Comparative Effects of Embryonic Metformin Exposure on Wild and Laboratory-Spawned Fathead Minnow (Pimephales promelas) Populations.

Metformin is routinely detected in aquatic ecosystems because of its widespread use as a treatment for Type 2 diabetes. Laboratory studies have shown that exposure to environmentally relevant concentrations of metformin can alter metabolic pathways and impact the growth of early life stage (ELS) fish; however, it is unknown whether these effects occur in wild populations. Herein, we evaluate whether findings from laboratory studies are representative and describe the relative sensitivities of both populations. Duplicate exposures (0, 5, or 50 µg/L metformin) were conducted using wild- and lab-spawned fathead minnow (Pimephales promelas) embryos. Apart from the water source, exposure conditions remained constant. Wild embryos were exposed to previously dosed lake water to account for changes in bioavailability, while reconstituted freshwater was used for the laboratory study. Developmental metformin exposure differentially impacted the growth and morphology of both cohorts, with energy dyshomeostasis and visual effects indicated. The fitness of wild-spawned larvae was impacted to a greater extent relative to lab-spawned fish. Moreover, baseline data reveal important morphological differences between wild- and lab-spawned ELS fatheads that may diminish representativeness of lab studies. Findings also confirm the bioavailability of metformin in naturally occurring systems and suggest current exposure scenarios may be sufficient to negatively impact developing fish.

Development of a thin-film solid-phase microextraction (TF-SPME) method coupled to liquid chromatography and tandem mass spectrometry for high-throughput determination of steroid hormones in white sucker fish plasma.

Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17ß-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150 ng/mL and from 0.020 to 0.500 ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract.

Assessing wild fish exposure to ligands for sex steroid receptors from pulp and paper mill effluents in the Biobio River Basin, Central Chile.

Bioactive substances in the Biobio River Basin in Chile were examined by deploying Semi Permeable Membrane Devices (SPMDs) upstream and downstream of 4 pulp mill effluent discharges. Androgenic and estrogenic activity of SPMD extracts were then evaluated using in vitro fish sex steroid receptor binding assays. The results indicated the occurrence of estrogenic type compounds associated with one of the mill discharges. A significant correlation among the presence of these compounds, an increase in gonadosomatic index GSI and induction of hepatic EROD activity of two native fish species was observed. However, no significant presence of mature oocytes in female gonads was detected. Although EROD induction was observed in sites impacted by mill effluents, an increase of its activity occurred towards the downstream areas, suggesting other non-mill sources. More research is needed to understand the environmental changes in context of the new technological improvements in treatment systems to MBBR (Moving Bed Biofilm Reactor) recently implemented by the pulp mill industries.

Development of environmental effects monitoring protocol in Brazil: a fish guide study of three river estuaries.

In Brazil, there are no unified and effective environmental monitoring models for bodies of water. Thus, several methodologies are used that result in information that is often difficult to compare, especially for stakeholders involved in regional water management. Studies in some countries such as Australia, Chile, the USA, and Sweden use the monitoring model implemented in Canada that was developed in the early 1990s. This model was designed to evaluate whether the current environmental regulations are sufficiently protective for pulp and paper effluents and for metal mining effluents. In this study, the Canadian Environmental Effects Monitoring methodologies were applied to three different Brazilian river basins, with the goal of constructing a framework for monitoring environmental effects. Pilot studies were carried out in the estuarine regions of the Benevente, Jucu, and Santa Maria da Vitória river basins, which are important rivers in the state of Espírito Santo. Evaluations included fish health, bioaccumulation studies, benthic invertebrate survey, and physical-chemical analyses of water and sediment. The quality of the environments was evaluated by means of seasonal samplings and comparisons between discharge, upstream, and downstream areas. This study made it possible to identify appropriate fish species to be used in environmental effects monitoring and the environmental quality of the rivers themselves as well as knowledge and policy gaps to implement such monitoring programs in Brazil. The study raises questions about the adequacy of Brazilian environmental legislation concerning tidal rivers.

Reduction of Intersex in a Wild Fish Population in Response to Major Municipal Wastewater Treatment Plant Upgrades.

