The effects of plastic additives on swimming activity and startle response in marine amphipod Echinogammarus marinus.

Plastic additives are widely used in plastic production and are found in the environment owing to their widespread applications. Among these additives, N-butyl benzenesulfonamide (NBBS) and triphenyl phosphate (TPHP) are under international watchlist for evaluation, with limited studies on amphipods. Di-ethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) are banned in some countries and categorised as substances of very high concern. This study aimed to investigate the effects of NBBS, TPHP, DEHP and DBP on the swimming activity of a coastal intertidal marine amphipod, Echinogammarus marinus. Furthermore, this study is the first to quantify startle response in E. marinus in response to light stimuli. Amphipods were exposed to 0, 0.5, 5, 50 and 500 µg/l concentrations of all test compounds. Swimming activity and startle responses were assessed by video tracking and analysis using an 8-min alternating dark and light protocol after exposure on days 7 and 14. We observed an overall compound and light effect on the swimming activity of E. marinus. A significant decrease in swimming distance was found in 500 µg/l NBBS and TPHP. We observed that the startle response in E. marinus had a latency period of >2 s and animals were assessed at 1 s and the sum of the first 5 s. There was a clear startle response in E. marinus during dark to light transition, evident with increased swimming distance. NBBS exposure significantly increased startle response at environmental concentrations, while significant effects were only seen in 500 µg/l TPHP at 5 s. We found no significant effects of DEHP and DBP on swimming behaviour at the concentrations assessed. The findings of this study affirm the necessity for a continuous review of plastic additives to combat adverse behavioural effects that may be transferable to the population levels.

Widespread psychoactive pollutant augments daytime restfulness and disrupts diurnal activity rhythms in fish.

Pharmaceutical pollution is a major driver of global change, with the capacity to alter key behavioural and physiological traits in exposed animals. Antidepressants are among the most commonly detected pharmaceuticals in the environment. Despite well-documented pharmacological effects of antidepressants on sleep in humans and other vertebrates, very little is known about their ecologically relevant impacts as pollutants on non-target wildlife. Accordingly, we investigated the effects of acute 3-day exposure of eastern mosquitofish (Gambusia holbrooki) to field-realistic levels (nominal concentrations: 30 and 300 ng/L) of the widespread psychoactive pollutant, fluoxetine, on diurnal activity patterns and restfulness, as indicators of disruptions to sleep. We show that exposure to fluoxetine disrupted diel activity patterns, which was driven by augmentation of daytime inactivity. Specifically, unexposed control fish were markedly diurnal, swimming farther during the day and exhibiting longer periods and more bouts of inactivity at night. However, in fluoxetine-exposed fish, this natural diel rhythm was eroded, with no differences in activity or restfulness observed between the day and night. As a misalignment in the circadian rhythm has been shown to adversely affect fecundity and lifespan in animals, our findings reveal a potentially serious threat to the survival and reproductive success of pollutant-exposed wildlife.

Effects of the antidepressant fluoxetine on the swimming behaviour of the amphipod Gammarus pulex: Comparison of short-term and long-term toxicity in the laboratory and the semi-field.

Fluoxetine is one of the worlds most prescribed antidepressant, and frequently detected in surface waters. Once present in the aquatic environment, fluoxetine has been shown to disrupt the swimming behaviour of fish and invertebrates. However, swimming behaviour is also known to be highly variable according to experimental conditions, potentially concealing relevant effects. Therefore, the aims of this study were two-fold: i) investigate the swimming and feeding behaviour of Gammarus pulex after exposure to the antidepressant fluoxetine (0.2, 2, 20, and 200 µg/L), and ii) assess to what degree the experimental test duration (short-term and long-term) and test location (laboratory and semi-field conditions) affect gammarid's swimming behaviour. We used automated video tracking and analysis to asses a range of swimming behaviours of G. pulex, including swimming speed, startle responses after light transition, acceleration, curvature and thigmotaxis. We found larger effects on the swimming behaviour of G. pulex due to experimental conditions than due to tested antidepressant concentrations. Gammarids swam faster, more straight and showed a stronger startle response during light transition when kept under semi-field conditions compared to the laboratory. Effects found for different test durations were opposite in the laboratory and semi-field. In the laboratory gammarids swam slower and spent more time at the inner zone of the arena after 2 days compared to 21 days while for the semi-field the reverse was observed. Fluoxetine had only minor impacts on the swimming behaviour of G. pulex, but experimental conditions influenced behavioural outcomes in response to fluoxetine exposure. Overall, our results highlight the importance of standardizing and optimizing experimental protocols that assess behaviour to achieve reproducible results in ecotoxicology.

Effects of the agricultural pollutant 17ß-trenbolone on morphology and behaviour of tadpoles (Limnodynastes tasmaniensis).

