Combined exposure to engineered nanoparticles (ENPs) and anthropogenic contaminants can lead to changes in bioavailability, uptake and thus effects of both groups of contaminants. In this study we investigated effects of single and combined exposures of silver (Ag) nanoparticles (AgNPs) and the synthetic hormone 17a-ethinylestradiol (EE2) on tissue uptake of both contaminants in juvenile turbot (Scophthalmus maximus). Silver uptake and tissue distribution (gills, liver, kidney, stomach, muscle and bile) were analyzed following a 14-day, 2-h daily pulsed exposure to AgNPs (2 mg L 1 and 200 mg L 1), Agþ (50 mg L 1), EE2 (50 ng L 1) and AgNP þ EE2 (2 or 200 mg L 1þ50 ng L 1). Effects of the exposures on plasma vitellogenin (Vtg) levels, EE2 and steroid hormone concentrations were investigated. The AgNP and AgNP þ EE2 exposures resulted in similar Ag concentrations in the tissues, indicating that combined exposure did not influence Ag uptake in tissues. The highest Ag concentrations were found in gills. For the Agþ exposed fish, the highest Ag concentrations were measured in the liver. Our results show dissolution processes of AgNPs in seawater, indicating that the tissue concentrations of Ag may partly originate from ionic release. Plasma EE2 concentrations and Vtg induction were similar in fish exposed to the single contaminants and the mixed contaminants, indicating that the presence of AgNPs did not significantly alter EE2 uptake. Similarly, concentrations of most steroid hormones were not significantly altered due to exposures to the combined contaminants versus the single compound exposures. However, high concentrations of AgNPs in combination with EE2 caused a drop of estrone (E1) (female fish) and androstenedione (AN) (male and female fish) levels in plasma below quantification limits. Our results indicate that the interactive effects between AgNPs and EE2 are limited, with only high concentrations of AgNPs triggering synergistic effects on plasma steroid hormone concentrations in juvenile turbots. | acceptedVersion

Threatened or endangered reptiles, such as sea turtles, are generally understudied within the field of wildlife toxicology, with even fewer studies on how contaminants affect threatened species reproduction. This paper aimed to better inform threatened species conservation by systematically and quantitatively reviewing available research on the reproductive toxicology of all reptiles, threatened and non-threatened. This review found 178 studies that matched our search criteria. These papers were categorized into location conducted, taxa studied, species studied, effects found, and chemicals investigated. The most studied taxa were turtles (n = 87 studies, 49%), alligators/crocodiles (n = 54, 30%), and lizards (n = 37, 21%). Maternal transfer, sex steroid alterations, sex reversal, altered sexual development, developmental abnormalities, and egg contamination were the most common effects found across all reptile taxa, providing guidance for avenues of research into threatened species. Maternal transfer of contaminants was found across all taxa, and taking into account the foraging behavior of sea turtles, could help elucidate differences in maternal transfer seen at nesting beaches. Sex steroid alterations were a common effect found with contaminant exposure, indicating the potential to use sex steroids as biomarkers along with traditional biomarkers such as vitellogenin. Sex reversal through chemical exposure was commonly found among species that exhibit temperature dependent sex determination, indicating the potential for both environmental pollution and climate change to disrupt population dynamics of many reptile species, including sea turtles. Few studies used in vitro, DNA, or molecular methodologies, indicating the need for more research using high-throughput, non-invasive, and cost-effective tools for threatened species research. The prevalence of developmental abnormalities and altered sexual development and function indicates the need to further study how anthropogenic pollutants affect reproductive output in threatened reptiles.

Plastics in the oceans can break up into smaller size and shape resembling prey or particles selected for ingestion by marine organisms. Plastic polymers may contain chemical additives and contaminants, including known endocrine disruptors that may be harmful for the marine organisms, in turn posing potential risks to marine ecosystems, biodiversity and food availability.This study assesses the presence of plastics in the contents of the gastrointestinal tract (GIT) of a commercial fish species, the Atlantic horse mackerel, Trachurus trachurus, sampled from two different fishing areas of central Mediterranean Sea. Adverse effect of plastics occurrence on T. Trachurus health were also assessed quantifying the liver expression of vitellogenin (VTG), a biomarker for endocrine disruption.A total of 92 specimens were collected and morphometric indices were analysed. A subgroup was examined for microplastics (MP < 1 mm) and macroplastics (MaP >1 cm) accumulation in the GIT and for VTG expression.Results indicated that specimens from the two locations are different in size and maturity but the ingestion of plastic is widespread, with microplastics (fragments and filaments) abundantly present in nearly all samples while macroplastics were found in the larger specimens, collected in one of the two locations. Spectroscopic analysis revealed that the most abundant polymers in MP fragments were polystyrene, polyethylene and polypropylene, whereas MP filaments were identified mainly as nylon 6, acrylic and polyester. MaP were composed mainly of weathered polyethylene or polypropylene.The expression of VTG was observed in the liver of 60% of all male specimens from both locations.The results of this study represent a first evidence that the ingestion of plastic pollution may alter endocrine system function in adult fish T. Trachurus and warrants further research.

