Changes in CpG Methylation of the Vitellogenin 1 Promoter in Adult Male Zebrafish after Exposure to 17α-Ethynylestradiol.

Numerous pharmaceutical and industrial chemicals are classified as endocrine-disrupting chemicals (EDCs) that interfere with hormonal homeostasis, leading to developmental disorders and other pathologies. The synthetic estrogen 17α-ethynylestradiol (EE2) is used in oral contraceptives and other hormone therapies. EE2 and other estrogens are inadvertently introduced into aquatic environments through municipal wastewater and agricultural effluents. Exposure of male fish to estrogens increases expression of the egg yolk precursor protein vitellogenin (Vtg), which is used as a molecular marker of exposure to estrogenic EDCs. The mechanisms behind Vtg induction are not fully known, and we hypothesized that it is regulated via DNA methylation. Adult zebrafish were exposed to either dimethyl sulfoxide or 20 ng/L EE2 for 14 days. Messenger RNA (mRNA) expression and DNA methylation were assessed in male zebrafish livers at 0, 0.25, 0.5, 1, 4, 7, and 14 days of exposure; and those of females were assessed at 13 days (n ≥ 4/group/time point). To test the persistence of any changes, we included a recovery group that received EE2 for 7 days and did not receive any for the following 7 days, in the total 14-day study. Methylation of DNA at the vtg1 promoter was assessed with targeted gene bisulfite sequencing in livers of adult male and female zebrafish. A significant increase in vtg1 mRNA was observed in the EE2-exposed male fish as early as 6 h. Interestingly, DNA methylation changes were observed at 4 days. Decreases in the overall methylation of the vtg1 promoter in exposed males resulted in levels comparable to those in female controls, suggesting feminization. Importantly, DNA methylation levels in males remained significantly impacted after 7 days post-EE2 removal, unlike mRNA levels. These data identify an epigenetic mark of feminization that may serve as an indicator of not only estrogenic exposure but also previous exposure to EE2. Environ Toxicol Chem 2024;00:1-10. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Assessing the Toxicity of Sea Salt to Early Life Stages of Freshwater Mussels: Implications for Sea Level Rise in Coastal Rivers.

Sea levels across the planet are rising, particularly along the eastern coast of the United States. Climate-induced sea level rise can result in the inundation and intrusion of seawater into freshwater drainages. This would alter salinity regimes and lead to the salinization of coastal freshwater ecosystems. Increased salinity levels in freshwater can negatively affect freshwater-dependent species, including native mussels belonging to the order Unionida, which are highly sensitive to changes in water quality. Sea salt is largely made up of sodium and chloride ions, forming sodium chloride, a known toxicant to freshwater mussels. However, sea salt is a mixture that also contains other major ions, including potassium, sulfate, calcium, strontium, and magnesium, among others. Freshwater mussels exposed to sea salt would be exposed to each of the sea salt ions at the same time, resulting in a mixture toxicity effect. The mixture toxicity of these ions on early life stages of freshwater mussels is largely unknown because most research to date has evaluated individual salt ions in relative isolation. Therefore, we conducted acute toxicity tests on early life stages (glochidia and juvenile) of three freshwater mussel species that inhabit Atlantic Slope drainages (nonsalinity-adapted Atlanticoncha ochracea, salinity-adapted A. ochracea, Sagittunio nasutus, and Utterbackiana implicata). Glochidia and juveniles of each species were exposed to a control and six concentrations of Instant Ocean® Sea Salt (IOSS), a synthetic sea salt that closely resembles the ionic composition of natural sea salt. Exposure concentrations were 1 part(s) per thousand (ppt), 2 ppt, 8.5 ppt, 12.5 ppt, 17 ppt, and 34 ppt. We calculated the median effect concentration (EC50) for each of the eight acute toxicity tests and found that glochidia were more sensitive than juveniles to IOSS. At hour 24 EC50s for the glochidia ranged from 0.38 to 3.6 ppt, with the most sensitive freshwater mussel being the nonsalinity-adapted A. ochracea, exhibiting an EC50 of 0.38 ppt (95% confidence interval [CI] 0.33-0.44). Juvenile freshwater mussels exhibited EC50s at hour 96 ranging from 5.0 to 10.4 ppt, with the least sensitive freshwater mussel being the nonsalinity-adapted A. ochracea, exhibiting an EC50 of 10.4 ppt (95% CI 9.1-12.0). Our results show that acute exposure to sea salt adversely affects freshwater mussel viability, particularly glochidia. This information can be used to enhance freshwater mussel conservation strategies in regions that are or will be impacted by climate-induced sea level rise and associated freshwater salinization. Environ Toxicol Chem 2023;42:2478-2489. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Method Development for a Short-Term 7-Day Toxicity Test with Unionid Mussels.

