No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Towards improved behavioural testing in aquatic toxicology: Acclimation and observation times are important factors when designing behavioural tests with fish
Abstract
The quality and reproducibility of science has recently come under scrutiny, with criticisms spanning disciplines. In aquatic toxicology, behavioural tests are currently an area of controversy since inconsistent findings have been highlighted and attributed to poor quality science. The problem likely relates to limitations to our understanding of basic behavioural patterns, which can influence our ability to design statistically robust experiments yielding ecologically relevant data. The present study takes a first step towards understanding baseline behaviours in fish, including how basic choices in experimental design might influence behavioural outcomes and interpretations in aquatic toxicology. Specifically, we explored how fish acclimate to behavioural arenas and how different lengths of observation time impact estimates of basic swimming parameters (i.e., average, maximum and angular velocity). We performed a semi-quantitative literature review to place our findings in the context of the published literature describing behavioural tests with fish. Our results demonstrate that fish fundamentally change their swimming behaviour over time, and that acclimation and observational timeframes may therefore have implications for influencing both the ecological relevance and statistical robustness of behavioural toxicity tests. Our review identified 165 studies describing behavioural responses in fish exposed to various stressors, and revealed that the majority of publications documenting fish behavioural responses report extremely brief acclimation times and observational durations, which helps explain inconsistencies identified across studies. We recommend that researchers applying behavioural tests with fish, and other species, apply a similar framework to better understand baseline behaviours and the implications of design choices for influencing study outcomes.
... On the other hand, a longer, yet timeconsuming, acclimation period may lead to reduced sample sizes and decreased power of statistical analysis, and also may be not suitable for rapid toxicity assessment or toxic effect tests (O'Neill et al. 2018). Only a few studies have emphasised the importance of the adequate duration of fish acclimation and observational time frames for the behavioural testing of such model fish species as the killifish (Fundulus heteroclitus), the adult mosquito fish (Gambusia holbrooki) and the guppy (Poecilia reticulate) (Kane et al. 2005;Melvin et al. 2017;O'Neil et al. 2018). Importantly, these studies have shown that selection of these parameters may influence interpretations of behavioural outcomes O'Neil et al. 2018). ...
... In contrast, we observed that the eurytopic fish species P. fluviatilis and G. aculeatus were less active than salmonid species after transfer to the new environment (open-field tank), especially during the first hours of acclimation. As reported by Melvin et al. (2017) andO'Neill et al. (2018), the locomotor activity of the tropic fish mosquitofish (G. holbrooki) and guppy (P. ...
... Some authors have emphasised the relevance of the appropriate fish acclimation duration to behavioural research due to its potential influence on fish behavioural outcomes (Kane et al. 2005;Melvin et al. 2017;O'Neill et al. 2018). O'Neill et al. (2018) reported the highest repeatability in guppy (P. ...
... Only a few studies have emphasized the importance of the adequate duration of fish acclimation and observational time frames for the behavioural testing of such model fish species as the killifish (Fundulus heteroclitus), the adult mosquito fish (Gambusia holbrooki) and the guppy (Poecilia reticulate) (Kane et al., 2005;Melvin et al., 2017;O'Neill et al., 2018). Importantly, these studies have shown that the selection of these parameters may influence interpretations of behavioural outcomes (Melvin et al., 2017;O'Neill et al., 2018). ...
... Only a few studies have emphasized the importance of the adequate duration of fish acclimation and observational time frames for the behavioural testing of such model fish species as the killifish (Fundulus heteroclitus), the adult mosquito fish (Gambusia holbrooki) and the guppy (Poecilia reticulate) (Kane et al., 2005;Melvin et al., 2017;O'Neill et al., 2018). Importantly, these studies have shown that the selection of these parameters may influence interpretations of behavioural outcomes (Melvin et al., 2017;O'Neill et al., 2018). Until now, changes in the behaviour of taxonomically and functionally different fish species during acclimation have not been studied. ...
... Some authors have emphasized the relevance of the appropriate fish acclimation duration to behavioural research because of its potential influence on fish behavioural outcomes (Kane et al., 2005;Melvin et al., 2017;O'Neill et al., 2018). O'Neill et al. (2018) ...
In the present study, the authors investigated the effect of acclimation duration (up to 4 h) on behavioural characteristics of taxonomically and functionally different fish species, i.e., the migratory rheophilic salmonids rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar), and the non‐migratory eurytopic European perch (Perca fluviatilis) and three‐spined stickleback (Gasterosteus aculeatus). Specifically, the authors explored fish behavioural patterns based on specific endpoints (average, maximum and angular velocity) during the acclimation period, and determined the acclimation period suitable for the tested fish species. The performed behavioural data analysis showed that the minimum time needed to adjust fish activity to a more stable (baseline) level should be at least 2 h for O. mykiss and S. salar and 1 h for G. aculeatus. Nonetheless, P. fluviatilis behaviour did not show significant changes during the 4 h acclimation. The results of this study revealed that the effect of the acclimation duration on such rheophilic species as O. mykiss and S. salar was greater than that on the eurytopic species P. fluviatilis and G. aculeatus, indicating that acclimation period is important in managing fish stress before behavioural observations. For all species, the highest variability was found in the endpoint of maximum velocity, and the lowest in that of angular velocity. This study showed that before starting actual toxicity testing experiments, it is important to determine an appropriate, species‐specific acclimation period.
... Fourth, laboratory behavior is heavily dependent on human interpretation of what situation in nature that are replicated by the behavior assay (Näslund et al., 2015), opening up for erroneous predictions as the relevance of laboratory tests are rarely validated in situ (Beekman and Jordan, 2017;Carter et al., 2013). Fifth, fish behavior in behavioral arenas are dependent on the acclimatization time, making it difficult to know if the initial behavior (early anxiety stage) or the later baseline behavior is representative of 'true' behavior (Melvin et al., 2017). Finally, complex natural environments, as opposed to simplified laboratory environments, can stimulate development of new behaviors through experience and adoption processes, and some behavioral traits have been shown to be non-repeatable after fish have adopted to natural environments (Wilson and Godin, 2009). ...
