Storage of chlorinated pesticides in human organs and tissues in Ferrara Province, Italy*

Bull. Org. mond. Santeh 1972, 46, 363-369Bull. Wld Hlth Org. ) Storage of chlorinated pesticides in human organs and tissues in Ferrara Province, Italy* L. PRATI,1 R. PAVANELLO,2 & F. GHEZZO8 Residues of organochlorine compounds in human fat, liver, brain, kidney, and spleen were determined by gas chromatography. All the samples were obtainedfrom residents of Ferrara Province, Italy, of both sexes and of ages ranging from 1 to 80 years who had no special exposure to insecticides. The following compounds were found: p,p'-DDE, o,p'- DDT, p,p'-DDT, HEOD, and heptachlor epoxide. DDT and its derivatives was found more frequently and at higher concentrations than the other compounds; the average weighted percentage ratio ofDDE (as DDT) to total equivalent DDT in fat was 73.7 %. The concentrations of residues in nonfatty tissues were considerably lower than in fatty tissue; in liver, and to a smaller extent in kidney, the average concentrations were higher than in other organs (brain and spleen). From comparisons of these results with those from other countries it appears that higher levels of chlorinated compounds are generally present in the Ferrara population. As part of an epidemiological investigation of the long-term effects of pesticides on human health (Paccagnella et al., 1971), the storage of chlorinated hydrocarbon residues in human tissues, both fatty and nonfatty, was studied in Ferrara Province, Italy, where the amounts of hydrocarbons used for agri- cultural purposes are among the highest in Italy. The results of analyses of 52 samples of fat obtained at autopsy, 33 samples of fat from biopsies, and 23 samples of liver, 15 of kidney, 15 of brain, and 13 of spleen, from 85 inhabitants of the province are presented in this report. The samples were supplied between 1966 and 1969 by the Institute of Pathological Anatomy, the Insti- tute of Surgical Pathology, and the Department of Obstetrics of Ferrara University, and were taken from subjects of both sexes who had had no occu- pational exposure to pesticides, and whose age ranged from 1 to 80 years. Autopsy material was obtained from cadavers with various pathologies (infections and cardiovascular, pulmonary, and * This investigation was supported jointly by the World Health Organization and the Ministry of Health, Italy. 1 Assistant Professor of Chemistry, Institute of Hygiene, University of Ferrara, Italy. 2 Chief, Environmental Pollution, WHO, Geneva. 3Assistant Professor, Institute of Hygiene, University of Ferrara, Italy. renal diseases). Samples of mesenteric fat were obtained during appendectomies or caesarian sec- tions. METHODS Pesticide residues were determined by gas chro- matography with electron-capture detection. All samples were homogenized and extracted with hexane in the presence of sodium sulfate following the method of de Faubert Maunder et al. (1964). Extracts were filtered through Whatman No. 1 paper and then frozen at - 20°C to obtain a first elimina- tion of the extracted lipids. After a second filtration, the organochlorine compounds were partitioned into dimethylformamide. The dimethylformamide layer was separated, made aqueous with 2% sodium sul- fate solution, and extracted with hexane. The extract was then concentrated by means of warm, dry air, eluted through a column of alumina (activated at 800°C for 4 hours), concentrated first in a Kuderna- Danish-type evaporation concentrator,4 and then by warm dry air to a known volume (5 ml) for analysis by gas-liquid chromatography. All these operations were checked separately with fresh hexane solutions containing known concen- ' Constructed according to the illustration in Zweig (1964). 