Comparative subchronic toxicity of copper and a tertiary copper mixture to early life stage rainbow trout (Oncorhynchus mykiss): impacts on growth, development, and histopathology.

This research aimed to characterize and compare the subchronic impacts of Cu to a Cu, Cd, and Zn mixture in early life stages of rainbow trout (Oncorhynchus mykiss) by examining uptake, survival, growth, development, and histopathology parameters. To accomplish this, rainbow trout were exposed for 31 days from eyed embryos to the swim-up fry life stage to waterborne Cu (31, 47, 70, and 104 µg/L) individually or as mixture containing Cd (4.1, 6.2, 9.3, and 14 µg/L) and Zn (385, 578, 867, and 1300 µg/L). Exposures elicited pronounced effects on survival when Cu was administered as a mixture (LC25 = 32.9 µg/L Cu) versus individually (LC25 = 46.3 µg/L Cu). Mixtures of Cu, Cd, and Zn also elicited more pronounced sublethal toxicity relative to equivalent Cu treatments with respect to reduced yolk sac resorption and increased incidence and/or severity of gill, liver, and kidney lesions. Our findings of reduced body weight (EC10, Cu = 55.0 µg/L Cu; EC10, Cu+Cd+Zn = 58.9 µg/L Cu), yolk sac resorption (LOECCu = 70 µg/L Cu; LOECCu+Cd+Zn = 70 µg/L Cu), coelomic fat (LOECCu = 47 µg/L Cu; LOECCu+Cd+Zn = 70 µg/L Cu), and increased hepatocellular cytoplasmic vacuolation (LOECCu = 70 µg/L Cu; LOECCu+Cd+Zn = 47 µg/L Cu) collectively indicate a complicated metabolic interference by metals in exposed fish. These lethal and sublethal effects observed in the laboratory could translate to reduced survival and fitness of wild salmonid populations inhabiting waterbodies receiving wastewater or runoff containing multiple metals at elevated concentrations.

Using Hepatic Gene Expression Assays in English Sole (Parophrys vetulus) to Investigate the Effects of Metro Vancouver Wastewater Effluents.

The present study has investigated the effects of Metro Vancouver's wastewater treatment plant (WWTP) effluents on English sole (Parophrys vetulus) hepatic gene expression using novel targeted gene expression assays to complement the 2017 Burrard Inlet Ambient Monitoring Program conducted by Metro Vancouver. Seven locations of varying distance to the WWTPs were included. Twelve genes involved in xenobiotic defense (CYP1A, HSP70), thyroid function (DIO1), lipid and glucose metabolism (FABP1, FASN, GLUT2, PPARδ, PPARγ), protein synthesis (18S rRNA, RPS4X), and reproduction (ERα, VTG) revealed several differences between these impacted sites. A key finding of the present study was that males exhibited VTG transcript levels either equivalent or exceeding female levels of this gene at all sites investigated, indicating widespread exposure of estrogenic contaminants throughout Burrard Inlet. Furthermore, the induction of hepatic CYP1A was observed due to possible downstream sites being subjected to a larger influx of certain planar halogenated and non-halogenated hydrocarbons from multiple industrial contributors. This study also revealed significant differences between the sites examined and in genes involved in transcriptional regulation and synthesis of proteins, lipids and glucose metabolism, and thyroid hormone metabolism. Collectively, this study demonstrates the potential of molecular biomarkers of urban contaminant exposure in wild caught English sole for use in diagnosing a broader range of adverse health effects when combined with conventional whole organism health indicators.

Acute Toxicity of 6PPD-Quinone to Early Life Stage Juvenile Chinook (Oncorhynchus tshawytscha) and Coho (Oncorhynchus kisutch) Salmon.