Intersex in fish downstream of municipal wastewater treatment plants (MWWTPs) is a global concern. Consistent high rates of intersex in male rainbow darter (Etheostoma caeruleum) have been reported for several years in the Grand River, in southern Ontario, Canada, in close proximity to two MWWTPs. The larger MWWTP (Kitchener) recently underwent upgrades that included the conversion from a carbonaceous activated sludge to nitrifying activated sludge treatment process. This created a unique opportunity to assess whether upgrades designed to improve effluent quality could also remediate the intersex previously observed in wild fish. Multiple years (2007-2012) of intersex data on male rainbow darter collected before the upgrades at sites associated with the MWWTP outfall were compared with intersex data collected in postupgrade years (2013-2015). These upgrades resulted in a reduction from 70 to 100% intersex incidence (preupgrade) to <10% in postupgrade years. Although the cause of intersex remains unknown, indicators of effluent quality including nutrients, pharmaceuticals, and estrogenicity improved in the effluent after the upgrades. This study demonstrated that investment in MWWTP upgrades improved effluent quality and was associated with an immediate change in biological responses in the receiving environment. This is an important finding considering the tremendous cost of wastewater infrastructure.

The Relationship between Organic Loading and Effects on Fish Reproduction for Pulp Mill Effluents across Canada.

This study builds upon the work of a multiagency consortium tasked with determining cost-effective solutions for the effects of pulp mill effluents on fish reproduction. A laboratory fathead minnow egg production test and chemical characterization tools were used to benchmark 81 effluents from 20 mills across Canada, representing the major pulping, bleaching, and effluent treatment technologies. For Kraft and mechanical pulp mills, effluents containing less than 20 mg/L BOD5 were found to have the greatest probability of having no effects. Organic loading, expressed as the total detected solvent-extractable components by gas chromatography/mass spectrometry (GC/MS), also correlated with decreased egg laying. Exceptions were found for specific Kraft, mechanical, and sulfite mills, suggesting yet unidentified causative agents are involved. Recycled fiber mill effluents, tested for the first time, were found to have little potential for reproductive effects despite large variations in BOD5 and GC/MS profiles. Effluent treatment systems across all production types were generally efficient, achieving a combined 82-98% BOD5 removal. Further reductions of final effluent organic loadings toward the target of less than 20 mg/L are recommended and can be realized through biotreatment optimization, the reduction of organic losses associated with production upsets and selecting best available technologies that reduce organic loadings to biotreatment.

Effects of metformin on wild fathead minnows (Pimephales promelas) using in-lake mesocosms in a boreal lake ecosystem.

Due to its widespread use for the treatment of Type-2 diabetes, metformin is routinely detected in surface waters globally. Laboratory studies have shown that environmentally relevant concentrations of metformin can adversely affect the health of adult fish, with effects observed more frequently in males. However, the potential risk to wild fish populations has yet to be fully elucidated and remains a topic of debate. To explore whether environmentally relevant metformin exposure poses a risk to wild fish populations, the present study exposed wild fathead minnows (Pimephales promelas) to 5 or 50 µg/L metformin via 2 m diameter in-lake mesocosms deployed in a natural boreal lake in Northern Ontario at the International Institute for Sustainable Development - Experimental Lakes Area (IISD-ELA). Environmental monitoring was performed at regular intervals for 8-weeks, with fish length, weight (body, liver and gonad), condition factor, gonadosomatic index, liver-somatic index, body composition (water and biomolecules) and hematocrit levels evaluated at test termination. Metabolic endpoints were also evaluated using liver, brain and muscle tissue, and gonads were evaluated histologically. Results indicate that current environmental exposure scenarios may be sufficient to adversely impact the health of wild fish populations. Adult male fish exposed to metformin had significantly reduced whole body weight and condition factor and several male fish from the high-dose metformin had oocytes in their testes. Metformin-exposed fish had altered moisture and lipid (decrease) content in their tissues. Further, brain (increase) and liver (decrease) glycogen were altered in fish exposed to high-dose metformin. To our knowledge, this study constitutes the first effort to understand metformin's effects on a wild small-bodied fish population under environmentally relevant field exposure conditions.

A survey of Canadian mechanical pulp and paper mill effluents: insights concerning the potential to affect fish reproduction.

