# Parallel assessment of male reproductive function in workers and wild rats exposed to pesticides in banana plantations in Guadeloupe.

Luc Multigner, Philippe Kadhel, Michel Pascal, Farida Huc-Terki, Henri Kercret, Catherine Massart, Eustase Janky, Jacques Auger, Bernard Jégou

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Luc Multigner, Philippe Kadhel, Michel Pascal, Farida Huc-Terki, Henri Kercret, et al.. Parallel assessment of male reproductive function in workers and wild rats exposed to pesticides in banana plantations in Guadeloupe.. Environmental health : a global access science source [electronic resource], 2008, 7, pp.40. 10.1186/1476-069X-7-40. hal-00669505

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# Parallel assessment of male reproductive function in workers and wild rats exposed to pesticides in banana plantations in Guadeloupe

Luc Multigner*¹,², Philippe Kadhel¹,²,³,⁴, Michel Pascal⁵, Farida Huc-Terki⁶, Henri Kercret¹,², Catherine Massart⁷, Eustase Janky³,⁴, Jacques Auger⁸ and Bernard Jégou¹,²

Address: ¹Inserm, U625, Rennes, France, ²Université Rennes 1, IFR 140, Rennes, France, ³Service Gynécologie-Obstétrique, CHU, Guadeloupe, ⁴Université des Antilles et de la Guyane, Guadeloupe, ⁵INRA, Station SCRIBE, Équipe Gestion des Populations Invasives, Rennes, France, ⁶CIST, Guadeloupe, ⁷Unité d'Hormonologie, CHU Ponchaillou, Rennes, France and ⁸Service d'Histologie-Embryologie et de Biologie de la Reproduction, CECOS, CHU Cochin, Paris, France

Email: Luc Multigner* - luc.multigner@rennes.inserm.fr; Philippe Kadhel - phil.kadhel@chu-guadeloupe.fr; Michel Pascal - Michel.Pascal@rennes.inra.fr; Farida Huc-Terki - f.huc-terki@cimt-gpe.com; Henri Kercret - henri.kercret@univ-rennes1.fr; Catherine Massart - catherine.massart@chu-rennes.fr; Eustase Janky - eustase.janky@chu-guadeloupe.fr; Jacques Auger - jacques.auger@cch.ap-hop-paris.fr; Bernard Jégou - bernard.jegou@rennes.inserm.fr

Published: 30 July 2008

Environmental Health 2008, 7:40 doi:10.1186/1476-069X-7-40

This article is available from: http://www.ehjournal.net/content/7/1/40

© 2008 Multigner et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: 22 February 2008

Accepted: 30 July 2008

## Abstract

**Background:** There is increasing evidence that reproductive abnormalities are increasing in frequency in both human population and among wild fauna. This increase is probably related to exposure to toxic contaminants in the environment. The use of sentinel species to raise alarms relating to human reproductive health has been strongly recommended. However, no simultaneous studies at the same site have been carried out in recent decades to evaluate the utility of wild animals for monitoring human reproductive disorders. We carried out a joint study in Guadeloupe assessing the reproductive function of workers exposed to pesticides in banana plantations and of male wild rats living in these plantations.

**Methods:** A cross-sectional study was performed to assess semen quality and reproductive hormones in banana workers and in men working in non-agricultural sectors. These reproductive parameters were also assessed in wild rats captured in the plantations and were compared with those in rats from areas not directly polluted by humans.

**Results:** No significant difference in sperm characteristics and/or hormones was found between workers exposed and not exposed to pesticide. By contrast, rats captured in the banana plantations had lower testosterone levels and gonadosomatic indices than control rats.

**Conclusion:** Wild rats seem to be more sensitive than humans to the effects of pesticide exposure on reproductive health. We conclude that the concept of sentinel species must be carefully validated as the actual nature of exposure may varies between human and wild species as well as the vulnerable time period of exposure and various ecological factors.

<footnote>* Corresponding author</footnote>
## Background

Over the last 20 years, a growing number of observations worldwide have warned of increases in the frequency of reproductive abnormalities in wild male vertebrates [1]. Increases in the frequency of several modifications to male reproductive function have also been reported in humans [1-4]. These include an apparent decline in semen quality and an increase in the incidence of both testicular cancer and congenital abnormalities, such as cryptorchidism and hypospadia. The reasons for these changes are unclear. The short time period over which they have occurred suggests a role for environmental rather than genetic factors. In recent decades, both animals and humans have been subjected to considerable changes in physical, chemical and biological aspects of the environment. Over the same period, sociocultural factors (e.g. diet and lifestyle) affecting humans have also changed considerably. The observed reproductive phenomena probably have multifactorial causes, but attention has principally focused on chemical agents. Chemical pollution has frequently been associated with reproductive effects in wildlife [5,6] and recent epidemiologic and occupational studies have also identified a possible syndromic role of chemical compounds [7,8]. The similarities and differences in reproductive abnormalities observed in humans and wildlife have stimulated fruitful interactions between toxicologists, epidemiologists, clinicians, ecotoxicologists and zoologists [4,9].

