Steven D. Melvin

The utility of behavioral studies for aquatic toxicology testing: a meta-analysis.

Behavioral responses have been applied for decades as tools for aquatic toxicity testing, but have received far less attention than studies assessing lethality, development or reproduction. With improved visual and non-visual assessment tools and increased knowledge of the importance of behavior for organism health and fitness, interest in behavioral analysis has increased in recent years. However, to our knowledge there has never been a quantitative assessment of the available techniques for organismal toxicity testing, so it is not clear whether behavioral studies represent valuable additions to environmental monitoring. We performed a meta-analysis comparing the relative sensitivities and average durations of behavioral studies to those assessing acute lethality, development and reproduction. Results demonstrate that the average duration of behavioral studies is consistently less than developmental or reproductive studies, and that behavioral endpoints are generally more sensitive than those assessing development or reproduction. We found effect sizes to be lower but power to be higher in behavioral and reproductive studies compared to studies assessing development, which likely relates to low sample sizes commonly used in developmental studies. Overall, we conclude that behavioral studies are comparatively fast and sensitive, and therefore warrant further attention as tools for assessing the toxicological effects of environmental contaminants. We suggest that research aimed at developing and optimizing techniques for behavioral analysis could prove extremely useful to the field of toxicology, but that future work must be directed at determining what specific behaviors are most sensitive to various classes of contaminants, and at understanding the relevance of changes to discrete behaviors for influencing organismal and population-level health and fitness.

Growth, development and incidence of deformities in amphibian larvae exposed as embryos to naphthenic acid concentrations detected in the Canadian oil sands region.

Naphthenic acids (NA) have been identified as harmful environmental contaminants that influence survival, growth and development of wildlife. Amphibian larvae are particularly susceptible to waterborne contaminants, but little information exists regarding exposure of amphibian embryos or tadpoles to NA. Our results demonstrate that embryos of Lithobates pipiens and Silurana tropicalis exposed to 2-4 mg/l of a commercial NA blend suffer significant reductions (32% and 25%, respectively) in growth and development upon hatching. Increased incidences of deformities were observed in exposed individuals of both species, but were only significant in L. pipiens. Embryos suffered 100% mortality following exposure to 6 mg/l NAs, and narcosis at lower concentrations. LC50 estimates were 4.10 mg/l for 72-h exposure in L. pipiens and 4.13, 3.51, and 2.95 mg/l for 24-, 48-, and 72-h exposure in S. tropicalis. These data suggest that exposure to NAs at environmentally realized concentrations may negatively affect tadpole populations.

Removal of trace organic contaminants from domestic wastewater: A meta-analysis comparison of sewage treatment technologies.

Trace organic contaminants (TrOCs), such as endocrine disrupting compounds (EDCs) and pharmaceuticals and personal care products (PPCPs), represent global threats to aquatic animals and ecosystems. A major source of TrOCs in the aquatic environment is via the discharge of treated sewage, so there is an urgent need to evaluate the comparative efficiencies of the most widely used sewage treatment technologies as regards elimination of these compounds from wastewater. To address this need, 976 published articles were compiled focusing on estimates of removal (%) for 20 common environmental TrOCs, from five major sewage treatment technologies: conventional activated sludge (CAS), oxidation ditch (OD), membrane bioreactor (MBR), ponds and constructed wetlands (PCW), and trickling biological filters (TBF). A quantitative meta-analysis was performed to compare standardized relative removal efficiencies (SREs) of the compounds amongst these technologies, and where possible potential sources of heterogeneity were considered (e.g., flow rates and chemical sorption potential). The results indicate that the most widely used CAS treatment and the less common TBF provide comparatively poor overall removal of common organic micropollutants. Membrane bioreactors appear to be capable of achieving the greatest overall removal efficiencies, but the sustainability and economic viability of this option has been questioned. Treatment with OD systems may be more economical while still achieving comparatively high removal efficiencies, and the analysis revealed OD to be the best option for targeting highly potent estrogenic EDCs. This study offers a unique global assessment of TrOC removal via leading sewage treatment technologies, and is an important step in the identification of effective options for treating municipal sewage.

