Joanna Yvonne Wilson

Anthropogenic activities can result in an unstable thermal environment which can alter key stages of fish development. Our study aimed to determine how incubation temperature influences the heat shock protein response and thermal tolerance in Yellow Perch, embryos and post-hatch fish. Yellow Perch are freshwater fish of economic, cultural, and recreational value. Embryos were incubated at 12, 15, and 18 ℃ and exposed to an acute 2 h thermal stress of + 3, 6, or 9 ℃ at key developmental stages (O1-O4, and 30 and 60 dph). Transcript abundance of key heat shock proteins was quantified and critical thermal maximum (CTmax) was determined for post-hatch fish. The magnitude and nature of the heat shock protein transcript abundance changed throughout development, with incubation temperature, and in response to both handling and thermal stress. Embryos at 12 and 18 ℃ were more sensitive to handling stress and significantly upregulated levels of hsp70-1, 70-2, and 47 with handling irrespective independent of thermal stress. By comparison, embryos at 15 ℃ did not increase hsp transcript levels with handling alone but they more readily increased hsp transcript abundance in response to thermal stress. Significant increased were detected between the + 6 or 9 ℃ treatments and the handling control in embryos incubated at 15 ⁰C. CTmax in post-hatch larvae (O4) from embryos incubated at 12 °C was higher than both 15 and 18 ⁰C groups. However, by 60 days post-hatch, there was no difference between 12 and 15 ⁰C incubations groups with both being significantly higher than Yellow Perch incubated at 18 ⁰C. Our research shows that early thermal environments can have a persistent influence on the nature of the heat shock protein response and its plasticity as well as whole animal thermal tolerance in fish 60 days post-hatch.

Across the world, there is an increasing desire for the replacement of whole-organism toxicity tests with new approach methods (NAMs) that refine, reduce, and replace animal use. These principles, collectively known as the 3Rs, are critical to consider when discussing NAM development and implementation. Despite the frequency at which NAMs are discussed as replacements to existing toxicity test methods, the application to whole-effluent toxicity (WET) monitoring is largely ignored in favour of their use for chemical assessments. These WET test methods are an ideal receptor for NAMs due to their volume of use and highly repetitive nature as legislative tools used in many areas of the world. In this paper, we investigate this with a focus on Canadian effluent regulation, first discussing the current tools used to regulate effluent toxicity in Canada and how NAMs can aim to fill these roles rapidly to address the changing landscape of Canada’s policy on vertebrate use in toxicity testing. We discuss three NAMs being developed with potential to be applied to whole-effluent toxicity testing, summarizing current trends in the literature, and identify areas where they may struggle to may struggle to meet the needs of environmental regulations. Further research needs are highlighted. Finally, we propose ways to integrate these NAMs into environmental regulations to robustly evaluate effluent toxicity should future data should future data collected for effluents follow existing trends.

Globally, there has been a push for the development of new approach methods that refine, reduce, and replace animal use in toxicity testing. A large source of this animal use is whole-effluent toxicity testing, which is widely used in regulation to determine the toxicity of effluents. The rainbow trout acute lethality test is one of the most widely used in North America for this purpose, as it empirically and irrefutably demonstrates effluent toxicity. Despite its strengths, this test is an ideal candidate for replacement due to its frequent use. One replacement is the RTgill-W1 assay, which uses a rainbow trout gill cell line to assess the toxicity of effluent samples. Despite its promise, the RTgill-W1 assay has rarely been investigated for its potential in replacing the rainbow trout acute lethality test. This study assessed the toxicity of pulp and paper mill effluents using the RTgill-W1 assay. Sixty-two effluents were sourced from three separate mills with a total of five unique sampling locations; an additional eight effluents were created by mixing effluents together. Of the 70 effluents, 50 were assessed with both the rainbow trout acute lethality test and the RTgill-W1 assay. Twenty of these caused > 50% mortality to the rainbow trout and > 50% decline in cell viability. An additional three effluents caused a >50% decline in cell viability. In effluents causing > 50% mortality and a decline in cell viability, there was a strong correlation in toxicity. Salt stratification and predator cues: Impacts on freshwater species depend on habitat choice (r = 0.786, p < 0.001). The alamarBlue endpoint was more sensitive in filtered effluents (p < 0.001). The effluent matrix did not influence the test endpoints, but un-ionized ammonia was observed to cause significant vacuolation of the cells. Overall, these results are promising for the implementation of the RTgill-W1 assay as a replacement to the rainbow trout acute lethality test for pulp mill effluents.

Temperate waters, such as the Great Lakes, are predicted to increase in temperature by 1°C every decade. Many poikilothermic fish thermoregulate behaviourally, moving to more suitable thermal environments. Embryos are incapable of locomotion and may be exposed to non-optimal temperatures during development. Previous work has suggested that temperature increases during embryogenesis can alter growth rates in fish. However, less is known of whether these early-life exposures to elevated temperatures can impart alterations to the phenotypic plasticity of performance traits, particularly in temperate species. We hypothesized that increased embryonic incubation temperature would diminish the larval performance of yellow perch (Perca flavescens), a critical cultural and ecological species of fish. We reared yellow perch embryos at 12, 15 or 18°C until hatching; after hatching, the temperature was raised to a common garden 18°C, their preferred post-hatch temperature. We assessed exploratory behaviour, metabolism (oxygen consumption) and cardiac performance throughout early development. At hatch, 12°C fish exhibited the greatest swimming activity, with 18°C fish consuming the least oxygen and possibly experiencing mitochondrial dysfunction. Cardiac development was more advanced at hatch in 18°C fish. Yet, warmer incubated fish had diminished movement and increased oxygen consumption at 20 days post-hatch, demonstrating long-term disruptions of increased temperature in the embryonic environment. Overall, elevations in rearing temperature may cause metabolic dysfunction and behavioural alterations, potentially impacting the survival of yellow perch.

