organo-chlorine insecticide adipose tissue in-south ... · brit. j. industr. med., 1965, 22, 220....

Brit. J. industr. Med., 1965, 22, 220.

ORGANO-CHLORINE INSECTICIDE CONTENT OF HUMANADIPOSE TISSUE IN-SOUTH-EASTERN ENGLAND

BY

J. ROBINSON, A. RICHARDSON, C. G. HUNTER, A. N. CRABTREE, and H. J. REES

From Shell Research Ltd., Tunstall Laboratory, Sittingbourne, Kent

(RECEIVED FOR PUBLICATION FEBRUARY 18, 1965)

One hundred specimens of human body fat, 50 necropsy and 50 biopsy, were collected in 1964from a semi-rural area in south-eastern England. The specimens were analysed by two methods andthe concentrations of seven organo-chlorine insecticides were determined. The results of the twoanalytical methods were in good agreement. Endrin and heptachlor epoxide were not detected inany of the specimens analysed for these compounds. The average concentrations of lindane,op'-DDT, and op'-DDE were very small: 0-015 p.p.m., 0 03 p.p.m., and 0-02 p.p.m. respectively.The average concentration of HEOD in the 100 specimens of fat (no significant difference in themeans of the necropsy and biopsy fats was detected) was 0-21 p.p.m., as in the 1961 survey. Theaverage concentration of total equivalent-DDT (DDT + DDE) in the 50 necropsy fats was 3-9p.p.m., as compared with 2-2 p.p.m. in 1961. In the biopsy fats the average concentration for maleswas 4 9 p.p.m., and for females 3-4 p.p.m. One method was suitable for the separate determinationof pp'-DDT and pp'-DDE. The average concentration of pp'-DDT was 1-0 p.p.m. in the 100necropsy and biopsy fats. The average concentration ofpp'-DDE was 2-2 p.p.m. in the 50 necropsyfats, and 3 1 p.p.m. and 2-0 p.p.m. in male and female biopsy fats respectively. The variability oftheconcentrations of these compounds in the necropsy specimens was generally greater than in thebiopsy specimens. No significant differences were found which were attributable to age or to thecause of death. It is considered that the amounts found of these compounds do not constitute ahazard to the population sampled in this survey. The design of surveys to determine the concentra-tions of organo-chlorine insecticides in man is discussed.

Organo-chlorine insecticides may be classified,according to their chemical composition, intothree groups: (a) diphenylethane derivatives, e.g.,pp'-DDT, pp'-DDE, Rhothane, Methoxychlor, etc.;(b) the y-isomer ofhexachlorocyclohexane, and toxa-phene, a chlorinated camphene; and (c) hexachloro-norbornene derivatives, e.g., aldrin, dieldrin, endrin,heptachlor, etc.*The various members of these groups of com-

pounds possess varying propensities for storage inthe body fat of experimental animals. The use ofthese insecticides in agriculture and public health,

with the consequent potential exposure ofman to thecompounds, has prompted a number of investiga-tions into the storage in man of, in particular, DDTand dieldrin. The presence of residues of thesecompounds in specimens ofnecropsy fat, obtained in1961, has already been reported (Hunter, Robinson,and Richardson, 1963). In this paper the results of asecond survey of specimens of human fat obtained in1964 are reported. The primary intention was toobtain information on the changes, if any, in theconcentrations in necropsy fats ofDDT and dieldrinderived materials during the three-year period, but

*The systematic names of the compounds discussed in this paper dieldrinare:

aldrin a product containing 95% of the compound1,2,3,4,10,10-hexachloro-l,4,4a,5,8,8a-hexa- endrinhydro-exo-1,4-endo-5,8-dimethano-naphthalene (HHDN)

pp'-DDT 1,1,1-trichloro-2,2-di-(4-chlorophenyl)ethane heptachlorpp'-DDE 1,1-dichloro-2,2-di-(4-chlorophenyl)ethyleneop'-DDT 1,1,1-trichloro-2,2-di-(2-chlorophenyl)ethane heptachlor epoxideop'-DDE 1 1-dichloro-2,2-di-(2-chlorophenyl)ethylene

220

a product containing 85% of the compound1,2,3,4, 10,lO-hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-exo-1,4-endo-5,8-dimethanonaphthalene (HEOD)

1,2,3,4,10,10-hexachloro-6,7-epoxy-I,4,4a,5,6,7,8,8a-octahydro-exo-1,4-exo-5,8-dimethanonaphthalene

1,4,5,6,7, 10,lO-heptachloro-4,7,8,9-tetrahydro-4,7-methyleneindene

1,4,5,6,7, 10,10-heptachloro-2,3-epoxy-2,3,4,7,8,9-hexahydro-4,7-methyleneindene

on January 5, 2020 by guest. Protected by copyright.