... Each fish was given one hour to acclimate in each arena before the testing began. This acclimation time is longer than the typical (median) 0.01 h used when measuring anxiety-like behavior, but is well within the initial four hour time-period when fish remain exploratory (and anxious) after being introduced in novel arenas (Melvin et al., 2017). The longer than typical acclimation time was motivated by the need for fish to adjust and thus, reduce eventual carry-over effects on behavioral between tests. ...
... Behavior differences between laboratory trials and within lake behavior, especially during the initial hours of the experiment, may be an artefact of handling stress prior to the release of the fishes. However, we found it very unlikely that the observed difference on habitat selection and home-range, effects measured over 11 days, were artefacts of fish handling prior to the release as anxious fish typically acclimatize after handling within an hourly time-scale (Melvin et al., 2017). Further, our randomization of behavioral tests makes it very unlikely that carryover effects between these test played any important role behind the seemingly counterintuitive correlation between laboratory and within lake behavior. ...
Behavioral traits measured in laboratory settings are commonly used when predicting ecological effects and evolutionary outcomes in natural systems. However, uncertainties regarding the relevance of simplified lab-based behavioral tests for complex natural environments have created doubts about the use of these tests within aquatic ecology
and ecotoxicology. In this study, we scrutinize the assumption that fish performance in six commonly applied behavioral assays have relevance for in situ behavior, by comparing individual behavior tracked in both artificial laboratory settings as well as in two natural lakes. We show that: i) commonly measured behavioral traits of individual fish (Perca
fluviatilis) have low predictive power for within-lake behaviors if interpreted alone, but that; ii) composite variables synthesized from several (six) behavioral assays explain important in situ measures such as swimming activity, dispersion, home-range size, and habitat preference. While our findings support recent criticisms against the use of single behavioral tests for predicting environmental effects, we provide empirical evidences suggesting that fish performances in multiple laboratory assays are highly relevant for fish behavior in nature.
... Therefore, observations of behavior give a unique ecological perspective, in which physiological and ecological consequences of environmental pollution are interrelated (Steinberg 2003). The most straightforward application of specialized behavioral analysis software involves the measurement of basic locomotor activity of characteristics such as swimming velocity, the distance traveled, frequency, and duration of fish movements (Tierney 2011;Benhaim et al. 2012;Melvin et al. 2017). In some cases, behavioral alterations in fish may be not noticed during short-term toxicity tests using one or a few endpoints. ...
... The following locomotor activity parameters were chosen for behavioral testing: average velocity (cm/s), maximum velocity (cm/s), angular velocity (deg/s), movement duration (%), and body mobility duration (%) ( Table 3). The choice of these endpoints for this experimental setup was predetermined by the fact that they are commonly reported in behavioral studies (Tierney 2011;Benhaim et al. 2012;Melvin et al. 2017). These parameters were recorded for 1 min at selected time intervals (every 10 min) in the course of 2-h exposure to selected leachate solutions using a digital video camera (PANASONIC HC-V770 Wi-Fi, 12.76 MP). ...
... On the other hand, detailed characterization of fish behavioral patterns may call for longer observation duration. Few studies in behavioral toxicology have paid attention to the importance of exploring and understanding the basic behavioral characteristics of fish in relation to time including such relevance factors as suitable observation duration and its potential influence on the outcomes and their interpretation (Melvin et al. 2017;O'Neill et al. 2018). Stankeviciūtė et al. (2018) showed that for the characterization of behavioral patterns of the Atlantic salmon (Salmo salar) juveniles exposed to complex metal mixtures at environmentally relevant concentrations, the duration of exposure has to be 2-h long. ...
The aim of the present study was to investigate changes in the locomotor activity of rainbow trout (Oncorhynchus mykiss) juveniles under the impact (2 h) of landfill leachate (as a multicomponent mixture) based on different endpoints such as average, maximum and angular velocities, movement duration, body mobility, and blood glucose level. Fish were exposed to five different sublethal leachate concentrations (0, 0.0625, 0.125, 0.25, and 0.5%). The locomotor activity of the leachate-exposed fish significantly decreased at 0.25 and 0.5% concentrations. Significant changes in fish behavior in response to sublethal leachate concentrations were determined during the first minutes of exposure. Angular velocity proved to be the most sensitive of all the endpoints tested. A positive correlation was observed among behavioral responses, but no correlation was established between the blood glucose level and behavioral endpoints. The blood glucose endpoint was found to be insensitive, and we suggest that it should be used only in combination with other endpoints to complement toxicity data. To enhance the understanding of rainbow trout behavioral characteristics in relation to time, and relations among behavioral endpoints of the fish under short-term exposure to a multicomponent mixture, in the current study, we investigated dynamics of the selected behavioral endpoints over time, relations among these endpoints and compared behavioral response rapidness and efficacy.
... There has been a call for more validation of behavioral assessments because animals are innately variable in their behaviors, and study design can influence outcomes, resulting in inconsistencies between studies (Sumpter et al. 2014;Melvin et al. 2017). We recommend further development, optimization, and standardization for juvenile frog behavioral assessments because information on this particular life stage is lacking yet highly influential in population sustainability (Biek et al. 2002;Vonesh and De la Cruz 2002). ...
... We recommend that our method be repeated with larger sample sizes, by other laboratories, and with other species of frogs to determine whether this type of assessment has a wider application and is relatively conserved between species. To further the use of the behavioral endpoints that have fitnessrelated consequences, such as our measures, baseline information on the intra-and interindividual variability is also required (Melvin et al. 2017). We did not conduct multiple trials with the same frog on the same day or as a repeated measure over time, and we therefore suggest that our study be considered an initial assessment that requires further validation and cautious data interpretation. ...
... Using our preliminary assessment, we would suggest a sample size of 50/treatment for followup studies based on a simple post hoc power analysis (85% active response for controls, $60% active response for imidacloprid treatments, a ¼ 0.05, b ¼ 0.2, power ¼ 0.8). Furthermore, our acclimation period of 1.5 min and observation period of 5 min was fairly arbitrary, and more work on the influence of these time frames may be worthwhile for the ultimate development of juvenile frog behavioral assessments (Melvin et al. 2017). ...