The breakdown product of the rubber tire antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD)-6-PPD-quinone has been strongly implicated in toxic injury and death in coho salmon (Oncorhynchus kisutch) in urban waterways. Whereas recent studies have reported a wide range of sensitivity to 6PPD-quinone in several fish species, little is known about the risks to Chinook salmon (Oncorhynchus tshawytscha), the primary prey of endangered Southern Resident killer whales (Orcinus orca) and the subject of much concern. Chinook face numerous conservation threats in Canada and the United States, with many populations assessed as either endangered or threatened. We evaluated the acute toxicity of 6PPD-quinone to newly feeding (~3 weeks post swim-up) juvenile Chinook and coho. Juvenile Chinook and coho were exposed for 24 h under static conditions to five concentrations of 6PPD-quinone. Juvenile coho were 3 orders of magnitude more sensitive to 6PPD-quinone compared with juvenile Chinook, with 24-h median lethal concentration (LC50) estimates of 41.0 and more than 67 307 ng/L, respectively. The coho LC50 was 2.3-fold lower than what was previously reported for 1+-year-old coho (95 ng/L), highlighting the value of evaluating age-related differences in sensitivity to this toxic tire-related chemical. Both fish species exhibited typical 6PPD-quinone symptomology (gasping, increased ventilation, loss of equilibrium, erratic swimming), with fish that were symptomatic generally exhibiting mortality. The LC50 values derived from our study for coho are below concentrations that have been measured in salmon-bearing waterways, suggesting the potential for population-level consequences in urban waters. The higher relative LC50 values for Chinook compared with coho merits further investigation, including for the potential for population-relevant sublethal effects. Environ Toxicol Chem 2023;42:815-822. © 2023 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Fisheries and Oceans Canada.

A cross-species comparative approach to assessing multi- and transgenerational effects of endocrine disrupting chemicals.

A wide range of chemicals have been identified as endocrine disrupting chemicals (EDCs) in vertebrate species. Most studies of EDCs have focused on exposure of both male and female adults to these chemicals; however, there is clear evidence that EDCs have dramatic effects when mature or developing gametes are exposed, and consequently are associated with in multigenerational and transgenerational effects. Several publications have reviewed such actions of EDCs in subgroups of species, e.g., fish or rodents. In this review, we take a holistic approach synthesizing knowledge of the effects of EDCs across vertebrate species, including fish, anurans, birds, and mammals, and discuss the potential mechanism(s) mediating such multi- and transgenerational effects. We also propose a series of recommendations aimed at moving the field forward in a structured and coherent manner.

Reproductive and thyroid endocrine axis cross-talk in rainbow trout (Oncorhynchus mykiss) alevins.

The goal of this study was to characterize morphological and molecular effects in rainbow trout alevins after waterborne exposures to 17ß-estradiol (E2; 0.0008 to 0.5 µg/L), triiodothyronine (T3; 0.52 to 65 µg/L), and various co-treatments for 21 to 23 days. Interestingly, there was no consistent evidence that E2 alone influenced growth, development or deformity rates, however, 65 µg/L T3 alone expedited development, and both 13 µg/L and 65 µg/L alone caused a unique opercular deformity not previously reported. In addition, some potentiation between E2 and T3 at lower concentrations suggests some cross-talk between these two hormonal pathways may also contribute to the development of this opercular deformity. Gene expression changes were observed, including induction of vtg in rainbow trout alevins at 0.02 µg/L concentration of E2, which is the lowest concentration reported to induce vtg in rainbow trout alevins. These data suggest low-level E2 does not negate abnormal growth and development caused by hyperthyroidism, and examining more time points is likely required to demonstrate a stronger response profile for individual hormones and endocrine axes cross-talk.

Investigating Acute and Subchronic Effects of Neonicotinoids on Northwestern Salamander Larvae.