2812 -363 L. PRATI ET AL. trations of active products (analytical grade) in order to evaluate (1) the possible presence of sub- stances in the solvents, reagents, and the equipment used for extraction, purification, and concentration that might interfere with the chromatographic deter- mination, and (2) the percentage recovery of the whole analytical process. Interfering substances were shown to be absent and the following mean values for recovery of active products were obtained: gamma-HCH, 88.1 %; heptachlor, 85.5%; hepta- chlor epoxide, 87.6 %; HHDN, 77.9 %; HEOD, 87.3 %; p,p'-DDE (1,1-dichloro-2,2-bis [p-chloro- phenyl]ethylene), 92.6%; o, p'-DDT, 87.3%; p,p'- DDT, 93.0%. Qualitative analysis was made only by comparing gas chromatographic retention times for the com- pounds detected analytically with those for the standards. The principal compounds and their metabolites were first separated in a chromatographic column packed with a 2:1 mixture of SE-30 and QF-1 (Table 1). By using the stationary phase OV-17 qualitative confirmation of the identity of the compounds in Table 1 was obtained, except for the beta-HCH isomer. It was found that the peak formerly identi- fied as beta-HCH, and always present in chromato- grams derived from analyses of fatty tissues, is significantly different and does not represent any other known organochlorine compound (Table 2); Table 1. Retention times relative to HHDN of various organochlorine compounds on a 200-cm column packed with 1 % SE-30 + QF-1 (2:1) on GC P Sil, 80-100 mesh Compound Retention time alfa-HCH 0.44 beta-HCH 0.48 gamma-HCH 0.55 heptachlor 0.81 HHDN 1.00 heptachlor epoxide 1.51 p,p'-DDE 2.17 HEOD 2.39 o,p'- DDT 2.95 p,p'-TDE 3.21 p,p'- DDT 4.06 Table 2. Retention times relative to HHDN of various organochlorine compounds on a 200-cm column packed with 1 % OV-17 on GC P Sil, 100-120 mesh Compound Retention time unknown substances (previously identified as beta-HCH) 0.44 alfa-HCH 0.48 gamma-HCH 0.64 beta-HCH 0.78 heptachlor 0.81 HHDN 1.00 heptachlor epoxide 1.56 HEOD 2.56 p,p'-DDE 2.67 o,p'- DDT 3.93 p,p'-TDE 4.27 p,p'- DDT 5.28 for this reason, the revised accordingly. analytical results have been Equipment The analytical equipment used was as follows: Instrument: Fractovap AID/f manufactured by C. Erba, Milan, Italy Detector: electron capture (63Ni) concentric tube, excited with a pulsing current of 100 ,us intervals, 5V potential Column: glass, with incorporated evaporator for direct injections, 200 cm in length and 3 mm inter- nal diameter Stationary (1) 1 % OV-17; (2) 1 % QF-1 (mainly for a phase: better determination of HEOD and p,p'- DDE; (3) 1 % SE-30+QF-1 (2:1) Support mesh: GCP Sil, 100-120 mesh Carrier gas: argon-methane (10 %), 1.5 kgf/cm2 inlet pres- sure. Operational temperature: column, 1 80°C; detector, 2300C The method of direct calibration was used for quantitative determinations, i.e., standard solutions of insecticide mixtures were injected at 4-hourly intervals during the analysis and the percentage quotients obtained by dividing the area of a sample peak by the background current and the area of a standard peak by the corresponding background 364 STORAGE OF CHLORINATED PESTICIDES IN HUMAN TISSUES current were compared. In this way small variations in the experimental conditions could be corrected. Since the distribution of the analytical data was positively skew, the geometrical mean on transform- ed original data; i.e., log (ppm x 000) was also calculated. The limit of sensitivity was considered to be 0.004 ppm. RESULTS AND DISCUSSION According to the