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ORGANO-CHLORINE INSECTICIDE CONTENT OF HUMAN ADIPOSE TISSUE 221

the investigation has been extended to include biopsyfats and also the determination of the residues ofother organo-chlorine insecticides. Specifically wehave analysed the specimens of fat, both necropsyand biopsy, for the presence of pp'-DDT, pp'-DDE(a metabolite of pp'-DDT), op'-DDT (an isomer ofpp'-DDT present in the technical material), y-BHC(lindane), heptachlor epoxide, endrin, and residuesderived from the insecticides aldrin and dieldrin.Both the latter insecticides give rise to residues of acompound which is conveniently abbreviated toHEOD; HEOD is the major component of technicaldieldrin and it is also a biochemical conversionproduct of the major component in technical aldrin.

Materials and Methods

Specimens of body fat were obtained from 50 consecu-tive necropsies of human subjects who died natural oraccidental deaths in the same semi-rural area in south-eastem England as that surveyed in 1961. In addition 50specimens of subcutaneous or omental fat were obtainedby laparotomy from hospital patients in the same ruralarea. All the patients were in a fair to good nutritionalstate. None of the persons from whom specimens wereobtained had had any known occupational exposure toorgano-chlorine insecticides. Of the 50 necropsy fats, 28were from men and 21 from women; the sex of the re-maining person was not reported to us. The samples ofbiopsy fats consisted of 23 specimens from men and 27from women. The age distributions of the two groups ofpersons are shown in Figure 1. The causes of death, inthe case of the necropsy specimens, were classified intofour categories, and the numbers of fats corresponding toeach of the categories are shown in Table 1.Each fat was analysed by two different methods. One

of these methods was the same as that used in the previoussurvey (Hunter et al., 1963), namely, alkaline hydrolysisof the fat followed by the determination of HEOD,

201 NECROPSY FATS

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CLASSIFICATION OFTABLE 1

NECROPSY FATS BY CAUSE OFDEATH

Number of Deaths Corresponding tothe Specified Cause of Death

Cause of DeathMales Females

Neoplasms 4 3

Cardiovascular diseaseincludir.g kidney disease 18 11

Infections 3 3

Accidents 3 4

pp'-DDE, and endrin in the unsaponifiable fraction bygas-liquid chromatography using an electron capture typedetector. The column size was 92 cm. x 0 4 cm., thestationary phase liquid was Oronite-Polybutene 128, 2%on silanized Celite, and the column temperature was168°C. The other analytical method did not involve ahydrolysis step and could therefore be used for the deter-mination of alkali-unstable materials. An extract of thefat in redistilled light petroleum (b.p. 64-67°) was pre-pared, and the organo-chlorine compounds were extractedby shaking into dimethylformamide. These compoundswere then re-extracted by light petroleum after an aqueoussodium sulphate solution had been added to the dimethyl-formamide. The hydrocarbon layer was separated fromthe aqueous phase, washed with distilled water, and driedwith anhydrous sodium sulphate. The dried lightpetroleum extract was then run through a Florisiladsorption column and the organo-chlorine compoundswere eluted with suitable solvent mixtures. The eluateswere then injected into a gas-liquid chromatographicapparatus fitted with an electron capture detector. Thecolumn conditions were the same as those used in thehydrolytic method. Using this technique it is possible todetermine y-BHC, heptachlor epoxide, aldrin and dieldrin-derived material (HEOD), endrin, pp'-DDE, pp'-DDT,op'-DDT, and pp'-DDD. Considerable difficulty was

BIOPSY FATS

2;

I4!0

7

60

FIG. 1.-Age distribution of persons fromwhom specimens of fat were obtained.

8b 100

AGE IN YEARS

0 20 40 601- I I


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experienced in the determination of pp'-DDT as a resultof its instability in the gas-liquid chromatographic con-ditions used; this instability appears to be mainly theresult of catalytic breakdown. Glass columns and glass-lined injection ports were used, and the temperature wasthe minimum compatible with an acceptable retentiontime. The performance of the columns deteriorated withuse: artefacts were produced which simulated op'-DDTas regards retention time, with a consequent reduction inthe apparentpp'-DDT content. It was found necessary tochange the columns frequently to ensure that reliablepp'-DDT results were obtained. Full details of theseanalytical techniques will be published elsewhere. Theextractable lipids were determined in about half the fatsby taking an aliquot of the light petroleum extract,evaporating the solvent, and weighing the residue.

