the flash-point the temperature read on the thermometer when a flash appears at any point on the surface of the liquid. The true flash must not be confused with a bluish halo that sometimes surrounds the test flame. - 280 - WHO/M/12 CRYSTALLIZING-POINT DETERMINATION OF CRYSTALLIZING-POINT 1. Special apparatus Method WHO/M/12 Approved 25 October 1965 Crystallizing-point apparatus equipped with a standard thermometer.2 A glass test-tube is placed inside a larger glass test-tube which acts as an air jacket. The wider tube is weighted with lead shot or similar material, and the inner tube is closed by means of a cork which carries a stirrer and, through its centre, the standard thermometer. The stirrer is a glass loop with a glass stem, the loop being arranged to surround the thermo- meter. The thermometer is so fixed in the cork that the bottom of the bulb is about 10 mm from the bottom of the inner tube. The cork pro- jects for such a distance (about 10 mm above the top of the inner tube) that the immersion mark on the thermometer is level with the top of the cork. The cooling liquid is contained in a 1000-ml beaker, approximately 150 mm in height and 100 mm in diameter at the base. The test-tube, with its jacket, is placed in the cooling-bath in such a manner that the level of the liquid in the bath is at least as high as the level of the sample in the inner tube. 2. Procedure Remove the inner tube of the apparatus from its jacket and introduce about 20 g of the sample. Determine the approximate crystallizing- point by a preliminary rapid cooling of the molten mixture. Partially immerse the tube in a bath about 50°C above the expected crystallizing- point, until all but the last traces of crystal are melted. Replace the inner tube in its jacket and assemble the apparatus as shown in Fig. 12, with the cooling-bath between 6°C and 8°C below the expected crystal- lizing-point. Read the thermometer at Yi-minute intervals, with continuous and gentle stirring, making sure that seed crystal is present as the temperature of the sample falls to that at which crystallization begins. The crystal- lizing-point corresponds to the average of the first five consecutive readings during which the temperature remains constant within 0.5°C. 1 A suitable apparatus is described in: Institute of Petroleum, London (1953) Standard methods for testing petroleum and its products, l3th ed., London, p. 475. 2 BS (British Standard) No. B.2IOC/100, or equivalent. - 281 - CRYSTALLIZING-POINT WHO/M/12 FIG. 12. APPARATUS FOR DETERMINATION OF CRYSTALLIZING-POINT c E. E E 0 F D G E E 5l I 100 mm I !--•- - ---------- ---- - - -, WHO 63~2 · A = inner test-tube, 25 mm in diameter B = larger test-tube (air jacket) , 50 mm in diameter D = standard thermometer, surrounded by stirrer E = cooling-bath thermometer F = lead shot C = cooling-bath (1000-ml beaker) G = stirrer Reproduced, by kind permission. from: Institute of Petroleum. London ( 1953) Standard methods for testing Detroleum and its products, JJth ed .• London, tJ . 475. - 282 - WHO/M/12 CRYSTALLIZING-POINT If supercooling takes place, the constant temperature may be observed immediately after the temperature rise. A temperature rise of 1 °C should be regarded as the maximum allowable. If a constant temperature is not obtained over the first five readings after the rise in temperature, six readings should be taken, commencing with the point at which the maximum temperature is first attained. The readings are plotted on graph paper against time intervals, the mid-points between the first and second and between the fifth and sixth readings con- nected, and the line thus obtained produced backwards until it intersects the portion of the curve before the temperature rise. The point of inter- section is then reported as the