approximately log-normal dis- tribution of the analytical data, the geometrical mean is considered to be the best central value of this distribution. In this discussion, however, the arithmetic mean is used for comparison with data from other studies, in most of which this parameter only was used. The frequency with which organochlorine com- pounds were found in the different tissues is shown in Table 3. It will be seen that (1) heptachlor epoxide was present in most samples of fat and liver but in only one spleen sample, and was not detected in kid- ney and brain samples; (2) HEOD was found in many samples of autopsy fat, in fewer samples of liver and biopsy fat, and in one sample each of spleen and brain; (3)p,p'-DDE was always present in samples of fat and liver, very often in samples of spleen, and moderately frequently in kidney and brain samples; (4) p,p'-TDE was found only in one sample of spleen; (5) o,p'-DDT was found quite often in kidney samples but seldom in other tissues: it was never found in autopsy fat and was very seldom found in biopsy fat (1 sample out of 33); (6)p,p'-DDT was found in almost all samples of fat, in many liver and kidney samples, and in few brain and spleen samples. From Table 4 it can be seen that DDT and its metabolites were always present at higher levels than either heptachlor epoxide or HEOD, while HCH, HHDN, and heptachlor were either absent or present in concentrations below the sensitivity of the analytical method used. No explanation can be given for the apparent differences in concentration between residues in autopsy and biopsy fats because of the great variability in the levels. p,p'-DDT and p,p'-DDE were found most frequently in the samples analysed. The average con- centrations of heptachlor epoxide, HEOD, and p, p'-DDT and its derivatives were higher than in most surveys, although the average concentration of HEOD in samples of body fat collected in another part of Italy (Liguria) were similar to those found in Ferrara. The constant presence of p,p'-DDE, indepen- dent of levels of total equivalent DDT, seems to be due in part to exogenous p,p'-DDE. The ratio of p,p'-DDE to total equivalent DDT was found to be 38-56% in complete diets in the USA (Durham et al., 1965), 60% in milk in England (Robinson & McGill, 1966) and 32% in 516 normal average diets in the USA (Duggan, 1969). In Ferrara, the ratio was 3.5% in fruits, 5.1 % in vegetables, 52.9% in cow's milk, and 55.5% in animal fats (Paccagnella et al., 1971). With regard to chlorinated compounds in non- fatty tissue, the results in Table 5 show that the average levels are always less than those in fatty tissue, and liver samples and, to a lesser degree, Table 3. Number of samples of fatty and nonfatty tissue containing organochlorine compounds Human No. of Heptachlor HEOD p,p'-DDE p,p'-TDE o,p'-DDT p,p'-DDT tissue samples No. % No. % No. % No. % No. % No. % fat (autopsy) 52 36 69.2 38 73.1 52 100.0 0 0 0 0 51 98.1 fat (biopsy) 33 18 54.5 6 18.2 33 100.0 0 0 1 3.0 28 84.8 liver 23 8 34.8 8 34.8 23 100.0 0 0 3 13.0 11 47.8 kidney 15 0 0 0 0 4 26.7 0 0 6 40.0 8 53.3 brain 15 0 0 0 0 4 26.7 0 0 2 13.3 4 26.7 spleen 13 1 7.7 1 7.7 8 61.5 1 7.7 2 15.4 2 15.4 total 151 63 41.7 53 35.1 124 82.1 1 0.7 14 9.3 104 68.9 365 L. PRATI ET AL. Table 4. Residues (ppm) of chlorinated organic compounds in fat taken at autopsy or biopsy Autopsy fat (52 samples) Biopsy fat (33 samples) Geometrical Compound total fat S.E. a Rangeb Geomean x S.E. a Range b Geometric (85 sam- mean ~~~~~~~ ~mean pIes) heptachlor opoxide 0.51 0.060 N.D.-1.58 0.140 0.24 0.053 N.D.-1.02 0.045 0.090 HEOD 0.91 0.115 N.D.-3.55 0.221 0.31 0.130 N.D.