Humans and wildlife are exposed to a number of pesticides in the environment. Many of these compounds have been shown to function as endocrine disruptors [10,11]. Other pesticides not formally identified as endocrine disruptors, such as dibromochloropropane (DBCP), may also damage the testes and spermatozoa directly or indirectly [12]. Nevertheless, epidemiologic and experimental studies in the field, particularly as concerns environmental exposure, have provided conflicting results [7]. The use of joint ecoepidemiologic and human epidemiologic surveys for the simultaneous collection and comparison of data is strongly recommended [13]. However, to our knowledge, no such study has yet been undertaken. As a consequence, the notion that wildlife species may be used as sentinel species for human syndromes remains largely theoretical and/or speculative.

In this study, we jointly assessed – during the same time period and at the same site – various endpoints of male reproductive function in humans and wild mammals, both chronically exposed to pesticides. This study was carried out in Guadeloupe (an island in the French West Indies), which has areas of intensive agriculture using large amounts of pesticides, particularly on banana plantations. We studied male employees working in banana plantations and male rats living in the banana fields.

Comparisons were made with men working in non-agricultural sectors and rats living in an area devoid of direct human sources of pollution.

## Materials and methods

### Study area

Guadeloupe consists of two main islands, Grande Terre and Basse Terre, separated by a narrow sea channel. Agriculture is the main economic activity and banana is the principal crop. The banana plantations are mostly located on the rainy slopes of Basse-Terre and intensive pesticide use controls insects, nematodes and fungi in these plantations. The National Park of Guadeloupe encompasses the central part of Basse-Terre and includes the Natural Reserve of Grand Cul-de-Sac Marin. While we are aware that no area can be totally pollution-free, this park has historically been protected from direct pollution by humans and provides an opportunity for research on wildlife less strongly affected by pollution.

### Human study

The study was carried out from 1999 to 2001. The study population consisted of 20- to 50-year-old male workers from Basse-Terre regularly followed up by the Guadeloupe Occupational Health Service. Information meetings were organized in the field, at which the men were informed that the study protocol included the collection of a semen sample, a blood sample, and physical and andrological examinations. Basic data, such as age and current job were collected for all men, including those who later declined participation in other aspects of the study. Each participating worker was given an appointment at the University Hospital of Pointe à Pitre. Blood samples were collected and medical examinations carried out, and information about occupational history and past and present exposure to chemical and physical agents was collected in face-to-face interviews. An indemnity of 80 Euros was paid to each participant who gave written informed consent and the Ethics Committee of Guadeloupe approved this study.

During the hospital visit, subjects underwent a physical and andrological examination, performed by a single physician unaware of the subject's exposure status. A detailed examination of external genitalia and staging of secondary sexual development were carried out according to WHO recommendations [14]. Testicular volumes were evaluated using the Lambert formula: volume (cm³) = 0.71 × major axis (cm) × minor axis (cm)². Abnormalities in testis thermoregulation are known to alter semen characteristics, so scrotal temperatures were measured in naked subjects in the supine position [15]. Demographic data, medical, surgical, urogenital and reproductive history, recent illnesses, fever and treatment and lifestyle data were
collected using a standardized questionnaire during a
face-to-face interview with the physician.

Pesticide exposure status was assessed with a question-
naire. Information was collected yearly concerning occu-
pational and non occupational exposure to chemical
substances (pesticides, solvents, metals and diesel exhaust
fumes) or physical agents (radiant heat and ionizing radi-
ation). The number of years of use and annual frequency
of use was recorded for each category of chemical sub-
stance or physical agent. We asked about whether pesti-
cides were applied in a banana plantation, sugar cane
plantation or other agricultural setting, and distinguished
between the agricultural use of pesticides and kitchen gar-
den activity or other domestic exposure. One occupa-
tional physician specifically trained in agricultural
activities assessed job descriptions blindly, to assess the
degree of correlation between job tasks and declared occu-
pational exposures.

Semen samples were collected by masturbation in the
hospital after a period of sexual abstinence for 2 to 7 days.
Semen analysis was performed according to WHO guide-
lines [16], at the Pathology Laboratory of the University
Hospital of Pointe à Pitre. Semen samples were analyzed
by 2 technicians blind to exposure status and specifically
trained on site. Intra- and inter-technician variations in
the assessment of sperm concentration and motility were
evaluated several times before and during the study and
were considered satisfactory, the coefficients of variation
being typically around 5 to 10%. Sperm morphology was
assessed from smears after Shorr staining, using the mod-
ified David classification [17], by a single technician at the
Reproductive Biology Laboratory, Cochin University Hos-
pital, Paris.

Blood samples were collected between 8 and 9 a.m. and serum samples were stored at -20°C until the analysis. FSH (follicle-stimulating hormone) and LH (luteinizing hormone) levels were determined by immunoradiometric assay (Immunotech, Marseilles, France). The dose-response curve of FSH was calibrated against the WHO international standard of 94/632, and the LH dose-response curve was calibrated against the 2nd IS international standard of 80/552. Total testosterone concentration was measured by radioimmunoassay (Immunotech, Marseilles, France) and dimeric inhibin B concentration was assessed by enzyme-linked immunosorbent assay (Oxford Innovation Ltd, Oxford, UK). Results were expressed as concentrations for testosterone (ng/ml), for inhibin B (pg/ml) and for FSH and LH (IU/l). Between- and within-assay coefficients of variation were < 8% for FSH and LH, < 11% for testosterone and < 17% for inhibin B.

