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Children’s Health | Article
Effect of Endosulfan on Male Reproductive Development
Habibullah Saiyed,1 Aruna Dewan,1 Vijay Bhatnagar,1 Udyavar Shenoy,2 Rathika Shenoy,2 Hirehall Rajmohan,3
Kumud Patel,1 Rekha Kashyap,1 Pradip Kulkarni,1 Bagalur Rajan,3 and Bhadabhai Lakkad 1
1National Institute of Occupational Health (Indian Council of Medical Research), Meghani Nagar, Ahmedabad, India; 2Department of
Pediatrics, Kasturba Medical College, Mangalore, India; 3Regional Occupational Health Research Centre, Bangalore, India


                                                                                                               dehydrogenase, providing further evidence
 There is experimental evidence of adverse effects of endosulfan on the male reproductive system, but          of effects on spermatogenesis (Khan and Sinha
 there are no human data. Therefore, we undertook a study to examine the relationship between envi-            1996; Sinha et al. 1995). Exposure of younger
 ronmental endosulfan exposure and reproductive development in male children and adolescents. The              animals (3 weeks old) showed marked depletion
 study population was composed of 117 male schoolchildren (10–19 years of age) of a village situated           of spermatid count as well as decreased daily
 at the foothills of cashew plantations, where endosulfan had been aerially sprayed for more than 20           sperm production at a dose of 2.5 mg/kg/day
 years, and 90 comparable controls with no such exposure history. The study parameters included                (Sinha et al. 1997), which was earlier seen only
 recording of clinical history, physical examination, sexual maturity rating (SMR) according to Tanner         at 5 mg/kg/day in adult rats by the same investi-
 stages, and estimation of serum levels of testosterone, luteinizing hormone (LH), follicle-stimulating        gators (Sinha et al. 1995). More recent studies
 hormone, and endosulfan residues (70 study and 47 control subjects). Mean ± SE serum endosulfan               have shown that exposure of pregnant rats
 levels in the study group (7.47 ± 1.19 ppb) were significantly higher (p < 0.001) than in controls (1.37       to endosulfan at 1 mg/kg/day from day 12
 ± 0.40 ppb). Multiple regression analysis showed that SMR scoring for development of pubic hair,              through parturition leads to decreased sper-
 testes, penis, and serum testosterone level was positively related to age and negatively related to aerial    matogenesis in offspring (Sinha et al. 2001).
 exposure to endosulfan (AEE; p < 0.01). Serum LH levels were significantly positively related to AEE           Dalsenter et al. (1999) reported similar observa-
 after controlling for age (p < 0.01). The prevalence of congenital abnormalities related to testicular        tions at 3 mg/kg/day but not at 1.5 mg/kg/day,
 descent (congenital hydrocele, undescended testis, and congenital inguinal hernia) among study and            and they attributed this to strain variation
 controls subjects was 5.1% and 1.1%, respectively, but the differences were statistically nonsignifi-          (Dalsenter et al. 2003). Thus, experimental
 cant. Our study results suggest that endosulfan exposure in male children may delay sexual maturity           studies suggest that endosulfan can affect the
 and interfere with sex hormone synthesis. Our study is limited by small sample size and nonparticipa-         male reproductive system and also that these
 tion. Key words: endocrine disruptor, endosulfan, luteinizing hormone, male reproductive develop-             effects are likely to be greater if exposure occurs
 ment, sexual maturity rating, testosterone. Environ Health Perspect 111:1958–1962 (2003).                     during the developmental phase.
 doi:10.1289/ehp.6271 available via http://dx.doi.org/ [Online 22 September 2003]                                  Environmental exposure to a single chemi-
                                                                                                               cal over a long period of time is very rare. We
                                                                                                               came across a situation where endosulfan
Endosulfan (6,7,8,9,10,10-hexachloro-                   experimental animals. No data are available            was the only pesticide that had been aerially
1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-              for subacute or chronic exposure to endosul-           sprayed two to three times a year for more
benzodioxathiepin-3-oxide) is a broad-                  fan in human subjects; however, the subacute           than 20 years on cashew nut plantations situ-
spectrum insecticide and acaricide first regis-          and chronic toxicity studies of endosulfan             ated on hilltops in some villages of northern
tered for use in the United States in 1954 to           in animals suggest that the liver, kidneys,            Kerala, India (Figure 1). The population living
control agricultural insect and mite pests on a         immune system, and testes are the main tar-            in the valley had a significant chance of expo-
variety of field, fruit, and vegetable crops.           get organs [Agency for Toxic Substances and            sure to this pesticide during aerial spray and
Technical-grade endosulfan is composed of               Disease Registry (ATSDR) 2000].                        subsequently through other contaminated
two stereochemical isomers, α-endosulfan and                In recent years, there has been growing            environmental media. This population, there-
β-endosulfan, in concentrations of approxi-             concern about toxicity of a number of chemi-           fore, provided a unique opportunity to study
mately 70% and 30%, respectively. Use data              cals, including pesticides, on the male repro-         the long-term health effects of endosulfan. In
from 1987 to 1997 indicate an average                   ductive system (Murray et al. 2001; Sharpe             this article, we report the effects of endosulfan
domestic use of approximately 1.38 million              2001). Reported effects of endosulfan on the           on male reproductive development.
pounds of active ingredient per year [U.S.              male reproductive system in experimental
Environmental Protection Agency (U.S. EPA)              animals have been variable, depending on               Materials and Methods
2002]. It has been found in at least 162 of the         species, age at exposure, dose, duration of            Selection of study and control areas. The
1,569 current National Priorities List sites by         exposure, and study end points. Routine gross          exposed population was defined as school-
the U.S. EPA (HazDat 2000). In India, it is             and histopathologic examination of the repro-          children who were permanent residents of the
widely used against a variety of agricultural           ductive organs of male mice that consumed
pests. During 1999–2000, about 81,000 met-              doses of 7.3 mg/kg/day for 13 weeks (Hoechst.          Address correspondence to H.N. Saiyed, Director,
ric tons of endosulfan was manufactured in              Unpublished data) or 2.5–5.0 mg/kg/day                 National Institute of Occupational Health, Meghani
India, and in terms of tonnage its production           for 2 years [Hack et al. 1995; Hoechst.                Nagar, Ahmedabad 380016, India. Telephone: 91-
                                                                                                               79-268-6351. Fax: 91-79-268-6110. E-mail:
was next only to mancozeb (103,000 metric               Unpublished data; National Cancer Institute
                                                                                                               saiyedhn@yahoo.com
tons) and monocrotophos (95,000 metric                  (NCI) 1978] revealed no toxic effects. Later             We gratefully acknowledge the Ministry of Health
tons) (Anonymous 2001).                                 on, more detailed studies in adult rats exposed        and Family Welfare, Government of India, for spon-
    Oral LD50 (lethal dose sufficient to kill           to 2.5, 5, and 10 mg/kg/day endosulfan for             soring this study; volunteers who participated in the
50% of population) endosulfan in rats is 80             5 days per week for 10 weeks showed reduced            study; and the pediatricians from Department of
mg/kg, and it has been classified as a moderately        intratesticular spermatid counts, sperm abnor-         Pediatrics, Kasturba Medical College, Mangalore,
                                                                                                               India.
hazardous (class II) pesticide [World Health            malities, and changes in the marker enzymes
                                                                                                                 The authors declare they have no competing
Organization (WHO) 2002]. Neurotoxicity is              of testicular activities, such as lactate dehy-        financial interests.
the major end point of concern in acute                 drogenase, sorbitol dehydrogenase, γ-glutamyl            Received 10 February 2003; accepted 22 September
endosulfan exposure in human beings and                 transpeptidase, and glucose-6-phosphate                2003.