-3.15 0.012 0.071 p,p'-DDE 9.94 1.131 0.41-40.50 6.888 7.04 1.332 0.73-41.17 5.052 6.108 o,p'-DDT - - - - - - N.D.-0.66 - - p,p'-DDT 4.04 0.615 N.D.-22.16 2.136 2.60 0.567 N.D.-16.50 0.838 1.485 DDT total equivalent 15.07 1.822 0.63-65.62 10.600 10.44 2.008 0.81-62.20 7.483 9.262 total 16.45 1.924 0.77-68.16 11.120 10.99 2.012 0.81-62.20 7.940 9.760 a S.E. = standard error. b N.D. = not detectable. kidney samples show residual concentrations higher than in other organs (spleen and brain). A comparison of the results obtained in this study with those reported by other authors (Casarett et al., 1968; de Vlieger et al., 1968) (Table 6) shows that: (1) higher levels of all chlorinated compounds were in liver samples; (2) higher levels ofp,p'-DDT were found in kidney samples than those reported from the USA, whereas the converse is true ofp,'p-DDE; (3) HEOD was not found in brain samples although it has been reported from the Netherlands and the USA. In the USA, the concentration of DDT was found to be higher than that ofDDE and our values for DDE were not higher than those in the USA; (4) the results show higher total average residual levels in spleen samples than those reported in the USA. Similarly, we found a generally higher level of chlorinated compounds in nonfatty tissues than other investigators (particularly in the Netherlands and the USA) have found. It should be noted, however, that these are generalizations only since the distribution of each compound was not recorded in every sample. Finally, for percentage ratios of DDE (measured as DDT) to total equivalent DDT, it was observed (Table 7) that for fatty tissues they show a low variability (from a minimum of 47,3% to a maximum of 100%), while those for liver and other organ tissues have a fairly scattered distribution (Table 8). It will be noted that in liver samples DDE was frequently found without DDT whereas in fat DDE and DDT were always associated; this is in accor- dance with findings reported from the Netherlands. It can therefore be assumed that fat is essentially a storage tissue while levels of chlorinated compounds in liver could indirectly indicate a progressive meta- bolic situation. It is also of interest to note the more frequent presence of DDE compared with DDT in spleen (of 13 samples, 7 showed DDE only and 1 DDT only), and of DDT compared with DDE in kidney (of 15 samples, 7 showed DDT only and 3 DDE only). Since samples were taken from many different subjects, it is not possible to reach a conclusion on the dynamic distribution of p,p'-DDT and p,p'- DDE in the different tissues. 366 STORAGE OF CHLORINATED PESTICIDES IN HUMAN TISSUES 367 Table 5. Residues (ppm) of chlorinated pesticides in nonfatty tissue samples Chlorinated organic compound b Heptachlor HEOD p.p'-DDE o,p'-DDT p,p'-DDT equivalent Total Liver (23 samples) x 0.07 0.09 0.55 0.07 0.63 1.31 1.47 S.E. 0.024 0.028 0.146 0.049 0.344 0.499 0.506 range N.D.-O.34 N.D.-0.40 0.03-2.83 N.D.-1.10 N.D.-8.00 0.03-11.32 0.03-11.32 geometric mean 0.015 0.016 0.293 0.007 0.039 0.476 0.566 Kidney (15 samples) x - - 0.02 0.08 0.43 0.54 0.54 S.E. - - 0.014 0.037 0.191 0.221 0.221 range - - N.D.-0.20 N.D.-0.54 N.D.-2.60 N.D.-2.92 N.D.-2.92 geometric mean - - 0.008 0.018 0.053 0.098 0.098 Brain (15 samples) xc - - 0.02 0.04 0.22 0.29 0.29 S.E. - - 0.010 0.029 0.140 0.161 0.161 range - - N.D.-0.13 N.D.-0.42 N.D.-2.00 N.D.-2.09 N.D.-2.09 geometric mean - - 0.008 0.007 0.015 0.022 0.022 Spleen (13 samples) x - - 0.10 0.02 0.04 0.19 0.22 S.E. - - 0.057 0.016 0.029 0.083 0.112 range N.D.-0.19 N.D.-0.27 N.D.-0.69 N.D.-0.19 N.D.-0.34 N.D.-0.95 N.D.-1.41 geometric mean - - 0.019 0.007 0.007 0.033 0.034 a S.E. = standard error. b N.D. = not detectable. 