Search

The oil sands area of northern Alberta has river sediments that contain natural bitumen. Eggs and fish in these rivers may be exposed to bitumen-related chemicals early in life. This paper assesses a short embryo-larval fish exposure to oil sands sediment and follows the fish behaviour as they mature in clean water and examines their breeding success as adults (5 months afterwards). The three different oil sands river sediments tested were: a sediment collected outside of the bitumen deposit (tested at 3 g/L, Reference sediment from upstream Steepbank River site), and two sediments collected within the deposit (each tested at low (1 g/L) and high (3 g/L) concentrations). The sediments within the bitumen deposit were from the Ells and Steepbank (Stp) Rivers, and both contained significant total PAHs (>170 ng/g wet weight sediment) and alkylated PAHs (>4480 ng/g). Fish were exposed to these sediments for 21 days (as eggs and larval fish), and then transferred permanently to clean water to mature and breed. There was a significant decrease in the number of egg clutches produced by fish exposed early in life to Stp downstream high sediment (compared to Reference sediment). There was also a decrease in overall cumulative egg production, with fish from Stp downstream high sediment producing just over 1000 eggs in total while fish exposed to Ref sediment produced nearly 6900 eggs. The fish with reduced egg production were also less social than expected as they matured, and they had a lower % of early vitellogenic eggs in their ovaries. Overall, the exposure shows that a single, brief exposure during early life stages to natural bitumen can affect fish in adulthood. Naturally occurring bitumen-derived PAHs can reduce fish reproductive output by complex mechanisms, measurable as lower ovary maturity and changes in social behaviour., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ussery, McMaster, Servos, Miller and Munkittrick.)

Changes in ecosystems after the removal of stress provide a rich source of information for conservation science. We used a long-term regional data set on the performance of white sucker (Catostomus commersonii) collected before and after the closure of a pulp mill to explore recovery in fish. Physiological indicators, including liver enzymes and plasma steroids, showed some compelling changes after the closure of the mill consistent with reduced exposure to pulp mill effluent but did not unequivocally demonstrate recovery. However, persistent signals in these fish may indicate effects of impoundment or discharge of sewage. We also used quantile regression with environmental covariates and bootstrap iteration to determine if systematic variation remained in relative body weight, liver weight, and gonad weight. In fish formerly exposed to pulp mill effluent, we found evidence of improvements (male gonad weight and liver weight of males and females), degradation (gonad weight of females), and no change (body weight). Although the observed patterns may be associated with closure of the mill, some differences were also found at regional locations, suggesting roles of additional stressors and challenging the clear association of change at the Mattagami River exposure site with the closure of the mill. However, fish captured at this location show responses consistent with regional locations, suggesting no residual impacts and highlights the challenges of identifying changes in fish even after large and known interventions. Environ Toxicol Chem 2021;40:162-176. © 2020 SETAC., (© 2020 SETAC.)

We explored tissue concentrations of polychlorinated biphenyls (PCBs), chlorinated pesticides, and relevant organochlorines and fish health in the following adult wild fish in the St. Clair River Area of Concern (Ontario, Canada): shorthead redhorse (Moxostoma macrolepidotum), yellow perch (Perca flavescens), and emerald shiner (Notropis atherinoides). We collected adult fish from sites within the river's industrial zone (Stag Island), a downstream site adjacent to Walpole Island (Chenal Écarte), and an upstream reference site in Lake Huron in 2002/2003 and 2014. We tested for trends in tissue concentrations of organic contaminants across sites and over time; we assessed the potential effects of contaminants on morphological indicators of fish health across sites by year. Over the 12-year period, the tissue concentrations of most PCBs declined at the river sites, except for some non-legacy PCBs (PCB11 and 185), which increased in yellow perch at Stag Island, a new observation for fish in the St. Clair River AOC. There was little difference between the concentrations of calculated toxic equivalents (TEQs) of the Lake Huron and the St. Clair River fish in 2014, except for emerald shiners from Stag Island which had elevated ΣPCB and TEQs. Each fish species at all sites exceeded the Canadian tissue residue guideline for PCBs for the protection of mammalian wildlife consumers of aquatic biota, but fish-derived TEQs indicated little potential health risk to fish. Over time, hexachlorobutadiene and hexachlorobenzene concentrations increased in some fish at Stag Island by about 8- and 4-fold, respectively, whereas they decreased at other sampling locations. Principal Component Analysis followed by Linear Discriminant Analysis of the 2014 SHRH data suggested that although the fish separated by site, tissue concentrations of PCB and organochlorine contaminants did not have consistent relationships to the morphological health indicators, including egg production in females, which implied the absence of causative relationships., Competing Interests: Declaration of competing interest The authors declare that they do not have actual or potential conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 (δ 13 C, δ 2 H) and radiocarbon (Δ 14 C) 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

The St. Lawrence River, at Cornwall Ontario, has accumulated sediment contaminants, mainly mercury (Hg) and polychlorinated biphenyls (PCBs), from industrial point sources over many years. Although those sources are past, the river at Cornwall remains an Area of Concern (AOC). Because of remediation and other changes in the AOC, improved knowledge of contaminants in wild-fish and their putative links to health effects could help decision makers to better assess the AOC's state. Thus, we compared tissue concentrations of Hg, PCBs, morphometric measures of health, and biomarkers of exposure, metabolic-, and reproductive health in native brown bullhead (Ameiurus nebulosus) from the AOC to those of upstream reference fish. Linear discriminant analysis separated the adult fish of both sexes among upstream and downstream sites without misclassification. Burdens of total-Hg (all sites) and PCB toxic equivalents (downstream sites) exceeded the guidance for the protection of wildlife consumers. There were subtle effects of site on physiological variables, particularly in female fish. Total-Hg in tissue correlated negatively to plasma testosterone and 17β-estradiol in female fish at Cornwall: moreover, concentrations of both hormones were lower within the AOC compared to reference site fish. A similar effect on vitellogenin, which was uncorrelated to E2/T at the downstream sites, indicated the potential for reproductive effects. Downstream fish also had altered thyroidal status (T 3 , TSH, and ratio of thyroid epithelial cell area to colloid area). Despite spatial and temporal variability of the endocrine-related responses, these subtle effects on fish health within the AOC warrant further study.

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., (© 2019 SETAC.)

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.