Perfluorooctane sulfonate (PFOS) has been reported to be widely distributed in the environment and wildlife with persistence. PFOS has various biological toxicity, especially disturbing the endocrine system. However, few studies have systematically evaluated its effect on sexual behaviors alteration and reproduction-related genes. This study was performed to assess the effect of PFOS exposure on sexual behaviors and genes in hypothalamic–pituitary–gonadal–liver (HPGL) axis in adult zebrafish.Male adult zebrafish were exposed to PFOS (0, 2, 20, and 200 μg/L) and 5 μg/L estradiol (E₂) continuously for 21 days. Sexual behaviors were analyzed by zebrafish behavior tracking system and the mRNA levels of HPGL-related genes was detected by RT-qPCR.Body weight of the fish was increased in 2, 200 μg/L PFOS and E₂ groups, and body length was increased with exposure to 2 μg/L PFOS and E₂. The hepatic-somatic index was decreased significantly after 2 and 20 μg/L PFOS treatments. Highest PFOS (200 μg/L) and E₂ exposure impaired standard zebrafish sexual behaviors significantly such as chasing, nose-tail and tail-touching. In brains, the genes gonadotropin-releasing hormone (GnRH), gonadotropin-releasing hormone receptor (GnRHr) were down-regulated with exposure to PFOS with linear trend and E₂ exposure, and follicle-stimulating hormone and luteinizing hormone were also down-regulated with exposure to 20 and 200 μg/L PFOS. In livers, the genes vitellogenin 1 and 3 were upregulated with some concentrations of PFOS and E₂, but estrogenic receptor α, β2 were upregulated in any concentration of PFOS and E₂. In testes, the expressions of follicle-stimulating hormone receptor, luteinizing hormone receptor, and androgen receptor genes were all significantly down-regulated with any exposure concentration of PFOS and E₂.PFOS may alter the zebrafish reproductive system by disrupting endocrine activity and impairing sexual behaviors.

The growing use of octocrylene (OC) in sunscreens has posed a great threat to aquatic organisms. In the present study, to assess its reproductive toxicity and mechanism, paired Japanese medaka (Oryzias latipes) (F0) were exposed to OC at nominal concentrations of 5, 50, and 500 μg/L for 28 d. Significant increases were observed in the gonadosomatic index (GSI) and hepatosomatic index (HSI) of F0 medaka at 500 μg/L OC (p < 0.05) without significant differences in fecundity. The fertility was significantly decreased at all treatments (p < 0.05). Significant increases in the percent of mature oocytes were observed at 5 and 500 μg/L OC, in which contrary to the percent of spermatozoa (p < 0.05). The plasma sex hormones and vitellogenin levels significantly increased in males at all treatments and in females at 50 and 500 μg/L OC (p < 0.05). In addition, the levels of fshβ and lhβ in the brains and the levels of fshr, lhr and cyp17α in the gonads were significantly upregulated in males at all treatments (p < 0.05), in line with those of ar, erα, erβ and cyp19β in the brains of male and female. The upregulation of vtg in male and female livers was observed only at 500 μg/L OC and upregulation of star and hsd3β was observed in testis at all treatments (p < 0.05). Continued exposure to OC significantly induced increases in the time to hatching, morphological abnormality rates, and cumulative death rates of F1 embryos, inconsistent with body length of F1 larvae (p < 0.05). Therefore, the responses of the exposed fish at the biochemical and molecular levels indicated reproductive toxicity and estrogenic activity of OC, providing insights into the mechanism of OC.

Current practices employed by most wastewater treatment plants (WWTP) are unable to completely remove endocrine disrupting compounds (EDCs) from reclaimed waters, and consistently discharge these substances to receiving systems. Effluent-dominated and dependent surface waters, especially during low instream flows, can increase exposure and risks to aquatic organisms due to adverse biological effects associated with EDCs. Given the ecological implications that may arise from exposure to such compounds, the present a multi-approach study examined spatio-temporal estrogenic potential of wastewater effluent to fish in East Canyon Creek (ECC), Utah, USA, a unique urban river with instream flows seasonally influenced by snowmelt. Juvenile rainbow trout (Oncorhynchus mykiss) were caged at different upstream and downstream sites from an effluent discharge during the summer and fall seasons. In the summer, where approximately 50% of the streamflow was dominated by effluent, fish from the upstream and a downstream site, located 13 miles away from the effluent discharge, presented significantly elevated concentrations of plasma vitellogenin (VTG). Similarly, significantly high 17β-estradiol to 11-ketotestosterone ratios were measured in the summer across all sites and time points, compared to the fall. In the laboratory, juvenile fish and primary hepatocytes were exposed to concentrated effluent and surface water samples. Quantification of VTG, although in significantly lower levels, resembled response patterns observed in fish from the field study. Furthermore, analytical quantification of common EDCs in wastewater revealed the presence of estriol and estrone, though these did not appear to be related to the observed biological responses, as these were more significant in sites were no EDCs were detected. These combined observations suggest potential estrogenicity for fish in ECC under continuous exposures and highlight the advantages of following weight-of-evidence (WoE) approaches for environmental monitoring, as targeted analytically-based assessments may or may not support the identification of causative contaminants for adverse biological effects.