Neonicotinoids are water‐soluble neurotoxic insecticides widely used in agriculture that are being detected in nontarget aquatic environments. Nontarget aquatic wildlife, such as amphibians, may be at risk of exposure. Studies using larval stages suggest neonicotinoids are a minor concern to amphibians; however, behavioral effects manifesting later in life are not often considered. Behavioral endpoints could further our understanding of potential sublethal neurotoxic effects after exposure has ended. Using juvenile wood frogs (Lithobates sylvaticus), we investigated the effects of chronic larval exposure to 3 concentrations (1, 10, and 100 μg/L) of formulations containing imidacloprid or thiamethoxam on the putative escape response to a simulated heron attack. We found that control frogs actively responded (i.e., moved or jumped) to the simulated predator attack but frogs exposed to imidacloprid at 10 and 100 μg/L were less likely to respond. The exposed frogs, specifically from the imidacloprid treatment at 10 μg/L (tendency at 100 μg/L) were less likely to leave the attack area compared with controls. However, frogs used refuge similarly among all treatments. Finally, there were no differences in locomotor performance, as measured by total number of jumps and distance traveled during a trial among treatments. In conclusion, our study suggests that exposure to neonicotinoids during amphibian larval development may affect a juvenile frog's ability to perceive or respond to a predator, potentially increasing their vulnerability to predation. Future studies should validate and explore this potential effect further. Environ Toxicol Chem 2018;9999:1–9. © 2018 Crown in the right of Canada. Published by Wiley Periodicals Inc. on behalf of SETAC.
... This study revels LC 50 value of the p', p' DDT (0.023 mg/L), and Chlorpyrifos (0.403 mg/L) on common carp fish observed less than the mean contamination (DDT 0.024 mg/L and chlorpyrifos 0.47 mg/L) level of these pesticides in the surface water of the study locations. Nevertheless, the toxic effects of the theses pesticide contamination not only depending on the quantity, but influenced by several other environmental and biological factors [35]. The present study estimated the contamination of excessively used run-off pesticide residues in surrounding water bodies and their toxic effects on aquatic organisms in coffee plantations. ...
... Sci. (Jan-Feb) 12(1):[32][33][34][35][36][37] ...
The study estimated the pesticide residues in 40 surface water samples from four locations of arabica coffee plantations in the Western Ghats. Residual analysis of water samples confirmed the contamination of both organochlorine (OC) and organophosphate (OP) pesticides in surface water. OC pesticide residues present in the range 0.01-1.10 µg/L, with the maximum presence of HCH, heptachlor, DDT, and DDT isomers. Chlorpyrifos present in the range of 0.02-.069 µg/L among the targeted OP pesticides. Acute toxicity estimated by using common carp fish for both p', p' DDT, and chlorpyrifos. LC50 value of p', p' DDT, and chlorpyrifos was 0.023 and 0.403 mg/L, respectively. This study confirmed the presence of excessive pesticide contamination in surface water and its toxic effect on common carp.
... Behavioral tests have come under greater scrutiny recently due to lack of consistency and comparability between data (Melvin et al., 2017). Our study followed two important protocol guidelines to make behavioral endpoint data robust. ...
... Bass were given 9 days to acclimate to exposure tanks during individual training, allowing ample adjustment time before experimental procedures began. In addition, 25 min periods were given for bass to chase and consume minnows, which allowed time for the fish to chase down prey if hungry (Melvin et al., 2017). Our experiments have demonstrated repeatability after comparison of results from three other researchers from our lab who have completed similar tests with different pharmaceuticals (Gaworecki and Klaine, 2008;Bisesi et al., 2014;Sweet, 2015;Bisesi et al., 2016). ...
Millions of pharmaceuticals are prescribed each year. Wastewater treatment plants fail to remove all pharmaceuticals from discharge leading to detectable concentrations entering aquatic ecosystems where the compounds can encounter nontarget organisms. Selective serotonin reuptake inhibitor (SSRI) class of antidepressants interact with transporters in the brain and peripheral nervous system to change serotonin levels in the synapse. Sublethal exposure to SSRIs can impact fish feeding behaviors, which can have impacts on ecological fitness. We exposed hybrid striped bass (Morone saxatilis x Morone chrysops) to low, medium, and high concentrations of sertraline (4.5 ± 0.84 µg/L, 35.4 ± 2.18 µg/L, and 96.8 ± 6.4 µg/L) over six days with six additional recovery days. Concentrations were chosen to compare results with a mixture study previously completed in our lab. Every three days we tracked how long each bass took to consume four fathead minnows (Pimephales promelas) and conducted destructive sampling to obtain brain and plasma samples. Brain and plasma samples were analyzed for sertraline levels and we calculated whole brain serotonin levels. During the exposure period, bass showed an increased time to capture prey, but time to capture prey returned to control levels during the six-day recovery period. Sertraline was detected in brain and plasma during the duration of the experiment, though not always in a dose-dependent fashion. While we demonstrated a relationship between time to capture prey and decrease whole brain serotonin levels, the decrease in time to capture prey during the recovery period suggests the serotonin levels in the brain are not solely responsible for the outward behavioral expression observed. Understanding individual pharmaceutical impacts on bass behavior can inform mixtures studies because aquatic organisms will encounter multiple toxicants at a time.
... Despite such promising advances, zebrafish locomotor activity tests often suffer from high inter-individual variability. This ultimately impairs both the robustness and repeatability within which small, but potentially important, biological effects can be detected [8][9][10][11][12][13][14] . ...
... Scientific research is constantly under intense scrutiny, specifically for the occurrence of irreproducible and non-comparable findings. In particular, high-throughput behavioral tests frequently result in inconsistent findings, which researchers attribute to poor quality science and non-standardized protocols [9][10][11][12][13] . However, this problem also strongly links to the lack of understanding of the variability of basic behavioral patterns, as fish fundamentally change their swimming behavior over time [1][2][3][4][5][6][7]92 . ...