In this study fathead minnow ( Pimephales promelas ) embryo-larval stages were exposed to two oil sands tailings pond sediments which had previously been shown to decrease the survival of embryo-larval larval stages of walleye ( Sander vitreus ) and northern pike ( Esox lucius ). Fathead minnow are standard test species and we wanted to compare their sensitivity to the other two species. Fathead minnow larvae were exposed for 20 days (5 days in the egg stage and 15 days in the larval stage) with daily renewal of sediments and waters. Sediments contained polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs (APAHs). Results from an earlier study showed that Sediment 1 contained 173 μg/g total PAHs + APAHs (97 % alkylated), and sediment 2 contained 401 μg/g total PAHs + APAHs (95 % alkylated). Fathead minnow larvae exposed to oil sands tailings pond sediments had decreased survival, decreased weight, and increased deformities. Fathead minnow survival was unaffected at the embryo stage and at hatch. Most deaths occurred at the larval stages 1-8 days after hatching, showing the importance of exposing the fish for at least a week after hatch. Toxicity was seen at 0.2 g/L of sediment, which was equivalent to the addition of 35 and 80 μg total PAHs + APAHs to 1 L of overlying water for sediment 1 and 2, respectively. When compared to embryo-larval northern pike and walleye results from previous studies, all three species of fish responded more strongly to sediment 2 compared to sediment 1. For effects on lethality, fathead minnow were equally sensitive to pike, but walleye were 5-28 times more sensitive to the lethal effects of the sediments compared to both fathead minnow and pike. The study (and comparisons to our previous studies) shows the difference in sensitivity between a model laboratory species (fathead minnow) and some species of wild fish that are highly relevant to the oil sands area of Alberta., (© 2019 Published by Elsevier Ltd.)

The aim of this study was to investigate the long-term effects of a short exposure to natural sediments within the Athabasca oil sand formation to critical stages of embryo-larval development in fathead minnows (Pimephales promelas). Three different sediments were used: Ref sediment from the upper Steepbank River tested at 3 g/L (containing 12.2 ng/g ∑PAHs), and two bitumen-rich sediments tested at 1 and 3 g/L; one from the Ells River (Ells downstream, 6480 ng/g ∑PAHs) and one from the Steepbank River (Stp downstream, 4660 ng/g ∑PAHs). Eggs and larvae were exposed to sediments for 21 days, then transferred to clean water for a 5-month grow-out and recovery period. Larval fish had significantly decreased survival after exposure to 3 g/L sediment from Stp downstream, and decreased growth (length and weight at 16 days post hatch) in Ells and Stp downstream sediments at both 1 and 3 g/L. Decreased tail length was a sensitive endpoint in larval fish exposed to Ells and Stp downstream sediments for 21 days compared to Ref sediment. After the grow-out in clean water, all growth effects from the bitumen-containing sediments recovered, but adult fish from Stp downstream 3 g/L sediment had significant increases in jaw deformities. The study shows the potential for fish to recover from the decreased growth effects caused by sediments containing oil sands-related compounds, but that some effects of the early-life sediment exposure occur later on in adult fish., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Although discovered more than a century ago, piscine rodlet cells (RCs) remain somewhat of a mystery to scientists in terms of their origin and function. Initially described as parasites, and later as potential secretory cells, the prevailing theory is that RCs are leucocyte-like cells that possess pathogen defence capabilities. The current case report involves a novel type of neoplasm discovered in the livers of two adult female white suckers (Catostomus commersonii) that were collected as part of a survey of fish from the St. Mary's River Area of Concern, in which sediment contaminated by polyaromatic hydrocarbons has been associated historically with a high prevalence of liver neoplasms in white suckers. The two tumours in this study were investigated by light microscopy, histochemical staining, immunohistochemical labelling for S100 protein and transmission electron microscopy. The evidence from these investigations suggests that these neoplasms may be derived from de-differentiated RCs or RC precursors. The unanticipated existence of these solid mesenchymal-like tumours may prompt a reassessment of the current dogma regarding the physiological function of RCs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: It has been postulated that swimming in heated indoor swimming pools in the first year of life is associated with the development of spinal deformity in children. We explored in pup mice whether exposure to certain disinfection by-products resulting from chlorination of heated pools would affect the future development of the spinal column., Methods: Mice, from birth and for 28 consecutive days, were exposed to chemicals known to be created by disinfection by-products of indoor heated swimming pools. The study made use of a body fluid analogue and a chlorine source to recreate the conditions found in municipal pools. A cohort of 51 wild-type C57B6 mice, male and female, were divided into two groups: experimental (n = 29) and controls (n = 22). 24 mice were observed for 8 months (32 weeks), with 27 culled at 4 months (16 weeks). Serial CT scanning was used to assess the spines., Results: Exposure to disinfection by-products resulted in an increase in the normal thoracic kyphotic spinal angle of the mice when compared with their controls at 10 weeks; experimental mice kyphosis range 35-82° versus 29-38° in controls. At 14 weeks the kyphosis of the experimental mice had reduced in size but never to that of the control group., Conclusion: We have demonstrated the ability to influence spinal development in pup mice through environmental factors and shown that the developmental deformity became evident only after a significant latent period., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

To assess the toxicity of winter-time atmospheric deposition in the oil sands mining area of Northern Alberta, embryo-larval fathead minnow (Pimephales promelas) were exposed to snowmelt samples. Snow was collected in 2011-2014 near (<7km) oil sands open pit mining operations in the Athabasca River watershed and at sites far from (>25km) oil sands mining. Snow was shipped frozen back to the laboratory, melted, and amended with essential ions prior to testing. Fertilized fathead minnow eggs were exposed (<24h post-fertilization to 7-16days post-hatch) to a range of 25%-100% snowmelt. Snow samples far from (25-277km away) surface mining operations and upgrading facilities did not affect larval fathead minnow survival at 100%. Snow samples from sites near surface mining and refining activities (<7km) showed reduced larval minnow survival. There was some variability in the potencies of snow year-to-year from 2011 to 2014, and there were increases in deformities in minnows exposed to snow from 1 site on the Steepbank River. Although exposure to snowmelt from sites near oil sands surface mining operations caused effects in larval fish, spring melt water from these same sites in late March-May of 2010, 2013 and 2014 showed no effects on larval survival when tested at 100%. Snow was analyzed for metals, total naphthenic acid concentrations, parent PAHs and alkylated PAHs. Naphthenic acid concentrations in snow were below those known to affect fish larvae. Concentrations of metals in ion-amended snow were below published water quality guideline concentrations. Compared to other sites, the snowmelt samples collected close to mining and upgrading activities had higher concentrations of PAHs and alkylated PAHs associated with airborne deposition of fugitive dusts from mining and coke piles, and in aerosols and particles from stack emissions., Capsule: Snow collected close to oil sands surface mining sites is toxic to larval fathead minnows in the lab; however spring melt water samples from the same sites do not reduce larval fish survival., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

