Potential endocrinedisruptor activity of drinking water samples

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Speciation of butyltin derivatives in surface sediments of three southern Brazilian harbors

For the first time, organotin compounds were determined in surface sediment samples collected from Sao Francisco do Sul, Itajai-Navegantes and Imbituba Harbors, located in Santa Catarina State, Brazil. Butyltins (BTs) were determined by gas chromatography with a pulsed flame photometric detector (GC-PFPD) after being modified using the Grignard derivatization method. The concentrations of BTs derivatives ranged from n.d. to 1136.6 ng (Sn) g(-1) of dry weight (dw) sediment for tributyltin (TOT), n.d. to 394.4 ng (Sn)g(-1) dw for dibutyltin (DOT) and n.d. to 312.2 ng (Sn)g(-1) dw for monobutyltin (MBT). The highest concentration of total BTs was found at the Itajai-Acu River dockyard, indicating intense inputs of antifouling paints to the environment. The relative difference in the BTs levels is a particular characteristic of sediments from harbors and may be related to the shipyards and the boat traffic which still use TBT-based antifouling paints.

IMPOSEX AS AN INDICATOR OF ORGANOTIN POLLUTION AT RIO DE JANEIRO SOUTH COAST: SEPETIBA AND ILHA GRANDE BAYS

Imposex is the development of male sexual characteristics, i.e., penis and/or vas deferens in females of dioecious gastropods. This phenomenon has been associated to the use of the organotin based marine antifouling paints and used worldwide as a bioindicator of pollution by the TBT (antifouling agent) as well as to evaluate the related ecological impact. This study reports imposex in Stramonita haemastoma at south coast of Rio de Janeiro State (Brazil), specifically at Sepetiba and Ilha Grande Bays. All the stations where snails were found have shown imposex. Ilha Grande Bay presented the highest values of imposex indices when compared to Sepetiba Bay. The results suggest that high concentration of shipping activities and coastal morphology are the principal causes at the most polluted sites.

Tributyltin in Crustacean Tissues: Analytical Performance and Validation of Method

The hermit crab Clibanarius vittatus is a typical organism from intertidal regions being considered as a good bioindicator of tributyltin presence at these environments. Thus this study presents the analytical performance and validation method for TBT quantification in tissues of C. vittatus by gas chromatography with pulsed flame photometric detector (GC-PFPD) after extraction with an apolar solvent (toluene) and Grignard derivatization. The limits of detection of the method (LOD) were 2.0 and 2.8 ng g(-1) for TBT and DBT (dibutyltin), respectively, and its limits of quantification (LOQ) were 6.6 and 8.9 ng g(-1) for TBT and DBT, respectively. The method was applied to samples from Santos Estuary, Sao Paulo State, Brazil. TBT and DBT concentrations ranged from 26.7 to 175.0 ng g(-1) and from 46.2 to 156.0 ng g(-1), respectively. These concentrations are worrisome since toxic effects (such as endocrine disruption) have been reported for other organisms even under lower levels of registred at this study.

Effects of tributyltin exposure in hermit crabs: Clibanarius vittatus as a model

Tributyltin (TBT) contamination affects the reproductive system of many species of invertebrates worldwide. The present study was designed to evaluate the effects of exposure to TBT pollution on the reproduction of the hermit crab Clibanarius vittatus. An orthogonal experiment was designed with two treatments: contamination (with or without TBT in the food) and crab sex (males and females). The animals were reared in the laboratory for nine months, and macroscopic and histological analyses of reproductive organs were carried out after the end of the experiment. Tributyltin was recorded in exposed crabs, but no morphological alterations were detected in the gonads of males, regardless of whether they were exposed to TBT. In contrast, females exposed to TBT displayed disorganization and atrophy of their ovaries, thus directly affecting reproduction in this hermit crab species. This effect observed in female hermit crabs may harm populations located in harbor regions, where TBT concentration is high, even after the worldwide TBT ban.