Pollutants, such as endocrine disrupting chemicals (EDCs), are increasingly being detected in organisms and ecosystems globally. Agricultural activities, including the use of hormonal growth promotants (HGPs), are a major source of EDC contamination. One potent EDC that enters into the environment through the use of HGPs is 17ß-trenbolone. Despite EDCs being repeatedly shown to affect reproduction and development, comparatively little is known regarding their effects on behaviour. Amphibians, one of the most imperilled vertebrate taxa globally, are at particular risk of exposure to such pollutants as they often live and breed near agricultural operations. Yet, no previous research on amphibians has explored the effects of 17ß-trenbolone exposure on foraging or antipredator behaviour, both of which are key fitness-related behavioural traits. Accordingly, we investigated the impacts of 28-day exposure to two environmentally realistic concentrations of 17ß-trenbolone (average measured concentrations: 10 and 66 ng/L) on the behaviour and growth of spotted marsh frog tadpoles (Limnodynastes tasmaniensis). Contrary to our predictions, there was no significant effect of 17ß-trenbolone exposure on tadpole growth, antipredator response, anxiety-like behaviour, or foraging. We hypothesise that the differences in effects found between this study and those conducted on fish may be due to taxonomic differences and/or the life stage of the animals used, and suggest further research is needed to investigate the potential for delayed manifestation of the effects of 17ß-trenbolone exposure.

The endocrine disruptor 17ß-trenbolone alters the relationship between pre- and post-copulatory sexual traits in male mosquitofish (Gambusia holbrooki).

It is now well-established that reproduction in wildlife can be disrupted by anthropogenic environmental changes, such as chemical pollution. However, very little is known about how these pollutants might affect the interplay between pre- and post-copulatory mechanisms of sexual selection. Here, we investigated the impacts of 21-day exposure of male eastern mosquitofish (Gambusia holbrooki) to a field-realistic level (average measured concentration: 11 ng/L) of the endocrine-disrupting chemical 17ß-trenbolone (17ß-TB) on pre- and post-copulatory reproductive traits. We examined male reproductive behaviour by testing the time spent near a female behind a partition, as well as the number of copulation attempts made, and the time spent chasing a female in a free-swimming context. Sperm traits were also assayed for all males. We found that exposure of male fish to 17ß-TB altered the relationship between key pre- and post-copulatory reproductive traits. Furthermore, 17ß-TB-exposed males had, on average, a higher percentage of motile sperm, and performed fewer copulation attempts than unexposed males. However, there was no overall effect of 17ß-TB exposure on either the time males spent associating with or chasing females. Taken together, our findings demonstrate the potential for chemical pollutants to affect both pre- and post-copulatory sexual traits, and the interplay between these mechanisms of sexual selection in contaminated wildlife.

High-Throughput Screening of Psychotropic Compounds: Impacts on Swimming Behaviours in Artemia franciscana.

Animal behaviour is becoming increasingly popular as an endpoint in ecotoxicology due to its increased sensitivity and speed compared to traditional endpoints. However, the widespread use of animal behaviours in environmental risk assessment is currently hindered by a lack of optimisation and standardisation of behavioural assays for model species. In this study, assays to assess swimming speed were developed for a model crustacean species, the brine shrimp Artemia franciscana. Preliminary works were performed to determine optimal arena size for this species, and weather lux used in the experiments had an impact on the animals phototactic response. Swimming speed was significantly lower in the smallest arena, whilst no difference was observed between the two larger arenas, suggesting that the small arena was limiting swimming ability. No significant difference was observed in attraction to light between high and low light intensities. Arena size had a significant impact on phototaxis behaviours. Large arenas resulted in animals spending more time in the light side of the arena compared to medium and small, irrespective of light intensity. The swimming speed assay was then used to expose specimens to a range of psychotropic compounds with varying modes of action. Results indicate that swimming speed provides a valid measure of the impacts of behaviour modulating compounds on A. franciscana. The psychotropic compounds tested varied in their impacts on animal behaviour. Fluoxetine resulted in increased swimming speed as has been found in other crustacean species, whilst oxazepam, venlafaxine and amitriptyline had no significant impacts on the behaviours measured. The results from this study suggest a simple, fast, high throughput assay for A. franciscana and gains insight on the impacts of a range of psychotropic compounds on the swimming behaviours of a model crustacean species used in ecotoxicology studies.

Transcriptome-wide changes associated with the reproductive behaviour of male guppies exposed to 17α-ethinyl estradiol.