1958                                                                              VOLUME   111 | NUMBER 16 | December 2003 • Environmental Health Perspectives
Children’s Health            |   Endosulfan and male reproductive development


village situated below the cashew plantations             Ethical aspects. This study was approved by                        of Occupational Health, Ahmedabad, India)
where endosulfan had been sprayed aerially.           the ethics committee of the National Institute                         for 2 hr. The organic layer was transferred
This village had 12 first-order streams origi-        of Occupational Health. Parents, who were                              into another graduated tube and was evapo-
nating from the cashew plantations. Most of           requested to accompany their children at the                           rated to dryness under a stream of nitrogen.
the habitations were along the valleys and            time of examination, were told the objectives of                       The final volume was made up with hexane
close to the stream banks. Most of the inhabi-        the study, and a consent form in local language                        corresponding to the expected concentration
tants depended on runoff water for irrigation         was read aloud to them. The children were                              of the residue. A suitable aliquot was injected
purposes. The control population was selected         examined only after one of the parents gave                            into a gas chromatograph with an electron
from schoolchildren of another village situated       written consent. In addition, special consent of                       capture detector. We calculated the recoveries
approximately 20 km away. The population of           the child was taken for the SMR study, and                             of endosulfan residues, which ranged from 88
this village was comparable with the exposed          only in willing cases were blood samples col-                          to 102%. In addition, fortified samples were
population in socioeconomic status, ethnicity,        lected. The SMR examination was carried out                            studied as a part of quality assurance and
and occupational characteristics but without          by pediatricians observing necessary privacy                           quality control.
any history of aerial endosulfan spray. The           required for this delicate examination.                                    Instrumentation and quantification. We
control village did not have streams.                     Collection, storage, and transport of blood                        used a gas chromatograph (model 6890)
     Selection of study and control subjects.         samples. Five milliliters of venous blood were                         equipped with a Micro Electron Capture
The main study was carried out to explore the         collected from each willing individual between                         Detector, a capillary column (HP 5, 60 m,
effects of endosulfan exposure on growth and          1000 and 1200 hr on the day of examination                             0.25 mm inner diameter, film thickness
development in 619 schoolchildren of both             and was centrifuged at 5,000 rpm for 5 min in                          0.25 µm; all these items from Hewlett Packard
sexes (5–19 years) and 416 comparable con-            the field laboratory. Serum was separated and                           Agilent Plus; Agilent Technologies, Little
trols. All male children (272 exposed and 135         stored at –20°C in a nearby hospital. The                              Fall, DE, USA), and N2 (ultra high purity,
controls) older than 10 years were asked to           serum samples were air-shipped under dry ice                           99.999% grade) as carrier gas for the quantifi-
participate in a sexual maturity rating (SMR)         to the laboratories at National Institute of                           cation of endosulfan residues. The initial oven
study; 117 (43%) exposed and 90 (67%) con-            Occupational Health, Ahmedabad, India. To                              temperature was 80°C with ramp rate of 20°C
trols participated in SMR examination. For            avoid observer bias, the samples were coded                            per min to 200°C. The injector port tempera-
the hormone study, every other student who            before being handed over for analysis.                                 ture was 220°C (splitless mode), and detector
participated in the SMR study was requested               Chemicals and standard control materials                           temperature was 275°C. We quantified the the
to donate a blood sample.                             for analysis of endosulfan in serum samples.                           samples by comparing the peak area of each
     Study parameters. The study parameters           All the chemicals and reagents used in the                             with those of their respective standards. The
included recording of clinical history in a           extraction and cleanup of endosulfan residues                          retention times of α-endosulfan, β-endosulfan,
specially designed pro forma physical examina-        were highly pure HPLC (high-performance                                and endosulfan sulfate were at 38.9, 52.5, and
tion, assessment of SMR by Tanner’s classi-           solvents filtered through 0.2-µ filters and                            67.2 min, respectively (Figure 2A,B).
fication (Marshell and Tanner 1969), and              packed under nitrogen)-grade obtained from                                 Hormone analyses. We estimated testos-
estimation of serum levels of testosterone,           Qualigens Fine Chemicals (Glaxo India Ltd.,                            terone, LH, and FSH in 50 µL and 100 µL
luteinizing hormone (LH), follicle-stimulating        Mumbai, India) and were checked for any                                serum samples of study and control subjects
hormone (FSH), and endosulfan residues.               pesticide contamination. Glassware used was                            using radioimmunoassay kits procured from
                                                      free from residue contamination. Standard                              Immunotech (Marseille, France). We used a
                                                      reference materials of α-endosulfan (99.0%),                           Wallac 1470 Wizard autogamma counter
                                                      β-endosulfan (99.0%), and endosulfan sulfate                           (Perkin Elmer, Turku, Finland) to count
                                                      (99.0%) were a gift from M/s Excel Ltd.,                               radioactivity with detection efficiency of 78%
                                                      (Mumbai, India), which is the largest manu-                            for I125 and negligible cross talk with other iso-
                                                      facturer of endosulfan in India.                                       topes. Equipment and glassware were segregated
                                                          Extraction of residues from serum.                                 to prevent cross-contamination. The hormones
                                                      Extraction was modified from techniques                                were estimated in serial dilutions of serum along
                                                      described in Dale et al. (1966) and U.S. EPA                           with parallel curves to standard. All the samples
                                                      (1980). Briefly, serum (0.5 mL) was pipetted                            for hormone estimations were processed in one
                                                      into a graduated stoppered centrifuge tube,                            assay to rule out interassay variations. We per-
                                                      6 mL of hexane was added, and it was rotated                           formed a linearity study to assess the sensitivity
                                                      in a slow-speed Roto-rac (National Institute                           of the hormone assays by serial dilution of a

                                                                   A                                                                    B
                                                      5.0 × 106                                                             6.5 × 105

                                                      4.0 × 106        α-Endosulfan
                                                                       RT = 38.8 min
                                                                                       β-Endosulfan                         6.0 × 105
                                                                                       RT = 52.5 min
                                                      3.0 × 106
                                                                                                        Endosulfan sulfate
                                                                                                          RT = 67.0 min    5.5 × 105    α-Endosulfan    β-Endosulfan         Endosulfan sulfate
                                                      2.0 × 106                                                                         RT = 38.8 min   RT = 52.6 min          RT = 67.1 min

                                                      1.0 × 106                                                             5.0 × 105

                                                              30            40          50             60           70                      40          50              60             70
                                                                                             Min                                                                Min
Figure 1. Cashew nut plantations on the hills and     Figure 2. Chromatograms of α-endosulfan, β-endosulfan, and endosulfan sulfate. RT, retention time. (A)
houses in the valley. Water streams formed in the     Standard chromatogram of α-endosulfan, β-endosulfan, and endosulfan sulfate. (B) Chromatogram of
hills pass through residential zones in the valley.   α-endosulfan, β-endosulfan, and endosulfan sulfate in blood sample of a study subject.