ResultsThe concentrations of organo-chlorine insecticides

determined by the two methods are summarized inthe form of histograms. Figure 2 gives the concen-tration distribution for HEOD by the two analyticalmethods, and Fig. 3 gives the distribution of totalequivalent-DDT. In Fig. 4 the results obtained forpp'-DDT andpp'-DDE respectively are summarized.A number of the specimens of fat were also analysedfory-BHC (lindane), endrin, heptachlor epoxide, andfor the op'-isomer of DDT; the results are given inTable 2. The concentrations of extractable lipiddetermined in some of the specimens are shown inthe histogram in Figure 5.

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NON-HYDROLYTIC METHOD,BIOPSY FATS

7 rm*2 0 4 0-6 a8 I 0

HYDROLYTIC METHOD,NECROPSY FATS

O 2 ' 0'4 ' 0'6 0'8 *i10

HEOD. ppm

FIG. 2.-Concentration of HEOD in specimens of human fat.

222

20iNON- HYDROLYTIC METHOD,NECROPSY FATS

151

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NON-HYDROLYTIC METHOD,BIOPSY FATS

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TOTAL EQUIVALENT- DDT, ppm

II 16 7 8 9, 10 11 12 13 14 IS 16

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TOTAL EQUIVALENT-DDT, ppm

FIG. 3.-Total equivalent-DDT in specimens of human fat.

Discussion

Comparison of Analytical Techniques.-Two ana-lytical methods were used for the determination ofHEOD and equivalent-DDT. These methods are notcompletely independent in that both include a finalstep using gas-liquid chromatographic equipmentwith an electron capture detector. However, theclean-up procedures before injection into the gaschromatographic equipment are quite different, andthe concordance of the results of the two methods isa measure of the validity of the methods in respect ofthe identity of the components. The concordance isgood considering the low levels found (Table 3), andthe overall picture of the distribution of the com-

pounds between the specimens of fat can be acceptedwith confidence. The amounts of HEOD estimatedby the two methods may differ because of thepresence of op'-DDE and pp'-dehydrochlorinated-DDD, neither of which would be completely separ-ated from HEOD in the hydrolytic method. Theywould not, however, interfere in the non-hydrolyticmethod.

Comparison of Organo-Chlorine InsecticideResidues in Various Specimens of Human Fat.-Asthe analytical results appear to be generally trust-worthy their implications can be discussed. Theresults obtained by the alkaline hydrolysis techniquewill be used unless otherwise stated.

Before examining the results for the human fats itis helpful to summarize the conclusions which maybe drawn from investigations with experimentalanimals. These conclusions are as follows. Theconcentration of an organo-chlorine insecticide inthe body fat of an animal is a function, firstly, of theamount of the compound in the food of the animal;secondly, of the period of exposure to the insecticide-containing diet; thirdly, of the dynamic equilibriumbetween the blood and tissues as measured by thepartition coefficients between them; and fourthly, ofthe rate of elimination of the compound. There isoften a tendency for the concentration in the fat offemale animals to differ from that in male animals.Finally, there are considerable variations betweenanimals of the same sex even when the exposure isconstant. But for a given animal the amount in the

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35-1 BIOPSY FATS

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TABLE 2CONCENTRATIONS OF LINDANE, ENDRIN, HEPTACHLOREPOXIDE, AND op'-DDT IN SPECIMENS OF HUMAN FAT

No. of Mean (arithmetic) Concentration ofInsecticide Specimens Insecticide (p.p.m.; range of

Analysed observations)

Lindane(y-BHC) 20 0 015 (<0 002-003)

Endrin -91 <0-02*

Heptachlorepoxide 20 <0 01*

op'-DDT 20 0 03 (<002-0050)

op'-DDE 20 0-02 (<0-01-0060)

*Less than experimental limits of detection.

fat is proportional to the amount in the food withinthe experimental error; and the relation between theconcentration in the fat and the period of exposureto the diet appears to be of the asymptotic type, i.e.,a steady level is finally reached when intake equalsexcretion. This has been fully established for HEODby Ludwig, Weis, and Korte (1964). It is also

FIG. 4.-Concentration of pp'-DDT andpp'-DDE in specimens ofhuman fat.

4 5 6 7 8 9 10

pp'-DDE, ppm

generally compatible with the experimental resultsfor rats, dogs, sheep, and monkeys althoughunequivocal proof for the attainment of a steadystate has not been obtained for all the organo-chlorine insecticides in all the species. The findingthat the concentrations in fat do not increasecontinually when animals are subjected to constantexposure is of obvious importance.

It must be remembered, however, in attempting toextrapolate the results of animal experiments to man

TABLE 3AVERAGE CONCENTRATIONS OF HEOD AND TOTAL

EQUIVALENT-DDT IN SPECIMENS OF HUMAN FAT

Mean (arithmetic) Concentrations ofOrgano-chlorine Insecticide (p.p.m.)

