Development of an exposure protocol for toxicity test (FEET) for a marine species: the European sea bass (Dicentrarchus labrax).

Regulatory assessment of the effects of chemicals requires the availability of validated tests representing different environments and organisms. In this context, developing new tests is particularly needed for marine species from temperate environments. It is also important to evaluate effects that are generally poorly characterized and seldom included in regulatory tests. In this study, we designed an exposure protocol using European sea bass (Dicentrarchus labrax) larvae. We examined classical toxicological values (LCx) as well as behavioral responses. By comparing different hatching and breeding strategies, we defined the optimal conditions of exposure as non-agitated conditions in 24- or 48-well microplates. Our exposure protocol was then tested with 3,4-dichloroaniline (3,4-DCA), a recommended reference molecule. Based on our results, the 96 h LC50 for 3,4-DCA corresponded to 2.04 mg/L while the 168 h LC50 to 0.79 mg/L. Behavioral analyses showed no effect of 3,4-DCA at low concentration (0.25 mg/L). In conclusion, the present work established the basis for a new test which includes behavioral analysis and shows that the use of sea bass is suitable to early-life stage toxicity tests.

Genotoxicity in the rivers from the Brantas catchment (East Java, Indonesia): occurrence in sediments and effects in Oreochromis niloticus (Linnæus 1758).

This paper reports the first data from an integrated study investigating genotoxicity in the Brantas River, Java, Indonesia. Results showed that organic sediment extracts from the sites in the Brantas Delta retained genotoxic compounds identified using the SOS Chromotest and that the Aloo River and, to a lesser extent, the Surabaya River were the most contaminated studied sites. This genotoxicity was attributable to compounds that did not require any bioactivation under the test conditions. Occurrence of genotoxic effects was further investigated in erythrocytes from Nile tilapia, Oreochromis niloticus. High numbers of micronuclei were counted, especially in fish sampled in the rivers of the Brantas Delta. Moreover, cytoplasmic alterations which could be indicative of the presence of lipofuscin were found in the cytoplasm of the fish blood cells, especially in fish from the Aloo, Surabaya and Kalimas rivers. Altogether, our data showed that genotoxicity is occurring in fish living in rivers of the delta of the Brantas River and suggest that sediments from these sites may constitute a major source of pollution and hazard for species living or feeding in the area.

Developmental instability is associated with estrogenic endocrine disruption in the Chilean native fish species, Trichomycterus areolatus.

Endocrine disrupting compounds (EDCs) are widespread contaminants that alter the normal functioning of the endocrine system. While they cause dysfunctions in essential biological processes, it is unclear whether EDCs also impact developmental stability. In the present study, we investigated the occurrence of estrogenic endocrine disrupting compounds in a small watershed of south-central Chile impacted by anthropogenic activities. Then, we assessed their relationship with internal levels of estrogenic active compounds and fluctuating asymmetry (FA), a proxy of developmental stability in organisms with bilateral symmetry, in a native fish species (Trichomycterus areolatus). Yeast estrogenic screen assays were performed to measure estrogenic activity in river sediments and in male fish tissues collected from 17 sites along the Chillán watershed, and geometric morphometrics used to estimate fluctuating asymmetry based on the shapes of 248 fish skulls. Estrogenic activity was detected both in sediments and male fish tissues at concentrations of up to 1005 ng and 83 ng 17ß-estradiol equivalent/kg dw, respectively. No significant correlation was found between the two. However, fish tissue estrogenicity, water temperature and dissolved oxygen explained >80% of the FA population variation. By showing a significant relationship between estrogenic activity and FA of T. areolatus, our results indicate that developmental stability can be altered by estrogenic endocrine disruption, and that FA can be a useful indicator of sub-lethal stress in T. areolatus populations.

Biomarkers as tools for monitoring within the Water Framework Directive context: concept, opinions and advancement of expertise.