We analyzed data for age, weight, height, body mass
index, smoking habits (past or current smoker vs. non
smokers), alcohol consumption (current drinker vs. non
drinker), recent illness (yes vs. no), fever or medical treat-
ment during the past 3 months (yes vs. no), fertility status
scored as at least one live-born child (yes vs. no) and, for
fertile participants, the total number of children and time
to pregnancy for the most recent child, days of sexual
abstinence before semen analysis, testicular volume and
scrotal temperature. In addition, the month in which the
semen analysis was performed was noted, to account for a
possible seasonal effect. The categories spring/summer vs.
autumn/winter were compared and, as there are no signif-
icant differences in temperature or day length between
seasons on Guadeloupe, we also considered summer/
autumn (wet season) vs. winter/spring (dry season). The
semen characteristics and hormones studied were: pH,
semen volume (ml), sperm concentration (millions/ml),
total sperm count (millions, the product of semen volume
and sperm concentration), sperm viability (the percentage
of live spermatozoa), "a"-type progressive motility (the
percentage of rapid and progressive spermatozoa),
"a"+"b"-type progressive motility (the percentage of rapid
and slow progressive spermatozoa), sperm morphology
(the percentage of morphologically normal spermatozoa)
and the multiple anomaly index (mean number of mor-
phological defects per abnormal spermatozoa). Semen
characteristics were also categorized as dichotomous vari-
ables, using WHO reference values [16] as thresholds: 2
ml for seminal volume, 20 millions/ml for sperm concen-
tration, 40 millions for total sperm count, 25% for "a"-
type progressive motility and 50% for "a" + "b"-type pro-
gressive motility. The threshold selected for sperm mor-
phology was the median percentage of morphologically
normal spermatozoa in non exposed men (13%).

Animal study

We selected two sites extremely different in terms of agri-
cultural activity. The "pesticide-exposed area", located in
Neuf-Chateau (south-east Basse-Terre), has been exclu-
sively devoted to banana cultivation since the mid-20th
Century. The "pesticide-free area" was Fajou Islet in the
Grand Cul-de-Sac Marin lagoon, within the marine
reserve of the National Park.

Trapping and field operations took place between the
18th and the 28th of January 2000, a period of high repro-
ductive activity in French West Indies rodent populations.
Ship rats (Rattus rattus), Norwegian rats (Rattus norvegicus)
and Javanese mongoose (Herpestes auropunctatus), all alien
species in the French West Indies [18], were trapped using
Manufrance® live traps baited with a mixture of peanut
butter, oat flakes and sardine oil [19]. The trapped ani-
mals were killed in the field by intrathoracic pentobarbital
injection. Blood was collected immediately by heart punc-
ture. Animals and blood samples were stored in a con-
tainer at + 4°C and transferred within 45 minutes to a
laboratory specifically installed for this study.

Mammals were sexed and males selected, weighed and
dissected. The testes, epididymis, ventral prostate and
seminal vesicles were removed and weighed. One testis
per animal was fixed by immersion in Bouin's solution for
histology, the second testis and one epididymis being
immediately frozen at – 20°C. Both eye balls were
removed and stored in 10% formaldehyde at room tem-
perature for more than 3 months to tan eye lenses. Eye
lenses were cleaned and dried at 105°C until their weight
stabilized. Lens pair weight was used as a surrogate for age
[20]. Each fixed testis was embedded in paraffin, and 5
µm sections were cut, stained with hematoxylin and eosin
and examined under a light microscope [21]. The number
of sperm in the testis and caudal epididymis was assessed
as previously described [22]. The total number of sperm
was defined as the sum of testicular and epididymal
sperm reserves. The gonadosomatic index was calculated
as (testis weight/body weight) × 100.

Serum fractions were stored at -20°C until use. Rat FSH
levels were determined by immunoradiometric assay
(BiocodeHycel, Belgium) and rat LH levels, by radioim-
munoassay (BiocodeHycel, Belgium). Dose-response
curves for FSH and LH were calibrated against the corre-
sponding NIH standard. Total rat testosterone levels were
determined by radioimmunoassay (Immunotech, Mar-
seilles, France). Results were expressed as concentrations
(ng/ml for testosterone, IU/l for FSH and LH). The intra-
and inter-assay coefficients of variation were < 6, 7 and 8
% for testosterone, FSH and LH, respectively.