This research investigated the adverse effects of neonicotinoids on the Northwestern salamander (Ambystoma gracile; NWS) after acute and subchronic exposures during early aquatic life stages via whole organism (i.e., growth, development) and molecular (i.e., gene expression) level endpoints. In a 96-h exposure, NWS larvae were exposed to four imidacloprid concentrations (250, 750, 2250, 6750 µg/L) and a water control treatment, and no effects on survival, body weight, snout-vent length (SVL), and total body length were observed. However, a significant 1.70- and 2.33-fold decrease in thyroid receptor ß (TRß) mRNA expression levels were detected in the larvae exposed to 750 and 2250 µg/L imidacloprid, respectively, compared with the larvae in the water control. In subsequent subchronic experiments, NWS larvae were exposed for 35 days to imidacloprid alone and an equal part mixture of neonicotinoids (imidacloprid, clothianidin, and thiamethoxam (ICT)) at three concentrations (10, 100 and 1000 µg total neonicotinoids/L) and a water control. In these experiments, there were no effects on larval survival, body weight, SVL, and total body length. However, advanced development of larvae in the 100 µg/L imidacloprid treatment was observed compared with the control after 35-day imidacloprid exposure, providing some evidence of disruption of the thyroid endocrine axis at an environmentally relevant concentration. Ultimately, there is a paucity of studies conducted examining the sensitivity of salamanders to pollutants; thus, this study reports novel findings that will contribute to understanding the sensitivity of a Caudate amphibian model to a common environmental pollutant.

Toxicity of the aquatic herbicide, reward®, on the fathead minnow with pulsed-exposure proteomic profile.

The objectives of this study were to assess the lethal and sub-lethal effects of the aquatic herbicide commercial formulation, Reward® (373 g/L DB), using application scenarios prescribed by the manufacturer. Specifically, a 14 d period between applications of Reward® in a water body undergoing treatment is required, yet the effects of these 'pulse' exposure scenarios on aquatic wildlife such as fish are unknown. In the first experiment early life stage FHM were exposed to a continuous DB concentrations from 0.105-12.6 mg/L which yielded a larval 7 d LC50 of 2.04 mg/L as well as a significant decrease in body mass (25.0 ±â€¯11.6%) at the 1.18 mg/L Reward® concentration. In a second experiment, FHM larvae were exposed for 24 h and then reared in clean water for 14 d followed by a second 24 h exposure to Reward®. The 16 d LC50 value was 4.19 mg/L. In a third experiment, adult FHM were exposed in a pulse/discontinuous manner to Reward® with a calculated 21 d LC50 value of 6.71 mg/L. No significant changes in gonadosomatic index or fecundity of the F1 generation's hatch success were found when eggs from exposed adults were then reared in clean water. Proteome analyses of whole FHM larvae from the discontinuous/pulse exposure showed the primary gene ontology molecular functions of the proteins in fish exposed to 3.78 mg/L DB that resulted in ~30% mortality with positive or negative differential abundance (p-value < .2) were: structural molecule activity; identical protein binding; structural constituent of cytoskeleton; ion binding; calcium ion binding; cytoskeletal protein binding; actin binding; and, ATP binding. These findings suggest that concentrations causing adverse effects occur above the maximum concentration predicted by the manufacturer when applied according to the label (i.e. >0.37 mg/L).

Toxicity of the aquatic herbicide, reward®, to the northwestern salamander.

Diquat dibromide (DB) is the active ingredient in several herbicide products used around the world for industrial and recreational control of terrestrial and aquatic pest plants. This study aimed to assess the adverse effects of the commercial formulation of the aquatic herbicide, Reward®, on the Pacific Northwest amphibian species, the northwestern salamander (Ambystoma gracile). Larvae were exposed to the Reward® herbicide in a 96-h acute bioassay (0.37-151.7 mg/L DB) and a continuous 21-day exposure (0.37-94.7 mg/L DB). The 96-h LC50 was 71.5 mg/L and the 21-day LC50 was 1.56 mg/L. Collectively, the results of this study demonstrate that early life stage A. gracile larvae appear largely insensitive to acute Reward® exposures compared to early life stage fish. However, A. gracile larvae are considerably more sensitive during sub-chronic exposure (21 days) with lethal and sub-lethal effects on growth occurring in the 1-2 mg/L range, which more closely resembles the larval fish lethal sensitivity to this active ingredient. This is the first study examining the toxicity of the aquatic herbicide formulation Reward® on A. gracile under acute and sub-chronic exposure scenarios.

