The assay of the molluscicide niclosamide using the ciliate protozoan Spirostomum ambiguum.

404 NOTES Table 1. Results of wet film examinations and BIIT of T. rhodesiense and T. brucei strains after in vitro incubation in human blood for 5 hours at 37°C Wet film examination BI 3ut of incubated samples BIT r3sults Parasite Number species of tests Normal, motile No trypanosomes trypanosomes + seen seen T. rhodesiense 100 60 40 100 - T. brucei 100 34 66 - 100 7. " Checker-board " titration ofman-tested T. brucei Since the conditions for the BIIT were established arbitrarily, it is necessary to define more precisely the points at which, in terms of the many factors in- volved in the test, T. brucei (sensu stricto) try- panosomes lose their infectivity to rats. Two of these factors, i.e., incubation time at 37°C and the ratio of human to positive rodent blood in vitro, were initially examined in checker-board titra- tions. These showed that, with incubation for 5 hours at 37°C, a human blood to positive rat blood ratio of 4: 1 was sufficient to prevent infection of the inoculated rats by any of the T. brucei strains used. When samples were tested at very high and very low levels of parasitaemia, the positivity patterns for the inoculated rats indicated that the infectivity level of the trypanosomes had, in both instances, been markedly diminished. This serves to underline the recommendation for the use, in the BIIT, of a medium and rising parasitaemia in the donor rats or mice of the strain under test (See note 4 above). REFERENCES Fairbairn, H. (1933) Ann. trop. Med. Parasit., 27, 185 Rickman, L. R. & Robson, J. (1970a) Bull. Wld Hlth Org., 42, 650 Rickman, L. R. & Robson, J. (1970b) Bull. Wld Hlth Org., 42, 911 The assay of the molluscicide niclosamide using the ciliate protozoan Spirostomum ambiguum R. MEREDITH 1 & G. C. MEREDITH 2 There is some evidence that the molluscicidal efficacy of the ethanolamine salt of niclosamide is affected by the composition of the water to which the molluscicide is applied. Fox, Ritchie & Frick (1963) and Strufe & Gdnnert (1962) have reported experi- ments that suggest that the molluscicidal activity of solutions of the ethanolamine salt of niclosamide may be influenced by the pH of the water and it is possible that other factors-e.g., suspended solids 1 Tropical Pesticides Research Headquarters and Informa- tion Unit, Foreign and Commonwealth Office, Overseas Development Administration, London, England. 2 Department of Zoology, University of Reading, Read- ing, Berks., England. 2815c and light-may lower the activity of solutions of the molluscicide. It is very difficult to determine these changes in activity, either in the field or in the laboratory. The chemical methods available (Strufe, 1962, 1965) are unsuitable for two reasons: 1. The determination is of the concentration of niclosamide. While there is no reason to suppose that the losses in activity are due to any changes in the structure of the niclosamide molecule, the niclosamide may be adsorbed on to particles in the water or be present as the free acid (which because of its low solubility is not likely to exhibit mollus- BIOASSAY OF THE MOLLUSCICIDE NICLOSAMIDE 405 cicidal properties as strong as those of the ethanol- amine salt). 2. In the field the accuracy of the chemical methods suffers because of interference from other substances present in the water (Strufe & Fikry El- Tawil, 1963). In many ways a bioassay technique would be more suitable for measuring changes in activity; however, of the two methods available, the bioassay with snails (Hopf & Muller, 1962) is either inaccurate or time-consuming and requires large numbers of snails, and the respiratory method of Strufe & G6nnert (1962) is unsuitable for use in the field. A method is required that will allow the activity of a molluscicidal solution to be measured accurately in the laboratory and that can also be used to extend the results of the laboratory to the field. In preliminary trials it was found that the ciliate protozoa of the genera Spirostomum and Euplotes were very sensitive to the presence of the ethanol- amine salt of niclosamide and Spirostomum ambiguum Ehrenberg var. minor was chosen for further studies for the following reasons: 1. The estimated LC50 of 0.2 ppm of niclosamide over 2 hours was very close to the 2-hour LC50 against Biomphalaria glabrata (Fletcher, 1965). 2. S. ambiguum var. major is too large to be easily handled by pipette. 