Tributyltin in crustacean tissues: analytical performance and validation of method

The hermit crab Clibanarius vittatus is a typical organism from intertidal regions being considered as a good bioindicator of tributyltin presence at these environments. Thus this study presents the analytical performance and validation method for TBT quantification in tissues of C. vittatus by gas chromatography with pulsed flame photometric detector (GC-PFPD) after extraction with an apolar solvent (toluene) and Grignard derivatization. The limits of detection of the method (LOD) were 2.0 and 2.8 ng g-1 for TBT and DBT (dibutyltin), respectively, and its limits of quantification (LOQ) were 6.6 and 8.9 ng g-1 for TBT and DBT, respectively. The method was applied to samples from Santos Estuary, São Paulo State, Brazil. TBT and DBT concentrations ranged from 26.7 to 175.0 ng g-1 and from 46.2 to 156.0 ng g-1, respectively. These concentrations are worrisome since toxic effects (such as endocrine disruption) have been reported for other organisms even under lower levels of registred at this study.

Long-term estrogen exposure of whitefish Coregonus lavaretus induces intersex but not Lake Thun-typical gonad malformations

A high prevalence of gonad morphological variations has been observed in whitefish Coregonus lavaretus from Lake Thun (Switzerland). To clarify the role of endocrine disruption as a possible cause of the gonad alterations, whitefish were reared in a long-term laboratory experiment under exposure to 17 beta-estradiol (E2). Fish were fed from first-feeding until 3 yr of age at a daily rate of 0 (control), 0.5 or 50 microg E2 kg(-1) fish. E2 exposure resulted in a time- and concentration-dependent increase of prevalence and intensity of intersex gonads, i.e. gonads that macroscopically appeared as either testis or ovary but microscopically contained both male and female germ cells. Four types of intersex could be distinguished: Types 1 and 2 were composed of mainly male tissue, with Type 1 containing single oocytes and Type 2 displaying an ovary-like lamellar structure of the tissue. In Type 3, an increased percentage of the tissue was occupied by female germ cells, while in Type 4, the majority of the gonad tissue consisted of female germ cells. Chronic E2 exposure additionally resulted in a concentration-dependent shift of the sex ratio towards females, a reduced condition factor, retarded gonad growth together with delayed maturation of germ cells, and elevated levels of hepatic vitellogenin mRNA. However, Lake Thun-typical alterations of gonad morphology were not induced by chronic E2 exposure. The results provide evidence that estrogen-active compounds unlikely play a role in the etiology of gonad malformations in Lake Thun whitefish.

Impact of environmental estrogens on Yfish considering the diversity of estrogen signaling.

Research on endocrine disruption in fish has been dominated by studies on estrogen-active compounds which act as mimics of the natural estrogen, 17β-estradiol (E2), and generally exert their biological actions by binding to and activation of estrogen receptors (ERs). Estrogens play central roles in reproductive physiology and regulate (female) sexual differentiation. In line with this, most adverse effects reported for fish exposed to environmental estrogens relate to sexual differentiation and reproduction. E2, however, utilizes a variety of signaling mechanisms, has multifaceted functions and targets, and therefore the toxicological and ecological effects of environmental estrogens in fish will extend beyond those associated with the reproduction. This review first describes the diversity of estrogen receptor signaling in fish, including both genomic and non-genomic mechanisms, and receptor crosstalk. It then considers the range of non-reproductive physiological processes in fish that are known to be responsive to estrogens, including sensory systems, the brain, the immune system, growth, specifically through the growth hormone/insulin-like growth factor system, and osmoregulation. The diversity in estrogen responses between fish species is then addressed, framed within evolutionary and ecological contexts, and we make assessments on their relevance for toxicological sensitivity as well as ecological vulnerability. The diversity of estrogen actions raises questions whether current risk assessment strategies, which focus on reproductive endpoints, and a few model fish species only, are protective of the wider potential health effects of estrogens. Available - although limited - evidence nevertheless suggests that quantitative environmental threshold concentrations for environmental protection derived from reproductive tests with model fish species are protective for non-reproductive effects as well. The diversity of actions of estrogens across divergent physiological systems, however, may lead to and underestimation of impacts on fish populations as their effects are generally considered on one functional process only and this may underrepresent the impact on the different physiological processes collectively.