Statistical analysis

We used chi-squared or Fisher's exact tests for categorical
variables and unpaired t-tests (equal variances) and anal-
ysis of variance/covariance for continuous variables, in
both human and animal studies. We normalized distribu-
tions by applying a log10 transformation or a square-root
transformation and then used the Kolmogorov-Smirnov
test to check that the resulting data were normally distrib-
uted. The effect of exposure on the categorized human
seminal outcomes was assessed, taking possible con-
founding factors into account, by logistic regression anal-
ysis to produce odds ratios (OR) and 95 percent
confidence intervals (95% CI). Factors were considered
confounding if their p values in univariate analysis were
below 0.2, and were included in the final model if they
modified the odds ratio of the outcome by more than 10
percent [23]. The potential confounding variables evalu-
ated were age, alcohol and tobacco consumption, solvent
exposure, past history of genital infection, sexual absti-
nence, and season of semen analysis. Age and duration of

abstinence were systematically included as known con-
founding factors for semen variables. Linear regression
was used to assess the relationship between eye lens
weight, body weight and total sperm reserve in the animal
study. All analyses were carried out using the Statview
software package (SAS Institute Inc., Cary, USA). All tests
were two-sided, and the results were considered signifi-
cant if P < 0.05.

Results

Human Study

In total, 101 of the 594 men approached for this study
(17%) agreed to participate. There was no difference in
age or proportion of banana plantation workers and
workers not employed by banana plantations between
those agreeing to and declining participation (data not
shown). Six men who agreed to participate were excluded
for one of the following reasons: past chemotherapy for
Hodgkins disease, current treatment for infertility, orchid-
ectomy, current active genital infection or sexual absti-
nence for less than 48 hours before semen analysis. Forty-
two were employed as banana plantation workers and
regularly used pesticides as part of their job. They worked
on 13 plantations but reported having applied pesticides
in 37 different plantations over the course of their careers,
corresponding to more than 80% of the local area under
banana. We listed the chemical products used in the
banana plantations from the notebooks used to record
pesticide applications. The pesticides in use at the time of
the study belonged to the organophosporus (cadusaphos,
ethoprophos, isazophos, pyrimiphos-ethyl, terbufos) and
carbamate (aldicarb) classes. The median duration of pes-
ticide use was 14 years for banana workers. The mean
annual frequency of application was 30 days, and was
similar for all banana plantation workers. A group of 45
manual and office workers enrolled in the study did not
apply pesticides in either occupational or non occupa-
tional circumstances. Finally, eight men were in contact
with pesticides under miscellaneous conditions, includ-
ing occupational exposure in sugarcane fields or vegetable
root plots or domestic applications in kitchen gardens.
This group was not analyzed further due to the small
number of subjects and their heterogeneity in terms of the
chemical family of pesticides used and the frequency and
intensity of application.

Table 1 presents the general characteristics of the 87 par-
ticipants included in the final analysis and the comparis-
ons between exposure groups. Banana plantation
workers were younger than unexposed men. The propor-
tion of smokers and men exposed to solvents was signifi-
cantly higher in the unexposed group than among banana
plantation workers. Likewise the proportion of drinkers
was slightly (not significantly) higher in unexposed men.
<table><caption>Table 1: General, lifestyle and occupational data for men</caption><thead><tr><th>Characteristics</th><th>Non exposed workers (n = 45)</th><th>Pesticide-exposed banana plantation workers (n = 42)</th><th>P</th></tr></thead><tbody><tr><td>Age, mean (SD)</td><td>38.4 (7.6)</td><td>34.8 (6.5)</td><td>0.02 *</td></tr><tr><td>Body mass index, mean (SD)</td><td>24.6 (3.3)</td><td>24.6 (3.3)</td><td>0.97 *</td></tr><tr><td>Body mass index, n (%)</td><td></td><td></td><td></td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;&lt; 25</td><td>28 (62)</td><td>26 (62)</td><td>0.86†</td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;25 – &lt; 30</td><td>15 (33)</td><td>13 (31)</td><td></td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;&gt; 30</td><td>2 (5)</td><td>3 (7)</td><td></td></tr><tr><td>Tobacco, n (%)</td><td></td><td></td><td></td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Non smoker</td><td>23 (51)</td><td>32 (76)</td><td>0.02†</td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Current or former smoker</td><td>22 (49)</td><td>10 (24)</td><td></td></tr><tr><td>Alcohol, n (%)</td><td></td><td></td><td></td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Non drinker</td><td>10 (22)</td><td>15 (36)</td><td>0.17†</td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Current or former drinker</td><td>35 (78)</td><td>27 (64)</td><td></td></tr><tr><td>Drugs, n (%)</td><td></td><td></td><td></td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Non consumers</td><td>41 (91)</td><td>37 (88)</td><td>0.64†</td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Current or former consumers</td><td>4 (9)</td><td>5 (12)</td><td></td></tr><tr><td>Heat exposure, n (%)</td><td></td><td></td><td></td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;No</td><td>43 (96)</td><td>38 (90)</td><td>0.35†</td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Yes</td><td>2 (4)</td><td>4 (10)</td><td></td></tr><tr><td>Solvent exposure, n (%)</td><td></td><td></td><td></td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;No</td><td>28 (62)</td><td>36 (86)</td><td>0.01†</td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Yes</td><td>17 (38)</td><td>6 (14)</td><td></td></tr><tr><td>Diesel exhaust exposure, n (%)</td><td></td><td></td><td></td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;No</td><td>38 (84)</td><td>36 (86)</td><td>0.87†</td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Yes</td><td>7 (16)</td><td>6 (14)</td><td></td></tr><tr><td>Season of semen analysis, n (%)</td><td></td><td></td><td></td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Spring or summer</td><td>28 (62)</td><td>20 (48)</td><td>0.17†</td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Autumn or winter</td><td>17 (38)</td><td>22 (52)</td><td></td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Dry season</td><td>23 (51)</td><td>30 (71)</td><td>0.05†</td></tr><tr><td>&nbsp;&nbsp;&nbsp;&nbsp;Wet season</td><td>22 (49)</td><td>12 (29)</td><td></td></tr><tr><td>Abstinence before semen analysis (days), mean (SD) mean (SD)</td><td>4.1 (1.3)</td><td>3.9 (1.0)</td><td>0.80 *</td></tr></tbody></table>

