Pharmaceuticals are increasingly being detected in aquatic ecosystems worldwide. Particularly concerning are pharmaceutical pollutants that can adversely impact exposed wildlife, even at extremely low concentrations. One such contaminant is the widely prescribed antidepressant fluoxetine, which can disrupt neurotransmission and behavioural pathways in wildlife. Despite this, relatively limited research has addressed the behavioural impacts of fluoxetine at ecologically realistic exposure concentrations. Here, we show that 28-day fluoxetine exposure at two ecologically relevant dosages—one representing low surface water concentrations and another representing high effluent flow concentrations—alters antipredator behaviour in Eastern mosquitofish (Gambusia holbrooki). We found that fluoxetine exposure at the lower dosage resulted in increased activity levels irrespective of the presence or absence of a predatory dragonfly nymph (Hemianax papuensis). Additionally, irrespective of exposure concentration, fluoxetine-exposed fish entered the predator ‘strike zone’ more rapidly. In a separate experiment, fluoxetine exposure reduced mosquitofish freezing behaviour—a common antipredator strategy—following a simulated predator strike, although, in females, this reduction in behaviour was seen only at the lower dosage. Together, our findings suggest that fluoxetine can cause both non-monotonic and sex-dependent shifts in behaviour. Further, they demonstrate that exposure to fluoxetine at environmentally realistic concentrations can alter antipredator behaviour, with important repercussions for organismal fitness.

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.

Hormonal growth promoters (HGPs), widely used in beef cattle production globally, make their way into the environment as agricultural effluent—with potential impacts on aquatic ecosystems. One HPG of particular concern is 17β-trenbolone, which is persistent in freshwater habitats and can affect the development, morphology and reproductive behaviors of aquatic organisms. Despite this, few studies have investigated impacts of 17β-trenbolone on non-reproductive behaviors linked to growth and survival, like boldness and predator avoidance. None consider the interaction between 17β-trenbolone and other environmental stressors, such as temperature, although environmental challenges confronting animals in the wild seldom, if ever, occur in isolation. Accordingly, this study aimed to test the interactive effects of trenbolone and temperature on organismal behavior. To do this, eastern mosquitofish (Gambusia holbrooki) were subjected to an environmentally-relevant concentration of 17β-trenbolone (average measured concentration 3.0 ± 0.2 ng/L) or freshwater (i.e. control) for 21 days under one of two temperatures (20 and 30 °C), after which the predator escape, boldness and exploration behavior of fish were tested. Predator escape behavior was assayed by subjecting fish to a simulated predator strike, while boldness and exploration were assessed in a separate maze experiment. We found that trenbolone exposure increased boldness behavior. Interestingly, some behavioral effects of trenbolone depended on temperature, sex, or both. Specifically, significant effects of trenbolone on male predator escape behavior were only noted at 30 °C, with males becoming less reactive to the simulated threat. Further, in the maze experiment, trenbolone-exposed fish explored the maze faster than control fish, but only at 20 °C. We conclude that field detected concentrations of 17β-trenbolone can impact ecologically important behaviors of fish, and such effects can be temperature dependent. Such findings underscore the importance of considering the potentially interactive effects of other environmental stressors when investigating behavioral effects of environmental contaminants.

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 17β-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 17β-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.

There is considerable interest in applying omics techniques, which have proven extremely valuable for laboratory-based toxicology studies, towards field-scale ecotoxicology and environmental monitoring. Concerns that confounding factors in natural ecosystems may exacerbate variability in omics datasets must be addressed to validate the transition from laboratory to field. This study explores how temporal variability related to seasonal and climatic trends influence qualitative and quantitative metabolomics outcomes, in fish from reference and metal(loid)-polluted wetlands in Australia. Female mosquitofish (Gambusia holbrooki) were sampled on two separate occasions, from a rehabilitated tailings wetland at the site of historic antimony (Sb) processing and a reference wetland with comparable water quality. The first sampling coincided with greater monthly rainfall and colder water temperature, whereas the second sampling was drier and water was warmer. Despite temporal changes and associated differences in metal(loid) concentrations, site differences in metabolite profiles were qualitatively very similar between sampling events. However, quantitative differences were observed, with a greater number of significantly altered metabolites identified during the second sampling event, which coincided with greater metal(loid) concentrations in both water and fish. The majority of identified metabolites were elevated in fish from the contaminated wetland, but with notable decreases in several metabolites that are known to play a role in various aspects of metal(loid) binding, detoxification and excretion. Specifically, decreased aspartate, histidine, myo-inositol, taurine and choline were observed in fish from the contaminated wetland, and may therefore represent a metabolite suite that is broadly indicative of metal toxicity. Quantitative differences between sampling events are suggestive of a dose-response relationship observable at the cellular level which, if harnessed, may be useful for assigning levels of concern based on the degree of change in a multi-parameter set of metabolite biomarkers.

Metal contamination causes multiple biological dysfunctions, including impairment of key physiological functions by targeting enzymes. This feature is a matter of concern, since it may imply significant disturbances in energy allocation, behaviour, reproduction, and survival. Inhibition of the cholinesterase (ChE) activity of aquatic organisms by metals has been described, and systematically used in biomonitoring studies as effect criterion of environmental exposure to these compounds. The present paper addresses the feasibility of using ChE inhibition to quantify the adverse acute and chronic effects of metals (copper, zinc, lead, and cadmium) on nervous tissue of Gambusia holbrooki. With the exception of acute exposure to copper, ChE activity was not significantly impaired. The meanings of the reported findings are further discussed, aiming at a more comprehensive use of this biomarker in environmental assessment. Based on the obtained results, the role of ChE inhibition in environmental metal contamination scenarios should be questioned or even discarded.