Environmental Health Perspectives     • VOLUME 111 | NUMBER 16 | December 2003                                                                                                        1959
Children’s Health                                    |   Saiyed et al.


high level with the zero standards. The sensitiv-                                       with respect to age were also comparable, indi-                                      Similar observations were made for the SMR
ity to testosterone, LH, and FSH was 2.5 ng/dL,                                         cating that the nonparticipation of the subjects                                     score for testicular (Figure 4) and penis devel-
0.45 IU/L and 0.02 IU/L, respectively.                                                  may not have had significant effects on the                                          opment (Figure 5).
     Statistical analysis. The statistical software                                     outcome of the study.                                                                     Serum hormone levels. Table 3 shows the
SPSS, release 6.1.4 (SPSS Inc., Chicago, IL,                                                Clinical examination revealed six (5.1%)                                         summary of the multiple regression analysis
USA), was used for statistical analysis of data                                         cases of congenital malformations in the study                                       of serum testosterone levels against age, AEE,
and for making graphs. For the multiple                                                 group, consisting of undescended testis (two),                                       and serum LH levels. The value of R2 is 0.61,
regression analysis, age and aerial exposure to                                         congenital hydrocele (three), and congenital                                         which means that 61% of the variations
endosulfan (AEE; study = 1, control = 0) were                                           inguinal hernia (one), and only one case                                             observed in serum testosterone levels can be
taken as independent variables, and parame-                                             (1.1%) of congenital inguinal hernia in the                                          explained on the basis of age, AEE, and serum
ters of SMR and serum hormone levels were                                               control group. The differences in prevalence                                         LH levels. Very small p-values (< 0.001 in all
taken as dependent variables. Exact age in                                              of congenital malformation were statistically                                        cases) of the regression coefficient indicate
years on the date of examination was used as                                            nonsignificant.                                                                       that statistically these parameters are signifi-
the age variable.                                                                           SMR score. A summary of the multiple                                             cant in determining the serum testosterone
     We used the following multiple regression                                          regression analysis of parameters of SMR                                             levels. The positive sign of the regression coef-
equation: SMR score = b0 + b1 age + b2 × AEE,                                           score on age and AEE is presented in Table 2.                                        ficient for age and LH indicates that with
where b0 is the regression constant and b1 and                                          The values of R2 corresponding to SMR of                                             increase in age and serum LH levels, there is
b2 are the regression coefficients of age and                                           pubic hair, testes, and penis are 0.48, 0.43,                                        an increase in the serum testosterone levels.
exposure, respectively, fitted for SMR scores of                                         and 0.43, respectively, are statistically signifi-                                    This is a well-known physiologic fact. On the
pubic hair, testes, and penis. Similarly, multi-                                        cant (p < 0.001), and indicate that consider-                                        other hand, the regression coefficient for AEE
ple regression equations were fitted for testos-                                         able proportion of variance of SMR scores can                                        has a negative sign, which means the serum
terone, LH, and FSH levels.                                                             be attributed to age and AEE. The statistically                                      testosterone levels of individuals belonging to
                                                                                        significant negative regression coefficient (b2)                                       the study group are statistically lower than
Results                                                                                 of AEE in all three equations signifies delayed                                       expected from age and serum LH levels. The
Table 1 shows the comparison of the mean ±                                              sexual maturity in the study group compared                                          levels of FSH were slightly higher for the age
SD values of the age and some of the growth-                                            with the control group. As expected, age had a                                       in study group; however, the differences were
related basic parameters in study and control                                           significant positive regression coefficient.                                           statistically nonsignificant.
subjects participating in the SMR study. There                                              These effects of AEE are further elaborated                                           In Figure 6, serum testosterone levels
are no statistically significant differences in the                                      graphically in Figures 3–5. In Figure 3, the                                         are plotted against age for study and control
mean values of these parameters in two groups.                                          findings for SMR for pubic hair is plotted                                           individuals. Regression lines drawn for the
The mean values of height, weight, body mass                                            against age for study and control individuals.                                       study and control individuals indicate that the
index, and skin-fold thickness of the nonpar-                                           The regression lines drawn for the study and                                         average serum testosterone levels for the study
ticipating subjects in study and control groups                                         control groups indicate that the SMR score                                           group are lower for the same age. Similarly, in
                                                                                        for the study group is lower for the same age.                                       Figure 7, serum LH levels are plotted against
Table 1. Comparison of the mean (± SD) age and
some of the growth-related basic parameters in                                          Table 2. Summary of the multiple regression analysis of parameters of SMR against age and history of
study and control subjects participating in the SMR                                     exposure to endosulfan through aerial spray.
examination.
                                                                                        Dependent                                                           Intercept                                      Age                                 AEE
Parameters                                  Control (n = 90)          Study (n = 117)   variable                                        R2                     (b0)                                b1                 SE                  b2         SE
Age (years)                                   13.10 ± 2.12             12.80 ± 2.07     SMR score
Height (cm)                                     141 ± 10.60              139 ± 13.30     Pubic hair                                    0.48**                –2.54**                      0.36**                     0.03            –0.43**         0.11
Weight (kg)                                   30.70 ± 7.44             29.50 ± 8.93      Testes                                        0.43**                –2.07**                      0.32**                     0.03            –0.32*          0.11
Body mass index                               15.30 ± 1.98             15.00 ± 2.11      Penis                                         0.43**                –2.00**                      0.32**                     0.03            –0.37*          0.12
Skin-fold
                                                                                        No. of observations: study = 117; control = 90.
 thickness (mm)                                   7.31 ± 2.15           7.40 ± 2.28     *p < 0.01. **p < 0.001.

                           5                                                                                   5       Study                                                                       5        Study
                                   Study                                                                               R 2 = 0.3611                                                                         R 2 = 0.3631
                                   R 2 = 0.4358
                                                                                                                       Control                                                                              Control
                                   Control                                                                             R 2 = 0.4786                                                                         R 2 = 0.4520
                                   R 2 = 0.4815
                           4                                                                                                                                                                       4
SMR score for pubic hair




                                                                                                               4
                                                                                                                                                                             SMR score for penis
                                                                                        SMR score for testes




                           3                                                                                   3                                                                                   3




                           2                                                                                   2                                                                                   2




                           1                                                                                   1                                                                                   1
                               8   10         12          14     16       18      20                               8   10         12         14        16       18      20                             8    10         12       14        16    18    20
                                                   Age (years)                                                                         Age (years)                                                                          Age (years)
Figure 3. SMR score for the development of pubic                                        Figure 4. SMR score for the development of testes                                    Figure 5. SMR score for the development of the
hair according to age and AEE.                                                          according to age and AEE.                                                            penis according to age and AEE.


1960                                                                                                                                         VOLUME   111 | NUMBER 16 | December 2003 • Environmental Health Perspectives
Children’s Health             |   Endosulfan and male reproductive development