Building on breakthroughs recently made at kraft mills, a survey of mechanical pulp and paper mill effluents was undertaken to gain insights concerning potential effects on fish reproduction. Effluents from seven Canadian mills were characterized chemically for conventional parameters such as biochemical oxygen demand (BOD) and total suspended solids (TSS). Each sample was further subjected to solvent extraction followed by gas chromatographic separation for the determination of resin/fatty acids and for the estimation of a gas chromatography (GC) profile index. Each mill effluent was assessed for the potential to affect fish reproduction in the laboratory using a five day adult fathead minnow (Pimephales promelas) egg production bioassay with exposures to 100% effluent. The seven effluents were found to have substantial variation both in terms of chemical characterization and effects on fish reproduction. Temporal variations were also noted in effluent quality at mills sampled on different occasions. Similar to what has been observed for kraft mills, a general trend of greater reductions in egg production caused by effluents with greater BOD concentrations and GC profile indices was noted. Effluents with BOD > 25 mg/L and GC Profile indices >5.0 caused a complete cessation of egg production. At the same time, about half of the total effluents sampled had BOD < 25 mg/L and GC profile indices <5.0 and caused no significant effects on egg production, suggesting these values may be useful as effluent quality targets for mechanical pulp and paper mills. However, 3 out of 14 effluents sampled had BOD < 25 mg/L and GC profile indices <5.0 and caused significant reductions in egg production. The reason(s) for reproductive effects caused by such effluents is presently unclear. The effluent quality parameters considered in this study may require further refinement to address their utility in predicting the adverse reproductive effects induced by effluents from mechanical pulp and paper mills.

Intersex manifestation in the rainbow darter (Etheostoma caeruleum): Are adult male fish susceptible to developing and recovering from intersex after exposure to endocrine active compounds?

For over a decade, intersex has been observed in rainbow darter (RD) (Etheostoma caeruleum) populations living downstream wastewater treatment plants (WWTPs) in the Grand River, Ontario, Canada. To further our understanding of intersex development in adult male fish, the current study addressed three objectives: i) can intersex be induced in adult male fish, ii) is there a specific window of exposure when adult male fish are more susceptible to developing intersex, and iii) can pre-exposed adult male fish recover from intersex? To assess intersex induction in adult male fish, wild male RD were exposed in the laboratory for 22 weeks (during periods of spawning, gonadal regression, and gonadal recrudescence) to environmentally relevant concentrations of 17α-ethinylestradiol (EE2) including nominal 0, 1, and 10 ng/L. Intersex rates and severity at 10 ng/L EE2 were similar to those observed historically in adult male populations living downstream WWTPs in the Grand River and confirmed previous predictions that 1-10 ng/L EE2 would cause these adverse effects. To assess a window of sensitivity in developing intersex, male RD were exposed to nominal 0, 1 or 10 ng/L EE2 for 4 weeks during three different periods of gonadal development, including (i) spawning, (ii) early recrudescence and (iii) late recrudescence. These short-term exposures revealed that intersex incidence and severity were greater when RD were exposed while gonads were fully developed (during spawning) compared to periods of recrudescence. To assess if RD recover from intersex, wild fish were collected downstream WWTPs in the Grand River and assessed for intersex both before and after a 22-week recovery period in clean water that included gonadal regression and recrudescence. Results showed that fish did not recover from intersex, with intersex rates and severity similar to those both before and after the transition to clean water. This study further advances our knowledge on intersex manifestation in adult male fish including their sensitivity to endocrine active compounds during different periods of their annual reproductive cycle and their limited ability to recover from intersex after onset of the condition.

Wild fish responses to wastewater treatment plant upgrades in the Grand River, Ontario.

Municipal wastewater treatment plant (WWTP) effluent is one of several point sources of contaminants (nutrients, pharmaceuticals, estrogens, etc.) which can lead to adverse responses in aquatic life. Studies of WWTP effluent impacts on rainbow darter (Etheostoma caeruleum) collected downstream of WWTPs in the Grand River, Ontario have reported disruption at multiple levels of biological organization, including altered vitellogenin gene expression, lower levels of in vitro steroid production, and high frequency of intersex. However, major upgrades have occurred at treatment plants in the central Grand River over the last decade. Treatment upgrades to the Waterloo WWTP were initiated in 2009 but due to construction delays, the upgrades came fully on-line in 2017/2018. Responses in rainbow darter have been followed at sites associated with the outfall consistently over this entire time period. The treatment plant upgrade resulted in nitrification of effluent, and once complete there was a major reduction in effluent ammonia, selected pharmaceuticals, and estrogenicity. This study compared several key responses in rainbow darter associated with the Waterloo WWTP outfall prior to and post upgrades. Stable isotopes signatures in fish were used to track exposure to effluent and changed dramatically over time, corresponding to the effluent quality. Disruptions in in vitro steroid production and intersex in the darters that had been identified prior to the upgrades were no longer statistically different from the upstream reference sites after the upgrades. Although annual variations in water temperature and flow can potentially mask or exacerbate the effects of the WWTP effluent, major capital investments in wastewater treatment targeted at improving effluent quality have corresponded with the reduction of adverse responses in fish in the receiving environment.

