Transcriptomic analysis reveals the endocrine toxicity of tributyltin and triphenyltin on the whelk Reishia clavigera and mechanisms of imposex formation.

Organotin compounds (OTs) are endocrine disruptors that induce imposex in hundreds of gastropods, but little is known about their underlying molecular mechanisms. This study aimed to investigate the endocrine toxicity and molecular responses to tributyltin (TBT) and triphenyltin (TPT) exposure in the whelk Reishia clavigera, which often serves as a biomonitor for OT contamination. Over a 120-day exposure to environmentally relevant concentrations of TBT (1000 ng L-1) and TPT (500 ng L-1), we observed a significant increase in penis length in both male and female whelks. Notably, TPT exhibited a stronger potency in inducing pseudo-penis development and female sterility, even at a half dose of TBT. Bioaccumulation analysis also revealed higher persistence and accumulation of TPT in whelk tissues compared to TBT. Differential expression analysis identified a substantial number of differentially expressed genes (DEGs), with TPT exposure eliciting more DEGs than TBT. Our results demonstrated that OTs induced xenobiotic metabolism and metabolic dysregulation in the digestive gland, impaired multiple cellular functions and triggered neurotoxicity in the nervous system, and disrupted lipid homeostasis and oxidative stress in the gonads. Furthermore, imposex was possibly associated with disturbances in retinoic acid metabolism, nuclear receptor signaling, and neuropeptide activity. When compared to TBT, TPT exhibited a more pronounced endocrine-disrupting effect, attributable to its higher bioaccumulation and substantial interruption of transcriptional regulation, OT detoxification, and biosynthesis of retinoic acids in R. clavigera. Our results, therefore, highlight the importance of considering the differences in bioaccumulation and molecular toxicity between TBT and TPT in future risk assessments of these contaminants. Overall, our study provided molecular insights into the toxicity and transcriptome profiles in R. clavigera exposed to TBT and TPT, shedding light on the endocrine-disrupting effects and reproductive impairment in female gastropods.

Intersex manifestation in the rainbow darter (Etheostoma caeruleum): Are adult male fish susceptible to developing and recovering from intersex after exposure to endocrine active compounds?

For over a decade, intersex has been observed in rainbow darter (RD) (Etheostoma caeruleum) populations living downstream wastewater treatment plants (WWTPs) in the Grand River, Ontario, Canada. To further our understanding of intersex development in adult male fish, the current study addressed three objectives: i) can intersex be induced in adult male fish, ii) is there a specific window of exposure when adult male fish are more susceptible to developing intersex, and iii) can pre-exposed adult male fish recover from intersex? To assess intersex induction in adult male fish, wild male RD were exposed in the laboratory for 22 weeks (during periods of spawning, gonadal regression, and gonadal recrudescence) to environmentally relevant concentrations of 17α-ethinylestradiol (EE2) including nominal 0, 1, and 10 ng/L. Intersex rates and severity at 10 ng/L EE2 were similar to those observed historically in adult male populations living downstream WWTPs in the Grand River and confirmed previous predictions that 1-10 ng/L EE2 would cause these adverse effects. To assess a window of sensitivity in developing intersex, male RD were exposed to nominal 0, 1 or 10 ng/L EE2 for 4 weeks during three different periods of gonadal development, including (i) spawning, (ii) early recrudescence and (iii) late recrudescence. These short-term exposures revealed that intersex incidence and severity were greater when RD were exposed while gonads were fully developed (during spawning) compared to periods of recrudescence. To assess if RD recover from intersex, wild fish were collected downstream WWTPs in the Grand River and assessed for intersex both before and after a 22-week recovery period in clean water that included gonadal regression and recrudescence. Results showed that fish did not recover from intersex, with intersex rates and severity similar to those both before and after the transition to clean water. This study further advances our knowledge on intersex manifestation in adult male fish including their sensitivity to endocrine active compounds during different periods of their annual reproductive cycle and their limited ability to recover from intersex after onset of the condition.