Human and ecological risk assessment of a crop protection chemical: a case study with the azole fungicide epoxiconazole.

Conventional risk assessments for crop protection chemicals compare the potential for causing toxicity (hazard identification) to anticipated exposure. New regulatory approaches have been proposed that would exclude exposure assessment and just focus on hazard identification based on endocrine disruption. This review comprises a critical analysis of hazard, focusing on the relative sensitivity of endocrine and non-endocrine endpoints, using a class of crop protection chemicals, the azole fungicides. These were selected because they are widely used on important crops (e.g. grains) and thereby can contact target and non-target plants and enter the food chain of humans and wildlife. Inhibition of lanosterol 14α-demethylase (CYP51) mediates the antifungal effect. Inhibition of other CYPs, such as aromatase (CYP19), can lead to numerous toxicological effects, which are also evident from high dose human exposures to therapeutic azoles. Because of its widespread use and substantial database, epoxiconazole was selected as a representative azole fungicide. Our critical analysis concluded that anticipated human exposure to epoxiconazole would yield a margin of safety of at least three orders of magnitude for reproductive effects observed in laboratory rodent studies that are postulated to be endocrine-driven (i.e. fetal resorptions). The most sensitive ecological species is the aquatic plant Lemna (duckweed), for which the margin of safety is less protective than for human health. For humans and wildlife, endocrine disruption is not the most sensitive endpoint. It is concluded that conventional risk assessment, considering anticipated exposure levels, will be protective of both human and ecological health. Although the toxic mechanisms of other azole compounds may be similar, large differences in potency will require a case-by-case risk assessment.

Dietary intake patterns and relationships to polybrominated diphenyl ether (PBDE) and phthalate body burden

Polybrominated diphenyl ethers (PBDEs) and phthalates are chemicals of concern because of high levels measured in people and the environment as well as the demonstrated toxicity in animal studies and limited epidemiological studies. Exposure to these chemicals has been associated with a range of toxicological outcomes, including developmental effects, behavioral changes, endocrine disruption, effects on sexual health, and cancer. Previous research has shown that both of these classes of chemicals contaminate food in the United States and worldwide. However, how large a role diet plays in exposure to these chemicals is currently unknown. To address this question, an exploratory analysis of data collected as part of the 2003-04 National Health and Nutrition Examination Survey (NHANES) was conducted. Associations between dietary intake (assessed by 24-hour dietary recalls) for a range of food types (meat, poultry, fish, and dairy) and levels PBDEs and phthalate metabolites were analyzed using multiple linear regression modeling. Levels of individual PBDE congeners 28, 47, 99, 100 as well as total PBDEs were found to be significantly associated with the consumption of poultry. Metabolites of di-(2-ethylhexyl) phthalate (DEHP) were found to be associated with the consumption of poultry, as well as with an increased consumption of fat of animal origin. These results, combined with results from previous studies, suggest that diet is an important route of intake for both PBDEs and phthalates. Further research needs to be conducted to determine the sources of food contamination with these toxic chemicals as well as to describe the levels of contamination of US food in a large, representative sample.^

Polybrominated diphenyl ether (PBDE) exposure in North Americans: Cause for concern?