Extraction _Technique Necropsy Fats Biopsy Fats

Total TotalHEOD Equivalent- HEOD Equivalent-

DDT DDT

Alkaline hydrolysis 0-28 5-2 0-25 4-5

Non-hydrolysis 0-26 4-5 0-25 4 5

224

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2°1 NECROPSY FATS (33)

5-

U)

z

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IL

0

z 4i40 60 80 100

zwUJ

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EXTRACTABLE LIPID 1°/O W/W)

FIG. 5.-Concentrations of extractable lipid in specimens of humanfat.

that there are a number of differences between theconditions of animal experiments and those prevail-ing in the case of man. In particular, the exposure ofthe animals is as uniform as possible and the environ-ment of the animals is usually closely controlled. Inman, his potential exposure varies as regards bothquantity and form. The distribution of residues inthe human diet is not uniform. It may vary from onetype of food to another and also from one season ofthe year to another, and in addition non-dietaryexposure may be occurring. Again, man's physicalenvironment is more variable. These differences inexposure between persons, combined with the differ-ences between persons in the dynamics of uptake,distribution, and elimination of organo-chlorineinsecticides will result in considerable variations inthe concentrations of these compounds in the bodyfat of different persons, a conclusion which is inaccordance with the observed variation in residues(see Figs 2, 3, and 4).The persons from whom the specimens were ob-

tained differed in regard to sex, age, extractable lipidcontent, dietary intake, nutritional status, and, in thecase of the necropsy fats, the cause of death. Thepossibility that the concentrations of organo-chlorineinsecticides in the specimens ofhuman fat are related,causally or otherwise, to these variables cannot beexcluded on a-priori grounds. In attempting toisolate the effects of these variables, the number ofspecimens in the corresponding sub-sets becomesquite small, and this limitation must be borne inmind when it is inferred that a given variable has no

significant effect on the concentration of organo-chlorine compounds in the body fat. However, anexamination of the results (by the Spearman rankcorrelation technique) for correlations between the

concentration of HEOD or total equivalent-DDTand the age of the person from whom the body fatwas obtained indic4ted that the concentration didnot depend on age or, in the case of necropy fats, oncause of death, classified as in Table 1. The extract-able lipid contents of the necropsy and biopsy fatswere not significantly different, and no significantcorrelations were found between the concentrationof the insecticides and the concentrations of lipid inthe specimens of fat. It was concluded, therefore,that the 100 specimens of fat could be classified intofour groups, namely, male or female necropsy fatsand male or female biopsy fats. In the analysis ofvariance of the necropsy fats classified according tocause of death it was found that the variances of eachgroup were different. Furthermore, the concentra-tions of HEOD etc. found were obviously notdistributed symmetrically about their averages, i.e.,the distributions were skew with a lot of low valuesbut a few relatively high ones. Statistical analysisconfirms that these distributions are skew, and insuch cases it is usual to carry out statistical tests interms of the logarithms of the concentrations. Thelogarithms are normally distributed, and compari-sons between the geometric means can be made bythe standard statistical methods.

HEOD, Distribution in Necropsy and BiopsyFats.-The geometric means of the necropsy fats ofmale and female origin are not significantly different,neither are the corresponding means of the biopsyfats. In the latter, however, the variance of thespecimens of female fat is significantly higher(p = 0-025) than that of the male fats. A comparisonof the results for necropsy and biopsy fats showedthat the geometric means are not significantly differ-ent for either the male or female fats, but that thevariance of the necropsy specimens is significantlygreater than that of the biopsy fats in the case of males(p = <0 001) though not in that of females. Thestatistical analysis for HEOD residues is summarizedin Table 4.The significantly higher variance of male necropsy

fats as compared with that of the biopsy fats appearsto indicate that the process of dying has an effect onthe concentration ofHEOD in the body fat of males.This effect appears to be independent of age at deathsince, as pointed out above, age and HEOD con-centration are not related. Why the variances of thefemale necropsy and biopsy fats are not significantlydifferent is obscure.The results obtained from the necropsy fats, male

and female, have been pooled for comparison withthe results from the specimens obtained in 1961(Hunter et al., 1963). No significant difference inmean values or variance between the two sets of


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TABLE 4CONCENTRATION OF HEOD IN NECROPSY AND BIOPSY

FATS: STATISTICAL ANALYSIS

Concentration ofHEODin Fat Expressed as Geometric

Type of Sex Year log10 (p.p.m. x 100) MeanFat _ (p.p.m.)

