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See detailInvestigating apical adverse effects of four endocrine active substances in the freshwater gastropod Lymnaea stagnalis
Giusti, Arnaud ULg; Lagadic, Laurent; Barsi, Alpar et al

in Science of the Total Environment (2014), 493

The hermaphroditic gastropod Lymnaea stagnalis is proposed as a candidate species for the development of OECD guidelines for testing of the reprotoxicity of chemicals, including endocrine active ... [more ▼]

The hermaphroditic gastropod Lymnaea stagnalis is proposed as a candidate species for the development of OECD guidelines for testing of the reprotoxicity of chemicals, including endocrine active substances (EASs). Up to now, only a few putative EASs have been tested for their reproductive toxicity in this species. In this study, we investigate the effects of four EASs with different affinities to the vertebrate estrogen and androgen receptors (chlordecone as an estrogen; cyproterone acetate, fenitrothion and vinclozolin as anti-androgens) on the reproduction of L. stagnalis in a 21-day semi-static test. Testosterone and 17α-ethinylestradiol (EE2) were used as the reference compounds. The tested EASs had no significant effect on growth and survival at the tested concentration ranges (ng to μg/L). Classical reproduction endpoints (i.e., oviposition and fecundity) were not responsive to the tested chemicals, except for chlordecone and 17α-ethinylestradiol, which hampered reproduction from 19.6 μg/L and 17.6 μg/L, respectively. The frequency of polyembryonic eggs, used as an additional endpoint, demonstrated the effects of all compounds except EE2. The molecular pathways, which are involved in such reproduction impairments, remain unknown. Our results suggest that egg quality is a more sensitive endpoint as compared to other reproductive endpoints commonly assessed in mollusk toxicity tests. [less ▲]

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See detailBioaccumulation of polybrominated diphenyl ethers (PBDEs) in Gammarus pulex: Relative importance of different exposure routes and multipathway modeling
Lebrun, Jérémie; Leroy, Delphine; Giusti, Arnaud ULg et al

in Aquatic Toxicology (2014), 154

Characterizing the exposure routes of an organism and its ability to regulate accumulated contaminants is a crucial step toward developing a biomonitor. To date, very little data are available on the ... [more ▼]

Characterizing the exposure routes of an organism and its ability to regulate accumulated contaminants is a crucial step toward developing a biomonitor. To date, very little data are available on the bioaccumulation kinetics of PBDEs in freshwater biota. This study aims at investigating the potential use of a litter-degrader widely distributed in European freshwaters, Gammarus pulex, as an indicator of exposure to PBDEs. In aquatic microcosms, gammarids were exposed to a mixture of brominated congeners (BDE-28, 47, 66, 85, 99, 100, 153, 154 and 183) to assess their ability to bioconcentrate PBDEs. Results show that all tested congeners are highly internalized by G. pulex and uptake rates of PBDEs are closely related to their partition coefficients (Kow). The determination of the elimination rate of BDE-47, the congener most readily accumulated by gammarids, indicated that metabolism and excretion of this congener are low in G. pulex, which argues in favor of its use as a quantitative biomonitor. Finally, bioaccumulation experiments were performed using contaminated leaves to determine the relative importance of dietary uptake in the contamination of gammarids. Even though water is the preeminent exposure route, a significant uptake of BDE-47 through food was observed (27%). We propose a biodynamic model that takes into account both exposure routes to describe BDE-47 bioaccumulation. This study supports the use of this ubiquitous amphipod as an early warning monitor of the bioavailable contamination of freshwaters by PBDEs. [less ▲]

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See detailProteomic Analysis of the Reproductive Organs of the Hermaphroditic Gastropod Lymnea stagnalis Exposed to Different Endocrine Disrupting Chemicals
Giusti, Arnaud ULg; Leprince, Pierre ULg; Mazzucchelli, Gabriel ULg et al

in PLoS ONE (2013), 8(11), 81086

Many studies have reported perturbations of mollusc reproduction following exposure to low concentrations (ng/L range) of endocrine disrupting chemicals (EDCs). However, the mechanisms of action of these ... [more ▼]