Temporal evolution of imposex and butyltin contamination in Gemophos viverratus from São Vicente (Cabo Verde) - a countercurrent trend on the world scenario.

Organotin (OT) based Antifouling Systems (AFS) were globally banned by the AFS Convention since 2008, but the Republic of Cabo Verde did not ratify this Convention, nor did it develop a national legislation to control OT-AFS. Gemophos viverratus imposex and butyltin tissue contamination were assessed around the São Vicente Island (Cabo Verde) in 2019 and compared with the data available from 2012. The vas deferens sequence index (VDSI), the relative penis length (RPL), the percentage of females with imposex (%I) and the percentage of sterilized females (%S) in 2019 ranged from 0 to 4.0, 0-84.4%, 0-100% and 0-5.1%, respectively, whilst TBT reached maximum values of ≈30 ng TBT-Sn g-1 dw in the whelk tissues. These values are very similar to those registered in 2012, which indicates that TBT pollution has not decreased over the years, in contrast to the declining trend observed worldwide.

The Occurrence of Intersex in Different Populations of the Marine Amphipod Echinogammarus marinus in North-West Brittany - A Longterm-Study.

In the past two decades, an increasing body of studies has been published on the intersex phenomenon in separate-sexed crustaceans from marine and freshwater ecosystems. Various causes are being considered that could have an influence on the occurrence of intersex. Besides genetic factors, environmental conditions such as photoperiodicity, temperature, salinity and parasitism, but also environmental pollution with endocrine disrupting chemicals (EDCs) are discussed. As part of a long-term monitoring (2012 - 2020) in north-west Brittany, we recorded the occurrence of intersex in the marine amphipod Echinogammarus marinus. We quantified the intersex incidence at marine and estuarine sites and analyzed the incidence in relation to the endocrine potential of the sediments. Intersex occurred with mean frequencies between 0.87% and 12%. It was striking that the incidence of intersex increased with increasing distance from the sea. Since the highest incidence was observed at the range boundary of this stenohaline species, we assume that intersex is triggered by endocrine potential and increasing stress due to increasing freshwater content - and thus an interplay of different environmental factors.

Imposex Incidence in Gastropod Species from Santa Marta Coastal Zone, Colombian Caribbean Sea.

Imposex is a phenomenon widely associated with environmental exposure to organotin compounds which were quite common components of antifouling paints applied on boats and ship hulls. Here we study the incidence of imposex in neogastropods and its relation with water quality and maritime traffic in the coastal strip of Santa Marta, Colombia. Imposex was determined via specialized indexes and related to the organisms' size, somatic conditions, variables of water quality and maritime traffic, in a space-time assessment. There was evidence of imposex in five species Plicopurpura patula, Vasula deltoidea, Stramonita haemastoma, S. floridana, and Gemophos auritulus. Purpura patula and Vasula deltoidea species were found in all sampling sites. The results have proved that imposex is highly influenced by the maritime traffic variable, with greater prevalence during the dry season, and with P. patula being more sensitive than V. deltoidea.

Reproductive viability of paradoxically masculinised Gambusia holbrooki generated following diethylstilbestrol (DES) treatment.