The US Environmental Protection Agency's short-term freshwater effluent test methods include a fish (Pimephales promelas), a cladoceran (Ceriodaphnia dubia), and a green alga (Raphidocelis subcapitata). There is a recognized need for additional taxa to accompany the three standard species for effluent testing. An appropriate additional taxon is unionid mussels because mussels are widely distributed, live burrowed in sediment and filter particles from the water column for food, and exhibit high sensitivity to a variety of contaminants. Multiple studies were conducted to develop a relevant and robust short-term test method for mussels. We first evaluated the comparative sensitivity of two mussel species (Villosa constricta and Lampsilis siliquoidea) and two standard species (P. promelas and C. dubia) using two mock effluents prepared by mixing ammonia and five metals (cadmium, copper, nickel, lead, and zinc) or a field-collected effluent in 7-day exposures. Both mussel species were equally or more sensitive (more than two-fold) to effluents compared with the standard species. Next, we refined the mussel test method by first determining the best feeding rate of a commercial algal mixture for three age groups (1, 2, and 3 weeks old) of L. siliquoidea in a 7-day feeding experiment, and then used the derived optimal feeding rates to assess the sensitivity of the three ages of juveniles in a 7-day reference toxicant (sodium chloride [NaCl]) test. Juvenile mussels grew substantially (30%-52% length increase) when the 1- or 2-week-old mussels were fed 2 ml twice daily and the 3-week-old mussels were fed 3 ml twice daily. The 25% inhibition concentrations (IC25s) for NaCl were similar (314-520 mg Cl/L) among the three age groups, indicating that an age range of 1- to 3-week-old mussels can be used for a 7-day test. Finally, using the refined test method, we conducted an interlaboratory study among 13 laboratories to evaluate the performance of a 7-day NaCl test with L. siliquoidea. Eleven laboratories successfully completed the test, with more than 80% control survival and reliable growth data. The IC25s ranged from 296 to 1076 mg Cl/L, with a low (34%) coefficient of variation, indicating that the proposed method for L. siliquoidea has acceptable precision. Environ Toxicol Chem 2021;40:3392-3409. © 2021 SETAC.

Brown watersnakes (Nerodia taxispilota) as bioindicators of mercury contamination in a riverine system.

Mercury (Hg) is a contaminant that enters the environment through natural or anthropogenic means. Ecological risk assessments have examined Hg bioaccumulation and effects in many taxa, but little is known about Hg dynamics in reptiles, or their potential use as bioindicator species for monitoring Hg in aquatic systems. Numerous snake species, like North American watersnakes (Nerodia spp.), are piscivorous and are exposed to Hg through their diet. The purpose of this study was to identify factors associated with Hg accumulation in a common watersnake species and compare Hg concentrations of the snakes to those in fish occupying the same habitats. To this end, we sampled brown watersnakes (Nerodia taxispilota) from the Savannah River, a major river system in the southeastern U.S., and compared N. taxispilota Hg accumulation trends to those of bass (Micropterus salmoides), catfish (Ictalurus and Ameiurus spp.), and panfish (Lepomis and Pomoxis spp.) collected from the same reach. Total Hg (THg) in N. taxispilota tail tips ranged from 0.020 to 0.431 mg/kg (wet weight; mean: 0.104 ± 0.008). Snake tail THg was significantly correlated with blood THg, which ranged from 0.003 to 1.140 mg/kg (0.154 ± 0.019). Snake size and site of capture were significantly associated with tail THg. Snake tail THg increased at sites along and downstream of the area of historic Hg pollution, consistent with fish THg. Snake muscle THg was predicted based on tail THg and ranged from 0.095 to 1.160 (0.352 ± 0.022). To gauge Hg biomagnification in N. taxispilota, we compared predicted snake muscle THg concentrations to THg in fish of consumable size. Average biomagnification factors for THg in N. taxispilota were 3.1 (panfish) and 5.4 (catfish), demonstrating N. taxispilota likely biomagnify Hg through their diet. These results reveal N. taxispilota to be an effective bioindicator species for monitoring Hg in aquatic environments.