A history of genital infection was more frequent among banana workers than among unexposed men (table 2). Similarly, a higher proportion of banana plantation workers than of unexposed men had conceived a child. No difference was observed between the two groups in time to pregnancy for the last conception. No significant difference between exposed and unexposed workers was found for any of semen characteristic or hormone level (Table 3). Although not significant after adjustment for covariates, sperm concentration and total sperm count were 20% lower in the pesticide-exposed banana plantation workers than in workers not exposed to pesticides. Based on WHO semen reference values, used as thresholds, banana plantation workers were found to have a slightly, but not significantly higher OR only for a low total sperm count (OR: 2.2, 95% CI 0.5 – 10.4). Following stratification for the number of years of pesticide application, the association between a low sperm concentration or total sperm count and having been exposed for more than 14

years became stronger, but remained non significant (Table 4).

## Animal Study

In total, 120 mammals were trapped: 75 in banana plantations and 45 in Fajou Islet. The very small number of trapped mongooses (n = 9) and the smaller numbers of Norwegian rats in Fajou Islet than in banana plantations (n = 19) ruled out the further analysis of these species. We captured 92 ship rats, and the proportion of males and females did not differ significantly between the sites (p = 0.29). The relationships between eye lens weight (a surrogate of age), body weight and sperm reserve were used to distinguish sexually mature from immature male ship rats. Five animals with a body weight less than 100 g (the cut-off generally used to distinguish between adult and non adult ship rats) with no epididymal and testicular spermatozoa had a dry eye lens weight below 20 mg. Three other animals with an absence of epididymal sper-

<footnote>*: unpaired t-test; †: χ² test</footnote>
<table><caption>Table 2: Medical, andrological and reproductive characteristics in men</caption><thead><tr><th>Characteristics</th><th>Non exposed<br>workers<br>(n = 45)</th><th>Pesticide-exposed<br>banana plantation<br>workers<br>(n = 42)</th><th>P</th></tr></thead><tbody><tr><td>Tanner, n (%)</td><td></td><td></td><td></td></tr><tr><td>3</td><td>2 (5)</td><td>1 (2)</td><td rowspan="3">0.75†</td></tr><tr><td>4</td><td>15 (33)</td><td>12 (29)</td></tr><tr><td>5</td><td>28 (62)</td><td>29 (69)</td></tr><tr><td>Testicular volume (ml), mean (SD)</td><td></td><td></td><td></td></tr><tr><td>Right testis</td><td>20.3 (8.3)</td><td>20.3 (8.7)</td><td>0.99*</td></tr><tr><td>Left testis</td><td>18.1 (7.7)</td><td>18.8 (6.4)</td><td>0.65*</td></tr><tr><td>Both testes</td><td>38.4 (15.3)</td><td>39.1 (12.6)</td><td>0.82*</td></tr><tr><td>Testicular temperature (°C), mean (SD)</td><td></td><td></td><td></td></tr><tr><td>Right testis</td><td>35.2 (0.7)</td><td>35.1 (0.7)</td><td>0.53*</td></tr><tr><td>Left testis</td><td>35.3 (0.7)</td><td>35.2 (0.6)</td><td>0.78*</td></tr><tr><td>Central temperature (°C), mean (SD)</td><td>36.9 (0.4)</td><td>36.9 (0.3)</td><td>0.76*</td></tr><tr><td>Past genital infection, n (%)</td><td></td><td></td><td></td></tr><tr><td>No</td><td>40 (89)</td><td>28 (67)</td><td rowspan="2">0.01†</td></tr><tr><td>Yes</td><td>5 (11)</td><td>14 (33)</td></tr><tr><td>Inguinal hernia (treated), n (%)</td><td></td><td></td><td></td></tr><tr><td>No</td><td>43 (96)</td><td>39 (93)</td><td rowspan="2">0.59†</td></tr><tr><td>Yes</td><td>2 (4)</td><td>3 (7)</td></tr><tr><td>Has conceived at least one child, n (%)</td><td></td><td></td><td></td></tr><tr><td>No</td><td>16 (36)</td><td>7 (17)</td><td rowspan="2">0.06†</td></tr><tr><td>Yes</td><td>29 (64)</td><td>35 (83)</td></tr><tr><td>Time to pregnancy (last conception), n (%)</td><td></td><td></td><td></td></tr><tr><td>≤ 12 months</td><td>26 (96)</td><td>28 (90)</td><td rowspan="2">0.37†</td></tr><tr><td>> 12 months</td><td>1 (4)</td><td>3 (10)</td></tr></tbody></table>