The Water Framework Directive (WFD) currently supports chemical and ecological monitoring programmes in order to achieve the good water surface status. Although chemical and ecological assessments are necessary, they have some limitations. Chemical approaches focus on certain substances identified as priorities, but they do not take into account other potentially harmful substances and also ignore the hazards related to contaminant cocktails. On the other hand, while ecological approaches provide holistic information on the impairment of biological communities in ecosystems, they do not distinguish the role of contaminants in these alterations, and consequently do not allow the establishment of contaminant impact reduction plans. Consequently, ecotoxicologists suggest the use of effect-based tools such as biomarkers. Biomarkers highlight the effect of potentially harmful substances (or a cocktail), and their specificity towards the chemicals makes it possible to properly discriminate the role of toxicants within biological community impairments. Thus, the integration of such tools (besides existing chemical and ecological tools) in the WFD could considerably improve its biomonitoring strategy. The B n' B project (Biomarkers and Biodiversity) exposes key objectives that will allow to (i) establish an inventory of the biomarkers developed by French laboratories; (ii) determine their methodological advancement and limits and, on this basis, formulate recommendations for biomonitoring use and future research needs; (iii) discuss the biomarkers' ecological significance, specificity to contaminants and interpretation capacity; (iv) establish, in fine, a selection of valuable biomarkers to enter the WFD; and (iv) propose integrative tools to facilitate the decision-taking by stakeholders.

Evidence for Bisphenol B Endocrine Properties: Scientific and Regulatory Perspectives.

BACKGROUND: The substitution of bisphenol A (BPA) by bisphenol B (BPB), a very close structural analog, stresses the need to assess its potential endocrine properties. OBJECTIVE: This analysis aimed to investigate whether BPB has endocrine disruptive properties in humans and in wildlife as defined by the World Health Organization (WHO) definition used in the regulatory field, that is, a) adverse effects, b) endocrine activity, and c) plausible mechanistic links between the observed endocrine activity and adverse effects. METHODS: We conducted a systematic review to identify BPB adverse effects and endocrine activities by focusing on animal models and in vitro mechanistic studies. The results were grouped by modality (estrogenic, androgenic, thyroid hormone, steroidogenesis-related, or other endocrine activities). After critical analysis of results, lines of evidence were built using a weight-of-evidence approach to establish a biologically plausible link. In addition, the ratio of BPA to BPB potency was reported from studies investigating both bisphenols. RESULTS: Among the 36 articles included in the analysis, 3 subchronic studies consistently reported effects of BPB on reproductive function. In rats, the 28-d and 48-week studies showed alteration of spermatogenesis associated with a lower height of the seminiferous tubules, the alteration of several sperm parameters, and a weight loss for the testis, epididymis, and seminal vesicles. In zebrafish, the results of a 21-d reproductive study demonstrated that exposed fish had a lower egg production and a lower hatching rate and viability. The in vitro and in vivo mechanistic data consistently demonstrated BPB's capacity to decrease testosterone production and to exert an estrogenic-like activity similar to or greater than BPA's, both pathways being potentially responsible for spermatogenesis impairment in rats and fish. CONCLUSION: The available in vivo, ex vivo, and in vitro data, although limited, coherently indicates that BPB meets the WHO definition of an endocrine disrupting chemical currently used in a regulatory context. https://doi.org/10.1289/EHP5200.

Molecular isolation and characterization of the kisspeptin system, KISS and GPR54 genes in roach Rutilus rutilus.

The reproduction of vertebrates is regulated by endocrine and neuro-endocrine signaling molecules acting along the brain-pituitary-gonad (BPG) axis. The understanding of the neuroendocrine role played in reproductive function has been recently revolutionized since the KiSS1/GPR54 (KiSS1r) system was discovered in 2003 in human and mice. Kisspeptins, neuropeptides that are encoded by the KiSS genes, have been recognized as essential in the regulation of the gonadotropic axis. They have been shown to play key roles in puberty onset and reproduction by regulating the gonadotropin secretion in mammals while physiological roles in vertebrates are still poorly known. In order to provide new knowledge on basic reproductive physiology in fish as well as new tools to assess impacts of endocrine disrupting compounds (EDCs), the neurotransmitter system, i.e., gene/receptor, KISS/GPR54 might constitute an appropriate biomarker. This study provides new understandings on the neuroendocrine regulation of roach reproduction as well as new molecular tools to be used as biomarkers of endocrine disruption. This work completes the set of biomarkers already validated in this species.

Regulatory identification of BPA as an endocrine disruptor: Context and methodology.