Wildfires are an environmental concern due to the loss of forest area and biodiversity, but also because their role as drivers of freshwater systems contamination by metals. In this context, the fish Gambusia holbrooki was used as a model, deployed for in situ exposure in watercourses standing within a recently burnt area and further assessment of toxic effects. The fish were exposed during 4 days at four different sites: one upstream and another downstream the burnt area and two within the burnt area. Biochemical biomarkers for oxidative stress and damage were assessed. The extent of lipoperoxidative damage was monitored by quantifying malondialdehyde and DNA damage evaluated through erythrocyte nuclear abnormalities observation. Chemical analysis revealed higher metal levels within the burnt area, and exposed fish consistently showed pro-oxidative responses therein, particularly an increase of gill glutathione peroxidase and glutathione reductase activity, the records doubling compared to samples from sites in the unburnt area; also the activity of glutathione-S-transferases comparatively increased (by 2-fold in the liver) in samples from the burnt area, and malondialdehyde was produced twice as much therein and in samples downstream the burnt area reflecting oxidative damage. Consistently, the frequency of erythrocyte nuclear abnormalities was higher at sites within and downstream the burnt area. This study supports the use of sensitive oxidative stress and genotoxicity biomarkers for an early detection of potentially noxious ecological effects of wildfires runoff.

Concerns about the negative effects of microplastics (MPs) on human health have led to increasing attention to the occurrence of MPs in the aquatic environment. Recent studies have focus on the spatio-temporal distribution of MPs in rivers for residential and agricultural areas. Qarasu River Basin, watershed to Gorgan Bay, is the site of many permanent rivers and the levels of MP pollution in those rivers are unknown. This research was conducted in three different types of land use: forest, residential, and agricultural along 8 different rivers. A total of 9 fish species were identified and the presence of MPs in 87% of fish was confirmed. The prevalence of MP among species was Liza sp. > Carassius gibelio > Gambusia holbrooki > Rutilus rutilus > Neogobius melanostomus > Cyprinus carpio = Vimba vimba > Rutilus frisii > Barbus sp. The highest frequency of MPs was observed in Qarasu River (1880 ± 251 n/kg) and Mohammadabad River (184 ± 1340 n/kg) in agricultural land use, and the lowest frequency was related to Baghu and Ziarat Rivers with 660 ± 77 and 600 ± 91 n/kg found in forest land use respectively. The highest type, color, and size of MP sediments were fragment, black, and 1–2 mm, respectively. The highest type, color, and size of MP in fishes were fiber, black, and 0.5–0.1 mm respectively. Overall according to the source tracing result, agricultural land use was the main source of MP pollution of Gorgan Bay. The Bay is strongly affected by the incoming rivers, the Qarasu River Basin as the most important river supplying water.

Arsenic (As) pollution in water has important impacts for human and ecosystem health. In freshwaters, arsenate (Asⱽ) can be taken up by microalgae due to its similarity with phosphate molecules, its toxicity being aggravated under phosphate depletion. An experiment combining ecological and ecotoxicological descriptors was conducted to investigate the effects of Asⱽ (130 μg L⁻¹ over 13 days) on the structure and function of fluvial biofilm under phosphate-limiting conditions. We further incorporated fish (Gambusia holbrooki) into our experimental system, expecting fish to provide more available phosphate for algae and, consequently, protecting algae against As toxicity. However, this protection role was not fully achieved. Arsenic inhibited algal growth and productivity but not bacteria. The diatom community was clearly affected showing a strong reduction in cell biovolume; selection for tolerant species, in particular Achnanthidium minutissimum; and a reduction in species richness. Our results have important implications for risk assessment, as the experimental As concentration used was lower than acute toxicity criteria established by the USEPA.

Sexually mature male mosquitofish (Gambusia holbrooki) were collected from various sites around Melbourne in 2009 to evaluate the performance of gonopodial indices as a biomarker for endocrine disruption in Melbourne's waterways. The mosquitofish indices assessed were body length (BL), gonopodial length (GL)/BL ratio, ray 4:6 ratio and the absence or presence of hooks and serrae, and these varied between sites. The study was complemented by measurements of estrogenic, retinoid, thyroid and aryl hydrocarbon (AhR) receptor activities of the water. Male mosquitofish were 16.3–21.5 mm in length, and although there was a statistically significant positive relationship showing that bigger fish had longer gonopodia than small fish (r2 = 0.52, p < 0.001), there were few significant differences in GL/BL ratio of fish between sites. Measured estrogenic activity was mostly in the range 0.1–1.7 ng/L EEQ, with one site having much higher levels (~12 ng/L EEQ). Aryl hydrocarbon (AhR) receptor activity was observed in all water samples (7–180 ng/L βNF EQ), although there was no consistent pattern in the level of AhR activity observed, i.e., ‘clean’ sites were as likely to return a high AhR activity response as urban or wastewater treatment plant (WWTP)-impacted sites. There was no correlation between measurements of receptor actvity and gonopodial length (GL):BL ratio and BL. We conclude that the mosquitofish gonopodia only fulfills part of the criteria for biomarker selection for screening. The mosquitofish indices assessed were cheap and easy-to-perform procedures; however, there is no baseline data from the selected sites to evaluate whether differences in the morpholical indices observed at a site were a result of natural selection in the population or due to estrogenic exposure.