Gonad metabolomics and blood biochemical analysis reveal differences associated with testicular oocytes in wild largemouth bass (Micropterus salmoides).

Adverse reproductive effects associated with gonadal intersex among freshwater fish could hold considerable implications for population sustainability. Presence of testicular oocytes (TO) is the most common form of intersex and is widespread among centrarchids (sunfishes) of North America and other freshwater teleosts. Placing TO within the toxicological context of adverse outcome pathways (AOPs) to assess ecological risk is a priority for ecotoxicologists due to the association of TO with harmful chemical exposure and adverse reproductive effects in some cases. However, key event relationships between EDC exposure, incidence of TO, and apical outcomes have yet to be fully elucidated - in part due to a lack of knowledge of relationships between intersex gonad physiology and fish health. Understanding the physiological status of intersex fish is critical to assess ecological risk, understand mechanisms of induction, and to establish biomarkers of intersex in fish. In the present study, features of gonad metabolite profiles associated with TO in largemouth bass (LMB, Micropterus salmoides) from an impoundment in Georgia (USA) were determined using GC-MS-based metabolomics. Clinical blood biochemical screens were used to evaluate markers of fish health associated with TO. Results suggest that physiological changes in energy expenditure as well as relatively 'feminized' gonad lipid and protein metabolism may be related to the occurrence of TO in male LMB, and highlight the need to understand relationships between intersex and physical stressors such as elevated temperature and hypoxia. These results provide novel insight to AOPs associated with TO and identify candidate analytes for biomarker discovery.

Comparative Toxicity of Nitrate to Common and Imperiled Freshwater Mussel Glochidia and Larval Fishes.

Growing human populations and increasingly intensive agriculture have resulted in widespread aquatic nitrate pollution. Freshwater mussel populations have been in decline for decades but often are underrepresented in data used for the development of ambient water quality criteria and acute toxicity of nitrate to mussel glochidia has not yet been established. Additionally, toxicity testing with aquatic species often is limited to a few model species; however, relatively little is known about how representative model species are of imperiled species. Therefore, to better define the acute toxicity of nitrate to common and rare aquatic species, we conducted 24-h nitrate acute toxicity tests with glochidia of four species of freshwater mussels, including a federally threatened species (Hamiota altilis) and 7-day tests with larval fish of three species: fathead minnow (Pimephales promelas), tricolor shiner (Cyprinella trichroistia), and tilapia (Oreochromis spp.), across a range of water hardness. Median effective concentrations (EC50s) in freshwater mussel glochidia ranged from 524 to 904 mg/L NO3-N in moderately hard water. In fish, median lethal concentrations (LC50s) ranged from 228 to 1725 mg/L NO3-N and varied with water hardness. Of the species tested, generally sensitivity of the common species was similar to the rare species, although relative sensitivity varied with water hardness. Based on these results, we can conclude that acute lethal effects are unlikely for the fish and mussel species considered here at current environmental levels, but the results of these standardized tests are useful for the development of ambient water quality criteria and other regulatory and management decisions regarding acute nitrate exposures.

Evaluation of the DGT technique for predicting uptake of metal mixtures by fathead minnow (Pimephales promelas) and yellow lampmussel (Lampsilis cariosa).

