Jim Sherry

Potential endocrine disruption of sexual development in free ranging male northern leopard frogs (Rana pipiens) and green frogs (Rana clamitans) from areas of intensive row crop agriculture

Intensive row crop agriculture (IRCA) for corn and soybean production is predominant in eastern and central North America. IRCA relies heavily on pesticide and nutrient inputs to maximize production under conventional systems. In 2003-2005, we assessed the occurrence of a suite of potential endocrine effects in amphibians inhabiting farm ponds and agricultural drains in IRCA areas of southwestern Ontario. Effects were compared to amphibians from two agricultural reference sites as well as four non-agricultural reference sites. Pesticide and nutrient concentrations were also determined in water samples from those sites. Atrazine and metolachlor were detected in most samples, exceeding 1 microg L(-1) at some sites. Blood samples were taken from northern leopard frogs (Rana pipiens) and green frogs (Rana clamitans) for analysis of circulating sex steroids and vitellogenin-like protein (Vtg-lp), a biomarker of exposure to environmental estrogens. Gonads were histologically examined for evidence of abnormalities. Some evidence of exposure to endocrine disrupting compounds was apparent from the data. The occurrence of testicular ovarian follicles (TOFS) in male R. pipiens was significantly higher (42%; p<0.05) at agricultural sites, particularly those in Chatham county compared to frogs from reference sites (7%). There was no difference in circulating sex steroid levels between frogs from agricultural and reference sites and sex steroid levels did not correlate with pesticide concentrations in the environment. No differences were detected in the gonadosomatic indices or stage of spermatogenesis between frogs from agricultural and non-agricultural regions (p>0.05). Plasma Vtg-lp was detected in only one male R. pipiens from an agricultural site. Neither gonad size, gonad maturity nor sex steroid levels differed between normal males and those with testicular oocytes. Although the proportion of testicular oocytes did not correlate directly with atrazine concentrations, it did correlate with a mixture of pesticides and nutrients, particularly atrazine and nitrate, while the number of pesticides detected at each site was also important.

Bioaccumulation of the pharmaceutical 17α-ethinylestradiol in shorthead redhorse suckers (Moxostoma macrolepidotum) from the St. Clair River, Canada.

17alpha-ethynylestradiol (EE2), a synthetic estrogen prescribed as a contraceptive, was measured in Shorthead Redhorse Suckers (ShRHSs) (Moxostoma macrolepidotum) collected near a wastewater treatment plant (WWTP) in the St. Clair River (Ontario, Canada). We detected EE2 in 50% of the fish samples caught near the WWTP (Stag Island), which averaged 1.6+/-0.6ng/g (wet weight) in males and 1.43+/-0.96ng/g in females. No EE2 was detected in the samples from the reference site (Port Lambton) which was 26km further downstream of the Stag Island site. Only males from Stag Island had VTG induction, suggesting the Corunna WWTP effluent as a likely source of environmental estrogen. EE2 concentrations were correlated with total body lipid content (R(2)=0.512, p<0.01, n=10). Lipid normalized EE2 concentrations were correlated with delta(15)N (R(2)=0.436, p<0.05, n=10), suggesting higher EE2 exposures in carnivores. Our data support the hypothesis of EE2 bioaccumulation in wild fish.

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.

Genotoxic potential of several naphthenic acids and a synthetic oil sands process-affected water in rainbow trout (Oncorhynchus mykiss)

The exploitation of oil sands has raised major environmental concerns, particularly regarding the presence of high concentration in contaminants such as polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) in oil sands process-affected water (OSPW). The purpose of this study was, first to evaluate the genotoxic impact of OSPW-related compounds such as NAs and PAHs in a salmonid species and secondly to assess if OSPW exposure leads to genotoxicity. For this purpose, rainbow trout hepatocytes were exposed in vitro to environmentally relevant concentrations of synthetic NAs, naphtalene, benzo(a)pyrene, and extracts of synthetic OSPW (generated by a laboratory bitumen extraction) and of oil sands leaching water (OSLW, mimicking leaching of oil sands in river water). Primary DNA damage was assessed by the formamidopyrimidine-DNA glycolyase (Fpg)-modified comet assay. Genotoxicity was observed in hepatocytes exposed to several NAs, mixture of them, OSPW and OSLW extracts. The chemical structure of NAs influences the genotoxicity potential: among the NAs tested, the most cyclic NA was the most genotoxic. It also appears that genotoxicity was more marked for OSPW than for OSLW. Because exposure to OSPW led to oxidative DNA damage, while after exposure to several NAs, these types of DNA damage were limited, the NAs tested in this study could not be qualified as the only major contaminants responsible for OSPW genotoxicity. Notwithstanding, it should be noteworthy that exposure to NAs resulted in genotoxic impact at concentrations lower than those documented by literature for fresh OSPW. Further research is needed to explore the relationships between the chemical structure of NAs and their genotoxicity in the light of the distribution of NAs in fresh OSPW samples as well as in surface waters.