Proteomic profiles of white sucker (Catostomus commersonii) sampled from within the Thunder Bay Area of Concern reveal up-regulation of proteins associated with tumor formation and exposure to environmental estrogens.

White sucker (Catostomus commersonii) sampled from the Thunder Bay Area of Concern were assessed for health using a shotgun approach to compile proteomic profiles. Plasma proteins were sampled from male and female fish from a reference location, an area in recovery within Thunder Bay Harbour, and a site at the mouth of the Kaministiquia River where water and sediment quality has been degraded by industrial activities. The proteins were characterized using reverse-phase liquid chromatography tandem to a quadrupole-time-of-flight (LC-Q-TOF) mass spectrometer and were identified by searching in peptide databases. In total, 1086 unique proteins were identified. The identified proteins were then examined by means of a bioinformatics pathway analysis to gain insight into the biological functions and disease pathways that were represented and to assess whether there were any significant changes in protein expression due to sampling location. Female white sucker exhibited significant (p = 0.00183) site-specific changes in the number of plasma proteins that were related to tumor formation, reproductive system disease, and neurological disease. Male fish plasma had a significantly different (p < 0.0001) number of proteins related to neurological disease and tumor formation. Plasma concentrations of vitellogenin were significantly elevated in females from the Kaministiquia River compared to the Thunder Bay Harbour and reference sites. The protein expression profiles indicate that white sucker health has benefited from the remediation of the Thunder Bay Harbour site, whereas white sucker from the Kaministiquia River site are impacted by ongoing contaminant discharges.

Effluent monitoring at a bleached kraft mill: directions for best management practices for eliminating effects on fish reproduction.

A long-term monitoring study was conducted on effluents from a bleached kraft pulp and paper mill located in Eastern Canada. The study was designed to gain insights into temporal effluent variability with respect to fish reproduction as it related to production upsets, mill restarts and conditions affecting biological treatment performance. Final effluent quality was monitored between February 2007 and May 2009 using biochemical and chemical oxygen demand, total suspended solids, resin and fatty acids, a gas chromatographic profiling index, and the presence of methyl substituted 2-cyclopentenones. Selected effluent samples were evaluated for effects on fish reproduction (egg production) using a shortened version of the adult fathead minnow reproductive test. The events relating to negative effects on fish reproduction were upsets of the pulping liquor recovery system resulting in black liquor losses, operational upsets of the hardwood line resulting in the loss of oxygen delignification filtrates, and conditions that reduced the performance of biological treatment (e.g., mill shutdown and low ambient temperatures). The reductions in egg production observed in fathead minnow were associated with biochemical oxygen demand values > 20 mg/L, GC profiling indices > 1.2 and the presence of methyl-substituted 2-cyclopentenones at concentrations > 100 µg/L. This study demonstrated the importance of both in-plant measures for controlling the loss of organics as well as the optimum operation of biological effluent treatment for eliminating effluent-related effects on fish reproduction (egg production) in the laboratory.

A 30-Year Study of Impacts, Recovery, and Development of Critical Effect Sizes for Endocrine Disruption in White Sucker (Catostomus commersonii) Exposed to Bleached-Kraft Pulp Mill Effluent at Jackfish Bay, Ontario, Canada.

Jackfish Bay is an isolated bay on the north shore of Lake Superior, Canada that has received effluent from a large bleached-kraft pulp mill since the 1940s. Studies conducted in the late 1980s found evidence of reductions in sex steroid hormone levels in multiple fish species living in the Bay, and increased growth, condition and relative liver weights, with a reduction in internal fat storage, reduced gonadal sizes, delayed sexual maturation, and altered levels of circulating sex steroid hormones in white sucker (Catostomus commersonii). These early studies provided some of the first pieces of evidence of endocrine disruption in wild animals. Studies on white sucker have continued at Jackfish Bay, monitoring fish health after the installation of secondary waste treatment (1989), changes in the pulp bleaching process (1990s), during facility maintenance shutdowns and during a series of facility closures associated with changing ownership (2000s), and were carried through to 2019 resulting in a 30-year study of fish health impacts, endocrine disruption, chemical exposure, and ecosystem recovery. The objective of the present study was to summarize and understand more than 75 physiological, endocrine, chemical and whole organism endpoints that have been studied providing important context for the complexity of endocrine responses, species differences, and challenges with extrapolation. Differences in body size, liver size, gonad size and condition persist, although changes in liver and gonad indices are much smaller than in the early years. Population modeling of the initial reproductive alterations predicted a 30% reduction in the population size, however with improvements over the last couple of decades those population impacts improved considerably. Reflection on these 30 years of detailed studies, on environmental conditions, physiological, and whole organism endpoints, gives insight into the complexity of endocrine responses to environmental change and mitigation.