age in study and control individuals. The                                                     plasma LH, FSH, and testosterone associated                                                    significantly higher in the study population
regression lines indicate that average serum                                                  with decrease in testicular testosterone in puber-                                             than in the control group even 10 months after
levels for the same age are higher in the study                                               tal rats exposed to endosulfan for 30 days.                                                    the last aerial spray (October 2001). Moreover,
group.                                                                                        Thus, our observations of low testosterone lev-                                                endosulfan residues were detected in water
    Endosulfan exposure. Endosulfan was                                                       els in male children conform with the animal                                                   (≥ 0.03 ppb) and pond sediments (≥ 0.3 ppb)
detected in serum samples of 78% of the chil-                                                 studies. Lower SMR scores appear to reflect                                                    only in the study area 1.5 years (June 2002)
dren in the study group and 29% of the chil-                                                  lower serum testosterone levels for age. In our                                                after the last aerial spray. This signifies that
dren in the control group. Table 4 shows the                                                  study, it is not possible to confirm disturbed                                                  low-level endosulfan exposures continued to
serum endosulfan levels in the study and con-                                                 spermatogenesis observed in animal studies.                                                    occur probably by translocation from the hill-
trol groups. The levels of endosulfan in the                                                       The higher prevalence of congenital abnor-                                                tops to the valley in the study area long after the
study group children are significantly higher                                                 malities related to testicular descent observed                                                aerial spray. This is supported by the report of
(p < 0.001).                                                                                  in the study group should not be overlooked                                                    Regional Remote Sensing Service Center
                                                                                              simply because it failed to achieve statistical                                                (RRSSC), Bangalore, India (Nageswara Rao
Discussion                                                                                    significance (which may be due to small sample                                                  PP. Personal communication). On the basis of
Our study results, after controlling for age,                                                 size), because there is indirect evidence of                                                   analysis of satellite pictures of the study area,
showed significantly lower SMR scores and                                                     endosulfan exposure associated with unde-                                                      the RRSSC reported, “The watershed charac-
serum testosterone levels and higher levels of                                                scended testes in a human population from                                                      teristics are favorable for any aerially sprayed
serum LH in the study group compared with                                                     Spain. Garcia-Rodriguez et al. (1996) reported                                                 toxicant to reach the soil-water-plant contin-
controls. To link these changes with endosul-                                                 a higher incidence of hospital admissions to                                                   uum in a very short span of time and get accu-
fan exposure, we should look at two issues:                                                   University of Granada Hospital for cryptorchi-                                                 mulated” (Nageswara Rao PP. Unpublished
biologic plausibility of the cause–effect rela-                                               dism from districts near the Mediterranean                                                     report). Endosulfan has a half life of 60–800
tionship and pathways of endosulfan exposure.                                                 coast, where there is intensive use of pesticides.                                             days in soil (ATSDR 2000). Frank et al. (1982)
     Biologic plausibility. There are reports of                                              A subsequent study reported endosulfan iso-                                                    have also reported that because of its persis-
testicular toxicity of endosulfan manifested as                                               mers and/or metabolites in adipose tissue of                                                   tence in soil, endosulfan residues were detected
decreased spermatogenesis and testicular hor-                                                 40% of children who were admitted to the                                                       in water samples throughout the year (outside
mone synthesis (steroidogenesis), as evidenced                                                same hospital for a variety of reasons (Olea et                                                the spray season) with storm runoff. The
by a decrease in spermatid count in testes and                                                al. 1999), indicating that significant endosulfan                                               results of several laboratory and greenhouse
in sperm count in the cauda epididymis and by                                                 exposures occurred in the region. In the pre-                                                  studies indicate that α- and β-endosulfan are
changes in marker enzymes for testicular                                                      sent study, there is a definite history of endo-                                                strongly adsorbed to soil (Bowman et al. 1965;
steroidogenesis in adult animals (Chitra et al.                                               sulfan exposure that is likely to have occurred                                                El Beit et al. 1981a, 1981b). The study area
1999; Singh and Pandey 1989, 1990; Sinha et                                                   during the prenatal period.                                                                    has an annual rainfall of 140 inches. Twelve
al. 1995). These effects were seen at much                                                         Pathways of endosulfan exposure in the                                                    first-order streams originate from the cashew
lower dosages and shorter durations if exposures                                              study population. In our study, we estimated                                                   plantations. It is likely that endosulfan sticking
occurred during the prenatal or prepubertal                                                   endosulfan residues in biologic and environ-                                                   on the soil is carried by runoff water during
periods (Dalsenter et al. 1999; Sinha et al.                                                  mental samples. The practice of aerial spraying                                                most of the year.
1995, 1997, 2001). Singh and Pandey (1990)                                                    of endosulfan was discontinued in December                                                         We have measured endosulfan levels only
also reported profound decreases in the levels of                                             2000. Serum endosulfan residue levels were                                                     once in serum samples. These individual endo-
                                                                                                                                                                                             sulfan measures may or may not accurately
Table 3. Summary of the multiple regression analysis for serum testosterone levels against age, history of
AEE, and serum LH levels.
                                                                                                                                                                                             reflect the chronic levels and/or levels during
                                                                                                                                                                                             critical developmental phases. However, the
Dependent                                                              Age                                                     Exposure                                    LH                effect of this would be to decrease power of the
variable                                                        b               SE                          b                               SE                    b                   SE
                                                                                                                                                                                             study (via an increase in random misclassifica-
Testosterone                                                  0.37**            0.06         –0.62**                                       0.21               1.09**                  0.20   tion of exposure) and thus bias toward the
No. of observations: study = 67; control = 46. Overall R2 = 0.61. **p < 0.001.                                                                                                               null. We believe that even single estimations of
                                                                                                                                                                                             serum endosulfan levels validate that children
                                10          Study                                                                                                                                            exposed to endosulfan via aerial spraying do, on
                                            R 2 = 0.5163                                                               4
                                                                                                                                    Study
                                                                                                                                    R 2 = 0.5150
                                                                                                                                                                                             average, have higher exposures than children in
                                            Control
                                            R 2 = 0.4626                                                                                                                                     the control group.
Serum testosterone levels (ng/dL)




                                    8                                                                                               Control
                                                                                                                                    R 2 = 0.1826                                                 Finally, it is important to discuss and
                                                                                                                       3                                                                     resolve the following weaknesses of the study.
                                                                                                                                                                                             First is nonparticipation in SMR study: 57%
                                                                                              Serum LH levels (IU/L)




                                    6
                                                                                                                                                                                             of the exposed and 33% of the control partici-
                                                                                                                                                                                             pants did not agree to undergo SMR examina-
                                                                                                                       2
                                    4
                                                                                                                                                                                             tion. However, growth-related end points
                                                                                                                                                                                             (height, weight, and skin-fold thickness) were
                                                                                                                                                                                             Table 4. Mean ± SE levels (ppb) of serum endosulfan
                                    2                                                                                  1
                                                                                                                                                                                             in study and control subjects.
                                                                                                                                                                                                                  Control (n = 45)   Study (n = 70)
                                    0                                                                                                                                                        α-Endosulfan           0.87 ± 0.23      4.24 ± 0.74**
                                        8   10         12       14        16    18     20                              0                                                                     β-Endosulfan           0.40 ± 0.17      1.77 ± 0.36**
                                                            Age (years)                                                    8        10         12        14           16    18         20    Endosulfan sulfate     0.10 ± 0.08      1.47 ± 0.33**
                                                                                                                                                    Age (years)                              Total endosulfan       1.37 ± 0.40      7.47 ± 1.19**
Figure 6. Serum testosterone levels according to
age and AEE.                                                                                  Figure 7. Serum LH levels according to age and AEE.                                            **p < 0.001.



Environmental Health Perspectives                                              • VOLUME 111 | NUMBER 16 | December 2003                                                                                                                   1961
Children’s Health           |   Saiyed et al.


comparable among the nonparticipating                                  chlorinated insecticides in a broad spectrum of soil types.      Murray TJ, Lea RG, Abramovich D R, Haites NE, Fowler PA. 2001.
groups. It is still difficult to envisage the over-                     J Agric Food Chem 13:360–365.                                         Endocrine disrupting chemicals: effects on human male
                                                                  Chitra KC, Latchoumycandane C, Mathur PP. 1999. Chronic                    reproductive health. Early Pregnancy 5:80–112.
all effect of nonparticipation. Second is one-                         effect of endosulfan on the testicular functions of rat. Asian   NCI. 1978. 78-Week Dietary Study in Osborne-Mendel Rats and
time collection of blood samples for hormone                           J Androl 1(4):203–206.                                                B6C3F1 Mice. NCI Study No. NCI-CG-TR62, Technical Report
analysis: Sex hormone secretion is pulsatile in                   Dale WE, Curley A, Cueto C Jr. 1966. Hexane extractable chlori-            Series No. 62. Bethesda, MD:National Cancer Institute.
                                                                       nated insecticides in human blood. Life Sci 5:47–54.             Olea N, Olea-Serrano F, Lardelli-Claret P, Rivas A, Barba-
nature, with a lot of diurnal variation. To                       Dalsenter PR, Dallegrave E, Mello JRB, Langeloh A, Oliveria RT,            Navarro A. 1999. Inadvertent exposure to xenoestrogens
minimize this effect, we collected blood sam-                          Faqi AS. 1999. Reproductive effects of endosulfan on male             in children. Toxicol Ind Health 15:151–158.
ples in all cases between 1000 and 1200 hr.                            offspring of rats exposed during pregnancy and lactation.        Sharpe RM. 2001. Hormones and testis development and the
                                                                       Hum Exp Toxicol 18:583–589.                                           possible adverse effects of environmental chemicals.
Because the variation in the blood hormone                        Dalsenter PR, de Aranjo SL, Silva de Assis HC, Andrade AJM,                Toxicol Lett 120:221–232.
levels is random, the overall effect of natural                        Dallegrave E. 2003. Pre- and postnatal exposure to endo-         Singh SK, Pandey RS. 1989. Gonadal toxicity of short term
variation is likely to be minimized. However,                          sulfan in Wistar rats. Hum Exp Toxicol 22:171–175.                    chronic endosulfan exposure to male rats. Indian J Exp Biol
                                                                  El Beit IOD, Wheelock JV, Cotton DE. 1981a. Factors affecting soil         27:341–346.
the increased random variability of hormone                            residues of dieldrin, endosulfan, gamma-HCH, dimethoate,         ———. 1990. Effect of sub-chronic endosulfan exposures on
would have decreased the power of the study                            and pyrolan. Ecotoxicol Environ Saf 5:135–160.                        plasma gonadotrophins, testosterone, testicular testos-
via an increase in random misclassification of                     ———. 1981b. Pesticide-microbial interaction in the soil. Int J             terone and enzymes of androgen biosynthesis in rat.
testosterone levels, which in turn would bias                          Environ Stud 16:171–179.                                              Indian J Exp Biol 28:953–956.
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toward the null.                                                       1982. Agriculture and water quality in the Canadian Great             ing fetal gonadal differentiation on spermatogenesis in rats.
     Our study results suggest that endosulfan                         Lakes Basin. V. Pesticide use in 11 agricultural watersheds           Environ Toxicol Pharmacol 10:29–32.
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fere with hormone synthesis in male children.                     García-Rodríguez J, García-Martín M, Nogueras-Ocaña M,                Sinha N, Narayan R, Shanker R, Saxena DK. 1995. Endosulfan-
To validate these findings, a study with larger                         Luna-del-Castillo JD, García MF, Olea, N, et al. 1996.                induced biochemical changes in the testis of rats. Vet Hum
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Bowman MC, Schechter MS, Carter RL. 1965. Behavior of                  changes in girls. Arch Dis Child 44:291–303.