<table><caption>Table 3: Semen characteristics and serum hormone levels in men</caption><thead><tr><th>Characteristics</th><th>Non exposed<br>workers<br>(n = 45)</th><th>Pesticide-exposed<br>banana plantation<br>workers<br>(n = 42)</th><th>P</th></tr></thead><tbody><tr><td><strong>Semen characteristics</strong></td><td></td><td></td><td></td></tr><tr><td>Volume of the ejaculate (mL), mean (SD)</td><td>3.8 (1.7)</td><td>3.4 (1.7)</td><td>0.44*</td></tr><tr><td>pH, mean (SD)</td><td>7.9 (0.2)</td><td>7.9 (0.2)</td><td>0.65*</td></tr><tr><td>Sperm concentration (millions/mL), mean (SD)</td><td>89.8 (81.1)</td><td>70.3 (59.4)</td><td>0.27*</td></tr><tr><td>Total sperm count (millions), mean (SD)</td><td>308 (236)</td><td>231 (229)</td><td>0.19*</td></tr><tr><td>Rapid and progressive sperm motility (%), mean (SD)</td><td>15.2 (8.6)</td><td>15.0 (6.8)</td><td>0.82*</td></tr><tr><td>Progressive sperm motility (%) mean (SD)</td><td>42.4 (11.9)</td><td>42.7 (13.9)</td><td>0.62*</td></tr><tr><td>Morphologically normal sperm (%), mean (SD)</td><td>13.9 (7.4)</td><td>13.0 (8.0)</td><td>0.36*</td></tr><tr><td>Multiple anomaly index, mean (SD)</td><td>1.71 (0.25)</td><td>1.73 (0.29)</td><td>0.79*</td></tr><tr><td>Live sperm (%), mean (SD)</td><td>54.1 (13.4)</td><td>54.5 (17.3)</td><td>0.56*</td></tr><tr><td><strong>Serum hormones</strong></td><td></td><td></td><td></td></tr><tr><td>FSH (IU/L), mean (SD)</td><td>6.3 (4.4)</td><td>5.9 (4.1)</td><td>0.81†</td></tr><tr><td>LH (IU/L), mean (SD)</td><td>4.7 (1.9)</td><td>5.5 (2.6)</td><td>0.16†</td></tr><tr><td>Testosterone (ng/mL), mean (SD)</td><td>7.5 (2.5)</td><td>6.8 (1.7)</td><td>0.33†</td></tr><tr><td>Inhibin B (pg/mL), mean (SD)</td><td>170 (72)</td><td>168 (67)</td><td>0.63†</td></tr></tbody></table>

<footnote>*: unpaired t-test; †: χ² test</footnote>

<footnote>* adjusted for age, tobacco use, solvents, genital infections, season of sperm analysis and sexual abstinence.</footnote>

<footnote>† adjusted for age and body mass index</footnote>
<table><caption>Table 4: Associations between semen characteristics and pesticide exposure in men</caption><thead><tr><th rowspan="2">Semen Characteristics</th><th>Non<br>exposed<br>workers<br>(n = 45)</th><th>Pesticide-exposed<br>banana plantation<br>workers<br>(&lt; 14 years)<br>(n = 21)</th><th>Pesticide-exposed<br>banana plantation<br>workers<br>(&gt; 14 years)<br>(n = 21)</th></tr><tr><th>OR</th><th>OR (95%CI) *</th><th>P</th><th>OR (95%CI) *</th><th>P</th></tr></thead><tbody><tr><td>Semen volume (&lt; 2 mL)</td><td>1.0</td><td>1.0 (0.2, 6.0)</td><td>0.99</td><td>1.4 (0.2, 11.3)</td><td>0.77</td></tr><tr><td>Sperm concentration (&lt; 20 millions/mL)</td><td>1.0</td><td>0.9 (0.2, 5.4)</td><td>0.92</td><td>2.0 (0.4, 10.1)</td><td>0.40</td></tr><tr><td>Total sperm count (&lt; 40. millions)</td><td>1.0</td><td>1.7 (0.2, 12.1)</td><td>0.58</td><td>3.2 (0.5, 22.0)</td><td>0.24</td></tr><tr><td>"a" progressive sperm motility (&lt; 25%)</td><td>1.0</td><td>1.6 (0.2, 17.4)</td><td>0.68</td><td>0.6 (0.1, 5.6)</td><td>0.59</td></tr><tr><td>"a" + "b" progressive sperm motility (&lt; 50%)</td><td>1.0</td><td>0.7 (0.2, 2.3)</td><td>0.60</td><td>1.0 (0.3, 3.6)</td><td>0.99</td></tr><tr><td>Sperm morphology (&lt; 13%)</td><td>1.0</td><td>0.9 (0.3, 2.8)</td><td>0.86</td><td>2.0 (0.6, 6.9)</td><td>0.28</td></tr></tbody></table>

matozoa but with testicular spermatozoa had eye lens weights below 21 mg. For these eight rats, testis histology indicated that there was no, or only focal, spermatogenesis. We therefore used a threshold eye lens weight of 21 mg w to distinguish sexually mature ship rats from immature ones.