Hormonal sex reversal can produce monosex fish stocks and provide insights into their gamity and reproductive physiology. However, paradoxical effects have been reported in several fish species that remain largely ignored as anomalies, particularly those of masculinisation. As a first step, this study examined reproductive viability of paradoxically masculinised Gambusia holbrooki produced following oral administration (20-100 mg/kg feed) of a feminizing hormone diethylstilbestrol (DES). Contrary to expectation, all treatment groups produced 100% male populations. Survival, mating behaviour, gamete production, breeding output as well as expression of anti-Mullerian hormone (amh), ovarian (cyp19a1a) and brain (cyp19a1b) aromatase of masculinised fish were also examined. Survival (≤ 54.1 ± 7.3%) at termination of DES treatment was significantly lower compared with controls (88.6 ± 4.3%) but remained unaffected post treatment. Gonopodium thrusting frequency (33 ± 9.8 per 10 min) was not significantly different to untreated males just as sperm abundance (3.9 ± 1.5 × 108/male) and their motility (88.6 ± 29.1%). Importantly, paradoxically masculinised fish mated with virgin females and produced clutch sizes (22 ± 4) and progeny survival (87.0 ± %) that were comparable to that of untreated males. Masculinised testes showed high amh and low cyp19a1a expression, a pattern resembling those of untreated males. Production of paradoxically sex-reversed males with a capability to produce viable offspring has not been reported previously in this or other fish species. The outcomes support a feed-back regulation of oestrogenic pathways in this viviparous fish and could be useful for ecological applications such as controlling invasive fish populations.

Reconnaissance of Surface Water Estrogenicity and the Prevalence of Intersex in Smallmouth Bass (Micropterus Dolomieu) Inhabiting New Jersey.

The observation of testicular oocytes in male fishes has been utilized as a biomarker of estrogenic endocrine disruption. A reconnaissance project led in the Northeastern United States (US) during the period of 2008-2010 identified a high prevalence of intersex smallmouth bass on or near US Fish & Wildlife Service National Wildlife Refuges that included the observation of 100% prevalence in smallmouth bass males collected from the Wallkill River, NJ, USA. To better assess the prevalence of intersex smallmouth bass across the state of New Jersey, a tiered reconnaissance approach was initiated during the fall of 2016. Surface water samples were collected from 101 (85 river, 16 lake/reservoir) sites across the state at base-flow conditions for estrogenicity bioassay screening. Detectable estrogenicity was observed at 90% of the sites and 64% were above the US Environmental Protection Agency trigger level of 1 ng/L. Median surface water estrogenicity was 1.8 ng/L and a maximum of 6.9 ng/L E2EqBLYES was observed. Adult smallmouth bass were collected from nine sites, pre-spawn during the spring of 2017. Intersex was identified in fish at all sites, and the composite intersex prevalence was 93.8%. Prevalence across sites ranged from 70.6% to 100%. In addition to intersex, there was detectable plasma vitellogenin in males at all sites. Total estrogenicity in surface water was determined at these fish collection sites, and notable change over time was observed. Correlation analysis indicated significant positive correlations between land use (altered land; urban + agriculture) and surface water estrogenicity. There were no clear associations between land use and organismal metrics of estrogenic endocrine disruption (intersex or vitellogenin). This work establishes a baseline prevalence of intersex in male smallmouth bass in the state of New Jersey at a limited number of locations and identifies a number of waterbodies with estrogenic activity above an effects-based threshold.

The Degree of Sex Reversal in Muscovy Ducks (Cairina moschata domestica) Induced by an Aromatase Inhibitor.

Under the same feeding conditions, the growth and development of male Muscovy ducks is significantly greater than that of females. Thus, controlling their sex expression can have economic benefits. However, reports on the degree of sex reversal in Muscovy ducks are scarce. In this study, we obtained sex-reversed Muscovy ducks by injecting letrozole before sex differentiation. We analyzed the degree of sex reversal in Muscovy ducks in terms of hormone levels, gonadal tissue development, and growth and found that the estradiol levels of AI-females (letrozole-induced female-to-male sex reversal) were not significantly different from those of normal males (p > 0.05), but the testosterone levels were significantly lower than those in normal males (p < 0.05). AI-female gonad tissue had changed, and the right gonad presented ovotestis tissue. The growth and development of AI-females was significantly less than that of normal males (p < 0.05) but was not significantly different from that of normal females (p > 0.05). Letrozole can induce female Muscovy ducks to convert into males, but the reversal cannot be completed. Thus, further studies are needed to elucidate how to entirely attain the change.