The present study examined in vitro 11-ketotestosterone and testosterone production by the testes of rainbow darter (Etheostoma caeruleum) collected from selected reference sites and downstream of 2 municipal wastewater treatment plants (MWWTPs; Waterloo and Kitchener) on the central Grand River (Ontario, Canada), over a 6-yr period (2011-2016). The main objective was to investigate if infrastructure upgrades at the Kitchener MWWTP in 2012 resulted in a recovery of this response in the post-upgrade period (2013-2016). Two supporting studies showed that the fall season is appropriate for measuring in vitro sex steroid production because it provides stable detection of steroid patterns, and that the sample handling practiced in the present study did not introduce a bias. Infrastructure upgrades of the Kitchener MWWTP resulted in significant reductions in ammonia and estrogenicity. After the upgrades, 11-ketotestosterone production by MWWTP-exposed fish increased in 2013 and it continued to recover throughout the study period of 2014 through 2016, returning to levels measured in reference fish. Testosterone production was less sensitive and it lacked consistency. The Waterloo MWWTP underwent some minor upgrades but the level of ammonia and estrogenicity remained variable over time. The production of 11-ketotestosterone and testosterone in rainbow darter below the Waterloo MWWTP was variable and without a clear recovery pattern over the course of the present study. The results of the present study demonstrated that measuring production of sex steroids (especially 11-ketotestosterone) over multiple years can be relevant for assessing responses in fish to environmental changes such as those resulting from major infrastructure upgrades. Environ Toxicol Chem 2018;37:501-514. © 2017 SETAC., (© 2017 SETAC.)

Concentrations of halogenated phenolic compounds were measured in the plasma of brown bullhead (Ameiurus nebulosus) from 4 Canadian Areas of Concern (AOCs), to assess exposure to suspected thyroid-disrupting chemicals. Hydroxylated polychlorinated biphenyls (OH-PCBs) were detected in every sample collected in 3 of the AOCs; the detection frequency was lower in samples from the Detroit River AOC. The OH-PCBs most frequently detected were pentachloro, hexachloro, and heptachloro congeners, which are structurally similar to thyroid hormones. Pentachlorophenol (PCP) was detected at highest concentrations (1.8 ng/g) in fish from Prince Edward Bay, the Bay of Quinte Lake reference site, and Hillman Marsh (the Wheatley Harbour reference site), suggesting local sources of contamination. Elevated PCP concentrations were also detected in the plasma of brown bullhead from exposed sites in the Toronto and Region AOC (0.4-0.6 ng/g). Triclosan was consistently detected in the Toronto and Region AOC (0.05-0.9 ng/g), consistent with wastewater emission. Greater concentrations were occasionally detected in the plasma of brown bullhead from the Bay of Quinte AOC. Concentrations of polybrominated diphenyl ethers were highest in the Toronto and Region AOC, and at 2 of the Bay of Quinte AOC exposed sites near Trenton and Belleville. Distribution patterns reflected the properties and usage of the compounds under investigation and the characteristics of each AOC. Environ Toxicol Chem 2017;36:2266-2273. © 2017 SETAC., (© 2017 SETAC.)

The present study measured hepatic transcriptome responses in male rainbow darter (Etheostoma caeruleum) exposed to 2 municipal wastewater-treatment plants (MWWTPs; Kitchener and Waterloo) over 4 fall seasons (2011-2014) in the Grand River (Ontario, Canada). The overall goal was to determine if upgrades at the Kitchener MWWTP (in 2012) resulted in transcriptome responses indicative of improved effluent quality. The number of differentially expressed probes in fish downstream of the Kitchener outfall (904-1223) remained comparable to that downstream of Waterloo (767-3867). Noteworthy was that year and the interaction of year and site explained variability in more than twice the number of transcripts than site alone, suggesting that year and the interaction of year and site had a greater effect on the transcriptome than site alone. Gene set enrichment analysis revealed a gradual reduction in the number of gene ontologies over time at exposure sites, which corresponded with lower contaminant load. Subnetwork enrichment analysis revealed that there were noticeable shifts in the cell pathways differently expressed in the liver preupgrade and postupgrade. The dominant pathways altered preupgrade were related to genetic modifications and cell division, whereas postupgrade they were associated with the immune system, reproduction, and biochemical responses. Molecular pathways were dynamic over time, and following the upgrades, there was little evidence that gene expression profiles in fish collected from high-impact sites postupgrade were more similar to those in fish collected from reference site. Environ Toxicol Chem 2017;36:2108-2122. © 2017 SETAC., (© 2017 SETAC.)

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 BOD 5 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 BOD 5 and GC/MS profiles. Effluent treatment systems across all production types were generally efficient, achieving a combined 82-98% BOD 5 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.

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.

Intersex is a condition that has been associated with exposure to sewage effluents in male rainbow darter (Etheostoma caeruleum). To better understand changes in the transcriptome that are associated with intersex, we characterized annual changes in the testis transcriptome in wild, unexposed fish. Rainbow darter males were collected from the Grand River (Ontario, Canada) in May (spawning), August (post-spawning), October (recrudescence), January (developing) and March (pre-spawning). Histology was used to determine the proportion of spermatogenic cell types that were present during each period of testicular maturation. Regression analysis determined that the proportion of spermatozoa versus spermatocytes in all stages of development (R2 ≥ 0.58) were inversely related; however this was not the case when males were in the post-spawning period. Gene networks that were specific to the transition from developing to pre-spawning stages included nitric oxide biosynthesis, response to wounding, sperm cell function, and stem cell maintenance. The pre-spawning to spawning transition included gene networks related to amino acid import, glycogenesis, Sertoli cell proliferation, sperm capacitation, and sperm motility. The spawning to post-spawning transition included unique gene networks associated with chromosome condensation, ribosome biogenesis and assembly, and mitotic spindle assembly. Lastly, the transition from post-spawning to recrudescence included gene networks associated with egg activation, epithelial to mesenchymal transition, membrane fluidity, and sperm cell adhesion. Noteworthy was that there were a significant number of gene networks related to immune system function that were differentially expressed throughout reproduction, suggesting that immune network signalling has a prominent role in the male testis. Transcripts in the testis of post-spawning individuals showed patterns of expression that were most different for the majority of transcripts investigated when compared to the other stages. Interestingly, many transcripts associated with female sex differentiation (i.e. esr1, sox9, cdca8 and survivin) were significantly higher in the testis during the post-spawning season compared to other testis stages. At post-spawning, there were higher levels of estrogen and androgen receptors (esr1, esr2, ar) in the testis, while there was a decrease in the levels of sperm associated antigen 1 (spag1) and spermatogenesis associated 4 (spata4) mRNA. Cyp17a was more abundant in the testis of fish in the pre-spawning, spawning, and post-spawning seasons compared to those individuals that were recrudescent while aromatase (cyp19a) did not vary in expression over the year. This study identifies cell process related to testis development in a seasonally spawning species and improves our understanding regarding the molecular signaling events that underlie testicular growth. This is significant because, while there are a number of studies characterizing molecular pathways in the ovary, there are comparatively less describing transcriptomic patterns in the testis in wild fish., Competing Interests: The authors have declared that no competing interests exist.