crystallizing-point. If a rise in temperature is not obtained, the readings are plotted against time intervals until there is a definite change in direction of the curve, after which another six points are plotted. The mid-points between the first and second and between the fifth and sixth readings are connected and the line thus obtained produced backwards until it intersects a line obtained by producing downwards the main portion of the curve before the change in direction. The point of intersection is reported as the crystallizing-point. - 283- EMULSION ST ABILITY TEST WHO/M/13 EMULSION STABILITY TEST 1. Special reagent Method WHO/M/13 Approved 25 October 1965 Standard hard water. Dissolve 0.304 g of anhydrous calcium chloride and 0.139 g of magnesium chloride hexahydrate in distilled water and make up to one litre. This provides water with a hardness of 342 parts per mil- lion, calculated as calcium carbonate. 2. Procedure Into a 250-ml beaker having an internal diameter of 6-6.5 cm and a I 00-ml calibration mark, pour 7 5-80 ml of water, 1 brought to a temperature of 30°C ± 1 °C. By means of a Mohr-type pipette add the required volume of the concentrate ( see section 1 . 2. 4 of appropriate specification), while stir- ring with a glass rod, 4-6 mm in diameter, at about four revolutions per second. The concentrate should be added to the water at the rate of 25- 30 ml per minute, with the point of the pipette 2 cm inside the beaker, the flow of the concentrate being directed towards the centre, and not against the side, of the beaker. Make up to 100 ml with water,1 stirring continuously, and immediately pour into a clean, dry 100-ml graduated cylinder. The stirring time should be 3 minutes from the beginning of the addition of the concentrate until the emulsion is poured into the l 00-ml cylinder. Keep at 29-31 °C for 1 hour and examine for any creaming 2 or separation. 1 Standard hard water for section l. 2. 4. 2 or distilled water for section l. 2 .4. 1. of the appropriate specification. 2 Creaming is defined as the formation at the top of the emulsion of a layer containing a proportion of the dispersed phase greater than that in the remainder of the emulsion. - 284 - WHO/M/14 TOXICITY TEST FOR RODENTICIDES TOXICITY TEST FOR RODENTICIDES Method WHO/M/14 1. Test animals The test animals shall consist of mature, healthy albino rats (Wistar strain or equivalent) weighing between 150 g and 250 g. 2. Preparation of bait Prepare a test bait containing 0.025% of active ingredient by mixing the appropriate quantity of the sample with crushed oats, breadcrumbs, or other suitable food. In a similar manner, prepare a standard bait from a sample of standard rodenticide.1 Fresh bait must be prepared every day. 3. Procedure Take five rat-cages, approximately 30 cm square, and place one female and one male rat in each. In each of the five cages, expose 100 g of the test bait, together with an equal amount of bait without rodenticide ; use low-form half-litre containers of such a shape and weight that rats can neither climb into them nor spill the bait out of them, but yet all the bait is accessible. Provide a supply of water in a bottle stoppered and fitted with a short glass tube projecting downwards into the cage. Offer freshly prepared baits each day, using clean containers and changing their relative locations each time. Run the test for 10 days, observing and record~ ing every 12 hours the deaths of the rats. Carry out exactly the same test on another five pairs of rats, using the standard bait in place of the test bait. 4. Interpretation of results The total time required for death of all the rats offered the bait under test should not exceed by more than 10% the corresponding time required with the standard bait. The biological effectiveness of the sample is then deemed to be not less than 90% of that of the standard material. 