The analysis of larval zebrafish locomotor behavior has emerged as a powerful indicator of perturbations in the nervous system and is used in many fields of research, including neuroscience, toxicology and drug discovery. The behavior of larval zebrafish however, is highly variable, resulting in the use of large numbers of animals and the inability to detect small effects. In this study, we analyzed whether individual locomotor behavior is stable over development and whether behavioral parameters correlate with physiological and morphological features, with the aim of better understanding the variability and predictability of larval locomotor behavior. Our results reveal that locomotor activity of an individual larva remains consistent throughout a given day and is predictable throughout larval development, especially during dark phases, under which larvae demonstrate light-searching behaviors and increased activity. The larvae’s response to startle-stimuli was found to be unpredictable, with no correlation found between response strength and locomotor activity. Furthermore, locomotor activity was not associated with physiological or morphological features of a larva (resting heart rate, body length, size of the swim bladder). Overall, our findings highlight the areas of intra-individual consistency, which could be used to improve the sensitivity of assays using zebrafish locomotor activity as an endpoint.
... However, studies have been met with some scepticism as to their repeatability due to varied results found in the literature (Sumpter, Donnachie & Johnson, 2014). This is potentially due to a lack of standardisation and validation of behavioural assays for the assessment of behavioural endpoints (Spruijt et al., 2014;Parker, 2016;Melvin et al., 2017). For example, the open field test has historically been implemented to measure multiple behaviours associated with anxiety and to assess the effects of behavioural modifying compounds in rodents. ...
... While the trial duration in this study was in accordance with other novelty based assays , our results do not confirm whether the trial duration in this study is sufficient for E. marinus. Melvin et al. (2017) assessed the effects of acclimation time to a test arena on a range of swimming behaviours in mosquito fish and concluded that a longer acclimation may be required to ensure that 'normal' baseline behaviours are being observed. It was also demonstrated that a longer acclimation time improved the statistical power of the behavioural assay. ...
The use of behaviour in ecotoxicology is expanding, however the lack of standardisation and validation of these assays currently presents a major drawback in moving forward in the development of behavioural assays. Furthermore, there is a current paucity of control data on test species, particularly invertebrate models. In this study we assessed a range of behaviours associated with spatial distribution and locomotion in relation to arena size and shape in two species of amphipod crustacean ( Echinogammarus marinus and Gammarus pulex ). Arena shape had significant effects on almost all behavioural parameters analysed. Increasing arena size resulted in an increased mean velocity and activity plus increased proportional use of the central zones. These results indicate that ‘ceiling effects’ may occur in some ecotoxicological studies resulting in potentially ‘false’ negative effects if careful consideration is not paid to experimental design. Differences in behaviours were observed between the two species of amphipod. For example, G. pulex spend approximately five times (∼20%) more of the available time crossing the central zones of the arenas compared to E. marinus (∼4%) which could have implications on assessing anxiolytic behaviours. The results of this study highlight several behaviours with potential for use in behavioural ecotoxicology with crustaceans but also underscore the need for careful consideration when designing these behavioural assays.
... The two experiments performed, A and B, are considered to contribute equally to the study of the system's behavior with different number of fish, and whether the experiment was performed by decreasing or by increasing the number of fish in the tank should not have a bearing on the results because the fish had been acclimated for a long enough period of time prior to the recordings. The acclimation periods we have used (12 and 2.5 days and 23 h) are longer or similar to most of those reported in the literature, for example Stienessen and Parrish (2013) used only 1 day and Melvin et al. (2017) indicated that these kind of studies should be preceded by an acclimation period of at least several hours to evaluate normal baseline behaviors. For the freshwater species they used, mosquitofish (Gambusia affinis), this period was 8 h. ...
... For the freshwater species they used, mosquitofish (Gambusia affinis), this period was 8 h. Moreover, in both Melvin et al. (2017) and our present work, the fish had been acclimated to the laboratory tanks for 3 months prior to the initiation of the experiment, which was carried out in similar tanks and conditions to those to which they had been acclimated to. ...
The present study aims at identifying the lowest number of fish (European seabass) that could be used for monitoring and/or experimental purposes in small-scale fish facilities by quantifying the effect that the number of individuals has on the Shannon entropy (SE) of the trajectory followed by the shoal’s centroid. Two different experiments were performed: (i) one starting with 50 fish and decreasing to 25, 13, and 1 fish, and (ii) a second experiment starting with one fish, adding one new fish per day during 5 days, ending up with five fish in the tank. The fish were recorded for 1h daily, during which time a stochastic event (a hit in the tank) was introduced. The SE values were calculated from the images corresponding to three arbitrary basal (shoaling) periods of 3.5 min prior to the event, and to the 3.5 min period immediately after the event (schooling response). Taking both experiments together, the coefficient of variation (CV) of the SE among measurements was largest for one fish systems (CV 37.12 and 17.94% for the daily average basal and response SE, respectively) and decreased concomitantly with the number of fish (CV 8.6–10% for the basal SE of 2 to 5 fish systems and 5.86, 2.69, and 2.31% for the basal SE of 13, 25, and 50 fish, respectively). The SE of the systems kept a power relationship with the number of fish (basal: R²= 0.93 and response: R²= 0.92). Thus, 5–13 individuals should be the lowest number for a compromise between acceptable variability (<10%) in the data and reduction in the number of fish. We believe this to be the first scientific work made to estimate the minimum number of individuals to be used in subsequent experimental (including behavioral) studies using shoaling fish species that reaches a compromise between the reduction in number demanded by animal welfare guidelines and a low variability in the fish system’s response.
... zero-inflated data sets). Because different observational timeframes may influence the overall conclusions of behavioural analysis (Melvin, 2017;Melvin et al., 2017), test durations should ideally be validated (i.e. robust and repeatable protocols) or justified in light of the species and life stage used, type, and overall context of the experiments that are conducted. ...