1962                                                                                               VOLUME    111 | NUMBER 16 | December 2003 • Environmental Health Perspectives

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Endosulfan does not affect the male reproductive system

  • 1. Children’s Health | Article Effect of Endosulfan on Male Reproductive Development Habibullah Saiyed,1 Aruna Dewan,1 Vijay Bhatnagar,1 Udyavar Shenoy,2 Rathika Shenoy,2 Hirehall Rajmohan,3 Kumud Patel,1 Rekha Kashyap,1 Pradip Kulkarni,1 Bagalur Rajan,3 and Bhadabhai Lakkad 1 1National Institute of Occupational Health (Indian Council of Medical Research), Meghani Nagar, Ahmedabad, India; 2Department of Pediatrics, Kasturba Medical College, Mangalore, India; 3Regional Occupational Health Research Centre, Bangalore, India dehydrogenase, providing further evidence There is experimental evidence of adverse effects of endosulfan on the male reproductive system, but of effects on spermatogenesis (Khan and Sinha there are no human data. Therefore, we undertook a study to examine the relationship between envi- 1996; Sinha et al. 1995). Exposure of younger ronmental endosulfan exposure and reproductive development in male children and adolescents. The animals (3 weeks old) showed marked depletion study population was composed of 117 male schoolchildren (10–19 years of age) of a village situated of spermatid count as well as decreased daily at the foothills of cashew plantations, where endosulfan had been aerially sprayed for more than 20 sperm production at a dose of 2.5 mg/kg/day years, and 90 comparable controls with no such exposure history. The study parameters included (Sinha et al. 1997), which was earlier seen only recording of clinical history, physical examination, sexual maturity rating (SMR) according to Tanner at 5 mg/kg/day in adult rats by the same investi- stages, and estimation of serum levels of testosterone, luteinizing hormone (LH), follicle-stimulating gators (Sinha et al. 1995). More recent studies hormone, and endosulfan residues (70 study and 47 control subjects). Mean ± SE serum endosulfan have shown that exposure of pregnant rats levels in the study group (7.47 ± 1.19 ppb) were significantly higher (p < 0.001) than in controls (1.37 to endosulfan at 1 mg/kg/day from day 12 ± 0.40 ppb). Multiple regression analysis showed that SMR scoring for development of pubic hair, through parturition leads to decreased sper- testes, penis, and serum testosterone level was positively related to age and negatively related to aerial matogenesis in offspring (Sinha et al. 2001). exposure to endosulfan (AEE; p < 0.01). Serum LH levels were significantly positively related to AEE Dalsenter et al. (1999) reported similar observa- after controlling for age (p < 0.01). The prevalence of congenital abnormalities related to testicular tions at 3 mg/kg/day but not at 1.5 mg/kg/day, descent (congenital hydrocele, undescended testis, and congenital inguinal hernia) among study and and they attributed this to strain variation controls subjects was 5.1% and 1.1%, respectively, but the differences were statistically nonsignifi- (Dalsenter et al. 2003). Thus, experimental cant. Our study results suggest that endosulfan exposure in male children may delay sexual maturity studies suggest that endosulfan can affect the and interfere with sex hormone synthesis. Our study is limited by small sample size and nonparticipa- male reproductive system and also that these tion. Key words: endocrine disruptor, endosulfan, luteinizing hormone, male reproductive develop- effects are likely to be greater if exposure occurs ment, sexual maturity rating, testosterone. Environ Health Perspect 111:1958–1962 (2003). during the developmental phase. doi:10.1289/ehp.6271 available via http://dx.doi.org/ [Online 22 September 2003] Environmental exposure to a single chemi- cal over a long period of time is very rare. We came across a situation where endosulfan Endosulfan (6,7,8,9,10,10-hexachloro- experimental animals. No data are available was the only pesticide that had been aerially 1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3- for subacute or chronic exposure to endosul- sprayed two to three times a year for more benzodioxathiepin-3-oxide) is a broad- fan in human subjects; however, the subacute than 20 years on cashew nut plantations situ- spectrum insecticide and acaricide first regis- and chronic toxicity studies of endosulfan ated on hilltops in some villages of northern tered for use in the United States in 1954 to in animals suggest that the liver, kidneys, Kerala, India (Figure 1). The population living control agricultural insect and mite pests on a immune system, and testes are the main tar- in the valley had a significant chance of expo- variety of field, fruit, and vegetable crops. get organs [Agency for Toxic Substances and sure to this pesticide during aerial spray and Technical-grade endosulfan is composed of Disease Registry (ATSDR) 2000]. subsequently through other contaminated two stereochemical isomers, α-endosulfan and In recent years, there has been growing environmental media. This population, there- β-endosulfan, in concentrations of approxi- concern about toxicity of a number of chemi- fore, provided a unique opportunity to study mately 70% and 30%, respectively. Use data cals, including pesticides, on the male repro- the long-term health effects of endosulfan. In from 1987 to 1997 indicate an average ductive system (Murray et al. 2001; Sharpe this article, we report the effects of endosulfan domestic use of approximately 1.38 million 2001). Reported effects of endosulfan on the on male reproductive development. pounds of active ingredient per year [U.S. male reproductive system in experimental Environmental Protection Agency (U.S. EPA) animals have been variable, depending on Materials and Methods 2002]. It has been found in at least 162 of the species, age at exposure, dose, duration of Selection of study and control areas. The 1,569 current National Priorities List sites by exposure, and study end points. Routine gross exposed population was defined as school- the U.S. EPA (HazDat 2000). In India, it is and histopathologic examination of the repro- children who were permanent residents of the widely used against a variety of agricultural ductive organs of male mice that consumed pests. During 1999–2000, about 81,000 met- doses of 7.3 mg/kg/day for 13 weeks (Hoechst. Address correspondence to H.N. Saiyed, Director, ric tons of endosulfan was manufactured in Unpublished data) or 2.5–5.0 mg/kg/day National Institute of Occupational Health, Meghani India, and in terms of tonnage its production for 2 years [Hack et al. 1995; Hoechst. Nagar, Ahmedabad 380016, India. Telephone: 91- 79-268-6351. Fax: 91-79-268-6110. E-mail: was next only to mancozeb (103,000 metric Unpublished data; National Cancer Institute saiyedhn@yahoo.com tons) and monocrotophos (95,000 metric (NCI) 1978] revealed no toxic effects. Later We gratefully acknowledge the Ministry of Health tons) (Anonymous 2001). on, more detailed studies in adult rats exposed and Family Welfare, Government of India, for spon- Oral LD50 (lethal dose sufficient to kill to 2.5, 5, and 10 mg/kg/day endosulfan for soring this study; volunteers who participated in the 50% of population) endosulfan in rats is 80 5 days per week for 10 weeks showed reduced study; and the pediatricians from Department of mg/kg, and it has been classified as a moderately intratesticular spermatid counts, sperm abnor- Pediatrics, Kasturba Medical College, Mangalore, India. hazardous (class II) pesticide [World Health malities, and changes in the marker enzymes The authors declare they have no competing Organization (WHO) 2002]. Neurotoxicity is of testicular activities, such as lactate dehy- financial interests. the major end point of concern in acute drogenase, sorbitol dehydrogenase, γ-glutamyl Received 10 February 2003; accepted 22 September endosulfan exposure in human beings and transpeptidase, and glucose-6-phosphate 2003. 1958 VOLUME 111 | NUMBER 16 | December 2003 • Environmental Health Perspectives