BPA is one of the most investigated substances for its endocrine disruptor (ED) properties and it is at the same time in the center of many ED-related controversies. The analysis on how BPA fits to the regulatory identification as an ED is a challenge in terms of methodology. It is also a great opportunity to test the regulatory framework with a uniquely data-rich substance and learn valuable lessons for future cases. From this extensive database, it was considered important to engage in a detailed analysis so as to provide specific and strong evidences of ED while reflecting accurately the complexity of the response as well the multiplicity of adverse effects. An appropriate delineation of the scope of the analysis was therefore critical. Four effects namely, alterations of estrous cyclicity, mammary gland development, brain development and memory function, and metabolism, were considered to provide solid evidence of ED-mediated effects of BPA.

High estradiol exposure disrupts the reproductive cycle of the clam Ruditapes decussatus in a sex-specific way.

Bivalve species may be susceptible to environmental estrogenic compounds including estradiol (E2). However, they are able to biotransform the hormone quite readily and inactivate its estrogenic action. To study the long-term effects of elevated free E2 tissue levels, we transiently exceeded the biotransformation capacity of the clam Ruditapes decussatus by exposing them with high E2 concentrations (400 ng/L) and subsequently study the consequences on gametogenesis during the following reproductive cycle. Exposure to 400 ngE2/L led to a significant increase in tissue free E2 levels, which reached 10-50 ng E2Eq/gww. No deleterious effect on gonado-somatic index (GSI), condition index (CI), or ability to respond to the stress on stress test could be detected after a month of exposure, suggesting the absence of negative effects on the clam's health. However, a marked increase in gametogenesis could be observed in both sexes during the exposure. Subsequent transplantation of the clams in the field allowed the normal development of the male clams and maturation of the gonads without any detrimental effect observed after 4 months. In contrast, in early July, all female clams formerly exposed to E2 showed lower health status, and only ovaries with atretic oocytes while all control and indigenous females were normal and mature. These results show a sex-specific effect of high E2 exposure and suggest either a direct or indirect role for E2 in R. decussatus' reproduction.

Endocrine disruption effects in male and intersex roach (Rutilus rutilus, L.) from French rivers: An integrative approach based on subcellular to individual responses.

Among the chemicals found in the aquatic environment, some have been reported to interact with the endocrine system of organisms and have been identified as endocrine disrupting compounds. In this study, we have assessed the biological effects due to environmental estrogenic compounds in adult male roach (Rutilus rutilus). Wild fish were collected in the Seine River, and one of its affluent, the Epte River in Normandy, France and impacts on endocrine and reproductive functions have been assessed. This study provides innovative data on the intersex condition in wild fish that are crucial to understand estrogenic contamination impacts and maintenance of sustainable fish population. The biomarker responses found in fish from contaminated sites included high plasmatic vitellogenin concentrations, altered steroid levels, histopathological and up to 50% intersex fish among the male. However, the overall sex ratio of the roach population indicated sign of feminization with some sites showing only 20% of males. The chemical organic contamination at the different location sites has been characterized by the levels of PAHs, PCBs and phthalates and total estrogenic activity was measured by the YES in vitro assay.

Integrated monitoring of chemicals and their effects on four sentinel species, Limanda limanda, Platichthys flesus, Nucella lapillus and Mytilus sp., in Seine Bay: A key step towards applying biological effects to monitoring.

The International workshop on Integrated Assessment of CONtaminants impacts on the North sea (ICON) provided a framework to validate the application of chemical and biological assessment thresholds (BACs and EACs) in the Seine Bay in France. Bioassays (oyster larval anomalies, Corophium arenarium toxicity assay and DR Calux) for sediment and biomarkers: ethoxyresorufin-O-deethylase (EROD) activity, acetylcholinesterase (AChE) activity, lysosomal membrane stability (LMS), DNA strand breaks using the Comet assay, DNA adducts, micronucleus (MN), PAH metabolites, imposex, intersex and fish external pathologies were analysed in four marine sentinel species (Platichthys flesus, Limanda limanda, Mytilus sp. and Nucella lapilus). Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and heavy metals were analysed in biota and sediment. Results for sediment and four species in 2008-2009 made it possible to quantify the impact of contaminants using thresholds (Environmental Assessment Criteria/EAC2008: 70% and EAC2009: 60%) and effects (EAC2008: 50% and EAC2009: 40%) in the Seine estuary. The Seine estuary is ranked among Europe's most highly polluted sites.

Sensitive periods for 17ß-estradiol exposure during immune system development in sea bass head kidney.