Municipal wastewater effluent (MWWE) and its constituents, such as chemicals of emerging concern, pose a potential threat to the sustainability of fish populations by disrupting key endocrine functions in aquatic organisms. While studies have demonstrated changes in biological markers of exposure of aquatic organisms to groups of chemicals of emerging concern, the variability of these markers over time has not been sufficiently described in wild fish species. The aim of this study was to assess the spatial and temporal variability of biological markers in response to MWWE exposure and to test the consistency of these responses between seasons and among years. Rainbow darter (Etheostoma caeruleum) were collected in spring and fall seasons over a 5-year period in the Grand River, Ontario, Canada. In addition to surface water chemistry (nutrients and selected pharmaceuticals), measures were taken across levels of biological organization in rainbow darter. The measurements of hormone production, gonad development, and intersex severity were temporally consistent and suggested impaired reproduction in male fish collected downstream of MWWE outfalls. In contrast, ovarian development and hormone production in females appeared to be influenced more by urbanization than MWWE. Measures of gene expression and somatic indices were highly variable between sites and years, respectively, and were inconclusive in terms of the impacts of MWWE overall. Robust biomonitoring programs must consider these factors in both the design and interpretation of results, especially when spatial and temporal sampling of biological endpoints is limited. Assessing the effects of contaminants and other stressors on fish in watersheds would be greatly enhanced by an approach that considers natural variability in the endpoints being measured., Competing Interests: The authors have declared that no competing interests exist.

Study Design: Prospective observational study., Objective: To evaluate the effect of internal thoracoplasty and anterior spine release combined with posterior spinal instrumentation in correcting and preventing a reoccurrence of the rib cage deformity in adolescent idiopathic thoracic scoliosis >70 degrees., Summary of Background Data: The rib cage deformity rather than the lateral curvature of the spine is usually the major cosmetic deformity in severe adolescent idiopathic thoracic scoliosis. This can be difficult to treat and assess the effect of surgery., Methods: The Integrated Shape Imaging System was used to assess rib cage deformity before surgery and during follow-up for more than 2 years in 37 patients with an adolescent idiopathic thoracic scoliosis (Lenke 1) >70° (mean 81°) treated by internal thoracoplasty and anterior spine release combined with posterior spinal instrumentation using a hybrid construct., Results: The mean Cobb angle was reduced from 81° to 30° (63% correction). The rib cage deformity was improved in 30 patients (81%) because of a combination of both a reduction of the convex rib hump and an elevation of the concave rib depression. However, the effect on patients with an angle of thoracic inclination <16° was not as reliable. One year after surgery, additional improvement of the chest deformity had occurred in 9 patients. In none of the 37 patients was there any further progression of the rib cage deformity., Conclusion: An internal thoracoplasty was effective in improving and/or stabilizing the rib cage deformity as well as achieving good correction of the scoliosis in patients with adolescent idiopathic thoracic scoliosis and severe deformity., (Copyright © 2016 Scoliosis Research Society. Published by Elsevier Inc. All rights reserved.)

Improved regulations for pulp and paper mill effluents and an industry shift away from elemental chlorine bleaching in the 1990s greatly reduced the release of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) into the environment. However, the high potential of these contaminants to persist in sediment and bioaccumulate in biota means that they have remained a concern. To document current contamination from bleached kraft pulp mill effluent, PCDD/Fs were measured in white sucker (Catostomus commersoni) collected from Jackfish Bay, Lake Superior. These values were contrasted to historically reported fish data as well as PCDD/F patterns from dated sediment cores. Patterns of PCDD/Fs in sediment cores from Jackfish Bay and reference sites demonstrated a relationship between contamination and mill process changes. During the peak PCDD/F contamination period (1991), when the mill was still using elemental chlorine, the contamination patterns in fish and sediment were distinct and dominated by 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran. Following the reduction in the use of elemental chlorine during the early 1990s, a rapid decline was observed in PCDD/F contamination of fish tissue, and levels are now approaching background conditions with congener patterns more reflective of atmospheric sources. Although surface sediments from Jackfish Bay continue to have elevated PCDD/Fs, with some locations exceeding sediment quality guidelines, they do not appear to be highly bioavailable to benthic fish., (© 2015 SETAC.)

Recovery of fish and wildlife populations after stressor mitigation serves as a basis for evaluating remediation success. Unfortunately, effectively monitoring population status on a routine basis can be difficult and costly. In the present study, the authors describe a framework that can be applied in conjunction with field monitoring efforts (e.g., through effects-based monitoring programs) to link chemically induced alterations in molecular and biochemical endpoints to adverse outcomes in whole organisms and populations. The approach employs a simple density-dependent logistic matrix model linked to adverse outcome pathways (AOPs) for reproductive effects in fish. Application of this framework requires a life table for the organism of interest, a measure of carrying capacity for the population of interest, and estimation of the effect of stressors on vital rates of organisms within the study population. The authors demonstrate the framework using linked AOPs and population models parameterized with long-term monitoring data for white sucker (Catostomus commersoni) collected from a study site at Jackfish Bay, Lake Superior, Canada. Individual responses of fish exposed to pulp mill effluent were used to demonstrate the framework's capability to project alterations in population status, both in terms of ongoing impact and subsequent recovery after stressor mitigation associated with process changes at the mill. The general approach demonstrated at the Jackfish Bay site can be applied to characterize population statuses of other species at a variety of impacted sites and can account for effects of multiple stressors (both chemical and nonchemical) and dynamics within complex landscapes (i.e., meta-populations including emigration and immigration processes)., (© 2015 SETAC.)