Diffusive gradients in thin films (DGT) were assessed for their predictive capability of fathead minnow and yellow lampmussel bioaccumulation in copper (Cu) and lead (Pb) mixed metal exposures. Nine treatments with a matrix of 3 Cu and 3 Pb concentrations were utilized. Exposures were coupled, with organisms and DGT exposed in tanks for 6 days. The Cu measured in fish, mussel, and DGT was found not to be influenced by Pb treatment, whereas Pb accumulation was impacted by the interaction of Cu and Pb treatment. The Pb accumulation increased with increasing Cu concentration, which was attributed to the different speciation of Cu and Pb in the water where Cu binds preferentially to ligands, decreasing its bioavailability and concomitantly displacing Pb from complexing sites. The DGT values were significantly correlated with accumulated Cu and Pb in the fish, but not with Pb in the mussel. In addition, DGT was determined to better predict aquatic organism bioaccumulation of Cu than the inorganic Cu fraction calculated by the speciation model, because DGT accumulated not only inorganic metal fractions but also complexes of metal and organic matter. The present study provides insights into metal speciation in polluted environments, extends the understanding of using DGT as a tool for estimating metal bioavailability, and provides implications for the selection of geochemical modeling, biological sampling, and passive sampling techniques for monitoring trace metal contamination. Environ Toxicol Chem 2019;38:61-70. © 2018 SETAC.

Effects of nitrate on freshwater mussel glochidia attachment and metamorphosis success to the juvenile stage.

Water quality and contaminants have been frequently identified as critical stressors for freshwater mussels, many species of which are highly imperiled throughout North America and the world. Nutrient pollution, specifically nitrate, has become one of the most prevalent causes of water quality degradation globally, with increasing anthropogenic input from suburban and agricultural runoff, municipal wastewater, and industrial waste. Nitrate acute toxicity is generally low for aquatic species, but the potential effects of nitrate exposure are largely unknown for freshwater mussels, particularly during the parasitic stage of their complex lifecycle. Therefore, this study was designed to determine the effects of short-term nitrate exposure at environmentally relevant concentrations on juvenile production in two freshwater mussel species. Lampsilis siliquoidea and L. fasciola glochidia were exposed to nitrate (0, 11, or 56 mg NO3-N/L) for 24 h before inoculation on a primary host, Largemouth Bass (Micropterus salmoides). Glochidia attachment, metamorphosis success, and total number of juveniles produced were monitored on individual fish. Exposure of L. siliquoidea glochidia to 56 mg NO3-N/L nitrate resulted in a significant (p = 0.02) 35% reduction of total juveniles produced, a combined result of moderate decreases in both glochidia attachment and metamorphosis success. A similar trend (28% reduction; p = 0.06) was evident with 11 mg NO3-N/L. No effects were apparent for L. fasciola, suggesting species-specific differences in responses even among closely related species. These results are the first to suggest that glochidia exposure to nitrate may adversely affect juvenile recruitment in some species. Findings from these studies are important for improving characterization of the hazards of nitrate pollution to aquatic life and this work will help better define the role of water quality in assessing habitat suitability for mussel conservation efforts.

Impact of natural organic matter and increased water hardness on DGT prediction of copper bioaccumulation by yellow lampmussel (Lampsilis cariosa) and fathead minnow (Pimephales promelas).

We conducted an exposure experiment with Diffusive Gradients in Thin- Films (DGT), fathead minnow (Pimephales promelas), and yellow lampmussel (Lampsilis cariosa) to estimate bioavailability and bioaccumulation of Cu. We hypothesized that Cu concentrations measured by DGT can be used to predict Cu accumulation in aquatic animals and alterations of water chemistry can affect DGT's predict ability. Three water chemistries (control soft water, hard water, and addition of natural organic matter (NOM)) and three Cu concentrations (0, 30, and 60 µg/L) were selected, so nine Cu-water chemistry combinations were used. NOM addition treatments resulted in decreased concentrations of DGT-measured Cu and free Cu ion predicted by Biotic Ligand Model (BLM). Both hard water and NOM addition treatments had reduced concentrations of Cu ion and Cu-dissolved organic matter complexes compared to other treatments. DGT-measured Cu concentrations were linearly correlated to fish accumulated Cu, but not to mussel accumulated Cu. Concentrations of bioavailable Cu predicted by BLM, the species complexed with biotic ligands of aquatic organisms and, was highly correlated to DGT-measured Cu. In general, DGT-measured Cu fit Cu accumulations in fish, and this passive sampling technique is acceptable at predicting Cu concentrations in fish in waters with low NOM concentrations.