Effects of atrazine and chlorothalonil on the reproductive success, development, and growth of early life stage sockeye salmon (Oncorhynchus nerka).

The effects of 2 currently used commercial pesticide formulations on Pacific sockeye salmon (Oncorhynchus nerka), from fertilization to emergence, were evaluated in a gravel-bed flume incubator that simulated a natural streambed. Embryos were exposed to atrazine at 25 µg/L (low atrazine) or atrazine at 250 µg/L (high atrazine) active ingredient (a.i.), and chlorothalonil at 0.5 µg/L (low chlorothalonil) or chlorothalonil at 5 µg/L a.i. (high chlorothalonil) and examined for effects on developmental success and timing, as well as physical and biochemical growth parameters. Survival to hatch was reduced in the high chlorothalonil group (55% compared with 83% in controls), accompanied by a 24% increase in finfold deformity incidence. Reduced alevin condition factor (2.9-5.4%) at emergence and elevated triglyceride levels were seen in chlorothalonil-exposed fish. Atrazine exposure caused premature hatch (average high atrazine time to 50% hatch [H50] = 100 d postfertilization [dpf]), and chlorothalonil exposure caused delayed hatch (high chlorothalonil H50 = 108 dpf; controls H50 = 102 dpf). All treatments caused premature emergence (average time to 50% emergence [E50]: control E50 = 181 dpf, low chlorothalonil E50 = 175 dpf, high chlorothalonil E50 = 174 dpf, high atrazine E50 = 175 dpf, low atrazine E50 = 174 dpf), highlighting the importance of using a gravel-bed incubator to examine this subtle, but critical endpoint. These alterations indicate that atrazine and chlorothalonil could affect survival of early life stages of sockeye salmon in the wild. Environ Toxicol Chem 2017;36:1354-1364. © 2017 SETAC.

Biological responses to phenylurea herbicides in fish and amphibians: New directions for characterizing mechanisms of toxicity.

Urea-based herbicides are applied in agriculture to control broadleaf and grassy weeds, acting to either inhibit photosynthesis at photosystem II (phenylureas) or to inhibit acetolactate synthase acetohydroxyacid synthase (sulfonylureas). While there are different chemical formulas for urea-based herbicides, the phenylureas are a widely used class in North America and have been detected in aquatic environments due to agricultural run-off. Here, we summarize the current state of the literature, synthesizing data on phenylureas and their biological effects in two non-target animals, fish and amphibians, with a primary focus on diuron and linuron. In fish, although the acutely lethal effects of diuron in early life stages appear to be >1mg/L, recent studies measuring sub-lethal behavioural and developmental endpoints suggest that diuron causes adverse effects at lower concentrations (i.e. <0.1mg/L). Considerably less toxicity data exist for amphibians, and this is a knowledge gap in the literature. In terms of sub-lethal effects and mode of action (MOA), linuron is well documented to have anti-androgenic effects in vertebrates, including fish. However, there are other MOAs that are not adequately assessed in toxicology studies. In order to identify additional potential MOAs, we conducted in silico analyses for linuron and diuron that were based upon transcriptome studies and chemical structure-function relationships (i.e. ToxCast™, Prediction of Activity Spectra of Substances). Based upon these analyses, we suggest that steroid biosynthesis, cholesterol metabolism and pregnane X receptor activation are common targets, and offer some new endpoints for future investigations of phenylurea herbicides in non-target animals.

Dietary selenium disrupts hepatic triglyceride stores and transcriptional networks associated with growth and Notch signaling in juvenile rainbow trout.