1 Samples of standard rodenticide may be obtained, on request, from the World Health Organization, Avenue Appia, 1211 Geneva, Switzerhnd. - 285 - 19 PARR PEROXIDE-BOMB (TOT AL CHLORINE) METHOD WHO/M/15 PARR PEROXIDE-BOMB (fOTAL CHLORINE) METHOD Method WHO/M/15 Approved 25 October 1965 1. Special apparatus Parr bomb, provided with a circular safety-shield. 2. Procedure Weigh accurately about 0.2 g of the sample in a No. 2 gelatine capsule. In the cup of the bomb place lO g of sodium peroxide and approximately 0.3 g of starch, and mix thoroughly with a tapered glass rod.1 By means of the rod, fashion a cavity in the peroxide and stand the capsule therein in a vertical position. Cover the capsule with 5 g of sodium peroxide. Brush the excess peroxide from the rim of the cup with a camel-hair brush. Provide the lid with a lead gasket and place on the cup. Place the cup in the bottom screw-clamp and screw on to the top screw-clamp, tightening up with the spanner and holder. Place the bomb in the safety-shield 2 and pour 2 ml of water into the recess formed by the top screw-clamp and the bomb lid. Heat the bomb over a Bunsen flame, adjusted in such a way that the tip of the flame-cone just touches the base of the bomb, until the water in the recess begins to boil. Discontinue the heating and cool the bomb in water, the level of which must be below the thread of the lower portion of the holder. Unscrew the holder and, with the lid still in position, wash the outside with distilled water. Dry with a piece of filter-paper. Remove the lid from the cup and rinse with distilled water, collecting the rinsings in an 800-ml low-form beaker. Place the cup on its side in the beaker and add warm distilled water. Carefully apply a slight swirling motion to the beaker so that the contents of the cup are transferred to the beaker. Remove the cup and wash thoroughly with distilled water. Boil the solution, if necessary, to decompose the excess sodium peroxide. During these operations keep the beaker covered to prevent any loss by spray. Cool, adjust the volume of the solution to 300 ml, and neutralize with dilute nitric acid, using methyl orange as indicator. Add 3 ml of l N nitric acid and titrate electrometrically with 0.1 N silver nitrate. 1 Goggles must be worn during the operation. Great care must be taken that no water is allowed to contaminate the reagents. 2 The bomb should be heated only when inside the safety-shield. - 286- WHO/M/15 PARR PEROXIDE-BOMB (TOT AL CHLORINE) METHOD Alternatively, the chlorine may be titrated by the Volhard method-i. e., the titration procedure used in the revised Stepanow (total organic chlorine method), WHO/M/16. 3. Calculation T l . hl . (% I ) a x o.3546 x J ota organic c onne content O w w = ------'- where a = volume (ml) of 0.1 N silver nitrate used w = weight (g) of sample T != A w where T = calculated value (%) for total chlorine in the substance being tested A = value found(%) for total chlorine in a recrystallized sample 1 of the substance by the above-described method and with the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 1 If the substance being tested is not available in pure, recrystallized form, f is evaluated on the basis of another chlorinated hydrocarbon, such as DDT. - 287 - REVISED STEPANOW (TOT AL ORGANIC CHLORINE) METHOD WHO/M/16 REVISED STEPANOW (TOTAL ORGANIC CHLORINE) METHOD (For HCH and Dieldrin and their Formulations) 1. Technical products l . l Procedure l . l. l Determination of total chlorine Method WHO/M/16 Approved 25 October 1965 Weigh accurately about l g of the sample, transfer to a 250-ml volu- metric flask, add 10 ml of chlorine-free and thiophene-free benzene to dissolve the sample, and then make up