Mean|M Variance

Necropsy Male 1964 1-3954 0-1670 0-25Necropsy Female 1964 1-2716 0-0846 0-19Biopsy Male 1964 1-4003 0-0230 0-25Biopsy Female 1964 1-3240 0 0518 0-21Necropsy Male and

femalecom-bined 1964 1-3295 0-1386 0-21

Necropsy Male andfemalecom-bined 1961 1-3222 0-1155 0 21

results was detected. This conclusion is of greatimportance. It implies that in the population survey-ed the intake and elimination of dieldrin (and/oraldrin) had reached a state of balance by 1961, andthat the daily intake has not changed significantlysince then. Factors other than these may, of course,be involved.

It is interesting to compare the results of thesurveys in the United Kingdom with those of theUnited States, the only other country for whichinformation has been published; these are summar-ized in Table 5.

TABLE 5CONCENTRATION OF HEOD IN HUMAN FAT:COMPARISON OF U.K. AND U.S.A. SAMPLES

MeanCountry Year No. of HEOD Reference

Specimens Content(P-P-m-)

U.K. 1961 131 0-21* Hunter et al. (1963)U.K. 1964 100 0-21* This communicationU.S.A. 1961/2 28 0-08* Dale & Quinby (1963)U.S.A. 1962/3 64 0llt Hoffman et al. (1964)

* Geometric mean; t arithmetic mean.

The specimens of human fat analysed in the 1961/2survey (Dale and Quinby, 1963) were obtained fromthree different areas of the United States of America.One of the areas (Louisville, Kentucky) gave resultswhich are significantly lower than those for the otherareas. For the other two (Wenatchee, Washingtonand Phoenix, Arizona) the geometric means, 0-15,are not significantly different from those obtained inthe two United Kingdom surveys in 1961 and 1964.The results of the survey in 1962/3 (Hoffman,Fishbein, Andelman, 1964) are similar to those forother surveys in the U.K. and U.S.A. but sufficientinformation is not available for detailed comparisonsto be made. It is also noteworthy that the results for

the two U.S. surveys appear, as in the case of theU.K. surveys, to have positively skew distribution.

Total Equivalent-DDT, Distribution in Necropsyand Biopsy Fats.-A comparison of the results fornecropsy fats from male and female subjects showsthat the geometric means are not significantly differ-ent, whereas for biopsy fats the mean concentrationin males is significantly higher than that in females.Necropsy and biopsy fats do not differ significantlyas regards the mean content of total equivalent-DDTfor either males or females, but the variances of thenecropsy fats, both male and female, are significantlygreater than those of biopsy fats. These results aresummarized in Table 6.

TABLE 6CONCENTRATION OF TOTAL EQUIVALENT-DDT INNECROPSY AND BIOPSY FATS: STATISTICAL ANALYSIS

Concentration of TotalEquivalent-DDT in Fat Geometric

Type of Sex of Expressed as MeanFat Person Year log10 (p.p.m. x 10) (p.p.m.)

Mean Variance

Necropsy Male 1964 1-6590 0-1963 4-6Necropsy Female 1964 15406 01064 3-5Biopsy Male 1964 1*6879 00301 4 9Biopsy Female 1964 1*5376 0-0370 3 4Necropsy Male and

femalecom-bined 1964 1-5952 0-1636 3 9

Necropsy Male andfemale

Necropsy Male andfemalecom-bined 11961 1 13444 0-1118 2-2

The mean results (male and female) for thenecropsy fats in 1964 are significantly higher(p = 0002) than for the 1961 samples. Thissignificant increase in the concentration of DDT-type material in the three-year period warrantsfurther investigation since it may be attributed toseveral causes. The most probable causes are eitherthat the exposure of the population surveyed in 1961and 1964 has increased in the three-year period, orthat the exposure has remained fairly constant butthe concentration in the body fat had not reachedequilibrium in 1961. In either case we have nogrounds for assuming that the concentration inhuman fat in this area of England in 1964 representsan equilibrium value; this can only be demonstratedby surveys in subsequent years.The concentration of DDT-type materials in

human fat has now been determined in a number ofcountries and the results are summarized in Table 7.

In our first paper attention was drawn to thesignificant correlation between the concentration ofHEOD and total equivalent-DDT in the body fat.

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TABLE 7CONCENTRATION OF TOTAL EQUIVALENT-DDT IN THE BODY FAT OF THE GENERAL POPULATION: COMPARISON

BETWEEN DIFFERENT COUNTRIES AND YEARS

No. of TotalCountry Year Specimens Type of Specimens Equivalent- Analytical Method Reference

DDT (ppm.)