Evaluation of diffusive gradients in thin films for prediction of copper bioaccumulation by yellow lampmussel (Lampsilis cariosa) and fathead minnow (Pimephales promelas).

Using a coupled method of diffusive gradients in thin films (DGT) exposure with aquatic organism bioassays, we assessed the use of DGT as a tool for estimating copper (Cu) bioavailability in contaminated waters. The DGT-accumulated Cu fraction could possibly be used as a surrogate for other assessments of metal bioavailability. The Cu concentrations in fathead minnow (Pimephales promelas) and yellow lampmussel (Lampsilis cariosa) soft tissue were compared with DGT-accumulated Cu after 2, 4, and 6 d of exposure to a Cu concentration series in static, water-only assays. The DGT-accumulated Cu was found to include free Cu ions, labile inorganic Cu complexes, and labile dissolved organic matter Cu complexes, compared with Cu speciation output from the biotic ligand model. Regressions of Cu concentrations between DGT and fathead minnow at 4 and 6 d of exposure demonstrated linear relationships. The Cu bioaccumulated in yellow lampmussel was overpredicted by DGT at Cu concentrations greater than 10 µg L-1 , which may be caused by internal regulation of Cu. The speciation component of the biotic ligand model predicted relationships between inorganic Cu and animal-accumulated Cu that were similar to predicted relationships between DGT-indicated Cu and animal-accumulated Cu at all deployment durations. Environ Toxicol Chem 2018;37:1535-1544. © 2018 SETAC.

Chronic nitrate exposure alters reproductive physiology in fathead minnows.

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.

Tissue Contaminant Burdens in San Francisco Estuary White Sturgeon (Acipenser transmontanus): Implications for Population Recovery.

The San Francisco Estuary (SFE) is heavily influenced by anthropogenic activities, including historic and chronic contaminant inputs. These contaminants can adversely affect SFE fish populations, particularly white sturgeon, because they are a benthic dwelling, long-lived species. We measured a suite of metals and organic contaminants in liver and gonad tissues of 25 male and 32 female white sturgeon as well as several physiological indicators of sturgeon health. Most sturgeon (68% of males and 83% of females) were estimated to be between 13 and 17 years of age. Sturgeon tissues had elevated concentrations of several metals, including As, Ba, Cd, Cu, Cr, Pb, Hg, Ni, Se, and Zn. The most frequently detected organic contaminants in sturgeon livers and gonads were DDE, PCBs, PBDEs, and galaxolide. Selenium was detected at levels similar to those shown to cause impaired liver physiology and reproductive success in white sturgeon. Observed Hg levels were higher than those shown to result in lower condition factor and gonadosomatic indices in white sturgeon. Liver galaxolide levels correlated with decreased plasma estradiol levels in female sturgeon. The Cd, As, and Cu warrant further investigation, because they were detected at levels known to impair fish health. Our results suggest contaminants are negatively affecting SFE white sturgeon health and fitness. Future SFE white sturgeon contaminant research is suggested.

Accumulation of coal combustion residues and their immunological effects in the yellow-bellied slider (Trachemys scripta scripta).