Individual and Mixture Toxicity of Pharmaceuticals Naproxen, Carbamazepine, and Sulfamethoxazole to Australian Striped Marsh Frog Tadpoles (Limnodynastes peronii)

Nonsteroidal human pharmaceuticals are prevalent in domestic wastewater and may find their way into the environment at low concentrations. Since most pharmaceuticals are designed to be biologically active at low concentrations, there is a risk that these compounds may affect aquatic wildlife. Of particular concern is the occurrence of pharmaceutical mixtures, which may lead to increased adverse effects compared to individual compounds. Interactive effects were previously demonstrated for amphibians exposed to pesticide mixtures, but no such studies investigating responses of amphibians to pharmaceutical mixtures are apparently available. Results demonstrated increased toxicity (loss of tactile response) of striped marsh frog (Limnodynastes peronii) tadpoles exposed to a mixture of naproxen, carbamazepine, and sulfamethoxazole, compared to exposures to the individual compounds. Significant time × treatment interactions were observed for tadpole development following chronic exposures to 10 or 100 μg/L of each compound and the mixture; however, responses were weak and main treatment effects were not significant. Despite minor effects at low exposure concentrations, results demonstrated a potential for mixtures of nonsteroidal pharmaceuticals commonly occurring in wastewater to influence amphibian development. With the vast numbers of pharmaceuticals that exist and are found in the environment, this work highlights a need for further research into mixtures of pharmaceutically active wastewater contaminants. Further, since pharmaceuticals exert extremely varied biological actions, it is suggested that future investigations would benefit from inclusion of endpoints that are indicative of physiological or metabolic performance, as well as assessment of sensitive behavioral responses.

Effects of the glyphosate-based herbicide Roundup WeatherMax ® on metamorphosis of wood frogs (Lithobates sylvaticus) in natural wetlands

Amphibian tadpoles develop in aquatic environments where they are susceptible to the effects of pesticides and other environmental contaminants. Glyphosate-based herbicides are currently the most commonly used herbicide in the world and have been shown to affect survival and development of tadpoles under laboratory and mesocosm conditions. In the present study, whole wetland manipulations were used to determine if exposure to an agriculturally relevant application of Roundup WeatherMax(®), a herbicide formulation containing the potassium salt of glyphosate and an undisclosed surfactant, influences the development of wood frog tadpoles (Lithobates sylvaticus) under natural conditions. Wetlands were divided in half with an impermeable curtain so that each wetland contained a treatment and control side. Tadpoles were exposed to two pulses of this herbicide at an environmentally realistic concentration (ERC, 0.21 mg acid equivalent (a.e.)/L) and the predicted maximum environmental concentration (PMEC, 2.89 mg a.e./L), after which abundance, growth, development, and mRNA levels of genes involved in tadpole metamorphosis were measured. Results present little evidence that exposure to this herbicide affects abundance, growth and development of wood frog tadpoles. As part of the Long-term Experimental Wetlands Area (LEWA) project, this research demonstrates that typical agricultural use of Roundup WeatherMax(®) poses minimal risk to larval amphibian development. However, our gene expression data (mRNA levels) suggests that glyphosate-based herbicides have the potential to alter hormonal pathways during tadpole development.

Tadpole mortality varies across experimental venues: do laboratory populations predict responses in nature?

Laboratory experiments are widely used to study how populations in nature might respond to various biological interactions, but the relevance of experiments in artificial venues is not known. We compiled mortality and growth data from 424 anuran populations carried out under laboratory, mesocosm, field enclosure, and field settings to determine if major differences exist amongst experimental venues and how this might influence experimental responses of tadpoles amongst venues. Our results show that there are fundamental differences in survival amongst venues, with the highest mortality occurring in field populations and the lowest in laboratory populations. Separation of mesocosm and field enclosure data based on the possibility of predatory interactions indicates that predation is an important factor leading to increased mortality in natural populations. Comparisons of size distributions across venues (although size data were limited for field populations) suggest that variation in tadpole size is low in natural populations compared to populations in artificial venues. We infer from this that mortality has a homogenizing effect on size in nature, resulting in natural populations that are not a random sample of hatched individuals. This finding suggests that populations reared under controlled laboratory conditions in the absence of predation (and other selective pressures) may not be representative of natural populations.

Effect of antidepressants on circadian rhythms in fish: Insights and implications regarding the design of behavioural toxicity tests.

Selective Serotonin Reuptake Inhibitors (SSRIs) and Serotonin and Norepinephrine Reuptake Inhibitors (SNRIs) are widely prescribed for the treatment of depression and anxiety disorders. Consequently, these compounds are frequently identified in global waterways where they may pose a hazard to aquatic biota. Evidence demonstrates these compounds to be capable of influencing the behaviour of fish, but the relevance of many reported behavioural endpoints is unclear and the value of some findings has been questioned. Since these compounds act on neuroendocrine-mediated pathways in vertebrates, the present study explored how exposure to two representative SSRIs (fluoxetine and sertraline) and an SNRI (venlafaxine) affect circadian rhythms in fish. Male mosquitofish (Gambusia holbrooki) were exposed to 1, 10 and 100μg/L concentrations of these compounds individually and when present as a full mixture, for a period of one week. Neither fluoxetine nor sertraline had an impact on diurnal activity patterns when fish were exposed to these compounds alone at any concentration, whereas venlafaxine significantly disrupted normal circadian rhythmicity but only at 100μg/L. When fish were exposed to the full mixture, significantly altered diurnal activity patterns were rapidly observed at nominal concentrations of 1 and 100μg/L, but there was no effect at 10μg/L. This sort of non-monotonic dose relationship is not altogether unusual for fish exposed to antidepressants, but it poses a problem when attempting to evaluate potential risks to the aquatic environment. To evaluate the possibility for misinterpretation when collecting behavioural data over short temporal scales, the data for each day of the experiment was analysed separately. The outcomes demonstrate the importance of longer periods of data collection, which may be necessary to capture the full range of natural behavioural variability that exists both amongst and within individual fish. More importantly, these findings may help reveal why discrepancies are commonly being reported in the literature with regards behavioural effects in fish exposed to antidepressants. It is thus suggested that research be aimed at documenting behavioural variability in fish species used in toxicity testing, to establish guidelines for quality control and where possible inform the development of standardised methodologies so that behavioural analysis can be more appropriately applied to the broad field of aquatic toxicology.