Many studies have reported perturbations of mollusc reproduction following exposure to low concentrations (ng/L range) of endocrine disrupting chemicals (EDCs). However, the mechanisms of action of these molecules on molluscs are still poorly understood. Investigation of the modifications of protein expression in organisms exposed to chemicals using proteomic methods can provide a broader and more comprehensive understanding of adverse impacts of pollution on organisms than conventional biochemical biomarkers (e.g., heat-shock proteins, metallothioneins, GST, EROD). In this study we have investigated the impacts of four chemicals, which exhibit different endocrine disrupting properties in vertebrates, on the proteome of the hermaphroditic freshwater pulmonate gastropod Lymnaea stagnalis after 21 days of exposure. Testosterone, tributyltin, chlordecone and cyproterone acetate were chosen as tested compounds as they can induce adverse effects on the reproduction of this snail. The 2D-DIGE method was used to identify proteins whose expression was affected by these compounds. In addition to modifying the expression of proteins involved in the structure and function of the cytoskeleton, chemicals had impacts on the expression of proteins involved in the reproduction of L. stagnalis. Exposure to 19.2 μg/L of chlordecone increased the abundance of ovipostatin, a peptide transmitted during mating through seminal fluid, which reduces oviposition in this species. The expression of yolk ferritin, the vitellogenin equivalent in L. stagnalis, was reduced after exposure to 94.2 ng Sn/L of tributyltin. The identification of yolk ferritin and the modification of its expression in snails exposed to chemicals were refined using western blot analysis. Our results showed that the tested compounds influenced the abundance of yolk ferritin in the reproductive organs. Alteration in proteins involved in reproductive pathways (e.g., ovipostatin and yolk ferritin) could constitute relevant evidence of interaction of EDCs with reproductive pathways that are under the control of the endocrine system of L. stagnalis. [less ▲]

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See detailEsterification of vertebrate like steroids in molluscs: A target of endocrine disruptors?
Giusti, Arnaud ULg; Joaquim-Justo, Célia ULg

in Comparative Biochemistry and Physiology. C : Comparative Pharmacology and Toxicology (2013), 158(4), 187-198

Alterations of the reproductive organs of gastropod molluscs exposed to pollutants have been reported in natural populations for more than 40 years. In some cases these impacts have been linked to ... [more ▼]

Alterations of the reproductive organs of gastropod molluscs exposed to pollutants have been reported in natural populations for more than 40 years. In some cases these impacts have been linked to exposure to endocrine disrupting chemicals (EDCs), which are known to induce adverse impacts on vertebrates, mainly by direct binding to steroid receptors or by altering hormone synthesis. Investigations on the mechanisms of action of endocrine disruptors in molluscs show that EDCs induce modifications of endogenous titres of androgens (e.g., testosterone, androstenedione) and oestrogens (e.g., 17ß-oestradiol). Alterations of the activity of enzymes related to steroid metabolism (i.e., cytochrome P450 aromatase, acyltransferases) are also often observed. In bivalves and gastropods, fatty acid esterification of steroids might constitute the major regulation of androgen and oestrogen homeostasis. The present review indicates that metabolism of steroid hormones to fatty acid esters might be a target of synthetic EDCs. Alterations of this process would impact the concentrations of free, potentially bioactive, form of steroids. [less ▲]

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See detailTestosterone levels and fecundity in the hermaphroditic aquatic snail Lymnaea stagnalis exposed to testosterone and endocrine disruptors
Giusti, Arnaud ULg; Ducrot, Virginie; Joaquim-Justo, Célia ULg et al

in Environmental Toxicology & Chemistry (2013), 32(8), 1740-1745

Endocrine disruptors are known to alter endogenous free and/or esterified levels of androgenic and estrogenic steroid hormones in aquatic molluscs. However, there is still a controversy on the origin of ... [more ▼]