to the mark with 99% isopropanol. Transfer a 25-ml aliquot to a 250-ml flask.1 Add 2.5 g of metallic sodium, in the form of ribbon or small pieces. Connect to a reflux condenser and boil gently for at least 2 hours, shaking the flask occasionally. Remove the excess metallic sodium by cautiously adding 10 ml of 50% aqueous isopropanol through the condenser, at the rate of 1-2 drops per second. Boil for an additional 10 minutes, and then add 60 ml of distilled water. Cool, add 2 or 3 drops of phenolphthalein indicator solution, neutralize by adding 50% nitric acid dropwise, and then add 10 ml in excess. Cool, if necessary, to room temperature, add a slight excess of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam- bath for half an hour, with frequent stirring. Cool, filter through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the total chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. l . l . 2 Determination of inorganic chlorine Weigh accurately about l g of the sample, dissolve in 10 ml of acetone, and add l 00 ml of distilled water. Keep at room temperature for 10 minutes and filter. Acidify the filtrate with 50% nitric acid, using phenolphthalein as indicator, then add a slight excess of 0.1 N silver nitrate solution and complete the determination as described in section l . l . 1. 1 Direct weighing of the sample may be substituted for the aliquoting, provided the weighing does not introduce an error of more than 0.1% in the sample weighed. - 288 - WHO/M/16 REVISED STEPANOW (TOT AL ORGANIC CHLORINE) METHOD 1 . 2 Calculation 1. Total organic chlorine content (%:w/w) = (a x __!_~ - _.?_ ') x 0.3546 x f W1 W2 where a volume (ml) of 0.1 N silver nitrate equivalent to the total chlorine b volume ( ml) of O. l N silver nitrate equivalent to the inorganic chlorine w1 = weight (g) of sample used for the total chlorine determination w2= weight (g) of sample used for the inorganic chlorine determina- tion T f = ---A where T = calculated value(%) for total chlorine in the active ingredient 1 A = value found (%) for total chlorine in a recrystallized sample of the substance by the above-described method and with the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2. Content of active ingredient (% w /w) = c x M where c = total organic chlorine content (% w /w) 100 M = -·;y and T has the definition given above.1 2. Water-dispersible powders and dusting-powders 2. I Procedure Weigh accurately an amount of the sample containing about I g of the active ingredient and extract it quantitatively with chlorine-free and thio- phene-free benzene. If a Soxhlet apparatus is used, care must be taken to ensure that extraction is complete and that channelling has not occurred. Concentrate the extract to such a volume that it can be transferred quan- titatively to a 100-ml volumetric flask, make up to the mark with benzene, and mix thoroughly. 1 For HCH, T = 73.14%, M = 1.37; for HEOD, T = 55.85%, M = 1.79. - 289 - REVISED STEPAN OW (TOT AL ORGANIC CHLORINE) METHOD WHO/M/16 Transfer a 10-ml aliquot to a 250-ml conical flask and add 25 ml of 99% isopropanol. Shake the flask, add 2.5 g of metallic sodium, in the form of ribbon or small pieces, and proceed as described in section 1. 1. 1 (second paragraph). 2. 2 Calculation Content of active ingredient (% w /w) a x f x M x 3.546 w where a = volume (ml) of 0.1 N silver nitrate used w = weight (g) of sample and M and f are as defined in section l . 2. 3. Emulsion concentrates 3 . 1 Procedure 3. l . l Determination of total chlorine Weigh accurately an amount of the sample containing about 0.1 g of the active ingredient and transfer to a 250-ml conical flask. Add 25 ml of 99% isopropanol, shake the flask, add 2.5 g of metallic sodium, in the form of ribbon or small pieces, and proceed as described in section 1.1. l (second paragraph). 