No significant difference was observed in the mean eye lens weight of ship rats captured in the two areas (table 5). This suggests that age structures of the two populations were similar. However, ship rats captured in banana plantations were significantly heavier than those captured in Fajou Islet. None of the absolute weights of reproductive organs differed significantly between ship rats from the two sites. However, the testis weight/body weight ratio, known as the gonadosomatic index, was lower in ship rats trapped in the banana plantations than for those trapped in Fajou Islet. This difference could not be accounted for

by a difference in the "relative age" of the animals, as eye lens weight was included as a covariate. Serum testosterone levels were also significantly lower in ship rats trapped in banana plantations than in those trapped in Fajou Islet, whereas gonadotropin levels were similar in rats from both sites.

## Discussion

There is widespread concern that reproductive function, particularly in males, is currently subject to the deleterious effects of chemical agents in the environment. According to the endocrine disruption hypothesis, exposure to chemicals, including pesticides, results in a range of reproductive disturbances in humans and in various wildlife species, some of which may be used as sentinel species indicating an increase in the threat to human reproduction and health [1,4,9,13].

<table><caption>Table 5: General and reproductive characteristics in male ship rats</caption><thead><tr><th>Characteristics</th><th>Pesticide-exposed area<br>(banana plantations)<br>(n = 21)<br>mean (SD)</th><th>Pesticide-free area<br>(Fajou Islet)<br>(n = 20)<br>mean (SD)</th><th>P</th></tr></thead><tbody><tr><td>Eye lens weight (mg)</td><td>29.4 (5.9)</td><td>29.0 (4.0)</td><td>0.92 †</td></tr><tr><td>Body weight (g)</td><td>218 (31)</td><td>166 (30)</td><td>&lt; 0.0001 †</td></tr><tr><td>Testis weight (g)</td><td>3.68 (0.72)</td><td>3.47 (0.36)</td><td>0.07 *</td></tr><tr><td>Epididymal weight (g)</td><td>1.22 (0.5)</td><td>1.28 (0.2)</td><td>0.39 *</td></tr><tr><td>Prostate weight (g)</td><td>0.21 (0.11)</td><td>0.20 (0.06)</td><td>0.79 *</td></tr><tr><td>Seminal vesicle weight (g)</td><td>0.82 (0.63)</td><td>0.88 (0.31)</td><td>0.50 *</td></tr><tr><td>Testicular sperm reserves (millions)</td><td>437(141)</td><td>419 (73)</td><td>0.57 *</td></tr><tr><td>Epididymal sperm reserves (millions)</td><td>427 (304)</td><td>477 (167)</td><td>0.30 *</td></tr><tr><td>Total sperm reserves (millions)</td><td>864 (413)</td><td>896 (223)</td><td>0.56 *</td></tr><tr><td>Gonadosomatic index</td><td>1.70 (0.31)</td><td>2.13 (0.29)</td><td>&lt; 0.0001 *</td></tr><tr><td>Testosterone (ng/mL)</td><td>11.2 (6.1)</td><td>19.1 (9.8)</td><td>0.02 *</td></tr><tr><td>LH (IU/L)</td><td>0.53 (0.46)</td><td>0.50 (0.18)</td><td>0.82 *</td></tr><tr><td>FSH (IU/L)</td><td>17.6 (3.6)</td><td>18.4 (6.3)</td><td>0.65 *</td></tr></tbody></table>

<footnote>* adjusted for age, tobacco use, solvents, genital infections, season of sperm analysis and sexual abstinence</footnote>

<footnote>†: unpaired t-test;</footnote>

<footnote>*: adjusted for eye lens weight as a surrogate for age</footnote>
Past chemical hazards to male reproductive function were
revealed in a dramatic manner, in a study showing that
banana plantation workers exposed to the nematocide
DBCP had severely impaired spermatogenesis, leading to
permanent infertility in some cases [12]. Other pesticides
used in banana crops, such as chlordecone and ethylene
dibromide, have also clearly been shown to cause sperma-
togenic damage in a laboratory setting [24,25].

The pesticides in use in banana plantations in Guade-
loupe at the time of this study included five organophos-
phorous agents – cadusafos, ethoprophos, isazophos,
pyrimiphos-ethyl and terbufos – and one carbamate
derivative, aldicarb. To our knowledge, until this study,
no epidemiological study had investigated the effects of
these pesticides on male fertility.

Human semen samples are generally difficult to obtain,
with participation rates generally below 20% [26]. The
participation rate of 17% in our study is consistent with
this observation. Our protocol allowed us to exclude all
subjects with identified infertility risk factors [27] and to
identify an occupationally exposed group that had
applied pesticides and been continuously exposed to
these agents over several years. While intrinsically
restricted by the expected limitations of cross-sectional
sperm studies and the small size of this study, our results
suggest that exposure to the pesticides currently used in
the banana plantations of Guadeloupe is not markedly
detrimental to male fertility.