  • 2. Children’s Health | Endosulfan and male reproductive development village situated below the cashew plantations Ethical aspects. This study was approved by of Occupational Health, Ahmedabad, India) where endosulfan had been sprayed aerially. the ethics committee of the National Institute for 2 hr. The organic layer was transferred This village had 12 first-order streams origi- of Occupational Health. Parents, who were into another graduated tube and was evapo- nating from the cashew plantations. Most of requested to accompany their children at the rated to dryness under a stream of nitrogen. the habitations were along the valleys and time of examination, were told the objectives of The final volume was made up with hexane close to the stream banks. Most of the inhabi- the study, and a consent form in local language corresponding to the expected concentration tants depended on runoff water for irrigation was read aloud to them. The children were of the residue. A suitable aliquot was injected purposes. The control population was selected examined only after one of the parents gave into a gas chromatograph with an electron from schoolchildren of another village situated written consent. In addition, special consent of capture detector. We calculated the recoveries approximately 20 km away. The population of the child was taken for the SMR study, and of endosulfan residues, which ranged from 88 this village was comparable with the exposed only in willing cases were blood samples col- to 102%. In addition, fortified samples were population in socioeconomic status, ethnicity, lected. The SMR examination was carried out studied as a part of quality assurance and and occupational characteristics but without by pediatricians observing necessary privacy quality control. any history of aerial endosulfan spray. The required for this delicate examination. Instrumentation and quantification. We control village did not have streams. Collection, storage, and transport of blood used a gas chromatograph (model 6890) Selection of study and control subjects. samples. Five milliliters of venous blood were equipped with a Micro Electron Capture The main study was carried out to explore the collected from each willing individual between Detector, a capillary column (HP 5, 60 m, effects of endosulfan exposure on growth and 1000 and 1200 hr on the day of examination 0.25 mm inner diameter, film thickness development in 619 schoolchildren of both and was centrifuged at 5,000 rpm for 5 min in 0.25 µm; all these items from Hewlett Packard sexes (5–19 years) and 416 comparable con- the field laboratory. Serum was separated and Agilent Plus; Agilent Technologies, Little trols. All male children (272 exposed and 135 stored at –20°C in a nearby hospital. The Fall, DE, USA), and N2 (ultra high purity, controls) older than 10 years were asked to serum samples were air-shipped under dry ice 99.999% grade) as carrier gas for the quantifi- participate in a sexual maturity rating (SMR) to the laboratories at National Institute of cation of endosulfan residues. The initial oven study; 117 (43%) exposed and 90 (67%) con- Occupational Health, Ahmedabad, India. To temperature was 80°C with ramp rate of 20°C trols participated in SMR examination. For avoid observer bias, the samples were coded per min to 200°C. The injector port tempera- the hormone study, every other student who before being handed over for analysis. ture was 220°C (splitless mode), and detector participated in the SMR study was requested Chemicals and standard control materials temperature was 275°C. We quantified the the to donate a blood sample. for analysis of endosulfan in serum samples. samples by comparing the peak area of each Study parameters. The study parameters All the chemicals and reagents used in the with those of their respective standards. The included recording of clinical history in a extraction and cleanup of endosulfan residues retention times of α-endosulfan, β-endosulfan, specially designed pro forma physical examina- were highly pure HPLC (high-performance and endosulfan sulfate were at 38.9, 52.5, and tion, assessment of SMR by Tanner’s classi- solvents filtered through 0.2-µ filters and 67.2 min, respectively (Figure 2A,B). fication (Marshell and Tanner 1969), and packed under nitrogen)-grade obtained from Hormone analyses. We estimated testos- estimation of serum levels of testosterone, Qualigens Fine Chemicals (Glaxo India Ltd., terone, LH, and FSH in 50 µL and 100 µL luteinizing hormone (LH), follicle-stimulating Mumbai, India) and were checked for any serum samples of study and control subjects hormone (FSH), and endosulfan residues. pesticide contamination. Glassware used was using radioimmunoassay kits procured from free from residue contamination. Standard Immunotech (Marseille, France). We used a reference materials of α-endosulfan (99.0%), Wallac 1470 Wizard autogamma counter β-endosulfan (99.0%), and endosulfan sulfate (Perkin Elmer, Turku, Finland) to count (99.0%) were a gift from M/s Excel Ltd., radioactivity with detection efficiency of 78% (Mumbai, India), which is the largest manu- for I125 and negligible cross talk with other iso- facturer of endosulfan in India. topes. Equipment and glassware were segregated Extraction of residues from serum. to prevent cross-contamination. The hormones Extraction was modified from techniques were estimated in serial dilutions of serum along described in Dale et al. (1966) and U.S. EPA with parallel curves to standard. All the samples (1980). Briefly, serum (0.5 mL) was pipetted for hormone estimations were processed in one into a graduated stoppered centrifuge tube, assay to rule out interassay variations. We per- 6 mL of hexane was added, and it was rotated formed a linearity study to assess the sensitivity in a slow-speed Roto-rac (National Institute of the hormone assays by serial dilution of a A B 5.0 × 106 6.5 × 105 4.0 × 106 α-Endosulfan RT = 38.8 min β-Endosulfan 6.0 × 105 RT = 52.5 min 3.0 × 106 Endosulfan sulfate RT = 67.0 min 5.5 × 105 α-Endosulfan β-Endosulfan Endosulfan sulfate 2.0 × 106 RT = 38.8 min RT = 52.6 min RT = 67.1 min 1.0 × 106 5.0 × 105 30 40 50 60 70 40 50 60 70 Min Min Figure 1. Cashew nut plantations on the hills and Figure 2. Chromatograms of α-endosulfan, β-endosulfan, and endosulfan sulfate. RT, retention time. (A) houses in the valley. Water streams formed in the Standard chromatogram of α-endosulfan, β-endosulfan, and endosulfan sulfate. (B) Chromatogram of hills pass through residential zones in the valley. α-endosulfan, β-endosulfan, and endosulfan sulfate in blood sample of a study subject. Environmental Health Perspectives • VOLUME 111 | NUMBER 16 | December 2003 1959