An increasing body of evidence suggests that sex steroids play an important role in the development and regulation of vertebrate immune defense. Therefore, compounds with estrogenic activity may influence the immune system via receptor-mediated pathways. The presence of estrogen receptors in immune cells and organs during the early stages of development may indicate that female steroid hormones are involved in the maturation of the fish immune system. This is of particular importance, as some marine fish are probably exposed to sources of exogenous estrogens while they reside in their estuarine nursery grounds. In this study, the influence of 17ß-estradiol (E2) on estrogen receptor and cytokine gene expression was assessed in juvenile sea bass (Dicentrarchus labrax) together with characterization of the head kidney leukocyte populations and corresponding phagocytic activity during organ regionalization from 98 to 239 dph. E2 exposure, beginning at 90 dph resulted in indirect and delayed modifications of interleukin 1ß and estrogen receptor α gene expression, which may affect B-lymphocyte proliferation in the sea bass head kidney. The E2 treatment of 120 dph fish led to an increase in estrogen receptor ß2 and a decrease in transforming growth factor ß1 gene expression, which coincided with decreased phagocytic activity of head kidney lymphocytes and monocytes/macrophages. Additionally, these changes were observed during developmental periods described as critical phases for B-lymphocyte development in mammals. Consequently, exogenous estrogens have the potential to modify the innate immune response in juvenile sea bass and to exert detrimental effects on head kidney development. Copyright © 2015 John Wiley & Sons, Ltd.

The endocrine-disrupting effect and other physiological responses of municipal effluent on the clam Ruditapes decussatus.

In order to document the potential endocrine disrupting and toxic effect of the municipal wastewater effluents discharged into the Sfax coastal area (South of Tunisia), specimens of clam R. decussatus were collected from a reference site and were in vivo exposed to treated sewage effluent for 30 days. To this end, estrogenic and androgenic activities were measured in the gills to assess potential accumulation and regulation of active compounds. After effluent exposure androgenic activity in organic extracts increased up to fivefold compared to controls and remained elevated, while estrogenic activity was not significantly affected by exposure. As a consequence, remarkable disruptions in the gametogenesis activity, glycogen content, and Vitellogenin-like protein levels in male clams were observed. A parallel analysis of heavy metals in clam tissues was determined. A significant uptake of Ni, Zn, and Pb in soft tissues of exposed clams was observed. The significant increase of malondialdehyde (MDA) concentrations as a function of exposure time implies that clams have been exposed to an oxidative stress probably due to the presence of high metal concentrations in sewage effluent. Correlation analysis has revealed a statistically significant and positive relationship between MDA levels and metal concentrations in clams' tissues. The acetylcholinesterase activity was not significantly affected by exposure. Altogether, these results showed that a short-term exposure to a mixture of chemical compounds released by the Sfax wastewater treatment plant induce adverse physiological and reproductive effects in R. decussatus. Further studies are underway in order to evaluate its long-term impacts on aquatic wildlife in the gulf of Gabes area.

TP53 and FGFR3 Gene Mutation Assessment in Urine: Pilot Study for Bladder Cancer Diagnosis.

AIM: To assess, in a prospective clinical research study, a new non-invasive and reliable test to accurately detect tumor protein 53 (TP53) and fibroblast growth factor receptor-3 (FGFR3) mutations in cells in urine. MATERIALS AND METHODS: TP53 mutations were analyzed using the functional analysis of separated allele in yeast (FASAY) method, which allows functional analysis of the P53 protein, and FGFR3 mutations were assessed with the SNaPshot system, detecting the eight most frequent point-mutations of this gene. Chi-square test or Fisher's exact test were used to compare TP53 and FGFR3 mutations in the tumors according to tumor stage and grade. RESULTS: TP53 and FGFR3 mutations in bladder tumors increased and decreased respectively with increasing tumor stage and cellular grade (p<0.05 and p<0.001, respectively). A total of 103 tumor/urinary sediment couples were analyzed. TP53 or FGFR3 mutations were observed in 76 tumors. The sensitivity for the detection of this type of mutation in urine was 46%, the specificity was 81%, the positive predictive value was 94% and the negative predictive value was 37%. CONCLUSION: Our original data confirmed the feasibility of TP53 and FGFR3 mutation detection in urine sediment. These measurements, together with urine cytology, may increase tumor detection. The sensitivity of the TP53/FGFR3 phenotype test in the urine was less than 50% and was not able to replace standard cystoscopy in the diagnosis of bladder tumors.