The RTgill-W1 in vitro assay is a new approach method designed as an alternative to one of the most widely used toxicity tests globally, the fish acute lethality test. The RTgill-W1 assay is standardized (Organisation for Economic Co-operation and Development; International Organisation of Standardization) but the test conditions could be optimized to allow for higher throughput, better replication, and lower costs. This study explores potential optimizations regarding the culturing conditions, plate format, and reference toxicant testing to make the RTgill-W1 assay more practical for widespread implementation. We demonstrate that the RTgill-W1 culture can be routinely split 1:3 without impacting test sensitivity (p = 0.207 to 0.612), which allows all work to be contained to a standard 5-day work week and 1.3x more tests over the current test methods. The test can adopt a 96-well plate format without impacting sensitivity (p = 0.672 to 0.889), dramatically improving the replication of the exposure wells and test controls and generating test data from a single plate. The fluorescent signal does not bleed across the smaller wells in the 96-well format to impact test endpoints (p = 0.465 to > 0.999). The reference toxicity test concentrations can be modified to increase confidence in the point estimate (EC50), allowing for more effective monitoring of assay performance. These optimizations improve the practicality and decrease the costs of the RTgill-W1 assay, which is particularly desirable for implementation in commercial and government laboratories that conduct regulatory toxicity testing.

Early life development in fishes is a period of high phenotypic plasticity. Water temperatures during embryogenesis can lead to alterations in growth and metabolic and morphological phenotypic variations. This study aimed to characterize the effects of temperature on the growth and morphology of yellow perch throughout early development, a species of significant ecological and economic importance in North America. Yellow perch (Perca flavescens) embryos were incubated at either constant temperature (12, 15, or 18°C) or under an ambient seasonal regime, where temperature increased throughout embryogenesis until it reached 18°C. All groups were gradually warmed at hatch until they reached 18°C, and morphology was assessed. Inverse relationships between temperature, length and yolk‐sac volume were found at the end of the embryonic period. Optimal embryonic growth was associated with the ambient seasonal regime. Fish reared at 15 or 18°C were larger and had higher growth rates when compared to those at 12°C following hatch. However, fish reared in cooler temperatures may possess more advantageous body shapes. Surprisingly, the ambient seasonal incubation had the lowest growth rate post‐hatch, even though this most closely mimicked natural incubation for this species. Because suitable larval morphology is related to survival and successful recruitment, these data suggest that yellow perch may be vulnerable to climate change and thermal pollution, although further work is needed to better predict the ecological implications of the phenotypes.

Anthropogenic impacts have led to increases in contaminants in marine habitats. The presence of compounds such as pharmaceuticals, personal care products, and pesticides is a concern, as these compounds have been shown to act as neurochemicals in aquatic organisms. However, screens of the multitude of chemicals found environmentally have yet to be carried out. Capitella teleta are marine annelid polychaete worms that live in the sediment of estuarian environments, acting as ecosystem engineers for marine habitats. This study aimed to develop a behaviour tool to identify neurochemical pathways involved in controlling locomotion that could be applied to screen the impacts of toxicants in the environment using this key invertebrate species at multiple life stages. Adult and juvenile life stages (2 weeks post-emergence) of Capitella teleta were observed in Petri dishes (adults only) or 6-well plates (adults and juveniles), and conserved behavioural responses were isolated, such as their velocity, distance travelled, the time to reach the edge of the arena, and time spent at the periphery. We exposed juvenile and adult worms to nicotine (acetylcholine agonist) as a proof of concept. We noted similar disruptions to locomotion at both life stages, with low doses of nicotine stimulating movement and higher doses reducing locomotion. From here, we exposed juvenile worms to fluoxetine (serotonin reuptake inhibitor), phenobarbital (GABA agonist), and apomorphine (dopamine agonist). The behaviour of juvenile worms can be altered by exposure to fluoxetine, phenobarbital, and apomorphine. Fluoxetine and phenobarbital exposure reduces movement at high doses, but fluoxetine influences the amount of time at the periphery of the arena. Apomorphine produced modest changes in locomotion compared to other chemicals tested. Genome searching, as well as transcriptomics, were used to confirm the presence of neurochemical pathways in Capitella teleta . Our results demonstrate that Capitella teleta is a viable model for behavioural work, that several neurochemical pathways contribute to locomotory behaviour, and that this assay may be useful as a screen for contaminants found in marine habitats.

Temperate waters, such as the Great Lakes, are predicted to increase by 1°C every decade. Poikilothermic fish thermoregulate behaviourally, moving to more suitable thermal environments. Embryos are incapable of locomotion and may be exposed to non-optimal temperatures during development. Increased temperature alters the normal development of the yellow perch ( Perca flavescens), however, whether altered incubation temperature influences the development of metabolism and function in these fish remains unknown. We hypothesized that increased embryonic incubation temperature would disturb cardiac and metabolic function and the behaviour of yellow perch larvae. We reared yellow perch embryos at 12°C, 15°C, or 18°C until hatching; after hatching, the temperature was raised to a common garden 18°C, their preferred post-hatch temperature. We assessed exploratory behaviour, metabolism (oxygen consumption), and cardiac performance throughout early development. At hatch, 12°C fish exhibited the greatest swimming activity, with 18°C fish consuming the least oxygen and possibly experiencing mitochondrial dysfunction. Cardiac development was more advanced at hatch in 18°C fish. Yet, warmer incubated fish had diminished movement and increased oxygen consumption at 20 days post-hatch, demonstrating long-term disruptions of increased temperature in the embryonic environment. Overall, elevations in rearing temperature may cause metabolic dysfunction and behavioural alterations, potentially impacting the survival of yellow perch.