Endocrine disruptors are known to alter endogenous free and/or esterified levels of androgenic and estrogenic steroid hormones in aquatic molluscs. However, there is still a controversy on the origin of steroids in these animals. In this study, free and esterified testosterone concentrations were measured in the hermaphroditic aquatic gastropod Lymnaea stagnalis exposed to molecules known for their androgenic (testosterone and tributyltin), anti-androgenic (cyproterone-acetate) and estrogenic (chlordecone) properties by reference to their mode of action in vertebrates. In parallel, snail oviposition and fecundity were followed over the 21-day exposure period. Testosterone exposure resulted in increased esterified testosterone levels whereas free testosterone concentrations remained stable. In contrast, cyproterone-acetate increased significantly the free form of testosterone with no changes in the esterified form whereas chlordecone showed a tendency to reduce, though not significantly, esterified testosterone concentrations without changes in free testosterone levels. Finally, tributyltin did not alter testosterone homeostasis. The production of egg-clutches and eggs was significantly reduced only in the snails exposed to the highest concentrations of chlordecone (19.6 µg/L) and tributyltin (94.2 ng Sn/L). Overall, this study demonstrates that uptake of testosterone from the exposure medium occurs in L. stagnalis. Moreover, it shows that cyproterone-acetate and, to a lesser extent, chlordecone can alter endogenous testosterone levels in this freshwater snail. However, relationship between hormonal changes and snail reproduction has not been established. [less ▲]

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See detailReproductive impacts of tributyltin (TBT) and triphenyltin (TPT) in the hermaphroditic freshwater gastropod Lymnaea stagnalis
Giusti, Arnaud ULg; Barsi, Alpar; Dugué, Maël et al

in Environmental Toxicology & Chemistry (2013), 32(7), 1552-1560

Tributyltin (TBT) and triphenyltin (TPT) are emblematic endocrine disruptors, which have been mostly studied in gonochoric prosobranchs. Although both compounds can simultaneously occur in the environment ... [more ▼]

Tributyltin (TBT) and triphenyltin (TPT) are emblematic endocrine disruptors, which have been mostly studied in gonochoric prosobranchs. Although both compounds can simultaneously occur in the environment, they have mainly been tested separately for their effects on snail reproduction. Because large discrepancies in experimental conditions occurred in these tests, the present study aimed at comparing the relative toxicity of TBT and TPT under similar laboratory conditions in the 0–600 ng Sn/L range. Tests were performed on the simultaneous hermaphrodite Lymnaea stagnalis, a freshwater snail in which effects of TPT were unknown. Survival, shell length and reproduction were monitored in a 21d semi-static test. Frequency of abnormal eggs was assessed as an additional endpoint. TPT hampered survival while TBT did not. Major effects on shell solidity and reproduction were observed for both compounds, reproductive outputs being more severely hampered by TBT than by TPT. Considering the frequency of abnormal eggs allowed increasing test sensitivity, since snail responses to TBT could be detected at concentrations as low as 19 ng Sn/L. However, the putative mode of action of the two compounds could not be deduced from the structure of the molecules or from the response of apical endpoints. Sensitivity of L. stagnalis to TBT and TPT was compared to the sensitivity of prosobranch molluscs with different habitats and different reproductive strategies. [less ▲]

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See detailImpacts and mechanisms of action of endocrine disrupting chemicals on the hermaphroditic freshwater gastropod Lymnaea stagnalis (Linnaeus, 1758)
Giusti, Arnaud ULg

Doctoral thesis (2013)

Adverse impacts exerted by molecules called endocrine disruptors on reproduction have been extensively described in vertebrates. However, endocrine-disrupting chemicals might alter reproduction of ... [more ▼]