  • 3. Children’s Health | Saiyed et al. high level with the zero standards. The sensitiv- with respect to age were also comparable, indi- Similar observations were made for the SMR ity to testosterone, LH, and FSH was 2.5 ng/dL, cating that the nonparticipation of the subjects score for testicular (Figure 4) and penis devel- 0.45 IU/L and 0.02 IU/L, respectively. may not have had significant effects on the opment (Figure 5). Statistical analysis. The statistical software outcome of the study. Serum hormone levels. Table 3 shows the SPSS, release 6.1.4 (SPSS Inc., Chicago, IL, Clinical examination revealed six (5.1%) summary of the multiple regression analysis USA), was used for statistical analysis of data cases of congenital malformations in the study of serum testosterone levels against age, AEE, and for making graphs. For the multiple group, consisting of undescended testis (two), and serum LH levels. The value of R2 is 0.61, regression analysis, age and aerial exposure to congenital hydrocele (three), and congenital which means that 61% of the variations endosulfan (AEE; study = 1, control = 0) were inguinal hernia (one), and only one case observed in serum testosterone levels can be taken as independent variables, and parame- (1.1%) of congenital inguinal hernia in the explained on the basis of age, AEE, and serum ters of SMR and serum hormone levels were control group. The differences in prevalence LH levels. Very small p-values (< 0.001 in all taken as dependent variables. Exact age in of congenital malformation were statistically cases) of the regression coefficient indicate years on the date of examination was used as nonsignificant. that statistically these parameters are signifi- the age variable. SMR score. A summary of the multiple cant in determining the serum testosterone We used the following multiple regression regression analysis of parameters of SMR levels. The positive sign of the regression coef- equation: SMR score = b0 + b1 age + b2 × AEE, score on age and AEE is presented in Table 2. ficient for age and LH indicates that with where b0 is the regression constant and b1 and The values of R2 corresponding to SMR of increase in age and serum LH levels, there is b2 are the regression coefficients of age and pubic hair, testes, and penis are 0.48, 0.43, an increase in the serum testosterone levels. exposure, respectively, fitted for SMR scores of and 0.43, respectively, are statistically signifi- This is a well-known physiologic fact. On the pubic hair, testes, and penis. Similarly, multi- cant (p < 0.001), and indicate that consider- other hand, the regression coefficient for AEE ple regression equations were fitted for testos- able proportion of variance of SMR scores can has a negative sign, which means the serum terone, LH, and FSH levels. be attributed to age and AEE. The statistically testosterone levels of individuals belonging to significant negative regression coefficient (b2) the study group are statistically lower than Results of AEE in all three equations signifies delayed expected from age and serum LH levels. The Table 1 shows the comparison of the mean ± sexual maturity in the study group compared levels of FSH were slightly higher for the age SD values of the age and some of the growth- with the control group. As expected, age had a in study group; however, the differences were related basic parameters in study and control significant positive regression coefficient. statistically nonsignificant. subjects participating in the SMR study. There These effects of AEE are further elaborated In Figure 6, serum testosterone levels are no statistically significant differences in the graphically in Figures 3–5. In Figure 3, the are plotted against age for study and control mean values of these parameters in two groups. findings for SMR for pubic hair is plotted individuals. Regression lines drawn for the The mean values of height, weight, body mass against age for study and control individuals. study and control individuals indicate that the index, and skin-fold thickness of the nonpar- The regression lines drawn for the study and average serum testosterone levels for the study ticipating subjects in study and control groups control groups indicate that the SMR score group are lower for the same age. Similarly, in for the study group is lower for the same age. Figure 7, serum LH levels are plotted against Table 1. Comparison of the mean (± SD) age and some of the growth-related basic parameters in Table 2. Summary of the multiple regression analysis of parameters of SMR against age and history of study and control subjects participating in the SMR exposure to endosulfan through aerial spray. examination. Dependent Intercept Age AEE Parameters Control (n = 90) Study (n = 117) variable R2 (b0) b1 SE b2 SE Age (years) 13.10 ± 2.12 12.80 ± 2.07 SMR score Height (cm) 141 ± 10.60 139 ± 13.30 Pubic hair 0.48** –2.54** 0.36** 0.03 –0.43** 0.11 Weight (kg) 30.70 ± 7.44 29.50 ± 8.93 Testes 0.43** –2.07** 0.32** 0.03 –0.32* 0.11 Body mass index 15.30 ± 1.98 15.00 ± 2.11 Penis 0.43** –2.00** 0.32** 0.03 –0.37* 0.12 Skin-fold No. of observations: study = 117; control = 90. thickness (mm) 7.31 ± 2.15 7.40 ± 2.28 *p < 0.01. **p < 0.001. 5 5 Study 5 Study Study R 2 = 0.3611 R 2 = 0.3631 R 2 = 0.4358 Control Control Control R 2 = 0.4786 R 2 = 0.4520 R 2 = 0.4815 4 4 SMR score for pubic hair 4 SMR score for penis SMR score for testes 3 3 3 2 2 2 1 1 1 8 10 12 14 16 18 20 8 10 12 14 16 18 20 8 10 12 14 16 18 20 Age (years) Age (years) Age (years) Figure 3. SMR score for the development of pubic Figure 4. SMR score for the development of testes Figure 5. SMR score for the development of the hair according to age and AEE. according to age and AEE. penis according to age and AEE. 1960 VOLUME 111 | NUMBER 16 | December 2003 • Environmental Health Perspectives
  • 4. Children’s Health | Endosulfan and male reproductive development age in study and control individuals. The plasma LH, FSH, and testosterone associated significantly higher in the study population regression lines indicate that average serum with decrease in testicular testosterone in puber- than in the control group even 10 months after levels for the same age are higher in the study tal rats exposed to endosulfan for 30 days. the last aerial spray (October 2001). Moreover, group. Thus, our observations of low testosterone lev- endosulfan residues were detected in water Endosulfan exposure. Endosulfan was els in male children conform with the animal (≥ 0.03 ppb) and pond sediments (≥ 0.3 ppb) detected in serum samples of 78% of the chil- studies. Lower SMR scores appear to reflect only in the study area 1.5 years (June 2002) dren in the study group and 29% of the chil- lower serum testosterone levels for age. In our after the last aerial spray. This signifies that dren in the control group. Table 4 shows the study, it is not possible to confirm disturbed low-level endosulfan exposures continued to serum endosulfan levels in the study and con- spermatogenesis observed in animal studies. occur probably by translocation from the hill- trol groups. The levels of endosulfan in the The higher prevalence of congenital abnor- tops to the valley in the study area long after the study group children are significantly higher malities related to testicular descent observed aerial spray. This is supported by the report of (p < 0.001). in the study group should not be overlooked Regional Remote Sensing Service Center simply because it failed to achieve statistical (RRSSC), Bangalore, India (Nageswara Rao Discussion significance (which may be due to small sample PP. Personal communication). On the basis of Our study results, after controlling for age, size), because there is indirect evidence of analysis of satellite pictures of the study area, showed significantly lower SMR scores and endosulfan exposure associated with unde- the RRSSC reported, “The watershed charac- serum testosterone levels and higher levels of scended testes in a human population from teristics are favorable for any aerially sprayed serum LH in the study group compared with Spain. Garcia-Rodriguez et al. (1996) reported toxicant to reach the soil-water-plant contin- controls. To link these changes with endosul- a higher incidence of hospital admissions to uum in a very short span of time and get accu- fan exposure, we should look at two issues: University of Granada Hospital for cryptorchi- mulated” (Nageswara Rao PP. Unpublished biologic plausibility of the cause–effect rela- dism from districts near the Mediterranean report). Endosulfan has a half life of 60–800 tionship and pathways of endosulfan exposure. coast, where there is intensive use of pesticides. days in soil (ATSDR 2000). Frank et al. (1982) Biologic plausibility. There are reports of A subsequent study reported endosulfan iso- have also reported that because of its persis- testicular toxicity of endosulfan manifested as mers and/or metabolites in adipose tissue of tence in soil, endosulfan residues were detected decreased spermatogenesis and testicular hor- 40% of