3. Euplotes moves too rapidly to be counted and is slightly less susceptible to niclosamide-ethanolamine. Materials and methods Spirostomum was cultured under the conditions recommended by Carter (1956). A sample contain- ing a few thousand animals was removed from the culture and centrifuged very gently in a hand cen- trifuge. The loose plug ofSpirostomum thus obtained was washed four times with artificially hardened water 1 (Hopf & Muller, 1962) and then suspended in sufficient artificially hardened water (AHW) to give a suspension of approximately 50 animals per ml. The suspension was adjusted to pH 7.2 with 0.005 M sodium hydrogen carbonate. A stock solution of 7.148 mg of niclosamide- ethanolamine per litre was made up in AHW and adjusted to pH 7.2. Then solutions of commercial niclosamide were prepared that gave concentrations I Approx. 2.9 m Eq/litre of hardness-producing ion. This is approximately equivalent to 145 mg CaCO,/litre= 10 English degrees of hardness. of the active ingredient (niclosamide) of between 0.02 ppm and 5 ppm in AHW at pH 7.2. Aliquots of 1 ml of each niclosamide solution were pipetted into 10-ml screw-capped bottles containing 1 ml of the suspension of Spirostomum. There were up to 4 replicates at each concentration. Controls containing 1 ml of Spirostomum and 1 ml of AHW at pH 7.2 were also set up. The tubes were then firmly capped and laid on the stage of a binocular dissecting microscope. The number of animals in the field of view was counted and the count was repeated after shaking the tube. A total of five counts were made for each tube (care being taken to ensure that the counts were made in the same part of the tube each time). The tubes were then placed in a water bath at 23°C. After 2 h the tubes were removed and the counting procedure was repeated. When they die the animals disintegrate, leaving the pellicle floating in the water surrounded by debris. The percentage mortality was, at any concentration, calculated from the sum of the 5 counts at the start of the assay and and sum of the counts after 2 h. This procedure was repeated with tubes at 10°C, 12°C, 150C, 17°C, 19°C, 20°C (twice), 210C, 22°C, and 25°C (twice) in order to determine the tem- perature dependence of the method. Results The relation between the log concentration and the probit of the mortality after exposure for 2 h at 23°C is shown in Fig. 1. The LC50 for the solution was calculated (Finney, 1952) as 0.196 ppm (95% confidence limits ±0.01 ppm where n = 40). The temperature dependence of the method is shown in Fig. 2, in which the reciprocal of the LC50 is plotted against temperature. The LC50 at 10°C was 0.5 ppm, at 20°C it was 0.292 ppm, and at 25°C it was 0.145 ppm. The LC50s at these temperatures were estimated by eye after plotting the probit per- centage mortality against log concentration. Discussion This method may be of value for estimating the concentration of the ethanolamine salt of niclos- amide both in the field and in the laboratory. At temperatures above 20°C it can be used to estimate concentrations of niclosamide above 0.2 ppm, which is approximately the level used in field trials, with an accuracy of about 5 %. The considerable change in sensitivity with tem- perature could cause some practical difficulties if the NOTES n 80 70 60 50 ._X v 40 a, C 30 4- )oLU _ 10 _ 0.17 0.1 0.19 0.2 0.21 0.22 0.24 Concentration of niclosamide (ppm) WHO 10929 assay were attempted in an environment with fluctuating temperatures, as it would be necessary to run a large number of standard solutions with the sample solutions. However, immersion of the sample tubes in the body of water undergoing treatment, or even placing the samples in a well-shaded place, would control the temperature fluctuations suffi- ciently so that the number of standards run would be determined by the degree of accuracy required. The Spirostomum assay is somewhat more accurate than the field chemical method (10% accuracy was claimed by Strufe & Fikry El-Tawil, 1963) but requires more time. However, the bioassay with Spirostomum measures the active niclosamide con- centration and may be preferable if it is considered that the effectiveness of the molluscicide is being lowered for some reason or if there are substances present in the water that give rise to interference with the chemical method. The effect of temperature on the activity of the ethanolamine salt of niclosamide was studied by Strufe & Gonnert (1962) using Biomphalaria glabrata. The results of these authors do not permit calculation of the gradient of the regression line showing probit percentage kill plotted against log concentration, nor do they indicate how this gradient changes with variations in temperature. However, the activity of the ethanolamine salt of niclosamide clearly in- creased with temperature. Fig. 2. The relation between temperature and the reciprocal of the LCso for niclosamide (as the ethanolamine salt) against Spirostomum. 