3 . 1 . 2 Determination of inorganic chlorine Weigh accurately about 1 g of the sample and transfer with 100 ml of distilled water to a 250-ml conical flask. Acidify with 50% nitric acid, using phenolphthalein as indicator, then add a slight excess of 0.1 N silver nitrate solution and complete the determination as described in section l . 1 . 1. 3 . 2 Calculation Content of active ingredient (% w/w) = (~ - ~) x 0.3546 x f x M W1 W2 where a = volume (ml) of 0.1 N silver nitrate equivalent to the total chlorine b volume (ml) of 0.1 N silver nitrate equivalent to the inorganic chlorine w1= weight (g) of sample used for the total chlorine determination w2= weight (g) of sample used for the inorganic chlorine determina- tion and M and f are as defined in section l. 2. - 290 - WHO/M/17 HCH CONTENT : HYDROL YSABLE CHLORINE METHOD HYDROLYSABLE CHLORINE METHOD FOR DETERMINATION OF HCH CONTENT 1. Technical HCH, refined HCH, or lindane 1 . 1 Procedure Method WHO/M/17 Approved 25 October 1965 1 . 1 . 1 Determination of hydrolysable plus inorganic chlorine Weigh accurately about 0.3 g of the sample into a 250-ml conical flask, add 50 ml of 0.5 N ethanolic potassium hydroxide, and reflux gently for half an hour. Wash down the condenser with distilled water and allow to cool. Add 20 ml of 2 N nitric acid and exactly 40 ml of 0.1 N silver nitrate, and coagulate the precipitated silver chloride by digesting on a steam-bath for half an hour, with frequent stirring. Cool, filter the coagu- lated silver chloride through a fast paper, and wash thoroughly with distilled water. Add 5 ml of 10% ferric alum solution and titrate the excess silver nitrate in the filtrate with 0.1 N potassium thiocyanate. The end-point is the appearance of the red ferric thiocyanate colour. Subtract the quantity of silver nitrate found in the filtrate from that originally added. The difference will be the quantity required to combine with the hydrolysable plus inorganic chlorine originally present in the sample. One millilitre of 0.1 N silver nitrate is equivalent to 0.003546 g of chlorine. Alternatively, the end-point may be determined electrometrically. 1 . 1 . 2 Determination of inorganic chlorine Weigh accurately about 1 g of the sample, dissolve in 10 ml of acetone, and add 100 ml of distilled water. Keep at room temperature for 10 minutes and filter. Acidify the filtrate with 50% nitric acid, add exactly 40 ml of 0.1 N silver nitrate, and complete the determination as described in section 1 . 1 . 1. 1 . 2 Calculation l,2,3,4,5,6-Hexachlorocyclohexane content(% w/w) = (!!_ - !!___) x f x o.968 W1 W2 - 291 - HCH CONTENT: HYDROL YSABLE CHLORINE METHOD WHO/M/17 where a volume (ml) of 0.1 N silver nitrate equivalent to the hydro- lysable plus inorganic chlorine b = volume (ml) of 0.1 N silver nitrate equivalent to the inorganic chlorine w1 = weight (g) of sample used for the hydrolysable plus inorganic chlorine determination w2= weight (g) of sample used for the inorganic chlorine determina- tion 36.57 f = -----x- where A = value found (%) for hydrolysable chlorine in recrystallized HCH by the above-described method and with the same reagents. The correction factor f is intended to allow for errors due to impurities in the reagents, as well as those inherent in the method itself or in its application by a given laboratory. Its value must lie within the range 0.98-1.02. 2. Water-dispersible powders and dusting-powders 2. 