Dietary Se has been shown to adversely affect adult fish by altering growth rates and metabolism. To determine the underlying mechanisms associated with these observations, we measured biochemical and transcriptomic endpoints in rainbow trout following dietary Se exposures. Treatment groups of juvenile rainbow trout were fed either control Lumbriculus variegatus worms or worms cultured on selenized yeast. Selenized yeast was cultured at four nominal doses of 5, 10, 20 or 40mg/kg Se dry weight (measured dose in the worms of 7.1, 10.7, 19.5, and 31.8mg/kgSedw respectively) and fish were fed for 60days. At 60 d, hepatic triglycerides, glycogen, total glutathione, 8-isoprostane and the transcriptome response in the liver (n=8/group) were measured. Fish fed the nominal dose of 20 and 40mg/kg Se dry weight had lower body weight and a shorter length, as well as lower triglyceride in the liver compared to controls. Evidence was lacking for an oxidative stress response and there was no change in total glutathione, 8-isoprostane levels, nor relative mRNA levels for glutathione peroxidase isoforms among groups. Microarray analysis revealed that molecular networks for long-chain fatty acid transport, lipid transport, and low density lipid oxidation were increased in the liver of fish fed 40mg/kg, and this is hypothesized to be associated with the lower triglyceride levels in these fish. In addition, up-regulated gene networks in the liver of 40mg/kg Se treated fish included epidermal growth factor receptor signaling, growth hormone receptor, and insulin growth factor receptor 1 signaling pathways. These molecular changes are hypothesized to be compensatory and related to impaired growth. A gene network related to Notch signaling, which is involved in cell-cell communication and gene transcription regulation, was also increased in the liver following dietary treatments with both 20 and 40mg/kg Se. Transcriptomic data support the hypothesis that dietary Se increases the expression of networks for growth-related signaling cascades in addition to those related to fatty acid synthesis and metabolism. We propose that the disruption of metabolites related to triglyceride processing and storage, as well as gene networks for epidermal growth factor and Notch signaling in the liver, represent key molecular initiating events for adverse outcomes related to growth and Se toxicity in fish.

Application of molecular endpoints in early life stage salmonid environmental biomonitoring.

Molecular endpoints can enhance existing whole animal bioassays by more fully characterizing the biological impacts of aquatic pollutants. Laboratory and field studies were used to examine the utility of adopting molecular endpoints for a well-developed in situ early life stage (eyed embryo to onset of swim-up fry) salmonid bioassay to improve diagnostic assessments of water quality in the field. Coastal cutthroat trout (Oncorhynchus clarki clarki) were exposed in the laboratory to the model metal (zinc, 40µg/L) and the polycyclic aromatic hydrocarbon (pyrene, 100µg/L) in water to examine the resulting early life stage salmonid responses. In situ field exposures and bioassays were conducted in parallel to evaluate the water quality of three urban streams in British Columbia (two sites with anthropogenic inputs and one reference site). The endpoints measured in swim-up fry included survival, deformities, growth (weight and length), vitellogenin (vtg) and metallothionein (Mt) protein levels, and hepatic gene expression (e.g., metallothioneins [mta and mtb], endocrine biomarkers [vtg and estrogen receptors, esr] and xenobiotic-metabolizing enzymes [cytochrome P450 1A3, cyp1a3 and glutathione transferases, gstk]). No effects were observed in the zinc treatment, however exposure of swim-up fry to pyrene resulted in decreased survival, deformities and increased estrogen receptor alpha (er1) mRNA levels. In the field exposures, xenobiotic-metabolizing enzymes (cyp1a3, gstk) and zinc transporter (zntBigM103) mRNA were significantly increased in swim-up fry deployed at the sites with more anthropogenic inputs compared to the reference site. Cluster analysis revealed that gene expression profiles in individuals from the streams receiving anthropogenic inputs were more similar to each other than to the reference site. Collectively, the results obtained in this study suggest that molecular endpoints may be useful, and potentially more sensitive, indicators of site-specific contamination in real-world, complex exposure scenarios in addition to whole body morphometric and physiological measures.

Molecular responses to 17ß-estradiol in early life stage salmonids.