In Guadeloupe, ship rats are a commensal species, proba-
bly introduced by Spanish explorers before 1635 [18]. Age
determination is a key element of population dynamics
studies in wild mammals of this kind [28]. It has been
shown that, for species with a life expectancy in the wild
of less than one year (as in rats), eye lens dry weight is a
suitable surrogate variable for the age of the animal [29].
Following the adjustment of values to account for differ-
ences in age between rats captured in pesticide-exposed
and unexposed areas, the only parameter significantly
affected was blood testosterone concentration. The
observed decrease in circulating testosterone concentra-
tion may reflect an anti-androgenic effect of the sub-
stances polluting the banana fields. Indeed, a number of
organophosphorus insecticides, including chlorphyri-
phos-methyl, malathion and diazinon, have been shown
in toxicological experiments to inhibit testosterone syn-
thesis/production [30-32]. It has also been suggested that
aldicarb, one of the pesticides in use at the time of the
study, acts in synergy with other pesticides as an endo-
crine disruptor in laboratory rats [33]. Furthermore, a
toxic interaction between testosterone and aldicarb has
been reported in the Japanese medaka fish [34]. However,
the lack of reproductive toxicity studies on the organo-

phosphorous pesticides used in Guadeloupe, and past
contamination of banana plantations with organochlo-
rine pesticides make it difficult to identify precisely the
causal agents reducing blood testosterone levels. The
affected rats displayed no significant change in LH con-
centration, possibly due to the fatness of rats trapped in
banana plantations, which were more than 20 % heavier
than Fajou Islet rats. This situation may have led to a
number of metabolic changes, including, for example, an
increase in circulating estradiol levels. However, this
hypothesis remains to be confirmed experimentally. This
difference in body weight reflects differences in food avail-
ability between the two habitats, as autopsy showed that
ship rats do eat bananas. Due to this discrepancy in body
weight, the gonadosomatic index was significantly higher
for rats from the unexposed area than for rats from the
pesticide-exposed area.

Several factors, such as the nature of the chemicals to
which humans and animals were exposed and major dif-
ferences in the generation time of humans and rats, must
be taken into account when comparing the impact of pes-
ticides on human and rat populations. For example,
DBCP was scarcely used in the French West Indies in the
past, but chlordecone was in widespread use until 1993
[35]. These chemicals were replaced by organophospho-
rus and carbamate insecticides. Thus, unlike humans, rats,
with their much shorter generation time, have been
exposed only to the most recently used pesticides. Other
considerations to be borne in mind when comparing the
human and rat populations is that rat populations are
strictly territorial and therefore remain permanently in
their habitat and that the periods of development (e.g.,
prenatally, adulthood) at which they are exposed may dif-
fer.

This study constitutes a prototype experiment. Major
logistic efforts were made: a thorough historical analysis
of environmental and agricultural conditions in Guade-
loupe, the setting up of a spermatology laboratory, with
the adequate training of local health technicians for
human sperm handling, processing and analysis, the set-
ting up of an animal autopsy laboratory, fully equipped
for the manipulation and storage of biological samples
and tissues and the coordination of several institutions for
the capture of wild animals and of researchers from differ-
ent disciplines including andrologists, wildlife specialists,
biologists and epidemiologists. For the further develop-
ment of such approaches, exposure measurements in both
humans and animals, larger numbers of human volun-
teers and the inclusion of several wildlife species would all
be relevant objectives. Furthermore, the relative impact of
ecological factors on reproductive success in wildlife spe-
cies should also be considered. These factors include hab-
itat restriction, stress due to the intrusion of humans and
changes in food supply. These, and subtle genetic differ-
ences in the population structure, could not be evaluated
here.

Conclusion

Wild rats seem to be more sensitive than humans to high
levels of pesticides (or of other unidentified factors) in the
environment. The concept of sentinel species is not as
simple as it may appear. Understanding the very complex
interactions between the plethora of potential contami-
nants and the organisms living in contaminated environ-
ments will require experiments including many
parameters and should, ideally, span the life cycle of some
of the species concerned.

Abbreviations

DBCP: Dibromochloropropane; FSH: Follicle stimulating hormone; LH: Luteinizing hormone; WHO: World Health Organization.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

LM, PK, MP, JA and BJ designed the study, supervised the overall project and were involved in the interpretation of the results and revision of the paper. LM was responsible for the overall running of the study and did the statistical analysis. PK, FHT and EJ were responsible for recruiting the human subjects and for medical data collection. LM, PK, MP, JA and BJ were involved in the field study. HK and CM were involved in hormonal analysis. All authors read and approved the final manuscript. LM and PK equally contributed to this work.

Acknowledgements

We thank AM Andrea, M Claise, X Delloue, A Ducros, G Fanget, A Lahmar,
C Lesaffre, S Meige, V Michel, N Nomal, J Thrace, AM Touzalin, ML
Ohresser, M Roudier, C Vuillaume for assistance. We also thank the
National Office for Hunting and Wildlife (ONCF), the French Agricultural
Research Centre for International Development (CIRAD), and the
National Park of Guadeloupe.

This work was funded by grants from the French Agency for Environmental and Occupational Health Safety (Afset), from the Clinical Research Hospital Program from the French Ministry of Health (PHRC), and through an unrestricted grant from the French Crop Protection Association (UIPP).

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