Short-term exposure to municipal wastewater influences energy, growth, and swimming performance in juvenile Empire Gudgeons (Hypseleotris compressa)

Effectively treating domestic wastewater is paramount for preserving the health of aquatic ecosystems. Various technologies exist for wastewater treatment, ranging from simple pond-based systems to advanced filtration, and it is important to evaluate the potential for these different options to produce water that is acceptable for discharge. Sub-lethal responses were therefore assessed in juvenile Empire Gudgeons (Hypseleotris compressa) exposed for a period of two weeks to control, 12.5, 25, 50, and 100% wastewater treated through a multi-stage constructed wetland (CW) treatment system. Effects on basic energy reserves (i.e., lipids and protein), growth and condition, and swimming performance were quantified following exposure. A significant increase in weight and condition was observed in fish exposed to 50 and 100% wastewater dilutions, whereas whole-body lipid content was significantly reduced in these treatments. Maximum swimming velocity increased in a dose-dependent manner amongst treatment groups (although not significantly), whereas angular velocity was significantly reduced in the 50 and 100% dilutions. Results demonstrate that treated domestic wastewater can influence the growth and swimming performance of fish, and that such effects may be related to alterations to primary energy stores. However, studies assessing complex wastewaters present difficulties when it comes to interpreting responses, as many possible factors can contribute towards the observed effects. Future research should address these uncertainties by exploring interaction between nutrients, basic water quality characteristics and relevant contaminant mixtures, for influencing the energetics, growth, and functional performance of aquatic animals.

Detectable effect size and bioassay power of mummichog (Fundulus heteroclitus) and fathead minnow (pimephales promelas) adult reproductive tests.

Although multiple reproductive tests have been developed in small-bodied fish to determine the effects of endocrine-disrupting substances, few direct comparisons have been made among the available tests. Side-by-side reproductive tests with mummichog (Fundulus heteroclitus) and fathead minnow (FHM; Pimephales promelas) were conducted with 0, 3, 10, and 30% effluent from a bleached kraft pulp mill in Saint John, New Brunswick, Canada. Egg production was significantly increased in mummichog exposed to 3% combined mill effluent, but no difference was observed in FHM. No differences were found in whole-body testosterone or estradiol levels in mummichog, and whole-body 11-ketotestosterone levels in males were increased in 3% exposed fish compared to those in 10% effluent. Male FHM exposed to 30% effluent had increased whole-body testosterone levels, and female FHM in 30% effluent had decreased testosterone. No differences in estradiol or 11-ketotestosterone were observed in FHM. Relatively limited response occurred in other endpoints. A comparison of the results of the present study to other published studies suggests that current reproductive bioassays are only sensitive for detecting magnitudes of change of greater than 50% and that differences exist in the sensitivities of fish. Future research should address methods of reducing variability within test populations and focus on understanding the comparative responses among species commonly used for endocrine-disrupting substance testing.

Toxicity of Naphthenic Acids to Wood Frog Tadpoles (Lithobates sylvaticus)

Increased incidences of mortality and adverse effects have been described for wildlife exposed to oil-sands-process-affected waters (OSPW). Naphthenic acids (NA) were identified as a primary toxic component of OSPW, yet little information exists regarding NA-induced toxicity in aquatic vertebrates. Amphibian larvae may be particularly susceptible to exposure to OSPW in groundwater surrounding oil sands regions, and increased frequency of mortality and adverse developmental effects were noted in exposed tadpoles. Despite this, there are no published studies investigating the effects of NA exposure on developing tadpoles. LC50 values of 4.76 mg/L NA were found for tadpoles at an early developmental stage (Gosner stage 28), and even greater toxicity with more developed tadpoles at 96 h, with an LC50 value of 3.04 mg/L in Gosner stage 36 tadpoles. These values are well below NA concentrations found in OSPW tailing ponds and similar to levels identified in groundwater in the Athabasca Oil Sands region.