0 Fig. 1. Relation between the probit percentage mor- tality and the concentration of niclosamide (as the ethanolamine salt) for Spirostomum at 23°C. :.2 406 BIOASSAY OF THE MOLLUSCICIDE NICLOSAMIDE 407 If the response shown by Spirostomum resembles the response of the snail, immediate study is war- ranted of the toxicity of the ethanolamine salt of niclosamide for the snail at different temperatures. A rise in temperature from 20°C to 25°C doubles the activity of the molluscicide and therefore halves the amount required (assuming no change in the gra- dient of the regression line of probit percentage kill plotted against log concentration). Investigations of the effect of temperature are now being carried out. REERENCES Carter, L. (1956) J. exp. Biol., 34, 71-84 Fox, I., Ritchie, L. S., & Frick, L. P. (1963) Hofrhenbr. Bayer PflSchutz. Nacht., 16, 269-277 Finney, J. D. (1947) Probit analysis, London, Cambridge University Press Fletcher, F. (1965) Hofchenbr. Bayer PflSchutz Nachr., 18, 153-155 Hopf, H. S. & Muller, R. L. (1962) Bull. Wld Hith Org., 27, 783-789 Strufe, R. (1962) Hofchenbr. Bayer PflSchutz Nachr., 15, 42-49 Strufe, R. (1965) Hofchenbr. Bayer PflSchutz Nachr., 18, 130-139 Strufe, R. & Fikry El-Tawil (1963) Hofchenbr. Bayer PflSchutz Nachr., 16, 231-243 Strufe, R. & G6nnert, R. (1962) Hofchenbr. Bayer PflSchutz Nachr., 15, 50-70 Observations of the guppy, Poecilia reticulata Peters, in Culex pipiens fatigans breeding sites in Bangkok, Rangoon, and Taipei ERNEST C. BAY 1 & LEE S. SELF 2 Sasa et al. (1965) reported the successful establish- ment of the guppy, Lebistes reticulatus (now Poecilia reticulata; Rosen & Bailey (1963)), in a number of shallow, highly polluted ground pools beneath low- income housing in Bangkok, Thailand. From this work, two questions logically arose: (1) had Bangkok guppies evolved a higher tolerance to organic pollu- tion than P. reticulata that had never been exposed to these conditions; and (2) what were the possibilities of using these fish elsewhere to control Culex pipiens fatigans, especially in Rangoon, Burma, where this mosquito is the vector of Wuchereria bancrofti. In order to explore these questions, the senior author first went to Bangkok in October 1967 to observe and collect guppies with the aid of the WHO Aedes Research Unit, and then to Rangoon for the purpose of making trial introductions of these gup- pies in conjunction with the WHO Filariasis Re- search Unit. During this assignment, visits concern- ing larvivorous fishes were also made to mosquito control teams in Hawaii, Japan, and Taiwan. This is an account of the observations made during and 1 Professor and Head, Department of Entomology, University of Maryland, College Park, Md., USA. 2 Entomologist, WHO Encephalitis Vector Research Unit, Seoul, Republic of Korea. 2815D since that trip, particularly in relation to the results of guppy introductions in Rangoon and in Taipei, Taiwan, during a 2-year period. Bangkok, Thailand Various C. p. fatigans breeding sites were visited in Bangkok with a view to determining the correct conditions for the release of P. reticulata in Rangoon. A special effort was made to visit the particular sites or locations described by Sasa et al. (1965), and also to find sites where C. p. fatigans larvae and P. reti- culata coexist. In the past, the coexistence of guppies and mosquito larvae has led some observers to doubt the importance of these fish in mosquito control. Sites of coexistence were found, as well as sites with- out guppies where there was very heavy C. p. fati- gans breeding, and sites with dense guppy popula- tions but no mosquito larvae although larvae could have been expected to be numerous. Except in Khlong Toei slum, where water was heavily silted by prevailing tidal conditions, all pol- luted ground pools were very similar in appearance. Water, although contaminated to a varying extent with domestic garbage (kitchen scraps) and occa- sional faecal matter, was usually clear enough for the bottom to be seen at depths of 10-20 cm. In some