Environmental estrogens (EE) are ubiquitous in many aquatic environments and biological responses to EEs in early developmental stages of salmonids are poorly understood compared to juvenile and adult stages. Using 17ß-estradiol (E2) as a model estrogen, waterborne exposures were conducted on early life stage rainbow trout (Oncorhynchus mykiss; egg, alevin, swim-up fry) and both molecular and physiological endpoints were measured to quantify the effects of E2. To investigate developmental stage-specific effects, laboratory exposures of 1 µg/L E2 were initiated pre-hatching as eyed embryos or post-hatching upon entering the alevin stage. High mortality (∼90%) was observed when E2 exposures were initiated at the eyed embryo stage compared to the alevin stage (∼35% mortality), demonstrating stage-specific sensitivity. Gene expression analyses revealed that vitellogenin was detectable in the liver of swim-up fry, and was highly inducible by 1 µg/L E2 (>200-fold higher levels compared to control animals). Experiments also confirmed the induction of vitellogenin protein levels in protein extracts isolated from head and tail regions of swim-up fry after E2 exposure. These findings suggest that induction of vitellogenin, a well-characterized biomarker for estrogenic exposure, can be informative measured at this early life stage. Several other genes of the reproductive endocrine axis (e.g. estrogen receptors and androgen receptors) exhibited decreased expression levels compared to control animals. In addition, chronic exposure to E2 during the eyed embryo and alevin stages resulted in suppressive effects on growth related genes (growth hormone receptors, insulin-like growth factor 1) as well as premature hatching, suggesting that the somatotropic axis is a key target for E2-mediated developmental and growth disruptions. Combining molecular biomarkers with morphological and physiological changes in early life stage salmonids holds considerable promise for further defining estrogen action during development, and for assessing the impacts of endocrine disrupting chemicals in vivo in teleosts.

The effects of the urea-based herbicide linuron on reproductive endpoints in the fathead minnow (Pimephales promelas).

Linuron is a widely used urea-based herbicide that has anti-androgenic activity in both fish and rodents. To further elucidate the potential mode of action (MOA) of linuron on the vertebrate endocrine system, adult male and female fathead minnows were exposed for 21 days to dechlorinated water, a solvent control, 17ß-estradiol (E2; 0.1 µg/L), dihydrotestosterone (DHT; 100 µg/L), linuron (1, 10, 100 µg/L) and one co-treatment of DHT (100 µg/L) and linuron (100 µg/L). There were no effects of linuron on egg hatching, 7 day egg survival, nuptial tubercle formation or gonadal histopathology. Administration of DHT and 1 and 100 µg/L linuron reduced plasma vitellogenin in females, while male plasma vitellogenin were induced after E2 exposure and co-exposure of DHT and linuron. Ovarian mRNA levels were examined for several genes involved in steroidogenesis (e.g. p450scc, cyp19a, star, tspo, hsd17b and hsd11b) and estrogen-mediated responses (esr1, esr2b, esr2a). Only p450scc mRNA was significantly decreased with DHT+linuron co-treatment. Clustering of steroidogenic mRNA transcript expression patterns revealed that patterns for linuron were more similar to E2 compared to DHT. Collectively, this study supports the hypothesis that linuron may not be a pure anti-androgen and may have multiple MOAs that affect vertebrate reproduction.

Triclosan exposure alters postembryonic development in a Pacific tree frog (Pseudacris regilla) Amphibian Metamorphosis Assay (TREEMA).