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants (BFRs) that have been widely produced and used as flame retardants since the 1970’s in many consumer products such as carpet and drape linings, plastics used in electronics, computer and television casings and polyurethane foam used in chairs, sofas and mattresses. PBDEs are persistent organic pollutants (POPs), which, by definition, are toxic in nature, persistent in the environment and accumulative in living organisms. Animal studies have found PBDEs to cause health defects such as fetal malformations, delayed onset of puberty, decreased sperm count, behavioral changes, permanent learning and memory impairment, endocrine disruption, as well as cancer at high doses. Recent research involving humans reported that elevated breast milk PBDEs levels in their mothers are associated with cryptorchidism (absence of one or both testes from the scrotum) in newborn boys and adverse birth outcomes as well as elevated serum PBDE levels in mothers are associated with low sperm count in young men. There are three commonly manufactured PBDE commercial mixtures: Penta-, Octa-, and Deca-BDEs. Two of them (Octa- and Penta-BDEs) have been banned by the European Union and are being voluntarily phased out in the United States. However, Deca continues to be manufactured, used, and imported in the United States. This MPH thesis consists of a literature review of peer reviewed scientific articles concerned with PBDEs in the environment and in humans, as well as a discussion concerning different routes of exposure to PBDEs and their blood, milk and tissue levels as surrogates for body burdens in North Americans and in people from other countries. Results of this literature review shows PBDE levels in human blood, milk and tissues are higher in North Americans than people from other countries worldwide. To date, the highest level of PBDEs was found in a toddler’s blood in a California study. Despite the fact that PBDEs are associated with adverse health effects, and highest levels of PBDEs in North Americans, Deca-BDE is still manufactured, used and imported in the United States. There is an urgent need of new federal regulatory policy to ban completely the production, importation and use of all commercial mixtures of PBDEs.^

The Effects of Brominated Flame Retardants on Thyroid Hormone Homeostasis in Human Placenta Tissues and Cell Culture

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants (BFRs) that have been heavily used in consumer products such as furniture foams, plastics, and textiles since the mid-1970’s. BFRs are added to products in order to meet state flammability standards intended to increase indoor safety in the event of a fire. The three commercial PBDE mixtures, Penta-, Octa-, and DecaBDE, have all been banned in the United States, however, limited use of DecaBDE is still permitted. PBDEs were phased out of production and added to the Stockholm Convention due to concerns over their environmental persistence and toxicity. Human exposure to PBDEs occurs primarily through the inadvertent ingestion of contaminated house dust, as well as though dietary sources. Despite the phase-out and discontinued use of PBDEs, human exposure to this class of chemicals is likely to continue for decades due to the continued use of treated products and existing environmental reservoirs of PBDEs. Extensive research over the years has shown that PBDEs disrupt thyroid hormone (TH) levels and neurodevelopmental endpoints in rodent and fish models. Additionally, there is growing epidemiological evidence linking PBDE exposure in humans to altered TH homeostasis and neurodevelopmental impairments in children. Due to the importance of THs throughout gestation, there is a great need to understand the effects of BFRs on the developing fetus. Specifically, the placenta plays a critical role in the transport, metabolism, and delivery of THs to the fetal compartment during pregnancy and is a likely target for BFR bioaccumulation and endocrine disruption. The central hypothesis of this dissertation research is that BFRs disrupt the activity of TH sulfotransferase (SULT) enzymes, thereby altering TH concentrations in the placenta.

In the first aim of this dissertation research, the concentrations of PBDEs and 2,4,6-TBP were measured in a cohort of 102 placenta tissue samples from an ongoing pregnancy cohort in Durham, NC. Methods were developed for the extraction and analysis of the BFR analytes. It was found that 2,4,6-TBP was significantly correlated with all PBDE analytes, indicating that 2,4,6-TBP may share common product applications with PBDEs or that 2,4,6-TBP is a metabolite of PBDE compounds. Additionally, this was the first study to measure 2,4,6-TBP in human placenta tissues.

In the second aim of this dissertation research, the placenta tissue concentrations of THs, as well as the endogenous activity of deiodinase (DI) and TH SULT enzymes were quantified using the same cohort of 102 placenta tissue samples. Enzyme activity was detected in all samples and this was the first study to measure TH DI and SULT activity in human placenta tissues. Enzyme activities and TH concentrations were compared with BFR concentrations measured in Aim 1. There were few statistically significant associations observed for the combined data, however, upon stratifying the data set based on infant sex, additional significant associations were observed. For example, among males, those with the highest concentrations of BDE-99 in placenta had T3 levels 0.80 times those with the lowest concentration of BDE-99 (95% confidence interval (CI): 0.59, 1.07). Whereas females with the highest concentrations of BDE-99 in placenta had T3 levels 1.50 times those with the lowest concentration of BDE-99 (95% CI: 1.10, 2.04). Additionally, all BFR analyte concentrations were higher in the placenta of males versus females and they were significantly higher for 2,4,6-TBP and BDE-209. 3,3’-T2 SULT activity was significantly higher in female placenta tissues, while type 3 DI activity was significantly higher in male placenta tissues. This research is the first to show sex-specific differences in the bioaccumulation of BFRs in human placenta tissue, as well as differences in TH concentrations and endogenous DI and SULT activity. The underlying mechanisms of these observed sex differences warrant further investigation.