Endocrine Disruptor Impacts on Fish From Chile: The Influence of Wastewaters.

Industrial wastewaters and urban discharges contain complex mixtures of chemicals capable of impacting reproductive performance in freshwater fish, called endocrine-disrupting compounds (EDCs). In Chile, the issue was highlighted by our group beginning over 15 years ago, by analyzing the impacts of pulp and paper mill effluents (PPME) in the Biobio, Itata, and Cruces River basins. All of the rivers studied are important freshwater ecosystems located in the Mediterranean region of Central Chile, each with a unique fish biodiversity. Sequentially, we developed a strategy based on laboratory assays, semicontrolled-field experiments (e.g., caging) and wild fish population assessments to explore the issue of reproductive impacts on both introduced and native fish in Chile. The integration of watershed, field, and laboratory studies was effective at understanding the endocrine responses in Chilean freshwater systems. The studies demonstrated that regardless of the type of treatment, pulp mill effluents can contain compounds capable of impacting endocrine systems. Urban wastewater treatment plant effluents (WWTP) were also investigated using the same integrated strategy. Although not directly compared, PPME and WWTP effluent seem to cause similar estrogenic effects in fish after waterborne exposure, with differing intensities. This body of work underscores the urgent need for further studies on the basic biology of Chilean native fish species, and an improved understanding on reproductive development and variability across Chilean ecosystems. The lack of knowledge of the ontogeny of Chilean fish, especially maturation and sexual development, with an emphasis on associated habitats and landscapes, are impediment factors for their conservation and protection against the threat of EDCs. The assessment of effects on native species in the receiving environment is critical for supporting and designing protective regulations and remediation strategies, and for conserving the unique Chilean fish biodiversity.

Signatures of contamination in invasive round gobies (Neogobius melanostomus): a double strike for ecosystem health?

The invasive round goby has a recognised role in transferring contaminants through foodwebs, but little work has been done on contaminant impacts on round gobies themselves. Here we present the first case study of contaminant biomarkers and subpopulation structure variation in round gobies, in relation to habitat contamination, within a Canadian Area of Concern, Hamilton Harbour. Copper and cadmium were elevated in livers of fish from contaminated habitats. Although catch abundances were similar across sites, fish were smaller, a greater proportion of fish were female and more males were in reproductive condition in contaminated sites. Fish from contaminated areas showed more fin loss. Males from contaminated sites showed intersex gonads and genitalia. Ethoxyresorufin-o-deethylase (EROD) activity was higher in fish collected near polycyclic aromatic hydrocarbon (PAH)-rich sediments. The results indicate that contaminants impact the characteristics of round goby populations, which could affect ecosystems beyond toxicant biomagnification. This study also confirms that round gobies can be abundant in polluted habitats, which may draw predators-facilitating mobilisation of contaminants in foodwebs.

Fish Performance Indicators Adjacent to Oil Sands Activity: Response in Performance Indicators of Slimy Sculpin in the Steepbank River, Alberta, Adjacent to Oil Sands Mining Activity.

Since 2009, the Canadian and Alberta governments have been developing monitoring plans for surface water quality and quantity of the lower Athabasca River and its tributaries (2010-2013). The objectives of the present study to the fish monitoring program were to 1) assess the current status of fish in a tributary of the lower Athabasca River, 2) identify existing differences between upstream reference and within the oil sands deposit exposure sites, and 3) identify trends/changes in fish performance indicators relative to historical studies. The present study examines the fish performance indicators in slimy sculpin (Cottus cognatus) in the Steepbank River, Alberta, in terms of growth, gonad size, condition, and hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity as an indicator of exposure to oil-sands-related compounds. The sampling program followed historical sampling methods (1999-2000) to provide comparable data over time with an additional upstream site (n = 2) added as development progressed. Consistent changes were documented in sculpin collected from downstream sections of the Steepbank River within the oil sands deposit (n = 2) in 2010 through 2013. Sculpin demonstrated increased liver size with corresponding induction of EROD activity consistent with historical data and reductions in energy investment relative to reproductive development and gonadal steroid production capacity. There was no consistent evidence of changes in fish performance indicators with increased surface mining development, particularly adjacent to the Steepbank River Mid site. Although physical development in the Steepbank watershed has increased over the last 15 yr, these results are consistent with historical data suggesting that the magnitude of the response in the aquatic environment adjacent to the development has not changed. Environ Toxicol Chem 2020;39:396-409. © 2019 SETAC.