U.S.A. 1950 75 Biopsy and necropsy 5 3* Schechter-Haller Laug et al. (1951)

U.S.A. 1955 49 Biopsy 19.9* Schechter-Haller Hayes et al. (1956)

U.S.A. 1954-56 61 Biopsy and necropsy 11-7* Schechter-Haller Hayes et al. (1958)

U.S.A. 1961-62 117 _ 12-9* - Quinby et al. (1963)

U.S.A. 1961-62 28 Biopsy and necropsy 10-7* Schechter-Haller Dale and Quinby (1963)

U.S.A. 1961-62 28 Biopsy and necropsy 6.7* G.L.C. micro-coulometric Dale and Quinby (1963)detector

U.S.A. 1962-63 282 Necropsy 11-2* G.L.C. micro-coulometric Hoffman et al. (1964)detector

U.S.A. (Alaska) 1960 20 Biopsy 3.0* Schechter-Haller Durham et al. (1961)

U.S.A. 1955-56 16 Biopsy (non-meat eaters) 5 9* Schechter-Haller Hayes et al. (1958)

Germany 1958-59 60 Necropsy 2-3* Schechter-Haller Maier-Bode (1960)

Canada 1959-60 62 Necropsy 4.9* Schechter-Haller Read and McKinley (1961)

France 1961 10 Necropsy 5.2* Schechter-Haller Hayes et al. (1963)

U.K. 1961-62 131 Necropsy 2.2t G.L.C. electron capture Hunter et al. (1963)detector

U.K. 1964 100 Biopsy and necropsy 4 Ot G.L.C. electron capture This communicationdetector

*arithmetic mean; t geometric mean.

An examination of the results for this survey showsthat the Spearman rank correlation coefficientsHEOD/total equivalent-DDT for the necropsy fatsis 0 53 (p = 0-001); for male biopsy fats it is 0 34(not significant); and for female biopsy fats 0-62(p = 0-001). From the significant correlation ob-tained for the 1961 survey it was tentatively inferredthat both HEOD and DDT had reached equilibriumlevels. This hypothesis cannot be invoked to accountfor the significant correlations in the 1964 survey.The organo-chlorine insecticides tend to be concen-trated in certain components of the diet (particularlythe lipoidal constituents) and an alternative hypo-thesis is that the correlation is a reflection of theamounts and sources of the fatty foods eaten by anygiven person.

Distribution of pp'-DDT and pp'-DDE.-Theconcentrations of these compounds could only bedetermined by the non-hydrolytic technique usingpartition between dimethyl-formamide and elutionthrough a Florisil column as the clean-up procedurebefore injection into the gas-liquid chromatographicequipment. The distribution of these compounds inthe biopsy and necropsy samples is shown in Figure4. No significant differences between the meanpp'-DDT concentration of the male and female fatswere found for either necropsy or biopsy specimens

or between necropsy and biopsy specimens. Thevariances of the biopsy fats, however, were signifi-cantly less than those of the necropsy fats (p = 0-025,males; and P = 0-05, females). In the case of pp'-DDE the mean concentrations in the male samples,both biopsy and necropsy, were significantly greaterthan those in the female samples (p = 0-05, necropsyP <0 005, biopsy). Once again the variances of thenecropsy samples were significantly greater than thoseof the biopsy samples (p = 0 025, males and females).The results are summarized in Table 8.A comparison of the results of this survey for the

concentrations ofpp-'DDT and pp'-DDE in humanfat and the results for surveys in other countries isgiven in Table 9.The results of a survey of the DDT content of

necropsy fats in Budapest have been summarized(Denes, 1962). The ratio of the concentration ofpp'-DDE to that of pp'-DDT shows some variationfrom one survey to another, but in all cases theconcentration of pp'-DDE is greater than that ofpp'-DDT. This is in contrast with the ratio obtainedin trials in which volunteers were given daily doses ofrecrystallized or technical DDT, when the averageratio ofDDE/DDT was about 0-2 (Hayes, Durham,and Cueto, 1956). It is possible therefore that theratio found in the general population indicates thatexposure to PP'-DDE as well as to pp'-DDT is


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22DBRITISH JOURNAL OF INDUSTRIAL MEDICINE

TABLE 8CONCENTRATION OF pp'-DDT AND pp'-DDE IN NECROPSY AND BIOPSY FATS COLLECTED IN 1964

Concentration of pp'-DDT Concentration of pp'-DDEin Fat Expressed as pp'-DDT in Fat Expressed as pp'-DDE

Type of Fat Sex logl, (p.p.m. x 10) (p.p.m.) log,0 (C x 10) (p.p.m.)Geometric Geometric