In the third aim of this dissertation research, the effects of BFRs were examined in a human choriocarcinoma placenta cell line, BeWo. Michaelis-Menten parameters and inhibition curves were calculated for 2,4,6-TBP, 3-OH BDE-47, and 6-OH BDE-47. 2,4,6-TBP was shown to be the most potent inhibitor of 3,3’-T2 SULT activity with a calculated IC50 value of 11.6 nM. It was also shown that 2,4,6-TBP and 3-OH BDE-47 exhibit mixed inhibition of 3,3’-T2 sulfation in BeWo cell homogenates. Next, a series of cell culture exposure experiments were performed using 1, 6, 12, and 24 hour exposure durations. Once again, 2,4,6-TBP was shown to be the most potent inhibitor of basal 3,3’-T2 SULT activity by significantly decreasing activity at the high and medium dose (1 M and 0.5 M, respectively) at all measured time points. Interestingly, BDE-99 was also shown to inhibit basal 3,3’-T2 SULT activity in BeWo cells following the 24 hour exposure, despite exhibiting no inhibitory effects in the BeWo cell homogenate experiments. This indicates that BDE-99 must act through a pathway other than direct enzyme inhibition. Following exposures, the TH concentrations in the cell culture growth media and mRNA expression of TH-related genes were also examined. There was no observed effect of BFR treatment on these endpoints. Future work should focus on determining the downstream biological effects of TH SULT disruption in placental cells, as well as the underlying mechanisms of action responsible for reductions in basal TH SULT activity following BFR exposure.

This was one of the first studies to measure BFRs in a cohort of placenta tissue samples from the United States and the first study to measure THs, DI activity, and SULT activity in human placenta tissues. This research provides a novel contribution to our growing understanding of the effects of BFRs on TH homeostasis within the human placenta, and provides further evidence for sex-specific differences within this important organ. Future research should continue to investigate the effects of environmental contaminants on TH homeostasis within the placenta, as this represents the most critical and vulnerable stage of human development.

BPA occupational exposure assessment in Europe: a scientific gap

Bisphenol A (BPA), 2,2-bis(4-hydroxyphenyl) propane one is of the greatest volume industrial chemicals utilized in the world with increased production every year. Environmental exposure to this xenoestrogen is considered a generalized phenomenon with a Tolerable Daily Intake (TDI) of4 µg/kg body weight/day established by the European Food Safety Authority. Several studies have focused in estimate human daily intake and potential associated health effects of environmental exposures, however despite of the massive BPA production and consumption in European countries, with policarbonate and epoxy resins as the major applications, occupational exposure to BPA have been overlooked and considered safe by the European authorities.

The bioaccumulation of persistent contaminants by zebra mussels and their effects on state-endangered common terns