However the physiological stress in aquatic organisms associated with hydropower plants (HPP) ecosystems has been previously investigated, no studies have so far assessed it on biochemical level. Therefore this study evaluated an oxidative stress and toxicity in the gibel carp Carassius auratus gibelio associated with a small-scale HPP in the West Ukraine. A battery of liver, brain and blood markers was evaluated individuals inhabiting upstream and downstream of the dam of the small-scale Kasperivtci HPP (KHPP; an installed capacity of 7.5 MW), and from a reference site. Number of alterations were noted in fish from the KHPP impoundment facility including signs of oxidative stress (a decrease in superoxide dismutase (SOD) activity and an increase in protein carbonyls) and cytotoxicity (an increase in micronucleated erythrocytes and caspase-3 activity). No changes in DNA fragmentation in hepatocytes or brain cholinesterase activity were detected. As demonstrated by the integral stress index, fish associated with downstream of the dam revealed the greatest alterations reflected by the combined oppression of antioxidant system (SOD, catalase) and pro-oxidants (thiobarbituric acid reactive substances and oxyradicals), low concentration of metallothioneins, but high cathepsin D activity (as markers of lysosomal dysfunction and autophagy) and increased vitellogenin concentration in males (indicating an endocrine disruption). The study highlights that fish inhabiting ecosystems associated with HPP, particularly downstream of the dam, may face additional stresses with long-term effects yet to be evaluated.

Environmental estrogens are capable of interfering with the spermatogenesis and fertility of fish. However in natural waters, these chemicals are more likely to occur as a combination rather than a single stressor. Whether and how the mixture of xenoestrogens with environmental relevant concentrations may affect fish spermatogenesis remains largely unknown. In this study, male zebrafish adults were administered to 17alpha-ethinylestradiol (EE2) and a mixture of xenoestrogens (Mix (E2, EE2, DES, 4-t-OP, 4-NP and BPA)), with the estrogenic potency equivalent to EE2. After a 60-day exposures, elevated mRNA levels of vitellogenin 1 (vtg1) and estrogen receptor 1 (esr1) in the liver of fish in both treated groups were observed. Moreover, the plasma level of E2 declined significantly in the Mix group and the ratio of 11-KT/E2 was significantly elevated in both treated groups. Consistently, the mRNA level of P450 side-chain cleavage (scc) in the EE2 group and ovarian type aromatase (cyp19a1a) in the Mix group was significantly suppressed. In addition, decreased gonadosomatic index and sperm count in the fish of Mix group were present. Furthermore, increased number of the proliferating germ cells (such as spermatogonia and spermatocytes) was observed in the fish of both groups, suggesting a stimulated germ cell proliferation and meiosis. Accordingly, both exposures significantly up-regulated the mRNA levels of genes in mitosis (cyclinb1) and meiosis (cyp26a1 in EE2 group, aldh1a2, cyp26a1, sycp3 and spo11 in Mix). In addition, decreased number of spermatozoa and increased number of TUNEL-positive signals were present in the testis of fish in the Mix group, indicating an enhanced apoptosis. Further analyses demonstrated the significant elevated expressions of tnfrsf1a and the ratio of tnfrsf1a/tnfrsf1b in the Mix group, suggesting an elevated apoptosis in the testis of fish in the Mix group via extrinsic pathway. The present study greatly extends our understanding of the underlying mechanisms of the reproductive toxicity of xenoestrogens on fish.

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.

Nitrate is a ubiquitous aquatic pollutant that is commonly associated with eutrophication and dead zones in estuaries around the world. At high concentrations nitrate is toxic to aquatic life but at environmental concentrations it has also been purported as an endocrine disruptor in fish. To investigate the potential for nitrate to cause endocrine disruption in fish, we conducted a lifecycle study with fathead minnows (Pimephales promelas) exposed to nitrate (0, 11.3, and 56.5 mg/L (total nitrate-nitrogen (NO3-N)) from <24 h post hatch to sexual maturity (209 days). Body mass, condition factor, gonadal somatic index (GSI), incidence of intersex, and vitellogenin induction were determined in mature male and female fish and plasma 11-keto testosterone (11-KT) was measured in males only. In nitrate-exposed males both 11-KT and vitellogenin were significantly induced when compared with controls. No significant differences occurred for body mass, condition factor, or GSI among males and intersex was not observed in any of the nitrate treatments. Nitrate-exposed females also had significant increases in vitellogenin compared to controls but no significant differences for mass, condition factor, or GSI were observed in nitrate exposed groups. Estradiol was used as a positive control for vitellogenin induction. Our findings suggest that environmentally relevant nitrate levels may disrupt steroid hormone synthesis and/or metabolism in male and female fish and may have implications for fish reproduction, watershed management, and regulation of nutrient pollution.