Behavioural analysis has been attracting significant attention as a broad indicator of sub‐lethal toxicity and has secured a place as an important subdiscipline in ecotoxicology. Among the most notable characteristics of behavioural research, compared to other established approaches in sub‐lethal ecotoxicology (e.g. reproductive and developmental bioassays), are the wide range of study designs being used and the diversity of endpoints considered. At the same time, environmental hazard and risk assessment, which underpins regulatory decisions to protect the environment from potentially harmful chemicals, often recommends that ecotoxicological data be produced following accepted and validated test guidelines. These guidelines typically do not address behavioural changes, meaning that these, often sensitive, effects are not represented in hazard and risk assessments. Here, we propose a new tool, the EthoCRED evaluation method, for assessing the relevance and reliability of behavioural ecotoxicity data, which considers the unique requirements and challenges encountered in this field. This method and accompanying reporting recommendations are designed to serve as an extension of the “Criteria for Reporting and Evaluating Ecotoxicity Data (CRED)” project. As such, EthoCRED can both accommodate the wide array of experimental design approaches seen in behavioural ecotoxicology, and could be readily implemented into regulatory frameworks as deemed appropriate by policy makers of different jurisdictions to allow better integration of knowledge gained from behavioural testing into environmental protection. Furthermore, through our reporting recommendations, we aim to improve the reporting of behavioural studies in the peer‐reviewed literature, and thereby increase their usefulness to inform chemical regulation.
... Development) Guidance Document (OECD 2002); moreover, it is cheap and easy to carry as limited manipulation of individuals is required. In addition, ecotoxicological studies usually require an important number of replicates for each treatment (e.g. 12 × 3 per experimental group in this study) related to the high inter-individual variability commonly observed (Melvin et al. 2017). The presented two-step procedure allows to reduce the number of individuals tested and allows to obtain a behavioural profile by testing the same individual under two different behavioural tests (i.e. ...
Endocrine-disrupting chemicals (EDCs) are widespread pollutants known to interfere with hormonal pathways and to disrupt behaviours. Standardised behavioural procedures have been developed in common fish model species to assess the impact of various pollutants on behaviours such as locomotor activity and anxiety-like as well as social behaviours. These procedures need now to be adapted to improve our knowledge on the behavioural effects of EDCs on less studied marine species. In this context, the European sea bass (Dicentrarchus labrax) is emerging as a valuable species representative of the European marine environment. Here, we designed and validated a two-step procedure allowing to sequentially assess anxiety-like behaviours (novel tank test) and social preference (visual social preference test) in sea bass. Thereafter, using this procedure, we evaluated whether social behavioural disruption occurs in 2-month-old larvae after an 8-day exposure to a xenoestrogen, the 17α-ethinylestradiol (EE2 at 0.5 and 50 nM). Our results confirmed previous studies showing that exposure to 50 nM of EE2 induces a significant increase in anxiety-like behaviours in sea bass larvae. On the contrary, social preference seemed unaffected whatever the EE2 concentration, suggesting that social behaviour has more complex mechanical regulations than anxiety.
... It is widely accepted that the choice of culture conditions for kept specimens represents a major challenge that potentially impacts results of any behavioral and physiological assay undertaken; however, no serious attempts have been made to standardize this type of research. Melvin et al. (2017) demonstrated that for every one of the 165 studies on fish behavior they analyzed, acclimation and behavioral observation periods differed. In contrast, for OA research, there are standardized guidelines (Riebesell et al., 2011) to ensure the comparability of chemical data obtained. ...
Chemosensory science, the study of how organisms produce and assess olfactory information, is central to our understanding of how organisms interact and gain information about their environment. Signaling cue identification in aquatic systems lags behind our knowledge in terrestrial insects due to analytical challenges in aqueous environments. Unambiguous, reliable, and fast behavioral assays to evaluate the biological activity and function of a chemosensory cue are critical to understand aquatic signaling systems and enable research into their ecology, evolution, and threats in a changing environment. Yet, a range of anthropomorphic assumptions made in this research field create additional challenges to interpret data generated. Here, we evaluate common challenges including assumed readiness of individuals to respond, lack of information on the animals’ physiological and social status, their pre-experimental cue exposure, the innate or learned character of the responses, the animals’ acclimation and habituation status, and the impact of the animals upon their own environment. These factors lead to significant variability in animals’ responses in bioassays, both in the field and in laboratory setups. In the light of our limited knowledge of aquatic chemosensory cues’ chemical structure, active concentrations in samples, and undetermined response thresholds, we evaluate methods of mitigation to minimize differences between studies. We conclude that currently it is nearly impossible to compare results from chemosensory behavioral studies undertaken in different ecosystems, laboratories, and time points. There is an urgent need for the standardization of behavioral methods, recording of environmental conditions, and individuals’ physiology, physical, and social status, to avoid conflicting and contradicting results when comparing studies. Including these parameters in experimental design and data interpretation will provide a deeper understanding of chemosensory communication, reduce unconscious bias in studies, and can help to explain the substantial individuality in animals’ responses to chemosensory cues and their acclimation to environmental stress.
... This behavior is commonly interpreted as an indicator of acute stress. To overcome handling/novelty stress, acclimation to the novel environment or direct recording in experimental set-ups are two alternatives, although there appears to be no consensus on the duration of acclimation from 1 to 60 min (Melvin et al. 2017). In our study, larvae were acclimated for 5 min prior to recordings. ...
The widespread use of 17α-ethinylestradiol (EE2), and other estrogenic endocrine disruptors, results in a continuous release of estrogenic compounds into aquatic environments. Xenoestrogens may interfere with the neuroendocrine system of aquatic organisms and may produce various adverse effects. The aim of the present study was to expose European sea bass larvae (Dicentrarchus labrax) to EE2 (0.5 and 50 nM) for 8 d and determine the expression levels of brain aromatase (cyp19a1b), gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), kisspeptins (kiss1, kiss2) and estrogen receptors (esr1, esr2a, esr2b, gpera, gperb). Growth and behavior of larvae as evidenced by locomotor activity and anxiety-like behaviors were measured 8 d after EE2 treatment and a depuration period of 20 d. Exposure to 0.5 nM EE2 induced a significant increase in cyp19a1b expression levels, while upregulation of gnrh2, kiss1, and cyp19a1b expression was noted after 8 d at 50 nM EE2. Standard length at the end of the exposure phase was significantly lower in larvae exposed to 50 nM EE2 than in control; however, this effect was no longer observed after the depuration phase. The upregulation of gnrh2, kiss1, and cyp19a1b expression levels was found in conjunction with elevation in locomotor activity and anxiety-like behaviors in larvae. Behavioral alterations were still detected at the end of the depuration phase. Evidence indicates that the long-lasting effects of EE2 on behavior might impact normal development and subsequent fitness of exposed fish.