Polycyclic aromatic compounds (PACs) in the Canadian environment: A review of sampling techniques, strategies and instrumentation.

A wide variety of sampling techniques and strategies are needed to analyze polycyclic aromatic compounds (PACs) and interpret their distributions in various environmental media (i.e., air, water, snow, soils, sediments, peat and biological material). In this review, we provide a summary of commonly employed sampling methods and strategies, as well as a discussion of routine and innovative approaches used to quantify and characterize PACs in frequently targeted environmental samples, with specific examples and applications in Canadian investigations. The pros and cons of different analytical techniques, including gas chromatography - flame ionization detection (GC-FID), GC low-resolution mass spectrometry (GC-LRMS), high performance liquid chromatography (HPLC) with ultraviolet, fluorescence or MS detection, GC high-resolution MS (GC-HRMS) and compound-specific stable (δ13C, δ2H) and radiocarbon (Δ14C) isotope analysis are considered. Using as an example research carried out in Canada's Athabasca oil sands region (AOSR), where alkylated polycyclic aromatic hydrocarbons and sulfur-containing dibenzothiophenes are frequently targeted, the need to move beyond the standard list of sixteen EPA priority PAHs and for adoption of an AOSR bitumen PAC reference standard are highlighted.

In vivo solid-phase microextraction sampling combined with metabolomics and toxicological studies for the non-lethal monitoring of the exposome in fish tissue.

Various environmental studies have employed the biomonitoring of fish in their aquatic ecosystems in order to identify potential metabolic responses to the exposome. In this study, we applied in vivo solid-phase microextraction (SPME) to perform non-lethal sampling on the muscle tissue of living fish to extract toxicants and various endogenous metabolites. Sixty white suckers (Catastomus commersonii) were sampled from sites upstream, adjacent, and downstream from the oil sands development region of the Athabasca River (Alberta, Canada) in order to track their biochemical responses to potential contaminants. In vivo SPME sampling facilitated the extraction of a wide range of endogenous metabolites, mainly related to lipid metabolism. The obtained results revealed significant changes in the levels of numerous metabolites, including eicosanoids, linoleic acids, and fat-soluble vitamins, in fish sampled in different areas of the river, thus demonstrating SPME's applicability for the direct monitoring of exposure to different environmental toxicants. In addition, several classes of toxins, including petroleum-related compounds, that can cause serious physiological impairment were tentatively identified in the extracts. In vivo SPME, combined with the analysis of contaminants and endogenous metabolites, provided important information about the exposome; as such, this approach represents a potentially powerful and non-lethal tool for identifying the mechanisms that produce altered metabolic pathways in response to the mixtures of different environmental pollutants.

Developing Triggers for Environmental Effects Monitoring Programs for Trout-Perch in the Lower Athabasca River (Canada).

Baseline variations in trout-perch energy use (growth, gonad size) and energy storage (condition, liver size) were characterized between 2009 and 2015 in 8 reaches of the Athabasca River (AB, Canada), including 2 reaches upstream of the city of Fort McMurray (AB, Canada) and 6 reaches downstream of Fort McMurray among existing oil sands operations. Generalized linear models, used to account for background variation, indicated that fork length, gonad size, and liver size decreased, whereas body weight increased, in relation to river discharge, for both male and female trout-perch. Air temperature was positively correlated with liver size and negatively correlated with gonad size and body weight for females, but only positively correlated with gonad weight for males. These linear models explained approximately 20 to 25% of the variation in adjusted body size, and upward of 80% of the variation in adjusted body weight, liver weight, and gonad weight. Residuals from linear models were used to estimate normal ranges of variation for each of the fish population performance measures. Combined, the models and normal ranges can be used to assess subsequent monitoring data, providing potential triggers for follow-up monitoring activities. Environ Toxicol Chem 2019;38:0-0. Environ Toxicol Chem 2019;38:1890-1901. © 2019 SETAC.

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.