children who were admitted to the in water samples throughout the year (outside mone synthesis (steroidogenesis), as evidenced same hospital for a variety of reasons (Olea et the spray season) with storm runoff. The by a decrease in spermatid count in testes and al. 1999), indicating that significant endosulfan results of several laboratory and greenhouse in sperm count in the cauda epididymis and by exposures occurred in the region. In the pre- studies indicate that α- and β-endosulfan are changes in marker enzymes for testicular sent study, there is a definite history of endo- strongly adsorbed to soil (Bowman et al. 1965; steroidogenesis in adult animals (Chitra et al. sulfan exposure that is likely to have occurred El Beit et al. 1981a, 1981b). The study area 1999; Singh and Pandey 1989, 1990; Sinha et during the prenatal period. has an annual rainfall of 140 inches. Twelve al. 1995). These effects were seen at much Pathways of endosulfan exposure in the first-order streams originate from the cashew lower dosages and shorter durations if exposures study population. In our study, we estimated plantations. It is likely that endosulfan sticking occurred during the prenatal or prepubertal endosulfan residues in biologic and environ- on the soil is carried by runoff water during periods (Dalsenter et al. 1999; Sinha et al. mental samples. The practice of aerial spraying most of the year. 1995, 1997, 2001). Singh and Pandey (1990) of endosulfan was discontinued in December We have measured endosulfan levels only also reported profound decreases in the levels of 2000. Serum endosulfan residue levels were once in serum samples. These individual endo- sulfan measures may or may not accurately Table 3. Summary of the multiple regression analysis for serum testosterone levels against age, history of AEE, and serum LH levels. reflect the chronic levels and/or levels during critical developmental phases. However, the Dependent Age Exposure LH effect of this would be to decrease power of the variable b SE b SE b SE study (via an increase in random misclassifica- Testosterone 0.37** 0.06 –0.62** 0.21 1.09** 0.20 tion of exposure) and thus bias toward the No. of observations: study = 67; control = 46. Overall R2 = 0.61. **p < 0.001. null. We believe that even single estimations of serum endosulfan levels validate that children 10 Study exposed to endosulfan via aerial spraying do, on R 2 = 0.5163 4 Study R 2 = 0.5150 average, have higher exposures than children in Control R 2 = 0.4626 the control group. Serum testosterone levels (ng/dL) 8 Control R 2 = 0.1826 Finally, it is important to discuss and 3 resolve the following weaknesses of the study. First is nonparticipation in SMR study: 57% Serum LH levels (IU/L) 6 of the exposed and 33% of the control partici- pants did not agree to undergo SMR examina- 2 4 tion. However, growth-related end points (height, weight, and skin-fold thickness) were Table 4. Mean ± SE levels (ppb) of serum endosulfan 2 1 in study and control subjects. Control (n = 45) Study (n = 70) 0 α-Endosulfan 0.87 ± 0.23 4.24 ± 0.74** 8 10 12 14 16 18 20 0 β-Endosulfan 0.40 ± 0.17 1.77 ± 0.36** Age (years) 8 10 12 14 16 18 20 Endosulfan sulfate 0.10 ± 0.08 1.47 ± 0.33** Age (years) Total endosulfan 1.37 ± 0.40 7.47 ± 1.19** Figure 6. Serum testosterone levels according to age and AEE. Figure 7. Serum LH levels according to age and AEE. **p < 0.001. Environmental Health Perspectives • VOLUME 111 | NUMBER 16 | December 2003 1961
  • 5. Children’s Health | Saiyed et al. comparable among the nonparticipating chlorinated insecticides in a broad spectrum of soil types. Murray TJ, Lea RG, Abramovich D R, Haites NE, Fowler PA. 2001. groups. It is still difficult to envisage the over- J Agric Food Chem 13:360–365. Endocrine disrupting chemicals: effects on human male Chitra KC, Latchoumycandane C, Mathur PP. 1999. Chronic reproductive health. Early Pregnancy 5:80–112. all effect of nonparticipation. Second is one- effect of endosulfan on the testicular functions of rat. Asian NCI. 1978. 78-Week Dietary Study in Osborne-Mendel Rats and time collection of blood samples for hormone J Androl 1(4):203–206. B6C3F1 Mice. NCI Study No. NCI-CG-TR62, Technical Report analysis: Sex hormone secretion is pulsatile in Dale WE, Curley A, Cueto C Jr. 1966. Hexane extractable chlori- Series No. 62. Bethesda, MD:National Cancer Institute. nated insecticides in human blood. Life Sci 5:47–54. Olea N, Olea-Serrano F, Lardelli-Claret P, Rivas A, Barba- nature, with a lot of diurnal variation. To Dalsenter PR, Dallegrave E, Mello JRB, Langeloh A, Oliveria RT, Navarro A. 1999. Inadvertent exposure to xenoestrogens minimize this effect, we collected blood sam- Faqi AS. 1999. Reproductive effects of endosulfan on male in children. Toxicol Ind Health 15:151–158. ples in all cases between 1000 and 1200 hr. offspring of rats exposed during pregnancy and lactation. Sharpe RM. 2001. Hormones and testis development and the Hum Exp Toxicol 18:583–589. possible adverse effects of environmental chemicals. Because the variation in the blood hormone Dalsenter PR, de Aranjo SL, Silva de Assis HC, Andrade AJM, Toxicol Lett 120:221–232. levels is random, the overall effect of natural Dallegrave E. 2003. Pre- and postnatal exposure to endo- Singh SK, Pandey RS. 1989. Gonadal toxicity of short term variation is likely to be minimized. However, sulfan in Wistar rats. Hum Exp Toxicol 22:171–175. chronic endosulfan exposure to male rats. Indian J Exp Biol El Beit IOD, Wheelock JV, Cotton DE. 1981a. Factors affecting soil 27:341–346. the increased random variability of hormone residues of dieldrin, endosulfan, gamma-HCH, dimethoate, ———. 1990. Effect of sub-chronic endosulfan exposures on would have decreased the power of the study and pyrolan. Ecotoxicol Environ Saf 5:135–160. plasma gonadotrophins, testosterone, testicular testos- via an increase in random misclassification of ———. 1981b. Pesticide-microbial interaction in the soil. Int J terone and enzymes of androgen biosynthesis in rat. testosterone levels, which in turn would bias Environ Stud 16:171–179. Indian J Exp Biol 28:953–956. Frank R, Braun HE, Van Hove Holdrinet M, Sirons GJ, Ripley BD. Sinha N, Adhikari N, Saxena DK. 2001. Effect of endosulfan dur- toward the null. 1982. Agriculture and water quality in the Canadian Great ing fetal gonadal differentiation on spermatogenesis in rats. Our study results suggest that endosulfan Lakes Basin. V. Pesticide use in 11 agricultural watersheds Environ Toxicol Pharmacol 10:29–32. exposure may delay sexual maturity and inter- and presence in stream water, 1975–1977. J Environ Qual Sinha N, Narayan R, Saxena DK. 1997. Effect of endosulfan on the 11:497–505. testis of growing rats. Bull Environ Contam Toxicol 58:79–86. fere with hormone synthesis in male children. García-Rodríguez J, García-Martín M, Nogueras-Ocaña M, Sinha N, Narayan R, Shanker R, Saxena DK. 1995. Endosulfan- To validate these findings, a study with larger Luna-del-Castillo JD, García MF, Olea, N, et al. 1996. induced biochemical changes in the testis of rats. Vet Hum sample size is essential. We do not know Exposure to pesticides and cryptorchidism: geographical Toxicol 37:547–549. evidence of a possible association. Environ Health U.S. EPA. 1980. Manual of Analytical Methods for the Analysis the significance of these findings. 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(endosulfan, phosphamidon and mancozeb). Mutagenesis 2000–2002. Geneva:World Health Organization, International ATSDR. 2000. Toxicological Profile for Endosulfan. Atlanta, 11:33–36. Programme on Chemical Safety/Inter-Organisation GA:Agency for Toxic Substances and Disease Registry. Marshell WA, Tanner JM. 1969. Variations in pattern of pubertal Programme for Sound Management of Chemicals. Bowman MC, Schechter MS, Carter RL. 1965. Behavior of changes in girls. Arch Dis Child 44:291–303. 1962 VOLUME 111 | NUMBER 16 | December 2003 • Environmental Health Perspectives