Adverse impacts exerted by molecules called endocrine disruptors on reproduction have been extensively described in vertebrates. However, endocrine-disrupting chemicals might alter reproduction of invertebrate species, at lower concentrations or through different modes of action, with consequences on population growth and stability. Among invertebrates, molluscs have been shown to be very sensitive to endocrine disruptors and were therefore proposed as model species for the development of OECD guideline for the testing and the assessment of reprotoxic effect of chemicals, including EDCs. The hermaphrodite gastropod species Lymnaea stagnalis is a candidate species for the development of such guidelines. In this context, the aim of this thesis is to provide a better understanding of the impacts and the mechanisms of action of endocrine disrupting chemicals on the reproduction of L. stagnalis. The chemicals tested in the present work were chosen based on their interactions with the endocrine system of vertebrates. Testosterone is the natural ligand of the androgen receptor in vertebrates. Three different molecules were selected for their antagonism of the androgen receptor in vertebrates: the synthetic steroid cyproterone acetate, the dicarbamide fungicide vinclozolin and the organophosphate insecticide fenitrothion. Chlordecone is an organochlorinated pesticide, which binds oestrogen receptors in vertebrates. Finally, two organotin compounds, triphenyltin and tributyltin, are able to induce the development of imposex, imposition of male sex organs in females, in gonochoric gastropod molluscs. Results of the present work are divided into two main sections. Firstly, we investigated the impacts of the selected chemicals on the reproduction of L. stagnalis. Our results displayed that TBT, TPT, and chlordecone are able to induce adverse impacts on the reproduction of L. stagnalis through a reduction of the cumulative number of eggs and clutches produced per individual. The assessment of the quality of eggs produced showed that exposure to the chemicals, except TPT, increases the frequency of abnormal eggs laid. The impacts on egg quality were observed at lower concentrations than those affecting the classical reproductive endpoints assessed in reprotoxicity tests in this species. Our results highlight that the implementation of egg quality as an endpoint should increase the sensitivity of the reprotoxicity test with L. stagnalis. The second section of results aims at providing new insights on the mechanisms of action of putative endocrine disruptors in L. stagnalis. Results from chapter 3 and 4 allowed us to select chemical treatments (testosterone, tributyltin, cyproterone acetate, and chlordecone) that induced adverse effects, by interacting with the endocrine system, on the reproduction of L. stagnalis. In Chapter 5, we analysed endogenous concentrations of testosterone, in its free and esterified forms, in the reproductive organs of L. stagnalis. Exposure to waterborne testosterone has confirmed that molluscs can take up this steroid from environmental media and that a homeostatic regulation of endogenous concentrations occurs through the esterification of steroids to fatty acid esters. Moreover, our results support that testosterone is endogenously synthesised in L. stagnalis, as it has been suggested in other mollusc species. However, the impacts on the reproduction and the modifications of endogenous concentrations of testosterone reported in this thesis could not be associated. In chapter 6, proteomic experiments were used to identify proteins differently expressed in reproductive organs of L. stagnalis exposed to testosterone, tributyltin, cyproterone acetate, and chlordecone. Modifications in the expression of three proteins involved in L. stagnalis reproduction (i.e., ovipostatin, yolk ferritin and PIWI) could be associated with the alterations of reproductive endpoints reported in chapters 3 and 4, therefore suggesting possible endocrine disruption mechanisms. Altogether, the results obtained in the present work provide evidences of interactions between chemicals and the endocrine system of this hermaphrodite species. Moreover, the additional sensitive reproductive endpoints described in this thesis (i.e., egg quality) as well as the identification of particular proteins (i.e., ovipostatin, yolk ferritin and PIWI) and their differential expression patterns following chemical exposure, provide reliable tools to screen potency of toxicants to be endocrine disruptors in a partial life cycle test on the freshwater gastropod Lymnaea stagnalis. [less ▲]

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See detailPersistent Organochlorine Pollutants with Endocrine Activity and Blood Steroid Hormone Levels in Middle- Aged Men
Emeville, Elise; Giton, Frank; Giusti, Arnaud ULg et al

in PLoS ONE (2013), 8(6), 66460

Abstract Background: Studies relating long-term exposure to persistent organochlorine pollutants (POPs) with endocrine activities (endocrine disrupting chemicals) on circulating levels of steroid hormones ... [more ▼]

Abstract Background: Studies relating long-term exposure to persistent organochlorine pollutants (POPs) with endocrine activities (endocrine disrupting chemicals) on circulating levels of steroid hormones have been limited to a small number of hormones and reported conflicting results. Objective: We examined the relationship between serum concentrations of dehydroepiandrosterone, dehydroepiandrosterone sulphate, androstenedione, androstenediol, testosterone, free and bioavailable testosterone, dihydrotestosterone, estrone, estrone sulphate, estradiol, sex-hormone binding globulin, follicle-stimulating hormone, and luteinizing hormone as a function of level of exposure to three POPs known to interfere with hormone-regulated processes in different way: dichlorodiphenyl dichloroethene (DDE), polychlorinated biphenyl (PCB) congener 153, and chlordecone. Methods: We collected fasting, morning serum samples from 277 healthy, non obese, middle-aged men from the French West Indies. Steroid hormones were determined by gas chromatography-mass spectrometry, except for dehydroepiandrosterone sulphate, which was determined by immunological assay, as were the concentrations of sex-hormone binding globulin, follicle-stimulating hormone and luteinizing hormone. Associations were assessed by multiple linear regression analysis, controlling for confounding factors, in a backward elimination procedure, in multiple bootstrap samples. Results: DDE exposure was negatively associated to dihydrotestosterone level and positively associated to luteinizing hormone level. PCB 153 was positively associated to androstenedione and estrone levels. No association was found for chlordecone. Conclusions: These results suggested that the endocrine response pattern, estimated by determining blood levels of steroid hormones, varies depending on the POPs studied, possibly reflecting differences in the modes of action generally attributed to these compounds. It remains to be investigated whether this response pattern is predictive of the subsequent occurrence of disease. [less ▲]