Municipal wastewater effluent (MWWE) contains emerging contaminants such as pharmaceuticals and personal care products (PPCPs) that have estrogenic properties. PPCPs are thought to be responsible for feminization of male fish in heavily urbanized areas around the globe. While many observations of feminized male fish have been made, the impact of feminization on reproductive success is not well understood. To address this lack of knowledge of the impacts of feminization, we examined the reproductive fitness of rainbow darter (Etheostoma caeruleum), a fish that is also known to have been feminized in some reaches of the Grand River, Kitchener-Waterloo, Ontario, Canada. In order to assess their reproductive health, somatic indices, gonadal steroid production, fecundity, and histological severity of intersex were measured in male rainbow darter collected through an urban gradient. Reproductive fitness was assessed by stripping milt and eggs from wild spawning fish, fertilizing eggs manually, and rearing embryos to hatch. The fertilization success and survival of embryos to hatch were compared among sites. In this study, it was found that rainbow darter collected at sites near a large municipal wastewater treatment plant (MWWTP) had decreased gonad size, increased severity of intersex, and decreased androgen production relative to other sites. Fish collected near the largest MWWTP also had lower fertilization success and survival to hatch. In contrast, fish collected near a second MWWTP farther upstream had comparable fertilization success, but lower survival to hatch relative to the upstream rural reference site. Intersex severity was negatively correlated with fertilization success, but not survival to hatch, suggesting that intersex is a good indicator of a population's fertilization success. Further investigation is required in order to determine if feminization will impact the sustainability of wild populations of fish., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To our knowledge there are no publications that have evaluated physical activities in relation to the etiopathogenesis of adolescent idiopathic scoliosis (AIS) other than sports scolioses. In a preliminary longitudinal case-control study, mother and child were questioned and the children examined by one observer. The aim of the study was to examine possible risk factors for AIS. Two study groups were assessed for physical activities: 79 children diagnosed as having progressive AIS at one spinal deformity centre (66 girls, 13 boys) and a Control Group of 77 school children (66 girls, 11 boys), the selection involving six criteria. A structured history of physical activities was obtained, every child allocated to a socioeconomic group and examined for toe touching. Unlike the Patients, the Controls were not X-rayed and were examined for surface vertical spinous process asymmetry (VSPA). Statistical analyses showed progressive AIS to be positively associated with social deprivation, early introduction to indoor heated swimming pools and ability to toe touch. AIS is negatively associated with participation in dance, skating, gymnastics or karate and football or hockey classes, which might suggest preventive possibilities. There is a significantly increased independent odds of AIS in children who went to an indoor heated swimming pool within the first year of life (odds ratio 3.88, 95% CI 1.77-8.48; p = 0·001). Furthermore fourteen (61%) Controls with VSPA compared with 9 (17%) Controls without VSPA had been introduced to the swimming pool within their first year of life (P < 0.001). Early exposure to indoor heated swimming pools for both AIS and VSPA, suggests that the AIS findings do not result from sample selection.

Rainbow darter (Etheostoma caeruleum; RBD) is a small benthic fish found in North America. This species is sensitive to sewage effluent, and intersex is found in up to 80% of males in near-field areas in the Grand River, Ontario, Canada. To learn more about the molecular signaling cascades associated with intersex, a developed customized oligonucleotide microarray (4×180 K) using next generation sequencing was developed to characterize the transcriptome in the gonad of male and female RBD. Gene expression profiling was performed in males and females from both a reference site and a polluted site. Males with and without intersex condition from the areas closest to effluent outfalls were compared to males and females from a reference site. Microarray analysis revealed that there was increased mRNA abundance for genes associated with oogenesis in intersex males (i.e. the presence of eggs within the testis), and a decrease in mRNA abundance for genes associated with spermatid development. In females exposed to effluent, cell processes related with hatching and ovulation were down-regulated, and genes involved in immune responses were increased in abundance. In the non-intersex males exposed to effluent, cell processes such as sperm cell adhesion were decreased at the transcript level relative to males from the reference site. Microarray analysis revealed that heat shock proteins (HSP) were significantly increased in non-intersex males exposed to effluent; however, HSPs were not differentially expressed in intersex males exposed to the effluent. Genes involved in sex differentiation (sox9, foxl2 and dmrt1) and reproduction (esr1, esrb, ar, vtg, cyp19a1 and cyp11a) were measured in males, females, and intersex individuals. Consistent with the intersex condition, many transcripts showed an intermediate expression level in intersex males when compared to phenotypic males and females. This study improves our knowledge regarding the molecular pathways that underlie the intersex condition and develops a suite of qPCR bioassays in RBD that are able to discriminate pollutant-exposed males without intersex from those males with intersex. Part A of this study reports on the effects of municipal wastewater effluents (MWWEs) on RBD in the Grand River and demonstrates that there are disruptions in higher level endpoints that include altered steroid levels. Here we develop a new tool for assessing and monitoring the intersex condition in RBD in polluted natural environments and begin to characterize gene networks that are associated with the condition., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Municipal wastewater effluents (MWWEs) contain anthropogenic substances that can exhibit endocrine-disrupting activity. These complex mixtures have been observed to exert adverse effects on fish. Rainbow darter (Etheostoma caeruleum, RBD) is a small benthic fish that is widespread throughout the Grand River, Ontario, Canada, and has been previously shown to be adversely affected by MWWE exposure in this watershed. The objectives of this study were to quantify biological responses in this sentinel species and intersex severity in male fish, in relation to the area of urbanization. It focused on RBD populations adjacent to wastewater outfalls in the Grand River watershed. In May 2011, nine sites across the urban gradient were selected to evaluate the impact of MWWEs. Endpoints for energy storage (i.e. condition factor, k; liversomatic index, LSI) as well as reproductive endpoints (i.e. gonadosomatic index, GSI; gonad development, hormone production), and intersex were assessed in the fish. Rainbow darter showed a high incidence of intersex downstream of the wastewater outfalls, especially below the largest treatment plant outfall at Kitchener (∼85%). We applied an intersex index (score from 0 to 7) that considers the number of eggs within the testis and the stage of maturation of the egg. RBD exposed downstream of the largest wastewater outfall at Kitchener had a score of 3.81±0.37 compared to upstream to the urban areas where there were no intersex males found other than a single individual with a score 1 (average intersex score of site 0.06±0.06). In addition, several fish associated with the Kitchener outfall had macroscopic vitellogenic eggs in the testes (intersex scores 5 and 6). The sub-population of fish located at the wastewater outfall also showed a tendency towards skewed sex ratios (greater proportion of females to male fish) compared to the population at the reference sites. Male fish inhabiting the urbanized area of the Grand River also showed reduced levels of testosterone (T) and 11-ketotestosterone (11KT). Intersex males had the lower levels of 11KT relative to the upstream reference fish but could not be distinguished from normal males collected at the exposed sites. Despite the high levels of intersex at these sites, no relationships were evident among intersex severity and other measured endpoints such as GSI, LSI or in vitro steroid production. The effects observed appear to be associated with urbanization and exposure to treated MWWEs in the watershed. Although intersex incidence and severity was a very good indicator of wastewater exposure, intersex could not be directly linked to other effects in this wild population. The effects of MWWEs on transcriptional changes in adult RBD exposed to the effluents are reported in the corresponding report (Part B)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The Bay of Quinte (BOQ) is an Area of Concern listed under the Great Lakes Water Quality Agreement. The presence of dioxins and dioxin-like PCBs in fish in the BOQ AOC has led to restrictions on fish consumption by humans, which is a beneficial use impairment. Adult yellow perch (Perca flavescens) and brown bullhead (Ameiurus nebulosus) were sampled from Trenton, Belleville, and Deseronto (reference site) in the BOQ. A suite of hormone assays and various measures of exposure and/or sublethal health effects were used to assess the health status of fish of both species and sex. Condition factor, hepatosomatic index, ethoxyresorufin-O-deethylase activity, circulating steroid and thyroid hormones, thyroid activation, oocyte size distribution, spermatogenic cell stages, and plasma vitellogenin were among the endpoints that were significantly (p < 0.05) affected by location. Many of those effects corresponded with significantly (p < 0.05) greater tissue concentrations of polychlorinated biphenyls (PCBs) at Belleville and Trenton. Hepatic extracts from brown bullhead sampled from Trenton had significantly (p < 0.05) greater binding activity to the androgen receptor and sex steroid binding protein. Taken together, these data and preliminary data from a concomitant study suggest that PCBs are likely being hydroxylated in vivo, resulting in enhanced bioactivity at endocrine receptors and measurable health responses. The present study supports the growing body of evidence that PCBs and their metabolites can affect fish thyroid and steroid hormone systems., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