Although many pharmaceutical compounds (and their metabolites) can induce harmful impacts at the molecular, physiological and behavioural levels, their underlying mechanistic associations have remained largely unexplored. Here, we utilized RNA-Seq to build a whole brain transcriptome profile to examine the impact of a common endocrine disrupting pharmaceutical (17α-ethinyl estradiol, EE2) on reproductive behaviour in wild guppies (Poecilia reticulata). Specifically, we annotated 16,791 coding transcripts in whole brain tissue in relation to the courtship behaviour (i.e. sigmoid display) of EE2 exposed (at environmentally relevant concentration of 8 ng/L for 28-days) and unexposed guppies. Further, we obtained 10,960 assembled transcripts matching in the non-coding orthologous genomes. Behavioural responses were assessed using a standard mate choice experiment, which allowed us to disentangle chemical cues from visual cues. We found that a high proportion of the RNAseq reads aligned back to our de novo assembled transcriptome with 80.59% mapping rate. Behavioural experiments showed that when males were presented only with female visual cues, there was a significant interaction between male treatment and female treatment in the time spent in the preference zone. This is one of the first studies to show that transcriptome-wide changes are associated with the reproductive behaviour of fish: EE2 exposed male guppies that performed high levels of courtship had a gene profile that deviated the most from the other treatment groups, while both non-courting EE2 and control males had similar gene signatures. Using Gene Ontology pathway analysis, our study shows that EE2-exposed males had gene transcripts enriched for pathways associated with altered immunity, starvation, altered metabolism and spermatogenesis. Our study demonstrates that multiple gene networks orchestrate courting behaviour, emphasizing the importance of investigating impacts of pharmaceuticals on gene networks instead of single genes.

Behavioural effects of psychoactive pharmaceutical exposure on European perch (Perca fluviatilis) in a multi-stressor environment.

With the ability to resist biodegradation and exert therapeutic effects at low concentrations, pharmaceutical contaminants have become environmental stressors for wildlife. One such contaminant is the anxiolytic oxazepam, a psychoactive pharmaceutical that is frequently detected in surface waters globally. Despite growing interest in understanding how wildlife respond to anxiolytics, synergistic effects of pharmaceuticals and other abiotic (e.g. temperature) and biotic (e.g. predation risk) stressors remain unclear. Here, using a multi-stressor approach, we investigated effects of 7-day oxazepam exposure (6.5 µg/L) on anxiety-related behaviours in juvenile European perch (Perca fluviatilis). The multi-stressor approach was achieved by exposing perch to oxazepam at two temperatures (10 °C and 18 °C), and at two predation risk regimes-generated using chemical cues from the northern pike (Esox lucius). Our exposures resulted in a successful uptake of the drug from the water, i.e., oxazepam was measured in perch muscle tissue at 50 ±â€¯17 ng/g (mean ±â€¯SD). We found significant oxazepam-induced effects on boldness, with 76.7% of the treated fish entering the white background (i.e. 'exposed' area where exposure to presumed risks are higher) within the first 5 min, compared to 66.6% of the control fish. We also found a significant effect of temperature on total time spent freezing (i.e. staying motionless). Specifically, fish in the low temperature treatments (oxazepam, predation) froze for longer than fish in high temperatures. Our multi-stressor study is the first to uncover how anxiety-related behaviours in wild juvenile fish are altered by changes in water temperature and perceived predation risk. Importantly, our findings highlight the need to focus on multiple stressors to improve understanding of how organisms not only survive, but adapt to, human-induced environmental change.

The pharmaceutical pollutant fluoxetine alters reproductive behaviour in a fish independent of predation risk.

Pharmaceutical pollutants constitute a major threat to wildlife because of their capacity to induce biological effects at low doses. One such pollutant is the antidepressant fluoxetine, which has been detected in surface waters globally at levels that recent studies suggest can alter physiology and behaviour in aquatic organisms. However, wildlife exposed to pharmaceutical contaminants are typically confronted with multiple stressors simultaneously, including predation risk, which is a particularly important natural stressor that can have direct (e.g. mortality) and indirect (e.g. changed prey behaviour) fitness effects. Accordingly, we investigated potential impacts of environmentally realistic fluoxetine exposure on reproductive behaviour in the guppy (Poecilia reticulata) under predation risk. Specifically, we tested whether fluoxetine exposure altered mating behaviour in male and female guppies in the presence of either a predatory spangled perch (Leiopotherapon unicolor) or a non-predatory rainbowfish (Melanotaenia splendida) control. We found that fluoxetine and the presence of a predatory spangled perch did not interact to affect reproductive behaviour. We also found that, independent of a predatory threat, fluoxetine exposure altered male mating strategy, with males in the high treatment conducting significantly more coercive 'sneak' copulations, whereas the number of courtship displays performed was not significantly affected. Moreover, while fluoxetine exposure did not significantly affect the amount of time that males and females spent following one another, we found that females, but not males, followed a potential partner less when in the presence of the predatory fish. Finally, both sexes reacted to the risk of predation by spending less time in close proximity to a predator than a non-predator. In combination, our findings highlight the capacity of fluoxetine to influence processes of sexual selection at field-realistic concentrations and emphasise the importance of considering multiple stressors when assessing impacts of pharmaceutical pollutants on the behaviour of wildlife.