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See detailEffects of tributyltin (TBT) and triphenyltin (TPT) on the reproduction of the freshwater snail Lymnaea stagnalis
Lagadic, Laurent; Giusti, Arnaud ULg; Barsi, Alpar et al

Conference (2012, December 10)

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See detailReproductive impact and proteomic analysis of androgenic and anti-androgenic disruptors on the hermaphroditic freshwater gastropod Lymnaea stagnalis
Giusti, Arnaud ULg; Ducrot, Virginie; Joaquim-Justo, Célia ULg et al

Poster (2011, November)

In this study, e ects of two androgens (tributyltin and testosterone), one antiandrogen (cyproterone acetate) and one estrogen (chlordecone) on growth and reproduction were investigated in the ... [more ▼]

In this study, e ects of two androgens (tributyltin and testosterone), one antiandrogen (cyproterone acetate) and one estrogen (chlordecone) on growth and reproduction were investigated in the hermaphrodite gastropod Lymnaea stagnalis. In this study, exposure to a range of concentrations (ng/l to μg/l) of each chemical was performed during 21 days. e number of clutches and the number of eggs per clutch were monitored. A decrease in clutches laid per snail was observed after exposure to tributyltin (540, 1180 and 2600 ng/l) and chlordecone (10, 22, 50 and 110 μg/l). A signi cant decrease in egg laid per snail was observed after exposure to tributyltin (540, 1180 and 2600 ng/l) and chlordecone (50 and 110 μg/l). An increase of egg abnormalities ratio in exposed snails (atrophied albumen, polyembryonicity,...) was observed in L. stagnalis following exposure to testosterone (2, 22, 50 and 110 ng/l), cyproterone acetate (4,5 and 50 μg/l), tributyltin (110 and 244 ng/l) and chlordecone (4,5 and 10 μg/l). Investigation of alteration in protein expression in exposed snails was performed using proteomic analysis such as 2D-DIGE. Mass spectrometry identi cation was performed on proteins with altered expression. We could establish correlation between reproductive endpoints and changes in proteins involved in egg formation and in egg laying were underlined. Egg yolk ferritin, the main protein of egg yolk, was shown to be reduced signi cantly in relationship with a decrease of egg yolk quality after exposure to tributyltin 540 ng/l and cyproterone acetate 4,5 μg/l. Ovipostatin, a protein proved to reduce egg masses, was signi cantly over expressed in snails exposed to 50 μg/l of chlordecone and were in relationship with a reduction of clutches laid by individuals. Further western blot analysis on those proteins involved in the reproduction are underway. ese analysis will enable us to con rm and re ne with more speci city the 2D-DIGE results for the selected proteins. e results of this study can help to establish new biomarkers of exposure of endocrine disruptors in freshwater environment and can provide new insight on mode of action of endocrine disruptors in L. stagnalis. [less ▲]

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See detailChlordecone Exposure and Risk of Prostate Cancer
Multigner, Luc; NDong, Jean-Rodrigue; Giusti, Arnaud ULg et al

in Journal of Clinical Oncology (2010)

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See detailEffects of androgenic and anti-androgenic substances on the freshwater gastropod Lymnaea stagnalis
Giusti, Arnaud ULg; Ducrot, Virginie; Joaquim-Justo, Célia ULg et al

Poster (2010, May 25)