Common terns currently are listed as endangered or threatened in many states, including Illinois, Vermont, Pennsylvania, Ohio, Wisconsin, Michigan, and New York, and a species of special concern by the U.S. Fish and Wildlife Service (USFWS, 2002). The sole remaining nesting colony in Illinois is located at the Naval Station Great Lakes (NSGL) in Lake County where intensive management by the Illinois Department of Natural Resources has reduced nest predation and increased the number of eggs that hatch. However, the overall reproductive success (the number of young successfully reaching independence) has not improved. Observations of gross deformities in hatchlings (i.e. compromised feather development and cross-bill), lethargic behavior of young birds, and lesions, suggested the influence of environmental contaminants (Jablonski et al., 2005). I investigated if there were significant levels of environmental contaminants in eggs and nestlings of common terns. While there were minimal concentration of selenium, mercury, lead, and cadmium, there were large concentration of polychlorinated biphenyls (PCBs) in both the eggs and nestlings. The greater amounts of PCBs in older chicks than younger chicks suggest local contamination. In order to potentially manage the factors responsible for exposing the terns to PCBs I investigated the pathway by which PCBs were exposed to terns. The two most likely biological pathways as determined by research on Great Lake fishes were investigated. The first pathway is through atmospheric deposition of PCBs and resuspension of PCB-ladel sediment which are subsequently acquired by filter-feeding fish (e.g. alewives, Alosa pseudoharengus) and then pelagic fish (e.g. lake trout, Salvelinus namaychus) or in this case terns. The second pathway explored was via the biodeposits of zebra mussels which are consumed by round gobies (Neogobius melanostromus) and ultimately littoral fish (e.g. small-mouthed bass, Micropterus dolomieui) or terns. Because common terns breed in near-shore sites where concentrations of zebra mussels are found, as well as forage in more pelagic environments it is possible that either or both pathways may be contributing to their PCB exposure. Field experiments and stable isotope analyses demonstrated that the most likely pathway by which terns are exposed to PCBs is via alewives, similar to how apex predators such as lake trout acquire PCBs. Biodeposits from zebra mussels do not appear to be a significant factor in PCB accumulation in terns. The impact of PCB exposure on birds can vary widely, however in this situation we choise to investigate one specific behavior often affected by PCB exposure, parental attentiveness. PCBs are known to cause endocrine disruption which ultimately results in reduced brooding of young and incubation of eggs. I used temperature sensors to quantify nest temperatures and parental attentiveness during incubation. High concentrations of PCBs in our study population appear to be leading to poor parental attentiveness, and extended periods of absence during incubation and brooding, ultimately leading to poor reproductive success. Common terns are perilously close to being extirpated in Illinois and management of PCB exposure will be difficult. I propose that additional testing should be conducted to locate a site with less PCB contamination and then to move the tern colony to this location, possibly using social cues as has been done with other tern species in Illinois. PCBs are having a profound impact on common tern populations in Illinois and without moving the colony it is likely that the population will continue to decline.

Analysis of sexually dimorphic growth in captive reared cobia (Rachycentron canadum) and the occurrence of intersex individuals

The growth of three cohorts of captive reared cobia, grown in a combination of flow-though and recirculating aquaculture systems, was progressively measured to determine the existence and extent of sexually dimorphic growth in cobia. Approximately 100 fish from each cohort were individually identified and regularly weighed until the average weight of the fish was approximately 5 kg. The sex of individuals was determined through gonadal observations at the conclusion of each trial and the gender fitted retrospectively to the growth data set. Intersex gonads were observed in the first two cohorts of cobia, with 16.9% incidence in cohort 1 and 6.8% in cohort 2. Cobia is considered a gonochoristic species. This was the first reported observation of intersex gonads in cobia and the first reported occurrence of intersex gonads from a gonochoristic fish species from Australian waters. Only one fish out of the 182 examined in the third cohort was identified as intersex. There was no sexually dimorphic growth in cobia when there was a relatively high incidence of the intersex anomaly, as seen in the first two cohorts of fish. In the relative absence of the intersex condition, female cobia was significantly larger than males from 2 kg onwards. The weight of female cobia was almost 30% more than that of males at 17 months of age when average weight of the cohort was 4.6 kg. It is likely that the first two cobia cohorts were exposed to endocrine disruption in some form, and the possible sources are discussed. Statement of relevance This study demonstrated that female cobia grow significantly faster than male fish and that investigations into monosex culture could lead to significant productivity gains for cobia aquaculture. It also demonstrated that cohorts containing intersex fish did not exhibit sexually dimorphic growth. It is likely that the reproductive anomaly is the result of disruption to the endocrine system.