Mean Variance Mean Mean Variance Mean

Necropsy M 1-0135 0-1237 1-0 1-3838 0-1479 2-4

Necropsy F 0 9800 0-1275 1.0 1*3154 0-1098 2-1

Biopsy M 1-0716 0 0475 1-2 1-4983 060558 3-1

Biopsy F 1-0116 0-0575 1-0 1-2981 0 0470 2-0

TABLE 9CONCENTRATION OF pp'-DDT and pp'-DDE IN THE BODYFAT OF THE GENERAL POPULATION: COMPARISON OF

SURVEYS IN DIFFERENT COUNTRIES

ConcentrationNo. of (p.p.m.) Ratio

Country Year Specimens pp'-DDE/pp'-DDT pp'-DDE pp'-DDT

U.S.A. 1950 75 5.3 - -

U.S.A. 1955 49 7-4 11-2 1-5U.S.A. 1954-56 61 4 9 6-1 1-2U.S.A. 1961-62 117 3-4 8-1 2-1U.S.A. 1961-62 28 2-4* 3-8 1-6U.S.A. 1962-63 282 2-9 7-4 2-6U.S.A.(Alaska) 1960 20 0-8 2-0 2-5U.S.A. (meatabstainers) 1955-63 16 2-3 3-2 1*4Canada 1959-60 62 1-6 3-0 1 9Germany 1958-59 60 1-0 1-2 1-2France 1961 10 1-7 3-1 1-8U.K. 1964 100 1-0 2-3 2-3

*op'-DDT + pp'-DDT.

occurring. This inference is in accordance with theobserved presence ofpp'-DDE in the human diet inthe U.S.A. (Fischbach, 1962; Mills, 1963; Williams,1964) and in the U.K. (Robinson, Richardson, andBush, unpublished).

Residues of Other Organo-chlorine Insecticides inHuman Fat.-Ninety-one of the specimens of fatwere analysed for endrin. None was found in any ofthem (experimental limit of detection, 0-02 p.p.m.).Similarly, analysis of 20 specimens for heptachlorepoxide also failed to reveal any detectable amountof this compound (experimental limit of detection,0-01 p.p.m.). The mean concentration of lindane in20 specimens was 0-015 p.p.m. This is much lowerthan the concentrations found in two surveys in theU.S.A. in which the average concentrations were 0-2and 0-6 p.p.m. lindane respectively (Dale and Quinby1963; Hoffman et al., 1964). The average concentra-tion of lindane in necropsy fats obtained in Francewas 1-2 p.p.m. (Hayes, Dale, and Le Breton, 1963).Twenty specimens of fat were also analysed for

op'-DDT and op'-DDE. In both cases the concentra-tions found were near the experimental limits of

detection, and the average concentrations were 003and 002 p.p.m. respectively. These results should beregarded as only tentative since the identities of thecompounds measured as op'-DDT and op'-DDEhave not been confirmed by other techniques.

Toxicological Significance of the Residues of Org-ano-chlorine Insecticides.-Direct knowledge of thepharmacodynamic behaviour and toxicity of thesecompounds in man is meagre. The concentration ofpp'-DDT in the body fat of volunteers given dailydoses (35 mg.) of this compound for periods of up to18 months have been reported (Hayes et al., 1956).No ill-effects were found in any of the men. Theaverage concentration of pp'-DDT in the body fatwas about 200 p.p.m. The study of men withoccupational exposure to these compounds is also ofassistance in interpreting the residues found in thegeneral population. The body fat of agriculturalworkers in the U.S.A. was found to have an averagecontent of 14 p.p.m. pp'-DDT, and one man withextensive occupational exposure had 648 p.p.m. ofpp'-DDT in his body fat. These values, and also theaverage values obtained for the general population inthe U.S.A. (2 to 7 p.p.m.), should be compared withthe average value of 1 p.p.m. found in this survey ofthe population in a semi-rural area of south-easternEngland.

In the case of dieldrin it has been found thatoccupationally exposed persons may have a concen-tration of HEOD in the blood which is some 30times that of the general population. These work-men appear to be in a normal state of health. It hasalso been shown that the concentration of HEOD inthe blood decreases when occupational exposureceases. Specimens of body fat from two men workingon an aldrin formulation plant contained 149 and44 p.p.m. of HEOD respectively. This picture ofincreased body burden when the exposure increases,and loss from the body when the exposure isremoved, indicates that there is no discontinuitybetween man and experimental animals in thepharmacodynamic behaviour of these compounds.

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Further, the comparison of the residues in the bodyfat of the general population with those in occupa-tionally exposed persons, who have not suffered anyapparent ill-effects as a result of their increased bodyburden, shows that the body burden of organo-chlorine insecticides in the general population is wellbelow that at which harmful effects would beexpected.