Gonad pathology and intersex severity in pelagic (Tilapia zilli) and benthic (Neochanna diversus and Clarias gariepinus) species from a pesticide-impacted agrarian catchment, south-south Nigeria.

Pesticides are distributed to different degrees in surface water and sediment, thus, risks of toxicity and adverse impacts to physiology of resident species could be determined by their microhabitat (sub-habitat) associations, either water column or sediment. River Owan receives pesticide input from diffuse sources from adjacent farmlands. Surface water, sediment and fish samples [(Pelagic: Tilapia zilli (n = 92) and benthic: Neochanna diversus (n = 59), Clarias gariepinus (n = 68) were collected within the catchment area across seasons for 18-months (August 2016-January 2018) and measured for pesticide levels. Testicular and Ovarian tissue samples across the three species were also examined for pathological alterations. Individual pesticide concentrations in surface water and sediment exceeded international allowable limits, while concentration of pesticide residues in tissues of benthic species was higher compared to pelagic fish. Histopathological assessment revealed a higher incidence of ovarian disruption including atretic follicles, intersex and disorganization of ovarian structure in benthic (bottom-dwelling) fish (C. gariepinus and N. diversus) compared to pelagic (water-column) fish (T. zilli). Males benthic fish species also recorded more severe anomalies, compared to pelagic fish. The damages and anomalies observed in ovarian and testicular tissue indicate chronic responses to pollutant exposure, and implicates the elevation of pesticide concentrations in surface water and sediment above permissible limits. The higher incidence of anomalies recorded for benthic species compared to pelagic species, indicates greater risks of reproductive disruption and could be associated with the microhabitat preferences (water-column or sediment).

Feminization, altered gonadal development, and liver damage in least killifish (Heterandria formosa) exposed to sublethal concentrations of 17α-ethinylestradiol.

The widespread use of the synthetic estrogen 17 α-ethinylestradiol (EE2) has resulted in elevated levels in aquatic environments, where it is known to act as an endocrine disrupting chemical affecting fish and other aquatic organisms. Examining changes in the structure of the fish' gonads and liver has proven to be an effective approach for assessing these impacts. While changes have been reported for various fish species, it is not clear whether impacts are equally severe in live-bearing fishes. The present study looked at gonadal and liver development in EE2-exposed least killifish (Heterandria formosa), a live-bearing Poeciliid. Exposures to 0, 5, or 25 ng/L EE2 began within six days of birth and continued until fish became sexually mature 12-23 weeks later. Exposure to 5 ng/L EE2 resulted in severe intersex in fish with external male characteristics, a slowdown of spermatogenesis in these intersex fish and a slowdown of oogenesis in the female fish. Moreover, these fish had a variety of liver injuries. Fish exposed to 25 ng/L EE2 exhibited intersex but at a lower frequency than occurred at 5 ng/L. In contrast, liver damage and slowdown of both oogenesis and spermatogenesis exhibited the typical dose-dependence. These findings illustrate the importance of including histological analyses when assessing endocrine disruption in fish, demonstrate that the live-bearing mode of reproduction appears to provide limited protection from the effects of waterborne EE2, and provide further evidence that EE2 has multiple impacts on fish health and reproduction that are severe enough to potentially affect fish populations.

The intersex phenomenon in Sarotherodon melanotheron from Lagos lagoon (Nigeria): Occurrence and severity in relation to contaminants burden in sediment.

