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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

Interaction of stilbene compounds with human and rainbow trout estrogen receptors.

Compounds with stilbene structures are widely used as pharmaceuticals and personal care products (PPCPs) and are present in plants. A suite of stilbene-related compounds, including PPCPs and plant-derived compounds were tested in vitro for interactions with the human and rainbow trout estrogen receptors and in vivo with rainbow trout using vitellogenin levels as a biomarker. Among the compounds with antagonistic activity, the common structural similarity was (in addition to the stilbene backbone) the presence of 4-hydroxy substitution. Stilbene-related compounds found to act as inhibitors at the estrogen receptor included the plant-derived compound resveratrol and two formulations of fluorescent whitening agents used in detergents, 4,4'-bis(2-sulfostyryl)biphenyl and diaminostilbene-1. In the yeast estrogenicity screening assay, the concentrations which caused a 50% inhibition in estrogenic response (IC50s) with the human estrogen receptor ranged from 2.56 x 10(-6) to 2.56 x 10(-6) M. In the rainbow trout estrogen receptor assay, the IC50s ranged from 7.75 x 10(-8) to 1.11 x 10(-5) M. However, in the in vivo rainbow trout vitellogenin assay, tamoxifen was the only stilbene of the compounds tested to have a significant effect as an inhibitor of estrogenicity.

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.