The present study describes the seasonal changes in reproductive endpoints of the greenside darter (Etheostoma blennioides) and its implications for environmental monitoring. Fish collections conducted at the appropriate time for the site-specific sentinel fish species can provide a wide variety of population-level information including recruitment, reproduction, and energy storage. The objectives of the present study were to: 1) characterize seasonal changes in reproductive endpoints of the greenside darter (both sexes) to determine the appropriate period for monitoring of this sentinel species; and 2) evaluate the effect of exposure of this sentinel species to tertiary treated municipal effluent at the selected monitoring period. Based on the selected parameters (gonadosomatic index [GSI], liver somatic index [LSI], condition factor, and in vitro gonadal steroid production [testosterone (T) in both sexes; estradiol (E2) in females; and 11-ketotestosterone (11KT) in males]), the present study provides evidence for the value of collecting darters during recrudescence (late fall/early winter) to ensure temporal stability, minimum variability, and stable steroid production capacity. Darters exposed to tertiary treated municipal effluent tended to be larger and heavier relative to reference fish but did not demonstrate any consistent responses in terms of condition or relative liver size. No effect on gonadal development was observed, even though these tertiary-effluent-exposed fish demonstrated a significant reduction in the ability to produce hormones. The present study suggests that although fish exposed to tertiary treated effluent demonstrate no population-level effects, they are still responding at a physiological level. Documentation of the reproductive cycle of sentinel species allows for selection of the most appropriate sampling period to reduce variability and greatly enhances the reliability and interpretation of biological responses., (© 2014 SETAC.)

Endocrine-disrupting chemicals are exogenous substances that can impact the reproduction of fish, potentially by altering circulating concentrations of 17β-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT). Common methods to measure steroids in plasma samples include radioimmunoassays (RIAs) and enzyme-linked immunosorbant assays (ELISAs). The present study examines variability in E2, T, and 11-KT across 8 laboratories measuring reference and pulp mill effluent-exposed white sucker (Catostomus commersoni) plasma. We examine the contribution of assay type (RIA vs ELISA), standardized hormone extraction, location of values on the standard curve (upper and lower limits), and other variables on the ability to distinguish hormone levels between reference and exposed fish and the impact of these variables on quantitation of hormones in different laboratories. Of the 8 participating laboratories, 7 of 8 and 7 of 7 identified differences between sites for female E2 and female T, respectively, and 7 of 7 and 4 of 5 identified no differences between male T and male 11-KT. Notably, however, the ng/mL concentration of steroids measured across laboratories varied by factors of 10-, 6-, 14-, and 10-fold, respectively. Within laboratory intra-assay variability was generally acceptable and below 15%. Factors contributing to interlaboratory variability included calculation errors, assay type, and methodology. Based on the interlaboratory variability detected, we provide guidelines and recommendations to improve the accuracy and precision of steroid measurements in fish ecotoxicology studies., (© 2013 SETAC.)

Rainbow darter (Etheostoma caeruleum) is a small benthic fish species found in North America that are abundant and distributed throughout the Grand River watershed, ON, Canada. Rainbow darter exhibit intersex in males at sites adjacent to municipal wastewater effluents (MWWE). In October 2010, female and male rainbow darter were collected at 3 sites (1 upstream reference and 2 downstream exposed sites) in the Grand River near the cities of Kitchener and Waterloo. The primary objectives of this research were (1) to characterize the responses of whole organism endpoints (i.e. condition factor (K), liversomatic (LSI) and gonadosomatic index (GSI), histopathology) to MWWEs and (2) to identify transcripts showing altered steady state abundance with exposure to MWWE in fish inhabiting municipal wastewater effluent-exposed areas. Genes measured in this study included vitellogenin, Sry-box containing protein 9 (sox9), forkhead box L2 (foxl2), doublesex and mab-3 related transcription factor 1 (dmrt1), cytochrome P450, family 11, subfamily A, polypeptide 1 (cyp11a) as well as estrogen (esr1, esrb) and androgen (ar) receptors. There were no changes in condition factor; however, there was a significant increase in LSI and a decrease in GSI in fish inhabiting downstream environments when compared with fish collected from the reference site. Males had a high incidence (∼ 70%) of intersex in downstream sites; characterized by the presence of oocytes within the testis. In the gonad, there were sex specific differences for genes related to sexual differentiation; dmrt1 was only expressed in males whereas foxl2 and sox9 were highly expressed in females compared to males. Expression levels of ar and esr1 were higher in females than males. Conversely, esrb was not differentially expressed between sexes or among sites. There were no differences detected for the genes investigated within sex among sites. This study is the first to report on gene expression changes in the rainbow darter, with emphasis on the differences in transcript abundance between sexes and how these changes relate to exposures to MWWEs. Molecular approaches are being investigated for their potential application to field ecotoxicology, and molecular bioassays for relevant, sentinel species in environmental monitoring programs are required to better understand the impact of anthropogenic impacts on species at risk in river systems., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The variability and extent of the intersex condition (oocytes in testes, or testis-ova) was documented in fish along an urban gradient in the Grand River, Ontario, Canada, that included major wastewater treatment plant outfalls. A method for rapid enumeration of testis-ova was developed and applied that increased the capacity to quantify intersex prevalence and severity. Male rainbow darters (Etheostoma caeruleum) sampled downstream of the first major wastewater outfall (Waterloo) had a significant increase, relative to 4 upstream reference sites, in the mean proportion of fish with at least 1 testis-oocyte per lobe of testes (9-20% proportion with ≤ 1 testis-oocyte/lobe vs 32-53% and >1.4 testis-oocyte/lobe). A much higher mean incidence of intersex proportion and degree was observed immediately downstream of the second wastewater outfall (Kitchener; 73-100% and 8-70 testis-oocyte/lobe); but only 6.3 km downstream of the Kitchener outfall, the occurrence of intersex dropped to those of the reference sites. In contrast, downstream of a tertiary treated wastewater outfall on a small tributary, intersex was similar to reference sites. Estrogenicity, measured using a yeast estrogen screen, followed a similar pattern, increasing from 0.81 ± 0.02 ng/L 17b-estradiol equivalents (EEq) (Guelph), to 4.32 ± 0.07 ng/L (Waterloo), and 16.99 ± 0.40 ng/L (Kitchener). Female rainbow darter downstream of the Kitchener outfall showed significant decreases in gonadosomatic index and liver somatic index, and increases in condition factor (k) relative to corresponding reference sites. The prevalence of intersex and alterations in somatic indices suggest that exposure to municipal wastewater effluent discharges can impact endocrine function, energy use, and energy storage in wild fish., (Copyright © 2013 SETAC.)