Field-realistic exposure to the androgenic endocrine disruptor 17ß-trenbolone alters ecologically important behaviours in female fish across multiple contexts.

The capacity of pharmaceutical pollution to alter behaviour in wildlife is of increasing environmental concern. A major pathway of these pollutants into the environment is the treatment of livestock with hormonal growth promotants (HGPs), which are highly potent veterinary pharmaceuticals that enter aquatic ecosystems via effluent runoff. Hormonal growth promotants are designed to exert biological effects at low doses, can act on physiological pathways that are evolutionarily conserved across taxa, and have been detected in ecosystems worldwide. However, despite being shown to alter key fitness-related processes (e.g., development, reproduction) in various non-target species, relatively little is known about the potential for HGPs to alter ecologically important behaviours, especially across multiple contexts. Here, we investigated the effects of exposure to a field-realistic level of the androgenic HGP metabolite 17ß-trenbolone-an endocrine-disrupting chemical that has repeatedly been detected in freshwater systems-on a suite of ecologically important behaviours in wild-caught female eastern mosquitofish (Gambusia holbrooki). First, we found that 17ß-trenbolone-exposed fish were more active and exploratory in a novel environment (i.e., maze arena), while boldness (i.e., refuge use) was not significantly affected. Second, when tested for sociability, exposed fish spent less time in close proximity to a shoal of stimulus (i.e., unexposed) conspecific females and were, again, found to be more active. Third, when assayed for foraging behaviour, exposed fish were faster to reach a foraging zone containing prey items (chironomid larvae), quicker to commence feeding, spent more time foraging, and consumed a greater number of prey items, although the effect of exposure on certain foraging behaviours was dependent on fish size. Taken together, these findings highlight the potential for exposure to sub-lethal levels of veterinary pharmaceuticals to alter sensitive behavioural processes in wildlife across multiple contexts, with potential ecological and evolutionary implications for exposed populations.

The antidepressant fluoxetine alters mechanisms of pre- and post-copulatory sexual selection in the eastern mosquitofish (Gambusia holbrooki).

Contamination of aquatic habitats with pharmaceuticals is a major environmental concern. Recent studies have detected pharmaceutical pollutants in a wide array of ecosystems and organisms, with many of these contaminants being highly resistant to biodegradation and capable of eliciting sub-lethal effects in non-target species. One such pollutant is fluoxetine, a widely prescribed antidepressant, which is frequently detected in surface waters globally and can alter physiology and behaviour in aquatic organisms. Despite this, relatively little is known about the potential for fluoxetine to disrupt mechanisms of sexual selection. Here, we investigate the impacts of 30-day exposure to two environmentally realistic levels of fluoxetine (low and high) on mechanisms of pre- and post-copulatory sexual selection in the eastern mosquitofish (Gambusia holbrooki). We tested 1) male mating behaviour in the absence or presence of a competitor male, and 2) sperm quality and quantity. We found that high-fluoxetine exposure increased male copulatory behaviour in the absence of a competitor, while no effect was detected under male-male competition. Further, fluoxetine exposure at both concentrations increased total sperm count relative to males from the control group, while no significant change in sperm quality was observed. Lastly, low-fluoxetine males showed a significant reduction in condition index (mass relative to length). Our study is the first to show altered mechanisms of both pre- and post-copulatory sexual selection in an aquatic species resulting from environmentally realistic fluoxetine exposure, highlighting the capacity of pharmaceutical pollution to interfere with sensitive reproductive processes in wildlife.

Sex in troubled waters: Widespread agricultural contaminant disrupts reproductive behaviour in fish.

Chemical pollution is a pervasive and insidious agent of environmental change. One class of chemical pollutant threatening ecosystems globally is the endocrine disrupting chemicals (EDCs). The capacity of EDCs to disrupt development and reproduction is well established, but their effects on behaviour have received far less attention. Here, we investigate the impact of a widespread androgenic EDC on reproductive behaviour in the guppy, Poecilia reticulata. We found that short-term exposure of male guppies to an environmentally relevant concentration of 17ß-trenbolone-a common environmental pollutant associated with livestock production-influenced the amount of male courtship and forced copulatory behaviour (sneaking) performed toward females, as well as the receptivity of females toward exposed males. Exposure to 17ß-trenbolone was also associated with greater male mass. However, no effect of female exposure to 17ß-trenbolone was detected on female reproductive behaviour, indicating sex-specific vulnerability at this dosage. Our study is the first to show altered male reproductive behaviour following exposure to an environmentally realistic concentration of 17ß-trenbolone, demonstrating the possibility of widespread disruption of mating systems of aquatic organisms by common agricultural contaminants.