Anthropogenic activities such as industrial processes often produce copious amounts of contaminants that have the potential to negatively impact growth, survival, and reproduction of exposed wildlife. Coal combustion residues (CCRs) represent a major source of pollutants globally, resulting in the release of potentially harmful trace elements such as arsenic (As), cadmium (Cd), and selenium (Se) into the environment. In the United States, CCRs are typically stored in aquatic settling basins that may become attractive nuisances to wildlife. Trace element contaminants, such as CCRs, may pose a threat to biota yet little is known about their sublethal effects on reptiles. To assess the effects of CCR exposure in turtles, we sampled 81 yellow-bellied sliders (Trachemys scripta scripta) in 2014-2015 from CCR-contaminated and uncontaminated reference wetlands located on the Savannah River Site (Aiken, SC, USA). Specific aims were to (1) compare the accumulation of trace elements in T. s. scripta claw and blood samples between reference and CCR-contaminated site types, (2) evaluate potential immunological effects of CCRs via bacterial killing assays and phytohaemagglutinin (PHA) assays, and (3) quantify differences in hemogregarine parasite loads between site types. Claw As, Cd, copper (Cu), and Se (all p ≤ 0.001) and blood As, Cu, Se, and strontium (Sr; p ≤ 0.015) were significantly elevated in turtles from CCR-contaminated wetlands compared to turtles from reference wetlands. Turtles from reference wetlands exhibited lower bacterial killing (p = 0.015) abilities than individuals from contaminated sites but neither PHA responses (p = 0.566) nor parasite loads (p = 0.980) differed by site type. Despite relatively high CCR body burdens, sliders did not exhibit apparent impairment of immunological response or parasite load. In addition, the high correlation between claw and blood concentrations within individuals suggests that nonlethal tissue sampling may be useful for monitoring CCR exposure in turtles.

Extending the toxicity-testing paradigm for freshwater mussels: Assessing chronic reproductive effects of the synthetic estrogen 17α-ethinylestradiol on the unionid mussel Elliptio complanata.

Surface water concentrations of the synthetic estrogen 17α-ethinylestradiol (EE2) as low as 1ng/L can cause adverse reproductive effects in fish under acute and chronic exposure conditions, whereas higher concentrations (> 5ng/L) in acute studies are necessary to elicit adverse effects in freshwater mussels. Prolonged chronic exposures of freshwater mussels to EE2 remain un-evaluated. An extended duration testing paradigm was used to examine reproductive and biochemical (carbohydrate, lipid, protein) effects of EE2 on the unionid mussel, Elliptio complanata, throughout its reproductive cycle. Mussels were exposed to a control and EE2 concentrations (5 and 50ng/L) in six discrete and sequential 28 d tests, and in one discrete and simultaneous 180 d test, from February through August. Foot protrusion and siphoning behavior were recorded daily, along with conglutinate releases and larval (glochidia) mortality. Gonad, hemolymph, and gonad fluid samples were taken for biochemical and vitellogenin-like protein (Vtg) analysis post-exposure. Female mussels released eggs and conglutinates during the months of April to June, indicating sexual maturation during this time. Conglutinates released in the 5ng/L treatment in 28 d exposures contained fewer glochidia and more eggs, and increased concentrations of Vtg in hemolymph were observed from April to August in the 5ng/L treatment during the 180 d exposure. Results indicate that the 180 d test approach, concurrent with the sequence of 28 d tests, enabled a more robust evaluation of mussel behavior and physiology than would have been possible with a single short-term (28 d) test.

Changing agricultural practices: potential consequences to aquatic organisms.

Agricultural practices pose threats to biotic diversity in freshwater systems with increasing use of glyphosate-based herbicides for weed control and animal waste for soil amendment becoming common in many regions. Over the past two decades, these particular agricultural trends have corresponded with marked declines in populations of fish and mussel species in the Upper Conasauga River watershed in Georgia/Tennessee, USA. To investigate the potential role of agriculture in the population declines, surface waters and sediments throughout the basin were tested for toxicity and analyzed for glyphosate, metals, nutrients, and steroid hormones. Assessments of chronic toxicity with Ceriodaphnia dubia and Hyalella azteca indicated that few water or sediment samples were harmful and metal concentrations were generally below impairment levels. Glyphosate was not observed in surface waters, although its primary degradation product, aminomethyl phosphonic acid (AMPA), was detected in 77% of the samples (mean = 509 µg/L, n = 99) and one or both compounds were measured in most sediment samples. Waterborne AMPA concentrations supported an inference that surfactants associated with glyphosate may be present at levels sufficient to affect early life stages of mussels. Nutrient enrichment of surface waters was widespread with nitrate (mean = 0.7 mg NO3-N/L, n = 179) and phosphorus (mean = 275 µg/L, n = 179) exceeding levels associated with eutrophication. Hormone concentrations in sediments were often above those shown to cause endocrine disruption in fish and appear to reflect the widespread application of poultry litter and manure. Observed species declines may be at least partially due to hormones, although excess nutrients and herbicide surfactants may also be implicated.