... Furthermore, one of the originalities of our study was to set the conditions to homogenize the gut microbiota diversity within amphioxus individuals, by one-week starvation, before running toxicity tests. It was also suggested as an important process in profiling physiological changes in toxicity tests to improve the ecological relevant and statistical robustness (Melvin et al., 2017). Finally, for each seawater change, the seawater was filtrated with 0.22 μm sterile filter, as a result of this, we could exclude the impact of nutrient source and bacterial community from the seawater. ...
Oceanic plastic pollution is of major concern to marine organisms, especially filter feeders. However, limited is known about the toxic effects of the weathered microplastics instead of the pristine ones. This study evaluates the effects of weathered polystyrene microplastic on a filter-feeder amphioxus under starvation conditions via its exposure to the microplastics previously deployed in the natural seawater allowing for the development of a mature biofilm (so-called plastisphere). The study focused on the integration of physiological, histological, biochemical, molecular, and microbiota impacts on amphioxus. Overall, specific alterations in gene expression of marker genes were observed to be associated with oxidative stresses and immune systems. Negligible impacts were observed on antioxidant biochemical activities and gut microbiota of amphioxus, while we highlighted the potential transfer of 12 bacterial taxa from the plastisphere to the amphioxus gut microbiota. Moreover, the classical perturbation of body shape detected in control animals under starvation conditions (a slim and curved body) but not for amphioxus exposed to microplastic, indicates that the microorganisms colonizing plastics could serve as a nutrient source for this filter-feeder, commitment with the elevated proportions of goblet cell-like structures after the microplastic exposure. The multidisciplinary approach developed in this study underlined the trait of microplastics that acted as vectors for transporting microorganisms from the plastisphere to amphioxus.
... Indeed, behavioral tests can be 10-1000 times more sensitive than lethality tests (Hellou, 2011;Hellou et al., 2008;Robinson, 2009). However, the potential exists for the experimental conditions themselves to influence behavior, especially acclimation and observation times (Melvin et al., 2017). Behavioral aquatic tests, therefore, should have a mechanistic basis, allow accurate predictions, represent what happens in the field, besides being reproducible and reliable. ...
An endless list of new chemicals are entering nature, which makes it an impossible task to assess all possible mixture combinations at all possible concentrations and conditions that are leading to the ubiquitous anthropogenic impacts on the aquatic environment resulting from deteriorating water quality. Therefore, ecotoxicology is moving more toward a mechanistic understanding of toxicological processes, using trait-based approaches and sublethal molecular and physiological endpoints to understand the mode of action of pollutants and the adverse outcomes at the organismal and population level. These molecular and physiological endpoints can be used as biomarkers, applicable in the field. This brings ecotoxicological research much closer to conservation physiology. Understanding the relationships between chemical reactivity in the water and in organisms, and assessing the consequences at higher levels, allows conservation physiologists and managers to take the right restoration measures for an optimal improvement of the aquatic habitats of concern. In this chapter we discuss the role which the promising approach of mechanistic-based Adverse Outcome Pathways (AOPs) can play in ecotoxicological research. It studies a pathway of events, from the direct interaction of a chemical with a molecular target, through subsequent intermediate events at cellular, tissue, organ and individual organism levels which then result in an Adverse Outcome (AO) relevant to ecotoxicological risk assessment and regulatory decision-making. In this context, we also discuss the importance of modeling, including bioavailability based and effect based models. Finally, we reflect on the possibilities that meta-analysis has to offer to detect unifying physiological processes, as well as interesting outliers.
... Swimming behavioural analysis is being largely applied in modern aquatic ecotoxicology [46,47]. The growing interest in behavioural video tracking is attributable to the relevance of swimming behavioural changes as an optimal sub-lethal endpoint [48], to technological advances in the past decade and to the easy accessibility of computational software [49]. The present work represents a first step towards understanding how the observation time can influence the analysis of the main behavioural parameters defining the swimming patterns of freshwater inbenthic microcrustaceans used to assess sub-lethal effects of pollutants. ...
The analysis of copepod behaviour gained an increasing impetus over the past decade
thanks to the advent of computer-assisted video analysis tools. Since the automated tracking consists
in detecting the animal’s position frame by frame and improving signals corrupted by strong background
noise, a crucial role is played by the length of the video recording. The aim of this study is
to: (i) assess whether the recording time influences the analysis of a suite of movement descriptive parameters;
(ii) understand if the recording time influences the outcome of the statistical analyses when
hypotheses on the effect of toxicants/chemicals on the freshwater invertebrate behaviour are tested.
We investigated trajectory parameters commonly used in behavioural studies—swimming speed,
percentage of activity and trajectory convex hull—derived from the trajectories described by the
inbenthic–interstitial freshwater copepod Bryocamptus pygmaeus exposed to a sub-lethal concentration
of diclofenac. The analyses presented in this work indicate that the recording time did not influence
the outcome of the results for the swimming speed and the percentage of activity. For the trajectory
convex hull area, our results showed that a recording session lasting at least 3 min provided robust
results. However, further investigations are needed to disentangle the role of concurrent factors, such
as the behavioural analysis of multiple individuals simultaneously, whether they are of the same or
opposite sex and the implications on sexual behaviour, competition for resources and predation.
... The maximum observation time for each group of tests was 40 min, and if the experimental fish failed to reach the vicinity of the water outlet after 40 min, the experimental fish were considered not successfully attracted. Nevertheless, such a short adaptation time and duration of the experiment may have caused some variation in the results [36,37]. To avoid the effects of light and time, the experiments were completed during well-lit periods, and the time of the experiments was kept the same as much as possible. ...