1 Procedure Weigh accurately an amount of the sample containing about l g of HCH and extract it quantitatively with chlorine-free and thiophene-free benzene. If a Soxhlet apparatus is used, care must be taken to ensure that extraction is complete and that channelling has not occurred. Concentrate the extract to such a volume that it can be transferred quantitatively to a 100-ml volumetric flask, make up to the mark with benzene, and mix thoroughly. Transfer a 10-ml aliquot to a 250-ml conical flask, add 40 ml of 0.5 N ethanolic potassium hydroxide, and reflux gently for half an hour. Wash down the condenser with distilled water and allow to cool. Add 20 ml of 2 N nitric acid and exactly 25 ml of 0.1 N silver nitrate, and complete the determination as described in section 1 . 1 . l. 2 . 2 Calculation 1,2,3,4,5,6-Hexachlorocyclohexane content (% w/w) a xf x 9.68 w - 292 - WHO/M/17 HCH CONTENT: HYDROL YSABLE CHLORINE METHOD where a = volume (ml) of 0.1 N silver nitrate used w = weight (g) of sample and f is as defined in section 1. 2. 3. Emulsion concentrate) 3 . 1 Procedure 3 . 1 . 1 Determination of total chlorine Weigh accurately an amount of the sample containing about 0.3 g of HCH and transfer to a 250-ml conical flask. Add 50 ml of acetone and 40 ml of 0.5 N ethanolic potassium hydrodroxide. Keep at 20-25°C for 15 minutes and add 50 ml of distilled water. Add 20 ml of 2 N nitric acid and exactly 40 ml of 0.1 N silver nitrate, and complete the determina- tion as described in section 1 . 1 . 1. 3 .1. 2 Determination of inorganic chlorine Weigh accurately about 1 g of the sample and transfer with 100 ml of distilled water to a 250-ml conical flask. Acidify with 50% nitric acid, add exactly 40 ml of 0.1 N silver nitrate, and proceed as described in section 1. 1. 1. 3 . 2 Calculation 1,2,3,4,5,6-Hexachlorocyclohexane content (% w /w) = (!!__ - -~) x f x 0.968 IV1 W2 where a volume (ml) of 0.1 N silver nitrate equivalent to the hydro- lysable plus inorganic chlorine · b volume (ml) of 0.1 N silver nitrate equivalent to the inorganic chlorine w1 = weight (g) of sample used for the hydrolysable plus inorganic chlorine determination w2 = weight (g) of sample used for the inorganic chlorine determina- tion and f is as defined in section l . 2. - 293 - TOXICITY TEST FOR LARVICIDAL OILS WHO/M/18 TOXICITY TEST FOR LARVICIDAL OILS Method WHO/M/18 Approved 25 October 1965 1. Special apparatus l. Glass beakers of 400 ml capacity and internal diameter of 7 .3 cm. 2. Enamel rearing pans, about 40 cm x 36 cm. 3. A colony cage of wood or other suitable material, for rearing adult mosquitos, provided in front with a wire screen and a cloth sleeve entrance, and large enough to allow the mosquitos sufficient space for swarming and mating; 60-cm cubical cages have been found satisfactory. 4. A small emergence cage for collecting the pupae. 5. A cage just large enough to confine a rabbit (or other suitable animal) inside the colony cage. 6. Pipettes for transfering larvae. 7. Pipettes for oil application. 2. Standard colony of mosquitos Obtain eggs from a known established colony, or place a gravid female mosquito in a 7 .5-cm specimen tube, the mouth of which is plugged with moist filter paper, where oviposition usually takes place. Transfer the eggs by means of a camel-hair brush to the rearing pans containing water maintained at 25°C ± l °C. Feed the larvae with dry yeast powder or any other suitable food 1 sprinkled on the water surface every morning, increasing the quantity of food as the larvae grow. To avoid overcrowding of the larvae, do not keep more than 750 larvae in each pan. Change the water in the pans on alternate days, shaking the fresh water thoroughly in a bottle before pouring it into the pans. Collect the pupae as they are formed and place them in the emergence cage. Transfer the hatched-out adults into the colony cage. Maintain a temperature of 24-27°C and a relative humidity of 75-80% inside the room and the colony cage (this is generally done by hanging wet cloths in the room and inside the colony cage). Keep cotton pads soaked in a 10% glucose solution inside the cage