Interaction of Galaxolide® with the human and trout estrogen receptor-α

Synthetic musks have been detected in sewage effluents, surface waters, and fish tissues where the polycyclic musk compound, HHCB (Galaxolide®) is the dominant compound in those matrices. In the present study, the Galaxolide® formulation was tested in the yeast estrogenicity screening (YES) assay, and also tested in in vitro and in vivo teleost systems to determine whether it interacts with the estrogen receptor as either an agonist or antagonist. In those tests, Galaxolide® did not act as an estrogen agonist, however there was strong evidence of antagonistic activity as Galaxolide® inhibited the estrogenic activity of 17β-estradiol (E2). In the YES assay based on a recombinant strain of yeast containing the human estrogen receptor (i.e. hERα), Galaxolide® inhibited the effects of E2 in a dose-dependent manner (IC50=1.63×10(-5)M). In a luciferase reporter gene assay based on the rainbow trout estrogen receptor (i.e. rtER) transfected into a rainbow trout gonadal (RTG-2) cell line, the IC50 for the antagonistic effect of Galaxolide® was 2.79×10(-9)M. In an in vivo assay based on modulation of vitellogenin in rainbow trout, Galaxolide® i.p. injected into trout at a dose of 3.64mg/kg caused inhibition of E2-induced vitellogenin production. That dose is within the range of concentrations of Galaxolide® that have been detected in tissues of fish from contaminated locations.

A subchronic in situ exposure method for evaluating effects in small‐bodied fish at contaminated sites

In situ fish‐caging studies at contaminated sites can provide information that is more realistic compared to traditional laboratory‐based studies. However, few methods have been developed for exposing sentinel fish species for subchronic durations, and fewer still are optimized for exposing small‐bodied fish while maintaining fish health and growth throughout the caging trial. Those methods typically lack a feeding regimen during the fish caging trial. While that may be acceptable or even appropriate for typical short‐term toxicity testing, it does limit the duration of the exposure, and may not be suitable when post‐caging trials or observations are necessary. Returning healthy fish to the lab following the in situ exposure would be important, for example, in studies designed to examine long‐term or multigenerational effects following an in situ exposure. In this article we describe a subchronic method for caging small fish at contaminated sites while maintaining growth and reproductive development. Fathead minnow (Pimephales promelas) were caged in situ for 6 weeks, after which time they were returned to the lab where they were evaluated for health and reproductive performance. Growth and reproductive endpoints revealed no adverse effect on fish due to fish caging and related handling, demonstrating the suitability of our caging and feeding method for long‐term caging studies.

Bioaccumulation of pharmaceuticals and personal care product chemicals in fish exposed to wastewater effluent in an urban wetland

The bioaccumulation of a broad range of pharmaceuticals and personal care product chemicals (PPCPs) was studied in Cootes Paradise Marsh (CPM), an urban wetland that receives tertiary treated municipal waste waters as well as urban storm runoff. We measured PPCPs in caged and wild goldfish, as well as wild carp, and compared observed bioaccumulation factors (BAFP) using concentrations in surface waters and fish blood plasma, with modeled BAFs. Thirty-two PPCPs were detected in water from the central CPM site (CPM3) while 64 PPCPs were found at higher concentrations at a site immediately downstream of the effluent outflow (CPM1). Following a 3-week deployment, 15 PPCPs were detected in the plasma of caged goldfish at CPM1, and 14 at CPM3, compared to only 3 in goldfish caged at a reference site. The highest BAFP in goldfish were for the antidepressant Σfluoxetine averaging 386u2009L/kg in caged and 906u2009L/kg in wild goldfish, respectively. In carp, ΣDiazepam (diazepam and oxazepam) had the highest BAFP (927u2009L/kg). This study identified a broader range of PPCPs in fish and surface waters than previously reported. However, modeled BAFs did not show good agreement with observed whole body or plasma BAFs, demonstrating that more work is needed to better explain bioaccumulation of PPCPs.