Municipalities utilize aquatic environments to assimilate their domestic effluent resulting in eutrophication, anoxia, toxicity and endocrine disruption of aquatic biota. The objective of this study was to assess the potential cumulative impacts of municipal wastewater effluent (MWWE) discharges in the Grand River on the health status of a sentinel species and the fish community downstream of 2 MWWE discharges. The fish communities downstream of the MWWE outfalls demonstrated differences in the abundance and diversity, species and family richness, % tolerance and % vulnerability when compared to the fish community upstream or further downstream of these points of effluent discharge. In both years studied, the fish community exposed to MWWE in the riffle-run habitats demonstrated reductions in the proportion of the most prominent fish (Rainbow Darter, Ethoestoma caeruleum) downstream of the outfalls, and a significant increase in the proportion of large mobile, tolerant-omnivorous fish species such as suckers and sunfish. There was less variability in the responses of the fish community to MWWE in the same season between years than between seasons within the same year. An examination of how impaired health of a sentinel species exposed to MWWE discharges parallels changes in the fish community is also conducted. This study successfully demonstrates the cumulative impact of urban development, including multiple outfalls of treated wastewater effluents on fish populations and communities. Municipalities are the major source of nutrients and pharmaceuticals and personal care products to aquatic systems, and they need to consider their impacts carefully with increasing urban population growth and ageing demographics., (Copyright © 2012 SETAC.)

Credible ecological risk assessments often need to include analysis of population-level impacts. In the present study, a predictive model was developed to investigate population dynamics for white sucker (Catostomus commersoni) exposed to pulp mill effluent at a well-studied site in Jackfish Bay, Lake Superior, Canada. The model uniquely combines a Leslie population projection matrix and the logistic equation to translate changes in the fecundity and the age structure of a breeding population of white sucker exposed to pulp mill effluent to alterations in population growth rate. Application of this density-dependent population projection model requires construction of a life table for the organism of interest, a measure of carrying capacity, and an estimation of the effect of stressors on vital rates. A white sucker population existing at carrying capacity and subsequently exposed to pulp mill effluent equivalent to a documented exposure experienced during the period 1988 to 1994 in Jackfish Bay would be expected to exhibit a 34% to 51% annual decrease in recruitment during the first 5 yr of exposure and approach a population size of 71% of carrying capacity. The Jackfish Bay study site contains monitoring data for biochemical endpoints in white sucker, including circulating sex steroid concentrations, that could be combined with population modeling to utilize the model demonstrated at the Jackfish Bay study site for investigation of other white sucker populations at sites that are less data-rich., (Copyright © 2013 SETAC.)

Background: Congenital scoliosis due to a unilateral failure of vertebral segmentation usually has a poor prognosis. However, not all curves progress to the same degree or develop the same spinal deformity., Methods: The medical records and spine radiographs of 171 patients with a scoliosis due to unilateral unsegmented bar were reviewed retrospectively. The mean patient age at diagnosis was 9.3 years. Thirty-six patients had immediate surgery, 103 immature patients were followed untreated or before surgery for a mean duration of 3.6 years, twenty-eight patients were seen untreated at skeletal maturity, and four patients had no follow-up., Results: The unsegmented bar occurred at all levels; the mean extent was three vertebrae (range, two to eight vertebrae). Before the age of ten years, patients had a mean rate of scoliosis progression without treatment for all regions of the spine of 2° to 3° per year. By the age of ten years, seventy-three patients who had been seen untreated or prior to treatment had a mean scoliosis of 50° (range, 18° to 100°). After the age of ten years, these patients had an increase in the mean rate of scoliosis progression, but this rate varied per year depending on the affected region of the spine: 7° for the thoracolumbar curve, 5° for the midthoracic curve, and 4° for the upper thoracic curve. Spine surgery was performed on seventy-four patients who had a mean age of 12.2 years and a mean scoliosis of 78° for thoracolumbar curves, 66° for midthoracic curves, and 54° for upper thoracic curves. Of the patients with midthoracic congenital scoliosis, 24% developed a lower structural compensatory curve, which became larger and more deforming than the congenital curve, 22% had congenital rib fusions, and 16% had occult intraspinal anomalies., Conclusions: Prognosis depends on growth imbalance at the site of the unsegmented bar as well as the location and extent of the bar, age at diagnosis, and spinal growth remaining., Level of Evidence: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Endocrine disruption is considered a highly relevant hazard for environmental risk assessment of chemicals, plant protection products, biocides and pharmaceuticals. Therefore, screening tests with a focus on interference with estrogen, androgen, and thyroid hormone pathways in fish and amphibians have been developed. However, they use a large number of animals and short-term alternatives to animal tests would be advantageous. Therefore, the status of alternative assays for endocrine disruption in fish and frogs was assessed by a detailed literature analysis. The aim was to (i) determine the strengths and limitations of alternative assays and (ii) present conclusions regarding chemical specificity, sensitivity, and correlation with in vivo data. Data from 1995 to present were collected related to the detection/testing of estrogen-, androgen-, and thyroid-active chemicals in the following test systems: cell lines, primary cells, fish/frog embryos, yeast and cell-free systems. The review shows that the majority of alternative assays measure effects directly mediated by receptor binding or resulting from interference with hormone synthesis. Other mechanisms were rarely analysed. A database was established and used for a quantitative and comparative analysis. For example, a high correlation was observed between cell-free ligand binding and cell-based reporter cell assays, between fish and frog estrogenic data and between fish embryo tests and in vivo reproductive effects. It was concluded that there is a need for a more systematic study of the predictive capacity of alternative tests and ways to reduce inter- and intra-assay variability.

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.

Whole-body polychlorinated biphenyls (ΣPCBs) and plasma hydroxylated PCBs (OH-PCBs) concentrations were determined in brown bullhead (Ameiurus nebulosus) from Wheatley Harbour, Ontario, Canada. Elevated ΣPCBs in Wheatley Harbour are suspected to have originated from industrial waste disposal and/or discharges from nearby fish processing through discarding of fish remains. Mean ΣPCB concentrations in brown bullhead from Wheatley Harbour were approximately 250 ng/g wet weight compared with approximately 40 ng/g wet weight for brown bullhead from the reference sites, Hillman Marsh and Turkey Creek (both in Ontario, Canada). A significant relationship was found between the concentrations of non-ortho and mono-ortho PCB concentrations (toxic equivalents) and liver mixed-function oxygenase in brown bullhead (r = 0.74, p < 0.001). Plasma OH-PCB concentrations were greater in Wheatley Harbour brown bullhead than in those from Hillman Marsh (3.6 vs 1.5 ng/g wet wt, p < 0.01), and were detected infrequently in those from Turkey Creek (0.1 ng/g wet wt, n = 2). The OH-PCB congeners most frequently detected were 4'-OH-CB172, 3'-OH-CB180, 4-OH-CB187, 4-OH-CB146, 3-OH-CB138, and 4-OH-CB130, which are structurally similar to the thyroid hormones. To test the hypothesis of fish waste as the cause of the observed PCB contamination of Wheatley Harbour brown bullhead, a principal component analysis (PCA) was used to compare the brown bullhead PCB congener data with equivalent data for Lake Erie walleye, Lake Erie sediment, and industrial Aroclor mixtures. The relative proportions of each Aroclor mixture were estimated using the conjugated gradient method. The high similarity between the congener signatures for Lake Erie walleye and Wheatley Harbour brown bullhead supports the hypothesis of contamination from the fish processing industry., (Copyright © 2012 Crown in the Right of Canada.)