for male mosquitos to feed on. In order 1 Finely-powdered dog biscuit ; powdered skimmed milk ; a mixture of 1 part of dehydrated blood-serum and 2 parts of litmus milk ; brewers' yeast ; powdered dried toast or bread-crumbs ; mature hay infusion (must be aerated vigorously every day) ; Spirogyra ; or chopped flies. -294 - WHO/M/18 TOXICITY TEST FOR LARVICIDAL OILS to provide the blood meal for female mosquitos, introduce each night into the colony cage the small cage containing a rabbit ( or other suitable animal) with shaved back.1 Put an enamel bowl with water inside the colony cage for oviposition. Keep the cage in a room illuminated for about 9 hours a day by a 60-watt lamp and kept dark the rest of the time. NOTE : The colony must be tested periodically against a standard larvicidal oil to ensure that it is behaving normally. A suitable standard is n-hexadecane. 3. Procedure The test shall be performed at 25°C ± 1 °C. Carry out 5 replicate tests simultaneously for each dosage of each test material. Transfer 25 early 4th instar larvae 2 from the standard colony to each of fifteen 400-ml glass beakers containing 250 ml of distilled water. Pipette not more than 0.01 ml of the sample 3 gently on to the surface of the water in each of 5 beakers and proceed similarly with doses of0.02 ml and 0.04 ml, using 5 beakers for each. The percentage mortality is recorded at the end of twenty-four hours. NOTE. The test dosage of 0.01 ml is equivalent, in terms of field dosage, to approximately 47 litres per hectare. 4. Recording of results The larvae shall be considered as dead if, at the end of 24 hours, they show no sign of swimming movements even after gently touching with a glass rod. Report the average of the individual test results. 1 If a suitable animal is not available, the human hand may be introduced into the cage for short periods. 2 The same test procedure can be used with pupae to evaluate pupacidal effectiveness of oils. 3 In those instances where the mortality is high at the 0.01-ml level, the dosage should be reduced to 0.005 ml or lower for precise determination of relative effectiveness. - 295 - NAMES OF PESTICIDES ANNEX 1 Annex 1 COMMON NAMES, TRADE NAMES AND CHEMICAL NAMES OF PESTICIDES In the list below, common names, trade names and chemical names of the pesticides for which specifications are given in Parts I-IV of this volume are shown in alphabetical order in the left-hand column. The common names approved by the International Organization for Stand- ardization (ISO) are printed in bold face, other common names and chemical names in light face lower case type. Trade names, where these could be ascertained, are shown in small capitals with an initial large capital. The right-hand column of the list contains synonyms and cross-references. 3-( cx-acetonylbenzyl)-4- -hydroxycoumarin 3-( cx-acetonyl-p-chloro- benzyl)-4-hydroxycoumarin BAYER 73 BAYLUSCIDE BAYTEX BHC 1 l, l-bis(p-chlorophenyl)- -2,2,2-trichloroethane cinerin coumachlor deet DDT DDVP diazinon see warfarin see coumachlor 2' ,5-dichloro-4' -nitrosalicylanilide, ethanol- amine salt 2' ,5-dichloro-4' -nitrosalicylanilide, ethanol- amine salt see fenthion see HCH DDT see pyrethrum 3-(cx-acetonyl-p-chlorobenzyl)-4-hydroxy- coumarin N,N-diethyl-m-toluamide (I, 1-bis-p-chlorophenyl)- 2,2,2-trichloroethane see dichlorvos 0 ,0-diethyl 0-(2-isopropyl-6-methyl-4- -pyrimidinyl) phosphorothioate 1 Both BHC and HCH have been approved by ISO, but HCH is normally preferred in WHO publications. - 296 - ANNEX 1 2' ,5-dichloro-4' -nitro- salicylanilide, ethanol- amine salt 2,2-dichlorovinyl dimethyl phosphate dichlorvos dieldrin 0 ,0-diethyl 0-(2-iso- propyl-6-methyl-4- -pyrimidinyl) phosphorothioate 0 ,0-diethyl 0-( p-nitro- phenyl) phosphorothioate N,N-diethyl-m-toluamide 0,0-dimethyl