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.

Contaminant biomonitoring augmented with a qPCR array indicates hepatic mRNA gene expression effects in wild-collected seabird embryos.

Birds can bioaccumulate persistent contaminants, and maternal transfer to eggs may expose embryos to concentrations sufficient to cause adverse effects during sensitive early-life stages. However, using tissue residue concentrations alone to infer whether contaminant effects are occurring suffers from uncertainty, and efficient, sensitive biomarkers remain limited in wildlife. We studied relationships between whole embryo contaminant concentrations (total mercury, organochlorine pesticides, perfluoroalkyl substances, polychlorinated biphenyls, and halogenated flame retardants) together with mRNA expression in embryonic liver tissue from a Pacific Ocean seabird, the rhinoceros auklet (Cerorhinca monocerata). Fresh eggs were collected, incubated under controlled conditions, and from the pre-hatch embryo, hepatic RNA was extracted for qPCR array analysis to measure gene expression (2-∆Cq), while the remaining embryo was analyzed for contaminant residues. Contaminant and gene expression data were assessed with a combination of multivariate approaches and linear models. Results indicated correlations between embryonic total mercury and several genes such as sepp1, which encodes selenoprotein P. Correlation between the biotransformation gene cyp1a4 and the C7 perfluoroalkyl carboxylic acid PFHpA was also evident. This study demonstrates that egg collection from free-living populations for contaminant biomonitoring programs can relate chemical residues to in ovo mRNA gene expression effects in embryo hepatic tissue.

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.

Risks of mining to salmonid-bearing watersheds.

Mining provides resources for people but can pose risks to ecosystems that support cultural keystone species. Our synthesis reviews relevant aspects of mining operations, describes the ecology of salmonid-bearing watersheds in northwestern North America, and compiles the impacts of metal and coal extraction on salmonids and their habitat. We conservatively estimate that this region encompasses nearly 4000 past producing mines, with present-day operations ranging from small placer sites to massive open-pit projects that annually mine more than 118 million metric tons of earth. Despite impact assessments that are intended to evaluate risk and inform mitigation, mines continue to harm salmonid-bearing watersheds via pathways such as toxic contaminants, stream channel burial, and flow regime alteration. To better maintain watershed processes that benefit salmonids, we highlight key windows during the mining governance life cycle for science to guide policy by more accurately accounting for stressor complexity, cumulative effects, and future environmental change.

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.

Effects of Avian Eggshell Oiling With Diluted Bitumen Show Sublethal Embryonic Polycyclic Aromatic Compound Exposure.

Breeding birds that become oiled may contaminate the shells of their eggs, and studies of conventional crude oil suggest that even small quantities can be absorbed through the eggshell and cause embryotoxicity. Unconventional crude oils remain untested, so we evaluated whether a major Canadian oil sands product, diluted bitumen (dilbit), would be absorbed and cause toxicity when applied to eggshells of two species, domestic chicken (Gallus gallus domesticus) and double-crested cormorant (Nannopterum auritum). We artificially incubated eggs and applied lightly weathered dilbit (Cold Lake blend) to the eggshells (0.015-0.15 mg g-1 egg in chicken; 0.1-0.4 mg g-1 egg in cormorant) at various points during incubation before sampling prehatch embryos. Polycyclic aromatic compound (PAC) residue in cormorant embryos was elevated only at the highest dilbit application (0.4 mg g-1 egg) closest (day 16) to sampling on day 22. In contrast, cormorant liver cytochrome P450 1a4 (Cyp1a4) mRNA expression (quantitative polymerase chain reaction assay) was elevated only in embryos treated with the earliest and lowest dilbit application (0.1 mg g-1 egg on day 4). These results confirm that dilbit can cross through the eggshell and be absorbed by embryos, and they imply rapid biotransformation of PACs and a nonmonotonic Cyp1a4 response. Despite evidence of exposure in cormorant, we found no detectable effects on the frequency of survival, deformity, and gross lesions, nor did we find effects on physiological endpoints indicative of growth and cardiovascular function in either chicken or cormorant. In ovo dilbit exposure may be less toxic than well-studied conventional crude oils. The effects of an oil spill scenario involving dilbit to bird embryos might be subtle, and PACs may be rapidly metabolized. Environ Toxicol Chem 2022;41:159-174. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Towards regulation of Endocrine Disrupting chemicals (EDCs) in water resources using bioassays - A guide to developing a testing strategy.