The correlation between endocrine active contaminants in the environment and alterations in reproductive development of Sarotherodon melanotheron from Lagos lagoon has been investigated. Sediment and a total of 155 fish (74 males and 81 females) were collected between November 2014-March 2015 from selected contaminated sites (Ikorodu, Oworonshoki, Makoko and Idumota) and a putative control site (Igbore) along the lagoon. Sediment contaminant analysis revealed, significantly higher concentration of lindane, dieldrin, 4-iso-nonylphenol, 4-t-octylphenol and monobutyltin cation at the contaminated sites. Examination of gross morphological and histological changes of fish gonads showed a 27.4% prevalence of intersex in the sampled fish, of which 78% were males (testes-ova) and 22% were females (ovo-testis). Quantitative PCR (qPCR) of liver transcripts revealed the presence of vitellogenin (vtg) levels in male fish from contaminated sites. Zona radiata proteins (zrp) mRNA levels were significantly higher in females, compared to male fish. In general, significantly lower vtg and zrp transcripts levels were recorded at Igbore (control site), compared with contaminated sites. Principal component analysis (PCA) showed site and sex relationship in biological responses and contaminants, including trace metals, demonstrating that measured endocrine responses in fish were associated with contaminant burden in sediment. In addition, positive relationships were observed in male fish from Idumota, Oworonshoki and Ikorodu with vtg and dieldrin/4-iso-nonyphenol, with higher levels in male fish, compared to females. Further, contaminants from the Makoko, Oworonshoki and Ikorodu sites were positively associated with higher GSI and zrp in females. More importantly, the severity of intersex and changes in vtg transcripts imply a progressive feminization of male fish with concomitant alteration in the reproductive health of fish inhabiting the Lagos lagoon.

Temporal evaluation of estrogenic endocrine disruption markers in smallmouth bass (Micropterus dolomieu) reveals seasonal variability in intersex.

A reconnaissance project completed in 2009 identified intersex and elevated plasma vitellogenin in male smallmouth bass inhabiting the Missisquoi River, VT. In an attempt to identify the presence and seasonality of putative endocrine disrupting chemicals or other factors associated with these observations, a comprehensive reevaluation was conducted between September 2012 and June 2014. Here, we collected smallmouth bass from three physically partitioned reaches along the river to measure biomarkers of estrogenic endocrine disruption in smallmouth bass. In addition, polar organic chemical integrative samples (POCIS) were deployed to identify specific chemicals associated with biological observations. We did not observe biological differences across reaches indicating the absence of clear point source contributions to the observation of intersex. Interestingly, intersex prevalence and severity decreased in a stepwise manner over the timespan of the project. Intersex decreased from 92.8% to 28.1%. The only significant predictor of intersex prevalence was year of capture, based on logistic regression analysis. The mixed model of fish length and year-of-capture best predicted intersex severity. Intersex severity was also significantly different across late summer and early spring collections indicating seasonal changes in this metric. Plasma vitellogenin and liver vitellogenin Aa transcript abundance in males did not indicate exposure to estrogenic endocrine disrupting chemicals at any of the four sample collections. Analysis of chemicals captured by the POCIS as well as results of screening discrete water samples or POCIS extracts did not indicate the contribution of appreciable estrogenic chemicals. It is possible that unreported changes in land-use activity have ameliorated the problem, and our observations indicate recovery. Regardless, this work clearly emphasizes that single, snap shot sampling for intersex may not yield representative data given that the manifestation of this condition within a population can change dramatically over time.

Imposex in Stramonita haemastoma from coastal sites of Cartagena, Colombia / Imposex em Stramonita haemastoma em locais costeiros de Cartagena, Colômbia

Abstract Imposex is the development of male sexual characteristics caused by the toxic effects of some chemicals that acts as an endocrinal disruptor. Antifouling paints contain these chemicals. Cartagena lacks studies to indicate the extent of imposex in its coastal waters. The aim of this study was to determine the prevalence of imposex in the gastropod Stramonita haemastoma in Cartagena, Colombia. Specimens were collected during 2013 from locations of high and low influence of port activity. Morphometric measurements and the frequency of the occurrence of imposex were registered. The comparison among morphometric variables showed statistically significant differences between the two sites studied. Furthermore, the females of the S. haemastoma species presented an imposex frequency of 93.1% in Birds' Island, Cartagena Bay, compared to 31.8% in La Bocana. The relative penis size index or RPLI (10.145 and 3.231) and vas deferens sequence index or VDSI (2.83 and 1.16), showed possible contamination by organotin compounds in both places.