S-[1,2-di- -( ethoxycarbonyl)ethyl] phosphorodithioate dimethyl l-hydroxy-2,2,2- -trichloroethylphosphonate 0 ,0-dimethyl 0-( 4- methylthio-m-tolyl) phosphorothioate 0 ,0-dimethyl 0-(p-nitro- phenyl) phosphorothioate DIPTEREX fenthion FRESCON gamma-BBC HCH HEOD BAYLUSCIDE BAYER 73 see dichlorvos NAMES OF PESTICIDES 2,2-dichlorovinyl dimethyl phosphate see HEOD see diazinon see parathion see deet see malathion see trichlorfon see fenthion see parathion-methyl see trichlorfon 0 ,0-dimethyl 0-( 4-methylthio-m-tolyl) phosphorothioate N-triphenylmethylmorpholine see lindane 1,2,3 ,4,5,6-hexachlorocyclohexane benzene hexachloride, BHC 1,2,3,4, 10, 10-hexachloro-6,7-epoxy-l ,4,4a, 5,6,7 ,8,8a-octahydro-endo-l ,4-exo-5,8- dimethanonaphthalene (dieldrin = 85% w/w HEOD) 1,2,3,4,5,6-hexachlorocyclo- see HCH hexane - 297 - NAMES OF PESTICIDES ANNEX 1 lindane gamma-isomer of 1,2,3,4,5,6-hexachloro- cyclohexane (99%) gamma-BHC, gamma isomer of BHC malathion O ,0-dimethyl S-[1,2-di( ethoxycarbonyl)ethyl] phosphorodithioate parathion O ,0-diethyl 0-(p-nitrophenyl) phosphorothioate parathion-methyl O ,0-dimethyl 0-(p-nitrophenyl) phosphorothioate 3-( o:-acetonylbenzyl)- see warfarin -4-hydroxycoumarin pindone 2-pivaloyl-1,3-indanedione P1v AL see pindone 2-pivaloyl-1,3-indanedione see pindone pyrethrin see pyrethrum pyrethrum preparation derived from pyrethrum flowers and consisting mainly of pyrethrin I, pyrethrin II, cinerin I and cinerin H trichlorofon dimethyl l-hydroxy-2,2,2-trichloroethyl- phosphonate N-triphenylmethyl- FRESCON morpholine warfarin 3-(o:-acetonylbenzyl)-4-hydroxycoumarin - 298- ANNEX 2 WHO EXPERT COMMITTEE ON INSECTICIDES Annex 2 COMPOSITION OF THE FIFTEENTH WHO EXPERT COMMITTEE ON INSECTICIDES* The specifications included in this volume have been revised in accord- ance with the recommendations contained in the report of the fifteenth WHO Expert Committee on Insecticides, which had the following com- position: Members: Dr E. Paulini, Chief, Chemical Laboratory, National Institute of Endemic Diseases, Belo Horizonte, Brazil Dr G. W. Pearce, Scientist Director, Chief, Biology/Chemistry Section, Technology Branch, Communicable Disease Center, USPHS, Savan- nah, Georgia, USA (Chairman) Monsieur l'Inspecteur general J. Prat, Institut National de Recherche Chimique Appliquee, Centre de Recherche de Vert-le-Petit, Vert- ie-Petit (Seine-et-Oise), France (Vice-Chairman) Mr A. K. Sengupta, Deputy Director, National Malaria Eradication Programme, National Institute for Communicable Diseases, Delhi, India Dr M. T. Shafik, College of Agriculture, Alexandria University, Alexandria, United Arab Republic Dr E. M. Thain, Assistant Director, Tropical Products Institute, London, England Dr J. Treboux, Research Laboratory, J. R. Geigy A. G., Basel, Switzerland (Rapporteur) Secretariat : Dr R. de B. Ashworth, Plant Pathology Laboratory, Ministry of Agriculture, Fisheries and Food, Harpenden, England (Consultant) * The Committee met in Geneva from 19 to 25 October 1965 and its report was issued as mimeographed document WHO/Vector Control/66.199. - 299 - WHO EXPERT COMMITTEE ON INSECTICIDES ANNEX 2 Dr J. W. Miles, Chief Chemistry Unit, Biology/Chemistry Section, Technology Branch, Communicable Disease Center, USPHS, Savannah, Georgia, USA (Consultant) Dr A. R. Stiles, Vector Control, WHO, Geneva (Co-Secretary) Mr J. W. Wright, Chief, Vector Control, WHO, Geneva (Co-Secretary) - 300 - WHO publications may be obtained through : APGHANISTAN I •• India, WHO .Retrlonal omce ARGENTINA: Editorial Su4amerlcana S.A,. Hum- berto 1° S4S, BUBNOS AIUa AUSTRALIA : Hunter Publlcationa, 23 Mc:Killop Stteet. Ml!LBouaNB C.1 AUSTRIA: Gerold & Co., I. Graben 31, VD!NNA 1 BELGIUM : Offlce lnlernational de Libralrle, 30 av. 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