Design of Surveys to Determine Distribution ofOrgano-chlorine Insecticides in Man.-The concentra-tions of organo-chlorine insecticides in the body fatof man may be used, firstly, as an index of the bodyburden of these compounds and consequently of thehazard to the general population and, secondly, toenable us to follow the secular variations, if any, inthe average body burden. There is also a thirdpurpose served by these surveys. Two officialenquiries ('Use of Pesticides', 1963; 'Review ofPersistent Organo-chlorine Pesticides', 1964) haverecommended that the accretion of persistentinsecticides in the environment should be controlled.The occurrence ofresidues of these compounds in thegeneral population is of adventitious origin and theirconcentration in human body fat therefore consti-tutes an index ofthe general environmental contamin-ation. In view of these three uses of the results ofsurveys, it is essential that the design of surveys ofinsecticide residues in man should be such that validconclusions can be drawn from results obtained byaccurate and reliable analysis of the specimens offat.The statistical principles underlying the design of

surveys is adequately discussed in standard statisticaltexts but it would be desirable to use the experiencegained so far in insecticide residue surveys to improve,if necessary, the design of future surveys. It appears,for example, that significant differences may existbetween the concentrations in the body fats of malesand females, and that there may be differencesbetween people in different geographical areas. Therelationships between age and insecticide content ofbody fat, or between cause of death and insecticidecontent, appear to be non-significant in suchinvestigations as have been made, but in view of thenumbers of specimens involved it would be advisableto investigate these possibilities more fully. Thesignificant difference between the variability ofresidues in necropsy and biopsy fats is perhapsrelevant in this context. A further variable, whichhas not so far been investigated, is that of social

class; the diets of the various social classes differfrom one another, and there may be consequentdifferences in the body burdens of insecticides inpersons of the different social classes. All thesedifferences, observed or potential, emphasize theheterogeneity of the population being sampled inthese surveys. If valid inferences are to be madeconcerning either the average body burden of thewhole population of a country, or the changes ininsecticide content with time, it is essential that thesampling schemes are suitably designed for suchheterogeneous populations. The most suitable typeof scheme, on the evidence available, appears to bestratified sampling, with perhaps proportional allo-cation for each of the strata. The characteristics forconstructing the strata would be such variables assex, geographical location, and/or social class etc.Once suitable strata have been chosen it is necessaryto ensure that the fat specimens (or other tissues)selected for analysis are representative of the variousstrata. Unless the sampling scheme is so designed itwill be difficult, particularly in the case of specimenscollected at various times, to draw conclusionsregarding the population as a whole and the signifi-cance of secular changes.

We express our gratitude to Mr. R. L. Canney and toDr. G. B. Forbes for their interest and assistance in thissurvey.

REFERENCES

Dale, W. E., and Quinby, G. E. (1963). Science, 142, 593.Denes, A. (1962). Nahrung, 6, 48.Durham, W. F., Armstrong, J. F., Upholt, W. M., and Heller, C.

(1961). Science, 134, 1880.Fischbach, H. (1962). Symposium of the Food Protection Committee.

Washington.Hayes, W. J., Jr., Durham, W. F., and Cueto, C., Jr. (1956). J. Amer.

med. Ass., 162, 890.Quinby, G. E., Walker, K. C., Elliott, J. W., and Upholt,W. M. (1958). Arch. industr. Hith, 18, 398.Dale, W. E., and LeBreton, R. (1963). Nature (Lond.), 199,1189.

Hoffman, W. S., Fishbein, W. I., and Andelman, M. B. (1964).Arch. environm. Hith, 9, 387.

Hunter, C. G., Robinson, J., and Richardson, A. (1963). Brit. med. J.,1, 221.

Laug, E. P., Kunze, F. M., and Prickett, C. S. (1951). Arch. industr.Hyg., 3, 245.

Ludwig, G., Weis, J., and Korte, F. (1964). Life Sciences, 3, 123.Maier-Bode, H. (1960). Med. exp. (Basel), 1, 146.Mills, P. A. (1963). J. Ass. agric. Chem., 46, 762.Quinby, G. E., Hayes, W. J., Jr., and Durham, W. F. (1963). (In

preparation.) Quoted by Hayes and others.Read, S. T., and McKinley, W. P. (1961). Arch. environm. Hlth, 3,

209.Review of Persistent Organo-chlorine Pesticides. H.M. Stationery

Office, London, 1964.Robinson, J., Richardson, A., and Bush, B. Unpublished work.Use of Pesticides. Report of the President's Science Advisory Com-

mittee, Washington, 1963.Williams, S. (1964). J. Ass. official agric. Chem., 47, 815.


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organo-chlorine insecticide adipose tissue in-south ... · brit. j. industr. med., 1965, 22, 220....

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