The damming of the river changes the structure of the original river ecosystem, and although fish passage plays an important role in maintaining the connectivity of the river ecosystem, the fish have difficulty finding the fish passage entrance during the upstream process. This paper studied the rheotaxis of fish under three different water flow conditions experimentally through recirculating water tanks. To better understand the response of Crucian carp (Carassius auratus) to water flow stimulation, the representative swimming trajectory, sensing success rate, attraction success rate, reaction time, and attraction time of the fish were analyzed by using a video monitoring system. The experimental results showed that fish responded differently to single-peak and lateral bimodal outflow conditions: (1) the single-peak outflow condition had a much better attraction effect than the lateral bimodal outflow condition, both in terms of sensing success rate and attraction success rate; (2) the fish swam mainly in the middle area of the lateral bimodal outflow condition, while the fish swam more evenly in the single-peak outflow condition. Therefore, setting the attraction current at the right time and near the entrance of the fish passage may help to improve the effect of fish attraction.
... Lack of feeding might have been caused by at least three different mechanisms: (1) fish might have been too small to feed on specific zooplankton species, i.e. gape limitation (DeVries et al., 1998), (2) fish may not react to, or chose to avoid, small prey perhaps due to insufficient energetic return (Sinervo, 1997), or (3) fish might not have been sufficiently acclimatized to the experimental setting (Melvin et al., 2017). The significance of the logistic parts of the zero-inflated models shows that the percentage of fish not feeding was nonrandomly distributed across fish sizes, fish species and prey species. ...
Invasion of non‐native species might alter food web structure and the strength of top‐down control within lake ecosystems. As top‐down control exerted by fish populations is often dominated by young of the year fish, the impact of new fish species might depend on the feeding rates of the juvenile fish. Here we provide comparative analyses of feeding rates of juvenile whitefish (Coregonus wartmanni) – a native and specialised planktivore and an invasive generalist (sticklebacks, Gasterosteus aculeatus). We studied feedings rates of whitefish and sticklebacks in aquaria experiments using 2 cm to 8 cm fish feeding on seven zooplankton species common to Lake Constance. As whitefish hatch several months earlier than sticklebacks, 0+ whitefish are larger than 0+ sticklebacks throughout the year and hence are predicted to have higher feeding rates on especially large zooplankton species. We show that sticklebacks as small as 2 cm were able to feed on the largest zooplankton species of Lake Constance. Further, stickleback feeding rates were similar to both the same size 0+ whitefish and the larger 0+ whitefish co‐occurring with smaller 0+ sticklebacks. Hence, 0+ sticklebacks will compete with 0+ whitefish for the same zooplankton species, therefore the invasion of sticklebacks is unlikely to change the relative feeding pressure by individual 0+ fish on zooplankton species. The juvenile period of fish is crucial for their establishment in a new habitat as well as for the grazing impact of fish on prey communities. Here, we show that juveniles of an invasive species, sticklebacks, do have similar feeding rates on various zooplankton species compared to native whitefish despite the latter are specialized planktivores and advanced sticklebacks in growth during the seasonal course. Our results suggest that the invasion of sticklebacks and partial replacement of whitefish should not result in changing relative feeding pressures on the various zooplankton species.
... Even if clove oil and tagging may have had an effect on fish behaviour, the protocol was strictly maintained in all fish in order to accurately compare behavioural parameters among the different experimental groups. Then, fish were let to recovered and acclimated in the testing tank for 1 h, according the acclimation time used in most of locomotor activity-related studies in several fish species (Melvin et al., 2017). After this acclimation period, fish were exposed to a mechano-sensorial vibrational stimulus, which consisted in an iron pendulum of 600 g that was dropped without any additional force against the tank wall from a distance of 71 cm. ...
N-3 long-chain polyunsaturated fatty acids (LC-PUFA), particularly docosahexaenoic acid (22:6n-3, DHA), are the most important fatty acids with physiological significance in brain function of all vertebrates. Recently, novel lipid sources are available for the industry, including products obtained from microorganisms. Dietary fatty acid profiles may affect tissue composition and cell functioning. To determine the effect of novel lipid sources on behaviour and neural function in gilthead sea bream (Sparus aurata), juveniles were fed three isoproteic and isoenergetic diets containing either (FO), a microalgae oil combined with poultry oil (DD) or only poultry oil (PO). Behaviour response, brain fatty acid composition and relative expression of neurogenesis and neural activity related-genes in telencephalon were evaluated during 5 months of feeding with the experimental diets. Brain of sea bream fed diet PO showed the highest DHA content as well as increased desaturation and elongation products, contrary to the dietary pattern. This, together with an increased telencephalic fads2 expression denoted the effective activation of LC-PUFA synthesis and retention as a compensatory mechanism for a dietary deficiency. Furthermore, neurod6, bdnf and nos1 were proportionally upregulated in relation to the neural DHA content. Locomotor performance during cruising and escape responses was not affected by the experimental diets. However, there was a tendency for escape latency to be longest in fish fed PO and DD diets, and a relationship between escape latency and brain n-6 docosapentaenoic acid (DPA) contents was suggested. Indeed, the relation of n-6 DPA with behavioural deficits was shown previously in mammals and might deserve further attention and corroboration in fish as well in the future.
... Changes in swimming behaviour of fish and other aquatic organisms induced by exposure to pollutants have a detrimental effect on animal behaviour including food search, consumption, and response to predators, predator avoidance, or successful reproduction (Steinberg 2003). The endpoints such as average, maximum, and angular velocities are commonly used to describe the swimming activity patterns of fish (Benhaïm et al. 2012;Melvin et al. 2017), the occurrence of their erratic movements (Kim and Wardle 2005), and stress responses including escape behaviour (Danos and Lauder 2012). ...