Knowledge on the impacts of endocrine disrupting chemicals (EDCs) on gastropods is scarce and their mechanisms of action are still poorly understood. In this study, effects of 3 androgens (tributyltin ... [more ▼]

Knowledge on the impacts of endocrine disrupting chemicals (EDCs) on gastropods is scarce and their mechanisms of action are still poorly understood. In this study, effects of 3 androgens (tributyltin, testosterone and fenitrothion), 2 anti-androgens (cyproterone acetate and vinclozolin) and 1 estrogen (chlordecone) on growth and reproduction were investigated in the hermaphrodite gastropod Lymnaea stagnalis. [less ▲]

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See detailImpacts of androgenic and antiandrogenic substances on the freshwater pulmonate gastropod Lymnaea stagnalis
Giusti, Arnaud ULg; Lagadic, Laurent; Joaquim-Justo, Célia ULg et al

Poster (2010, May 23)

Knowledge on the impacts of endocrine disruptors on gastropods is scarce and their mechanism of action poorly understood especially the impacts of androgenic and antiandrogenic compounds. In this study ... [more ▼]

Knowledge on the impacts of endocrine disruptors on gastropods is scarce and their mechanism of action poorly understood especially the impacts of androgenic and antiandrogenic compounds. In this study effects of 5 androgenics and antiandrogenics endocrine disruptors were investigated on the reproduction and life traits of the freshwater pulmonate gastropod Lymnaea stagnalis. The chemicals tested were Tributyltin, Cyproterone acetate, Methyltestosterone, Vinclozolin and Fenitrothion. Tributyltin was used as androgenic positive control. The other compounds tested are androgenic and antiandrogenic compounds either steroids or non steroids. Adult snails were exposed to 3 concentrations of each chemical for 30 days. The size and reproductive organs development were monitored throughout exposure. The number of clutches, the number of eggs per clutch were assessed during the first 10 days. The clutches were kept individually in clear water except for the clutches of the eighth day which were divided in two. Half was kept individually in clear water and the other half was individually reared in contaminated water following the same exposure as their parents. The percentage of hatching per clutch of these clutches was measured. The development size, mortality, and development of reproductive organs of the juveniles were assessed until their first clutch was laid. [less ▲]

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See detailPesticide exposure of pregnant women in Guadeloupe: Ability of a food frequency questionnaire to estimate blood concentration of chlordecone
Guldner, Laurence; Multigner, Luc; Héraud, Fanny et al

in Environmental Research (2010), 110(2), 146-151

Context Chlordecone, an environmentally persistent organochlorine insecticide used intensively in banana culture in the French West Indies until 1993, has permanently polluted soils and contaminated ... [more ▼]

Context Chlordecone, an environmentally persistent organochlorine insecticide used intensively in banana culture in the French West Indies until 1993, has permanently polluted soils and contaminated foodstuffs. Consumption of contaminated food is the main source of exposure nowadays. We sought to identify main contributors to blood chlordecone concentration (BCC) and to validate an exposure indicator based on food intakes.Material and methods We used a food frequency questionnaire (FFQ) completed by a sample of 194 pregnant women to estimate their dietary exposure to chlordecone and compared it to blood levels. In a first approach, chlordecone daily intake was estimated as the product of daily eaten quantity of 214 foodstuffs, multiplied by their chlordecone content, and summed over all items. We then predicted individual blood chlordecone concentration with empirical weight regression models based on frequency of food consumption, and without contamination data.Results Among the 191 subjects who had BCC determination, 146 (76%) had detectable values and mean BCC was 0.86†ng/mL (range < LOD-13.2). Mean per capita dietary intake of chlordecone was estimated at 3.3†[mu]g/day (range: 0.1-22.2). Blood chlordecone levels were significantly correlated with food exposure predicted from the empirical weight models (r=0.47, p<0.0001) and, to a lesser extent, with chlordecone intake estimated from food consumption and food contamination data (r=0.20, p=0.007). Main contributors to chlordecone exposure included seafood, root vegetables, and Cucurbitaceous.Conclusion These results show that the Timoun FFQ provides valid estimates of chlordecone exposure. Estimates from empirical weight models correlated better with blood levels of chlordecone than did estimates from the dietary intake assessment. [less ▲]

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