Resumo Imposex é o desenvolvimento de características sexuais masculinas causadas por poluentes tóxicos de alguns produtos químicos que atuam como desreguladores endócrinos. Tintas anti-incrustantes são as que contêm estes produtos químicos. Cartagena carece de estudos para indicar a extensão do imposex nas suas águas costeiras. O objetivo deste estudo foi determinar a prevalência de imposex no gastrópode Stramonita haemastoma em Cartagena, Colômbia. Os espécimes foram coletados durante 2013 de locais de alta e baixa influência da atividade portuária. Foram registradas as medidas morfométricas e a frequência da ocorrência do imposex. A comparação entre as variáveis morfométricas mostrou diferenças estatisticamente significantes entre os dois locais estudados. Além disso, as fêmeas da espécie S. haemastomaapresentaram uma frequência de imposex de 93,1% na Ilha das Aves, Baía das Cartagena, em comparação com 31,8% em La Bocana. O índice do comprimento relativo do pênis ou RPLI (10,145 e 3,231) e o índice da sequência do vaso deferente ou VDSI (2,83 e 1,16), mostraram possível contaminação por compostos organoestânicos em ambos os locais.

Indicators of exposure to estrogenic compounds at Great Lakes Areas of Concern: species and site comparisons.

Adverse effects resulting from potential exposure of wild fishes to estrogenic endocrine disruptors were assessed at seven United States Great Lakes Areas of Concern using biomarkers ranging from organismal (gonadosomatic indices) to tissue/plasma (histology, plasma vitellogenin) and molecular (hepatic gene transcripts) levels. Biomonitoring was conducted on pelagic, top predator species, largemouth Micropterus salmoides and smallmouth M. dolomieu bass and benthic, omnivorous white sucker Catostomus commersonii. Seasonal (spring and fall) comparisons were conducted at select sites. Intersex (testicular oocytes), plasma vitellogenin, and hepatic vitellogenin transcripts were commonly observed in bass species. Testicular oocyte severity was positively, although weakly, correlated with plasma vitellogenin, hepatic transcripts of vitellogenin, estrogen receptor α, and estrogen receptor ß2, while negatively correlated with androgen receptor ß and phosphoenolpyruvate carboxykinase. No testicular oocytes were observed in white sucker; however, plasma vitellogenin and hepatic vitellogenin transcripts were commonly detected in the males. The results demonstrate the importance of utilizing multiple endpoints to assess exposure to estrogenic compounds as well as the importance of choosing sensitive species.

The reduction in sexual behavior induced by neonatal immune stress is not related to androgen levels in male rats.