Simultaneous extraction and cleanup of high-lipid organs from white sturgeon (Acipenser transmontanus) for multiple legacy and emerging organic contaminants using QuEChERS sample preparation.

The objective of this research was to utilize the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method to extract a broad range of persistent organic pollutants from sturgeon organs (liver and gonad) as indicators of exposure. The analyte list was prioritized to include carcinogenic polyaromatic hydrocarbons (PAHs), the most commonly occurring polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs), persistent bioaccumulative and toxic chemicals (PBTs), and emergent contaminants of concern (ECCs) as indicators of human sewage exposure. White sturgeon (Acipenser transmontanus) were selected for this study to support a larger ecotoxicological study to monitor contaminants as an assessment of fish health. Organ tissues contained high lipid content with percentages of 15% and 34% for liver and gonad, respectively. Overall recoveries from fortified sturgeon tissues were high, 71-98% for PAHs, 60-107% for PBDEs and PCBs, 86-107% for PBT chemicals, and 88-107% for ECCs with the exception of octinoxate (28%) from liver tissues. Analyte recovery trends decreased as analyte lipophilicity and molecular weight increased. These recoveries demonstrate that extraction using QuEChERS can be used for screening of the most common bioaccumulating organic compounds in high lipid fish tissue using a single extraction and analysis.

Metabolomic, behavioral, and reproductive effects of the aromatase inhibitor fadrozole hydrochloride on the unionid mussel Lampsilis fasciola.

Androgen-induced masculinization of female aquatic biota poses concerns for natural population stability. This research evaluated the effects of a twelve day exposure of fadrozole hydrochloride on the metabolism and reproductive status of the unionid mussel Lampsilis fasciola. Although this compound is not considered to be widespread in the aquatic environment, it was selected as a model aromatase (enzyme that converts testosterone to estradiol) inhibitor. Adult mussels were exposed to a control and 3 concentrations of fadrozole (2µg/L, 20µg/L, and 50µg/L), and samples of gill tissue were taken on days 4 and 12 for metabolomics analysis. Gills were used because of the variety of critical processes they mediate, such as feeding, ion exchange, and siphoning. Daily observed mussel behavior included female mantle display, foot protrusion, siphoning, and larval (glochidia) releases. Glochidia mortality was significantly higher in the 20µg/L treatment. Fewer conglutinate (packets of glochidia) releases were observed in the 50µg/L treatment, and mortality was highly correlated to release numbers. Foot protrusion was significantly higher in females in nearly all treatments, including the control, during the first 4days of observations. However, this sex difference was observed only in the 50µg/L treatment during the last 8days. Generally, metabolites were significantly altered in female gill tissue in the 2µg/L treatment whereas males were mostly affected only at the highest (50µg/L) treatment. Both sexes also revealed significant reductions in fadrozole-induced metabolic effects in gill tissue sampled after 12days compared to tissue sampled after 4days, indicating time-dependent mechanisms of disruptions in metabolic pathways and homeostatic processes to compensate for such disruptions.

Metabolomic, behavioral, and reproductive effects of the synthetic estrogen 17 α-ethinylestradiol on the unionid mussel Lampsilis fasciola.