368 L. PRATI ET AL. Table 6. Average residues (ppm) of chlorinated pesticides in nonfatty human tissues: comparison of the results of de Vlieger et al. (1), Casarett et al. (2), and the present study (3) No. of Heptachlor DDE TDE DDT totalTissue samples Hepoxide HEOD as as o,p'-DDT p,p'-DDT equivalentsamples ~~~~DDT DDT (1) 11 - 0.034 0.14 - - - 0.14 Liver (2) 42 0.0019 0.0037 0.222 0.0362 - 0.0467 0.3049 (3) 23 0.07 0.09 0.62 - 0.07 0.63 1.31 (2) 38 0.0009 0.0056 0.232 0.0024 - 0.0827 0.3171 Kidney (3) 15 - - 0.03 - 0.08 0.43 0.54 (l)a 28 - 0.006 0.023 (1) b 28 - 0.009 0.034 Brain (2) 32 0.0002 0.0031 0.0922 0.0022 - 0.0105 0.1049 (3) 15 - - 0.03 - 0.04 0.22 0.29 (2) 27 - 0.0021 0.0339 0.0034 - 0.0112 0.0485 Spleen (3) 13 0.01 0.02 0.11 0.01 0.02 0.04 0.19 a Grey matter. b White matter. Table 7. Weighted values for ratio of DDE (as DDT) Table 8. Residues of DDE and DDT in various organs; to total equivalent DDT number of positive samples Tissue No. of x S.E. n Range Fat Liver Kid- Brain SpleenTissue (autopsy)Lie ney kidney 11 0.28 0.139 0 -1.00 DDE only 1 12 3 2 7 brain 6 0.36 0.204 0 -1.00 DDT only 0 0 7 2 1 liver 23 0.76 0.290 0.24-1.00 DDE and DDT present 51 11 1 2 1 fat (autopsy) 52 0.76 0.095 0.47-1.00 DDE and DDT absent 0 0 4 9 4 spleen 9 0.80 0.396 0 -1.00 total no. of samples examined 52 23 15 15 13 a S.E. = standard error. STORAGE OF CHLORINATED PESTICIDES IN HUMAN TISSUES 369 RESUME ACCUMULATION DE PESTICIDES ORGANOCHLORES DANS LES ORGANES ET LES TISSUS HUMAINS DANS LA PROVINCE DE FERRARE (ITALIE) On a etudie les modalites de I'accumulation de residus de pesticides organochlores dans les tissus humains dans la province de Ferrare, region choisie parce que les taux d'utilisation de ces produits en agriculture y sont parmi les plus eleves d'Italie. Les analyses, effectuees par chromatographie gazeuse, ont porte sur une serie d'echantillons de tissus ou d'organes (tissu adipeux, foie, rein, rate et cerveau) preleves lors d'autopsies ou par biopsie chez 85 habitants des deux sexes, ages de 1 a 80 ans, qui n'avaient pas 6te specialement exposes aux insecticides. Les composes suivants ont ete identifies: p,p'-DDE,o,p'- DDT,p,p'-DDT, HEOD et heptachlore. Le DDT et ses derives ont e decel6s plus souvent et a des concentrations plus fortes que les autres composes. Les taux de residus 6taient beaucoup moins eleves dans les tissus non adi- peux que dans les tissus adipeux. Les teneurs moyennes en residus du foie, et dans une moindre mesure du rein, etaient plus fortes que celles des autres organes examines (cerveau, rate). La comparaison des donnees recueillies au cours de cette etude avec les observations faites dans d'autres pays montre que dans l'ensemble les tissus et organes des habitants de la province de Ferrare con- tiennent davantage de pesticides organochlores. REFERENCES Casarett, L. J., Fryer, G. C., Yauger, W. L., Jr& Klemmer H. W. (1968) Arch. environm. Hlth, 17, 306-311 de Faubert Maunder, M. J., Egan, H., Godly, E. W., Hammond, E. W., Roburn, J. Thomson, J. (1964) Analyst, 89, 168-174 de Vlieger, M., Robinson, J., Baldwin, M. K., Crabtree, A. N. & van Dijk, M. C. (1968) Arch. environm. Hith, 17, 759-767 Duggan, R. E. (1969) Ann. N. Y. Acad. Sci., 160, 173-182 Durham, W. F., Armstrong, J. F., Quinby, G. E. (1965) Arch. environm. Hlth, 11, 641-647 Paccagnella, B., Ghezzo, F., Prati, L., Fedrazzoni, U. & Belloni, G., (1971) Bull. Wld Hith Org., 45, 181-199 Robinson, J., McGill, A. E. J. (1966) Nature (Lond.), 212, 1037-1038 Zweig, G., ed. (1964) Analytical methods for pesticides, plant growth regulators, andfood additives, New York, Academic Press, vol. 2, p. 432