PURPOSE: It is known that various types of stress in early life increase the incidence of diabetes, myocardial infarctions, and psychiatric disorders in adulthood. We examined the mechanism by which neonatal immune stress reduces sexual behavior in adult male rats. METHODS: Male rats were randomly divided into 3 groups: the control (n = 17), postnatal day 10 lipopolysaccharide (PND10LPS) (n = 31), and PND25LPS (n = 16) groups, which received intraperitoneal injections of LPS (100 µg/kg) or saline (injection volume: ≤0.1 ml/g) on postnatal days 10 and 25. In experiment 1, male rats (age: 11 to 12 weeks) were put together with female rats in a one-to-one setting for mating, and sexual behavior (mounting, intromission, and ejaculation) was monitored for 30 minutes. The serum levels of luteinizing hormone (LH) and testosterone (T) and the hypothalamic mRNA expression levels of factors related to sexual behavior were examined. After experiment 1 finished, the remaining 37 male rats were used for experiment 2: the control group (n = 8), PND10 LPS group (n = 21) and PND25LPS group (n = 8) these rats had been given an i.p. injection of the saline during the expriment1. All of the rats were orchidectomized at 14 weeks of age. After a 3-week recovery period, a silastic tube containing crystalline T was subcutaneously implanted into the back of each rat. The rats' sexual behavior, serum hormone concentrations, and hypothalamic mRNA expression levels were assessed. RESULTS: In experiment 1, preputial separation occurred significantly later in the PND10LPS group than in the control group. The frequency of sexual behavior was significantly lower in the PND10LPS group than in the control group. The serum T concentrations of the PND10LPS and PND25LPS groups were significantly lower than that of the control group, but the serum LH concentrations of the 3 groups did not differ significantly. The hypothalamic mRNA expression levels of progesterone receptor B (PRB) and gonadotropin-releasing hormone (GnRH) were significantly lower in the PND10LPS and PND25LPS groups than in the control group, whereas the hypothalamic PRA + B mRNA expression levels of the 3 groups did not differ significantly. In experiment 2, after T supplementation the frequency of sexual behavior was significantly lower in the PND10LPS and PND25LPS groups than in the control group, although there were no significant differences in the serum T or LH concentrations or the hypothalamic PRB, PRA + B, or GnRH mRNA expression levels of the 3 groups. CONCLUSION: In male rats, immune stress in the early neonatal period delayed sexual maturation, reduced sexual behavior, suppressed the serum T concentration, and downregulated the hypothalamic mRNA expression levels of GnRH and the PR in adulthood. The delayed sexual maturation was presumed to have been caused by the reduction in the serum T concentration. However, the rats that experienced neonatal stress exhibited reduced sexual behavior irrespective of their serum T concentrations.

Modeling the exposure of wild fish to endocrine active chemicals: Potential linkages of total estrogenicity to field-observed intersex.

Decades of studies on endocrine disruption have suggested the need to manage the release of key estrogens from municipal wastewater treatment plants (WWTP). However, the proposed thresholds are below the detection limits of most routine chemical analysis, thereby restricting the ability of watershed managers to assess the environmental exposure appropriately. In this study, we demonstrated the utility of a mechanistic model to address the data gaps on estrogen exposure. Concentrations of the prominent estrogenic contaminants in wastewaters (estrone, estradiol, and ethinylestradiol) were simulated in the Grand River in southern Ontario (Canada) for nine years, including a period when major WWTP upgrades occurred. The predicted concentrations expressed as total estrogenicity (E2 equivalent concentrations) were contrasted to a key estrogenic response (i.e., intersex) in rainbow darter (Etheostoma caeruleum), a wild sentinel fish species. A predicted total estrogenicity in the river of ≥10 ng/L E2 equivalents was associated with high intersex incidence and severity, whereas concentrations <0.1 ng/L E2 equivalents were associated with minimal intersex expression. Exposure to a predicted river concentration of 0.4 ng/L E2 equivalents, the environmental quality standard (EQS) proposed by the European Union for estradiol, was associated with 34% (95% CI:30-38) intersex incidence and a very low severity score of 0.6 (95% CI:0.5-0.7). This exposure is not predicted to cause adverse effects in rainbow darter. The analyses completed in this study were only based on the predicted presence of three major estrogens (E1, E2, EE2), so caution must be exercised when interpreting the results. Nevertheless, this study illustrates the use of models for exposure assessment, especially when measured data are not available.

Critical analysis of the relationship between imposex and butyltin body burden in Nassarius reticulatus and Nucella lapillus.