The endocrine disrupting effects of estrogenic compounds in surface waters on fish, such as feminization of males and altered sex ratios, may also occur in aquatic invertebrates. However, the underlying mechanisms of action and toxicity, especially in native freshwater mussels (Order Unionoida), remain undefined. This study evaluated the effects of a 12-day exposure of 17 α-ethinylestradiol (EE2), a synthetic estrogen in oral contraceptives commonly found in surface waters, on the behavior, condition, metabolism, and reproductive status of Lampsilis fasciola. Adult mussels of both sexes were exposed to a control and two concentrations of EE2 (0 ng/L, 5 ng/L considered to be environmentally relevant, and 1,000 ng/L designed to provide a positive metabolic response), and samples of gill tissue were taken on days 4 and 12; gills were used because of the variety of critical processes they mediate, such as feeding, ion exchange, and siphoning. Observations of mussel behavior (mantle display, siphoning, and foot movement) were made daily, and condition of conglutinates (packets of eggs and/or glochidia) released by females was examined. No significant effects of EE2 on glochidia mortality, conglutinate condition, female marsupial gill condition, or mussel foot extension were observed. However, exposure to both concentrations of EE2 significantly reduced male siphoning and mantle display behavior of females. Metabolomics analyses identified 207 known biochemicals in mussel gill tissue and showed that environmentally relevant EE2 concentrations led to decreases in glycogen metabolism end products, glucose, and several essential fatty acids in females after 12 days, indicating reductions in energy reserves that could otherwise be used for growth or reproduction. Moreover, males and females showed significant alterations in metabolites involved in signal transduction, immune response, and neuromodulation. Most of these changes were apparent at 1,000 ng/L EE2, but similar metabolites and pathways were also affected at 5 ng/L EE2. Components of the extracellular matrix of gill tissue were also altered. These results demonstrate the utility of metabolomics when used in conjunction with traditional physiological and behavioral toxicity test endpoints and establish the usefulness of this approach in determining possible underlying toxicological mechanisms of EE2 in exposed freshwater mussels.

Chronic fluoxetine exposure alters movement and burrowing in adult freshwater mussels.

The antidepressant fluoxetine is commonly found in aquatic fauna living near or downstream from point-sources of municipal waste effluent. Continuous release of fluoxetine results in increased effective exposure duration in surface waters, resulting in a chronic exposure for animals downstream, particularly in effluent dominated ecosystems. Fluoxetine is known to cause disruptions in reproductive behavior of freshwater mussels (order Unionoida), including stimulating release of gametes, parturition of glochidia (larvae), and changes in lure display and foot protrusion. However, the ecological relevance of these effects at environmental concentrations is unknown. We conducted a 67-d exposure of adult Lampsilis fasciola to fluoxetine concentrations of 0, 0.5, 2.5, and 22.3µg/L and assessed impacts on behavior (lateral movement, burrowing, and filtering) and metabolism (glycogen storage and respiration). Mussels treated with 2.5 and 22.3µg/L fluoxetine displayed mantle lures significantly (p<0.05) more than controls. Animals treated with 22.3µg/L fluoxetine were statistically more likely to have shorter time-to-movement, greater total movement, and initiate burrowing sooner than control animals. These observations suggest that increased activity of mussels exposed to fluoxetine may result in increased susceptibility to predators and may lead to a reduction in energy stores.

Assessment of toxicity test endpoints for freshwater mussel larvae (glochidia).

The objectives of the present study were to determine if the viability of freshwater mussel larvae (glochidia) is an ecologically relevant endpoint for toxicity tests and to define the appropriate duration of those tests. The authors assessed 1) how viability (the shell closure response to sodium chloride) compares with infectivity (ability to attach to a host fish and successfully metamorphose to the juvenile stage), and 2) the decline of viability and infectivity over time after glochidia were released from female mussels. Glochidia of 7 mussel species were isolated from females, placed in water, and subsampled daily for 2 d to 5 d. Viability, when ≥90%, was generally a good predictor of infectivity; however, when viability was <90%, infectivity was often disproportionately low, especially for glochidia collected near the end of the brooding period. Viability and infectivity declined more rapidly in natural water and sediment compared to reconstituted water. Following 24-h exposure to a toxicant (sodium chloride or copper), infectivity of the viable glochidia did not differ among concentrations of toxicants. The results indicate that viability is a valid proxy for infectivity and an ecologically relevant endpoint for standard toxicity tests with freshwater mussels for any test duration with control viability >90%.