Altered expression of metabolites and proteins in wild and caged fish exposed to wastewater effluents in situ

Population growth has led to increased global discharges of wastewater. Contaminants that are not fully removed during wastewater treatment, such as pharmaceuticals and personal care products (PPCPs), may negatively affect aquatic ecosystems. PPCPs can bioaccumulate causing adverse health effects and behavioural changes in exposed fish. To assess the impact of PPCPs on wild fish, and to assess whether caged fish could be used as a surrogate for resident wild fish in future monitoring, we caged goldfish in a marsh affected by discharges of wastewater effluents (Cootes Paradise, Lake Ontario, Canada). We collected plasma from resident wild goldfish, and from goldfish that we caged in the marsh for three weeks. We analyzed the plasma proteome and metabolome of both wild and caged fish. We also compared proteomic and metabolic responses in caged and wild fish from the marsh to fish caged at a reference site (Jordan Harbour Conservation Area). We identified significant changes in expression of over 250 molecules that were related to liver necrosis, accumulation and synthesis of lipids, synthesis of cyclic AMP, and the quantity of intracellular calcium in fish from the wastewater affected marsh. Our results suggest that PPCPs could be affecting the health of wild fish populations.

Reduced anxiety is associated with the accumulation of six serotonin reuptake inhibitors in wastewater treatment effluent exposed goldfish Carassius auratus

Pharmaceuticals and personal care products (PPCPs) have been found in wastewater treatment plant (WWTP) effluents and their recipient watersheds. To assess the potential of WWTP effluents to alter fish behaviour, we caged male goldfish (Carassius auratus) for 21-days at three sites along a contamination gradient downstream from a WWTP which discharges into Cootes Paradise Marsh, on the western tip of Lake Ontario. We also included a fourth caging site as an external reference site within Lake Ontario at the Jordan Harbour Conservation Area. We then measured concentrations of PPCPs and monoamine neurotransmitters in caged goldfish plasma, and conducted behavioural assays measuring activity, startle response, and feeding. We detected fifteen different PPCPs in goldfish plasma including six serotonin reuptake inhibitors (amitriptyline, citalopram, fluoxetine/norfluoxetine, sertraline, venlafaxine, and diphenhydramine). Plasma concentrations of serotonin were significantly greater in plasma of fish caged closer to the WWTP effluent outfall site. The fish caged near and downstream of the WWTP effluent were bolder, more exploratory, and more active overall than fish caged at the reference site. Taken together, our results suggest that fish downstream of WWTPs are accumulating PPCPs at levels sufficient to alter neurotransmitter concentrations and to also impair ecologically-relevant behaviours.

An evaluation of germline mutations and reproductive impacts in fathead minnow (Pimephales promelas) exposed to contaminated sediment

Polycyclic aromatic hydrocarbons (PAHs) have become ubiquitous in the aquatic environment. Some PAHs are mutagenic, potentially causing germline mutations in fish that inhabit PAH contaminated waters. We evaluated the effect of exposure to sediment-borne PAHs on reproduction and germline mutation rates in fathead minnows (Pimephales promelas). Exposure to the contaminated sediment had no significant impact on the reproductive endpoints measured in this study. Germline mutations rates at three microsatellite DNA loci were 1.69u202f×u202f10-3 in fish exposed to PAH-contaminated sediment and 0.55u202f×u202f10-3 in control fish, with zero mutations being observed in fish exposed to sediment from a reference site. While the difference in mutation rates between treatments was not statistically significant for the sample size used (15-19 families per treatment), the observed mutations rates enabled us to estimate the sample size required to detect a significant effect. To our knowledge, this is the first report of germline mutation rates in fathead minnow exposed to an environmental contaminant, providing baseline data for use in the design of future experiments.