The Amphibian Metamorphosis Assay (AMA), developed for Xenopus laevis, is designed to identify chemicals that disrupt thyroid hormone (TH)-mediated biological processes. We adapted the AMA for use on an ecologically-relevant North American species, the Pacific tree frog (Pseudacris regilla), and applied molecular endpoints to evaluate the effects of the antibacterial agent, triclosan (TCS). Premetamorphic (Gosner stage 26-28) tadpoles were immersed for 21 days in solvent control, 1.5 µg/L thyroxine (T(4)), 0.3, 3 and 30 µg/L (nominal) TCS, or combined T(4)/TCS treatments. Exposure effects were scored by morphometric (developmental stage, wet weight, and body, snout-vent and hindlimb lengths) and molecular (mRNA abundance using quantitative real time polymerase chain reaction) criteria. T(4) treatment alone accelerated development concomitant with altered levels of TH receptors α and ß, proliferating cell nuclear antigen, and gelatinase B mRNAs in the brain and tail. We observed TCS-induced perturbations in all of the molecular and morphological endpoints indicating that TCS exposure disrupts coordination of postembryonic tadpole development. Clear alterations in molecular endpoints were evident at day 2 whereas the earliest morphological effects appeared at day 4 and were most evident at day 21. Although TCS alone (3 and 30 µg/L) was protective against tadpole mortality, this protection was lost in the presence of T(4). The Pacific tree frog is the most sensitive species examined to date displaying disruption of TH-mediated development by a common antimicrobial agent.

Hepatic protein expression networks associated with masculinization in the female fathead minnow (Pimephales promelas).

Endocrine disruptors that act via the androgen receptor (AR) are less well studied than environmental estrogens, and there is evidence that treatment with AR agonists can result in masculinization of female fish. In this study, female fathead minnows (FHM) were exposed to the model nonaromatizable androgen 5-alpha dihydrotestosterone (DHT) (100 µg/L), the ureic-based herbicide linuron (LIN) (100 µg/L), and a mixture of DHT and LIN (100 µg/L each) to better characterize androgen action in females. LIN was used because of reports that this chemical has an antiandrogenic mode of action in fish. After 21d, DHT and LIN treatments resulted in a significant depression of plasma vitellogenin (Vtg) and DHT and DHT+LIN increased the prevalence of nuptial tubercles in female FHMs indicating masculinization. Using iTRAQ and an LTQ Orbitrap Velos, ∼2000 proteins were identified in the FHM liver and the number of proteins quantified after exposures was >1200. Proteins that significantly and consistently changed in abundance across biological replicates included prostaglandin E synthase 3, programmed cell death 4a, glutathione S transferases, canopy, selenoprotein U, and ribosomal proteins. Subnetwork enrichment analysis identified that interferon and epidermal growth factor signaling were regulated by DHT and LIN, suggesting that these signaling pathways are correlated to depressed plasma vitellogenin. These data provide novel insight into hepatic protein networks that are associated with the process of masculinization in teleosts.

The occurrence of steroidal estrogens in south-eastern Ontario wastewater treatment plants.

We measured steroidal estrogens in wastewater in Ottawa and Cornwall (Ontario, Canada) to determine removal efficiency of these steroids during the treatment process, and whether removal varies during a seasonal cycle. Estrone (E1), 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2) were found at maximum concentrations in raw sewage (RS), at 104, 66.9 and 5.7 ng L(-1), respectively. For the Ottawa wastewater treatment plant (WWTP), there was sufficient data to show that E1 concentrations in RS correlated with both ambient air temperature and mean daily flow of the WWTP (R(2)=0.792, p=0.003 and R(2)=0.757, p=0.005). E1 removal was correlated with the percent difference in cBOD from RS to FE (final effluent) (R(2)=0.435, p=0.075). However estrogenic potency, as determined by a sensitive in vitro reporter gene assay, did not decrease during the water treatment process, suggesting that many estrogenic chemicals are conserved in FE. E1 and EE2 were found in river water, both upstream and downstream of the WWTPs, and at much lower concentrations than in FE. Our study demonstrates the persistence of steroidal estrogens and estrogenic potency in Ontario WWTP effluents and surface waters, and has uncovered temporal patterns of release that may be used to help predict risks to aquatic organisms in these environments.

Lumiestrone is Photochemically Derived from Estrone and may be Released to the Environment without Detection.