The overall health and endocrine function of wild brown bullhead (Ameiurus nebulosus) and goldfish (Carassius auratus) from the Wheatley Harbour Area of Concern (Lake Erie, Ontario, Canada) was assessed using a suite of physiological and biochemical endpoints. Smaller gonads were detected in female brown bullhead and goldfish from Wheatley Harbour compared with Hillman Marsh (Ontario, Canada) reference fish. Female brown bullhead exhibited decreased in vitro synthesis of 17β-estradiol. Female goldfish had decreased plasma vitellogenin concentrations. Plasma testosterone and 11-ketotestosterone were significantly depressed in males of both species. Perturbations in the thyroid status were detected, but varied between sexes and species. Observed differences included lower plasma concentrations of thyroid hormones and/or elevated liver deiodinase activity. Histological evaluation of the thyroid tissue indicated that in the case of female goldfish, those perturbations stimulated the thyroid (as indicated by increased thyroid epithelial cell height) and partially depleted the thyroxine reserves, as indicated by decreased colloid and elevated thyroid activation index. Increased mixed-function oxygenase activity in brown bullhead from Wheatley Harbour was consistent with exposure to planar aromatic contaminants. A principal component analysis of selected variables showed the separation of fish by collection site. The endpoints most strongly associated with the separation were generally those exhibiting significant differences between sites. The results of the present study indicate that the health of fish populations within Wheatley Harbour warrants continued attention., (Copyright © 2012 Crown in the Right of Canada.)

Background: Over the past 10-15 years, a substantial amount of work has been done by the scientific, regulatory, and business communities to elucidate the effects and risks of pharmaceuticals and personal care products (PPCPs) in the environment., Objective: This review was undertaken to identify key outstanding issues regarding the effects of PPCPs on human and ecological health in order to ensure that future resources will be focused on the most important areas., Data Sources: To better understand and manage the risks of PPCPs in the environment, we used the "key question" approach to identify the principle issues that need to be addressed. Initially, questions were solicited from academic, government, and business communities around the world. A list of 101 questions was then discussed at an international expert workshop, and a top-20 list was developed. Following the workshop, workshop attendees ranked the 20 questions by importance., Data Synthesis: The top 20 priority questions fell into seven categories: a) prioritization of substances for assessment, b) pathways of exposure, c) bioavailability and uptake, d) effects characterization, e) risk and relative risk, f ) antibiotic resistance, and g) risk management., Conclusions: A large body of information is now available on PPCPs in the environment. This exercise prioritized the most critical questions to aid in development of future research programs on the topic.

The oil sands region of northern Alberta represents the world's largest reserves of bitumen, and the accelerated pace of industrial extraction activity has raised concern about the possible impacts on the Athabasca River and its tributaries. An ecotoxicogenomic study was undertaken on Oncorhynchus mykiss trout hepatocytes exposed to extracts of water samples near the oil sand development area, as well as to oil sands process-affected water (OSPW) extracts using the quantitative reverse transcriptase polymerase chain reaction technique. The expression of the following genes (mRNA) was monitored to track changes in xenobiotic biotransformation (CYP1A1, CYP3A4, glutathione S-transferase, multi-drug resistance transporter), estrogenicity (estrogen receptor and vitellogenin), oxidative stress (superoxide dismutase and metallothionein) and DNA repair activity (DNA ligase). The extent of DNA-aromatic hydrocarbon adducts was also determined in cells by immuno-staining. A comparative analysis of gene expression between the river/lake and OSPW samples revealed that CYP3A4, metallothioneins, DNA ligase and GST genes, were specifically expressed by OSPW. Cells exposed to OSPW, commercial naphthenic acids, and benzo(a)pyrene showed increased polyaromatic hydrocarbon DNA-adducts, as determined by cell immunofluorescence analysis. Other genes were induced by all types of water samples, although the induction potential was stronger in OSPW most of the time (e.g., VTG gene was expressed nearly 15-fold by surface waters from the lake and river samples but increased to a maximum of 31-fold in OSPW). A multivariate discriminant function analysis revealed that the lake and river water samples were well discriminated from the OSPW. The CYP3A4 gene was the most highly expressed gene in cells exposed to OSPW and responded less to the lake or river water in the Athabasca River area. This study identified a suite of gene targets that responded specifically to OSPW extracts, which could serve as toxicogenomic fingerprints of OSPW contamination., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

During the winter low flow periods, Wascana Creek, Saskatchewan, Canada, can be 100% treated municipal wastewater downstream of the City of Regina's Wastewater Treatment Plant. The objective of this study was to determine if exposure to municipal effluent affects the health and reproductive development of fish in an effluent-dominated stream. Field studies were conducted on post-spawning (August 2006), spawning (June 2007), recrudescent (October 2007) and pre-spawning (May 2008) sentinel fish [Fathead Minnow Pimephales promelas and Brook Stickleback Culaea inconstans] to assess responses in terms of growth (condition factor), reproduction (in vitro sex steroid biosynthetic capacity, and gonadosomatic indices, histology) and survival associated with the effluent outfall. Sentinel species demonstrated varying responses depending on the season of field collections. While Stickleback collected downstream of the sewage discharge were often longer, heavier and had greater condition, Fatheads from the same site were shorter and lighter. Exposed fish of both species exhibited delayed spawning and altered gonadal development depending on the season. Exposed male Fathead Minnows also had significantly lower scores of secondary sexual characteristics (fewer nuptial tubercles, little or no development of the dorsal pad, and the lack of presence of a dorsal fin dot). Histopathology of exposed Fathead Minnows revealed thickening of the gill lamellae and alterations in structure of the kidneys (inflammation of the proximal tubules and Bowman's capsule). It is not known if the effluents are affecting natural reproduction and recruitment into this population or if these impacted populations rely on immigration from upstream reaches to sustain the populations. Climate change and human population growth will further challenge this effluent-dominated stream's ability to assimilate nutrients and contaminants which may further impair the performance of fish in this arid environment., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)