Endocrine disrupting chemicals (EDCs) are found in every environmental medium and are chemically diverse. Their presence in water resources can negatively impact the health of both human and wildlife. Currently, there are no mandatory screening mandates or regulations for EDC levels in complex water samples globally. Bioassays, which allow quantifying in vivo or in vitro biological effects of chemicals are used commonly to assess acute toxicity in water. The existing OECD framework to identify single-compound EDCs offers a set of bioassays that are validated for the Estrogen-, Androgen-, and Thyroid hormones, and for Steroidogenesis pathways (EATS). In this review, we discussed bioassays that could be potentially used to screen EDCs in water resources, including in vivo and in vitro bioassays using invertebrates, fish, amphibians, and/or mammalians species. Strengths and weaknesses of samples preparation for complex water samples are discussed. We also review how to calculate the Effect-Based Trigger values, which could serve as thresholds to determine if a given water sample poses a risk based on existing quality standards. This work aims to assist governments and regulatory agencies in developing a testing strategy towards regulation of EDCs in water resources worldwide. The main recommendations include 1) opting for internationally validated cell reporter in vitro bioassays to reduce animal use & cost; 2) testing for cell viability (a critical parameter) when using in vitro bioassays; and 3) evaluating the recovery of the water sample preparation method selected. This review also highlights future research avenues for the EDC screening revolution (e.g., 3D tissue culture, transgenic animals, OMICs, and Adverse Outcome Pathways (AOPs)).

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.

Morphometric and proteomic responses of early-life stage rainbow trout (Oncorhynchus mykiss) to the aquatic herbicide diquat dibromide.

The objective of this study was to examine the acute toxicity and sub-lethal effects of the commercial formulation of diquat dibromide, Reward® Landscape and Aquatic Herbicide, on multiple life stages of rainbow trout. The continuous exposure 96 h LC50 derived for juvenile feeding fry aged 85 d post-hatch was 9.8 mg/L. Rainbow trout eyed embryos and juvenile feeding fry were also exposed to concentrations of Reward® ranging from 0.12 to 10 mg/L during two 24 h pulse exposures separated by 14 d of rearing in fresh water to mimic the manufacturers instructions for direct applications to water bodies. Decreased survival and body morphometrics were evident at 9.3 mg/L during the embryo/alevin exposures, but not in feeding juveniles, indicating a higher sensitivity of the early life stage fish. Quantitative proteomics and subnetwork enrichment analyses were conducted in the livers for both life stages to evaluate protein profiles after exposure to 0.37 mg/L diquat via Reward® exposure. Unique protein profiles were revealed for pre-feeding swim-up fry and for feeding juvenile fish, reflecting differences between the two life stages in sub-cellular responses after diquat dibromide exposure. Hepatic proteome effects were more dramatic in the pre-feeding swim-up fry with 315 proteins differentially expressed between the control and exposed fish while in the later life stage feeding fry, only 84 proteins were different after Reward® exposure. Exposure to Reward® significantly increased RNA/mRNA processes, induced activation of Atk/mTOR and caspase activity, and altered energy homeostasis. Proteomic alterations are associated with reduced growth observed in embryo/alevin at higher exposure concentrations, offering insight into key events underlying growth impairment within the adverse outcome pathway framework. This study is the first to report the sub-cellular and whole organism level effects of diquat dibromide in a commercial formulation and demonstrates that concentrations based on aquatic application rates alter the hepatic proteome.

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).

Sub-lethal effects of a neonicotinoid, clothianidin, on wild early life stage sockeye salmon (Oncorhynchus nerka).

One of the categories of environmental contaminants possibly contributing to declining sockeye salmon (Oncorhynchus nerka) in the Fraser River, British Columbia, Canada is pesticides. In this 4-month study, the effects of environmentally relevant concentrations of a waterborne neonicotinoid, clothianidin (0.15, 1.5, 15 and 150 µg/L), on embryonic, alevin and early swim-up fry sockeye salmon derived from four unique genetic crosses of the Pitt River, BC stock were investigated. There were no significant effects of clothianidin on survival, hatching, growth or deformities, although genetic variation significantly affected these endpoints. Clothianidin caused a significant 4.7-fold increase in whole body 17ß-estradiol levels in swim-up fry after exposure to 0.15 µg/L, but no effects were observed on testosterone levels. In addition, hepatic expression of the gene encoding glucocorticoid receptor 2 was also impacted at the highest concentration of clothianidin tested, and was found to be ∼4-fold lower compared to the sockeye reared in control water. These results indicate additional examination of clothianidin and its effects on salmonid gonad development and the reproductive and stress endocrine axes in general, is warranted.

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.