Endocrine disrupting chemicals are adversely affecting the reproductive health and metabolic status of aquatic vertebrates. Estrone is often the dominant natural estrogen in urban sewage, yet little is known about its environmental fate and biological effects. Increased use of UV-B radiation for effluent treatments, and exposure of effluents to sunlight in holding ponds led us to examine the effects of environmentally relevant levels of UV-B radiation on the photodegradation potential of estrone. Surprisingly, UV-B-mediated degradation leads to the photoproduction of lumiestrone, a little known 13α-epimer form of estrone. We show for the first time that lumiestrone possesses novel biological activity. In vivo treatment with estrone stimulated estrogen receptor (ER) α mRNA production in the male goldfish liver, whereas lumiestrone was without effect, suggesting a total loss of estrogenicity. In contrast, results from in vitro ER-dependent reporter gene assays indicate that lumiestrone showed relatively higher estrogenic potency with the zebrafish ERß2 than zfERα, suggesting that it may act through an ERß-selectivity. Lumiestrone also activated human ERs. Microarray analysis of male goldfish liver following in vivo treatments showed that lumiestrone respectively up- and down-regulated 20 and 69 mRNAs, which was indicative of metabolic upsets and endocrine activities. As a photodegradation product from a common estrogen of both human and farm animal origin, lumiestrone is present in sewage effluent, is produced from estrone upon exposure to natural sunlight and should be considered as a new environmental contaminant.

Environmental factors affecting ultraviolet photodegradation rates and estrogenicity of estrone and ethinylestradiol in natural waters.

The environmental fate and persistence of steroidal estrogens is influenced by their photodegradation. This can potentially occur both in the presence of the ultraviolet (UV) portion of solar radiation and in tertiary wastewater treatment plants that use UV radiation for disinfection purposes. To determine patterns of UV photodegradation for estrone (E1) and 17α-ethinylestradiol (EE2), water samples containing these compounds were exposed to levels of UVB radiation that would simulate exposure to ambient sunlight. E1 degraded with a pseudo-first-order rate law constant that was directly proportional to UVB radiation intensity (R² = 0.999, P < 0.001) and inversely proportional to dissolved organic carbon (DOC) concentration (R² = 0.812, P = 0.037). DOC acted as a competitive inhibitor to direct photolysis of E1 by UV. In contrast to E1, EE2 was more persistent under similar UVB treatment. A reporter gene assay showed that the estrogenicity of UVB-exposed estrogens did not decrease relative to non-UVB-exposed estrogens, suggesting that some of the photoproducts may also have estrogenic potency. These results show that environmental degradation rates of steroidal estrogens are predictable from the UV intensity reaching surface waters, and the DOC concentrations in these surface waters.

Sex- and tissue-specific effects of waterborne estrogen on estrogen receptor subtypes and E2-mediated gene expression in the reproductive axis of goldfish.

This research examined the gene expression profile of three goldfish estrogen receptor (ER) subtypes in multiple tissues in relation to mRNA levels of aromatase B and vitellogenin (VTG) following waterborne estrogen exposures. The protocol consisted of: i) adult male goldfish in late gonadal recrudescence exposed to 1 nM 17beta-estradiol (E2); ii) adult male and female goldfish in early sexual regression exposed to 1 nM E2 for 3, 6, 12 and 24h; and, iii) sexually mature, adult male goldfish exposed to 0.3 nM 17alpha-ethynylestradiol (EE2) for 24h. Liver produced the most consistent response with up-regulation of ERalpha in sexually regressed, mature and recrudescing males and in sexually regressed females. The dose and length of exposure, reproductive state and sex affected the auto-regulation of ERbeta1 by E2. ERbeta2 was not affected in any experiments suggesting it may not be auto-regulated by E2. Aromatase B and VTG gene expression were affected by E2, but also by other experimental conditions. EE2 induced liver ERalpha and VTG mRNA levels indicating that high environmental EE2 levels induce E2-mediated gene expression in a model teleost. These studies reveal a more complicated action of estrogenic compounds that has important implications on estrogenic endocrine disruptors in teleosts.