Imposex is a disorder caused by organotins, mainly tributyltin, which results in the appearance of male sexual characteristics in females of gastropod mollusks. The main objective of this work was to make a critical analysis of the relationship between imposex and butyltin body burdens in Nucella lapillus and Nassarius reticulatus. Specifically, this study evaluates possible additive effects among butyltins, proposes scales of effects based on robust statistical criteria as alternatives to existing ones and defines the body burdens of TBT in N. lapillus and N. reticulatus corresponding to the assessment classes (ACs) of the Vas Deferens Sequence Index (VDSI) established by OSPAR. Data of organotin body burdens and biological effects was retrieved from the ICES Dataset and from scientific literature. All responses, except the percentage of females displaying Imposex (IMPF) in Nucella lapillus, showed a sigmoidal profile regarding to the body burden of mono- (MBT), di- (DBT) and tributyltin and sum of butyltins (SumBTs). TBT and the SumBTs were better indicators of the VDSI or Relative Penis Size Index/Relative Penis Length Index (RPSI/RPLI) responses than MBT or DBT in most cases. From a statistical point of view, RPSI/RPLI and VDSI were better indicators of contamination by TBT than IMPF, although both RPSI and RPLI showed lower sensitivity than VDSI. The model used for describing the joint effect of butyltins provided a statistically significant fitting to the data assuming a null effect for both MBT and DBT for N. lapillus, and a lower toxic contribution of MBT and DBT with respect to TBT for N. reticulatus. RPSI or RPLI values, equivalent to the ACs for VDSI, were proposed as alternative criteria when measuring moderate to high levels of imposex. TBT concentrations in N. reticulatus and N. lapillus tissues, corresponding to the ACs were calculated and provided valuable information for cross-species comparisons.

First assessment of the effectiveness of the international convention on the control of harmful anti-fouling systems on ships in Tunisia using imposex in Hexaplex trunculus as biomarker.

The global ban on the use of tributyltin (TBT) forced in September 2008 was ratified in Tunisia as late as June 2011. In this context, the present study aims to highlight the consequences of TBT contamination by monitoring 22 Tunisian sites before (2007) and after the ban (2012 and 2016) using as biomarker the occurrence of imposex in Hexaplex trunculus. From 2004 to 2016, complete recovery from imposex was reported in 11 sites. All average imposex indices calculated for each sampling year also decreased: imposex incidence from 61 to 27%, VDSI from 2 to 0.7, and RPLI from 15.7 to 1.6%. Ecological Quality Ratios (EQR) revealed that the sampling sites are in moderate to good ecological status. Overall, the present study confirms the effectiveness of the enacted legislation in reducing the impact of TBT pollution along the Tunisian coast.

Recovery of the New Zealand muricid dogwhelk Haustrum scobina from TBT-induced imposex.

Levels of imposex in the muricid dogwhelk Haustrum scobina (Quoy & Gaimard, 1833) were assessed in two major New Zealand ports between 2015 and 2017, 12+years after the banning of TBT-based antifouling paints. In the 1990s imposex was common adjacent to port facilities and marinas in Waitemata and Tauranga Harbours, and several populations were found to be largely sterile and in decline. By 2015-17 imposex was largely absent from Tauranga Harbour and considerably reduced in Waitemata Harbour. In Waitemata Harbour imposex remained in areas adjacent to hull cleaning facilities and marinas, but at low levels and frequencies. These data highlight the success of the 2003 banning of TBT-based antifouling paints in reducing levels of imposex in New Zealand ports. However, these reductions in antifouling chemicals and subsequent population recoveries may have had unforseen effects within these harbours including changes in community structure and the arrival of non-indigenous species.

Extended imposex monitoring in N Atlantic Spain confirms punctual attainment of European environmental objectives for TBT.

Legislation in the European Union (EU) aimed at reaching by 2015 a Good Ecological Status in regard to tributyltin (TBT, the biocide used in traditional antifouling paints). With a view to check such an achievement in N Atlantic Spain, baseline monitoring of gastropod imposex (the recommended assessment tool) was extended up to that date. In Galicia (the Western part of the study area) the use of the rock snail Nucella lapillus since 1996 had shown this environmental objective to be met as soon as 2009, but new surveys reveal no further improvement thereafter. As for the Eastern Cantabrian coast, imposex levels in the mud snail Nassarius reticulatus progressively declined from 2006 to 2015, when records finally complied with expectations. Both data sets are confronted and discussed in relation to the diverse environmental factors that may be determining the distribution of gastropods in these regions.