Intersex related gene expression profiles in clams Scrobicularia plana: Molecular markers and environmental application.

Intersex, the appearance of female characteristics in male gonads, has been identified in several aquatic species. It is a widespread phenomenon in populations of the bivalve, Scrobicularia plana, from the southwest coast of the U.K. Genes previously identified as differentially expressed (ferritin, testicular haploid expressed gene, THEG, proliferating cell nuclear antigen, PCNA; receptor activated protein kinase C, RACK; cytochrome B, CYB; and cytochrome c oxidase 1, COX1) in intersex clams relative to normal male clams, were selected for characterisation and an environmental survey of the Channel region. Transcripts were significantly differentially expressed at sites with varying intersex incidence and contaminant burdens. Significant correlations between specific gene expressions, key contaminants and sampling locations have been identified, though no single gene was associated with intersex incidence. The results highlight the difficulty in understanding the intersex phenomenon in molluscs where there is still a lack of knowledge on the control of normal reproduction.

Recommendations to design environmental monitoring in the European bullhead, Cottus sp., based on reproductive cycle and immunomarker measurement.

European bullhead is a relevant fish species to assess adverse effects of environmental stress on wild fish. Nevertheless, their complex reproductive cycle is very different between sites and could interfere with many physiological processes. Thus, prior to use biomarker to statute on environmental quality of rivers, we wanted to characterize reproductive profile (spawn number, GSI, gonad development). The major results demonstrated that the two types of reproductive cycle shown were strongly correlated to water temperature variation. In a second time, even if innate immunomarkers are highly relevant on biomonitoring program, hormonal variation seems to impact severely their responses. Thus, the link between reproductive status and immune activity (leucocyte distribution, cellular mortality, respiratory burst, phagocytosis activity) must also be study. Nonetheless, in the present work, immune capacities seems to be more correlated with season and environmental factors than reproduction.

Widespread contamination of coastal sediments in the Transmanche Channel with anti-androgenic compounds.

This study analysed the levels of androgen receptor antagonist activity in extracts of coastal sediments sampled from estuaries in southern UK and northern France. Anti-androgenic (AA) activity varied between <0.2 and 224.3±38.4µg flutamide equivalents/g dry weight of sediment and was significantly correlated with the total organic carbon and silt content of samples. AA activity was detected in tissues extracts of clams, Scrobicularia plana, sampled from a contaminated estuary, some of which was due to uptake of a series of 4 or 5 ring polycyclic aromatic hydrocarbons (PAHs). Initial studies also indicated that fractionated extracts of male, but not female, clams also contained androgen receptor agonist activity due to the presence of dihydrotestosterone in tissues. This study reveals widespread contamination of coastal sediments of the Transmanche region with anti-androgenic compounds and these contaminants should be investigated for their potential to disrupt sexual differentiation in aquatic organisms.

Cellular immune responses and phagocytic activity of fishes exposed to pollution of volcano mud.

Since May 29, 2006, a mud volcano in the Brantas Delta of the Sidoarjo district has emitted mud that has inundated nearby villages. Pollution in this area has been implicated in detrimental effects on fish health. In fishes, leukocyte and phagocytic cells play a vital role in body defenses. We report for the first time the effect of "LUSI" volcano mud on the immune systems of fish in the Brantas Delta. The aim of this study was to find biomarkers to allow the evaluation of the effects of volcanic mud and anthropogenic pollution on fish health in the Brantas Delta. The study took places at the Brantas Delta, which was polluted by volcano mud, and at reference sites in Karangkates and Pasuruan. Leukocyte numbers were determined using a Neubauer hemocytometer and a light microscope. Differential leukocyte counts were determined using blood smears stained with May Grunwald-Giemsa, providing neutrophil, lymphocyte and monocyte counts. Macrophages were taken from fish kidney, and their phagocytic activity was measured. In vitro analyses revealed that leukocyte and differential leukocyte counts (DLC) were higher in Channa striata and Chanos chanos caught from the polluted area. Macrophage numbers were higher in Oreochromis mossambicus than in the other species, indicating that this species is more sensitive to pollution. In areas close to volcanic mud eruption, all specimens had lower phagocytic activity. Our results show that immune cells were changed and phagocytic activity was reduced in the polluted area indicating cytotoxicity and alteration of the innate immune system in fishes exposed to LUSI volcano mud and anthropogenic pollution.