Sticklebacks (Gasterosteiformes) are increasingly used in ecological and evolutionary research and have become well established as role model species for biologists. However, ecotoxicology studies concerning behavioural effects in sticklebacks regarding stress responses, mainly induced by chemical mixtures, have hardly been addressed. For this purpose, we investigated the swimming behaviour (including mortality rate based on 96-h LC50 values) of two ecologically similar three-spined (Gasterosteus aculeatus) and nine-spined sticklebacks (Pungitius pungitius) to short-term (up to 24 h) metal mixture (MIX) exposure. We evaluated the relevance and efficacy of behavioural responses of test species in the early toxicity assessment of chemical mixtures. Fish exposed to six (Zn, Pb, Cd, Cu, Ni, and Cr) metals in the mixture were either singled out by the Water Framework Directive as priority or as relevant substances in surface water, which was prepared according to the environmental quality standards (EQSs) of these metals set for inland waters in the European Union (EU) (Directive 2013/39/EU). The performed behavioural analysis showed the main effect on the interaction between time, species, and treatment variables. Although both species exposed to MIX revealed a decreasing tendency in swimming activity, these species’ responsiveness to MIX was somewhat different. Substantial changes in the activity of G. aculeatus were established after a 3-h exposure to MIX solutions, which was 1.43-fold lower, while in the case of P. pungitius, 1.96-fold higher than established 96-h LC50 values for each species. This study demonstrated species-specific differences in response sensitivity to metal-based water pollution, indicating behavioural insensitivity of P. pungitius as model species for aquatic biomonitoring and environmental risk assessments.
... The locomotor activity of fish is the most frequently assessed sublethal endpoint when determining behavioural changes in response to contaminants in toxicity tests and, therefore, it is considered to be a critical character when establishing fish survival in natural environment (Gui et al., 2014). Such endpoints as average velocity and angular velocity that are commonly used in behavioural studies provide useful information for the basic swimming performance assessment, and when combined, indicate behavioural complexity and occurrences of erratic movements (Melvin et al., 2017;Benhaim et al., 2012;Tierney, 2011). Since behaviour integrates biochemical, physiological, and ecological processes, it may be ideal for studying environmental pollution effects (Scott and Sloman, 2004). ...
The present study tested the biological consequences of exposure to a multimetal mixture as a multiple chemical stressor on Oncorhynchus mykiss at molecular, cellular, physiological and whole-organism levels and on biomarker responses of this fish during the depuration period. To represent environmentally relevant multiple chemical stressors, in our study, we used the mixture of Zn, Cu, Ni, Cr, Pb and Cd at the concentrations corresponding to Maximum-Permissible-Concentrations (MPCs) acceptable for the EU inland waters. This study was undertaken with a view to elucidate if changes in the MPC of the test mixture components (Ni, Pb, Cd) could cause significantly different biomarker responses in O. mykiss from those previously determined in the carnivorous and omnivorous fishes exposed to the mixture of the same metals but at different MPCs of Ni, Pb and Cd. This study has revealed that exposure to mixtures of metals at MPC produces genotoxic effects in fish blood erythrocytes and a lethargic effect on O. mykiss behaviour, and, also, significantly increases the levels of Cd, Cr and Ni accumulated in the gills tissue. O. mykiss successfully depurated Cr and Ni in less than 28 days, however, the level of Cd decreased by only approximately 40% over the same period. A significant capacity of O. mykiss to restore its DNA integrity (Comet assay) after exposure to metal mixtures was revealed. However, the 28-day recovery period proved to be insufficiently long for erythrocytes with nuclear abnormalities to recover to the unexposed level. In conclusion, changes in the MPCs of Ni, Pb and Cd in the test mixture produce biological effects similar to those previously determined in S. salar, R. rutilus and P. fluviatilis exposed to the mixture of the same metals but at lower MPCs of Ni and Pb and at higher MPC of Cd.
... Researchers have highlighted a lack of baseline data to be a source of variability in the results of behavioural studies in ecotoxicology [68]. This has been supported by Melvin et al. [69] who explored how fish acclimate to behavioural arenas and how different lengths of observation time impact estimates of basic swimming parameters. They concluded that researchers need to establish a basic knowledge about the baseline behavioural characteristics for a model species, as this could influence study outcomes of behavioural ecotoxicology experiments. ...
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.
... They could have an aberrant response to the arena, despite its small size (6.4-mm) and despite their lack of eye pigment. That response could represent a form of acclimation (Melvin, Petit, Duvignacq, & Sumpter, 2017;Teske, Perez-Leighton, Billington, & Kotz, 2014), with initial exploration followed by settling down to their baseline quiescent phenotype. Second, CASK mutants might be unable to sustain even moderate walking, perhaps because of metabolic insufficiency. ...
Mutations in hundreds of genes cause neurodevelopmental disorders with abnormal motor behavior alongside cognitive deficits. Boys with fragile X syndrome (FXS), a leading monogenic cause of intellectual disability, often display repetitive behaviors, a core feature of autism. By direct observation and manual analysis, we characterized spontaneous-motor-behavior phenotypes of Drosophila dfmr1 mutants, an established model for FXS. We recorded individual 1-day-old adult flies, with mature nervous systems and prior to the onset of aging, in small arenas. We scored behavior using open-source video-annotation software to generate continuous activity timelines, which were represented graphically and quantitatively. Young dfmr1 mutants spent excessive time grooming, with increased bout number and duration; both were rescued by transgenic wild-type dfmr1+. By two grooming-pattern measures, dfmr1-mutant flies showed elevated repetitions consistent with perseveration, which is common in FXS. In addition, the mutant flies display a preference for grooming posterior body structures, and an increased rate of grooming transitions from one site to another. We raise the possibility that courtship and circadian rhythm defects, previously reported for dfmr1 mutants, are complicated by excessive grooming. We also observed significantly increased grooming in CASK mutants, despite their dramatically decreased walking phenotype. The mutant flies, a model for human CASK-related neurodevelopmental disorders, displayed consistently elevated grooming indices throughout the assay, but transient locomotory activation immediately after placement in the arena. Based on published data identifying FMRP-target transcripts and functional analyses of mutations causing human genetic neurodevelopmental disorders, we propose the following proteins as candidate mediators of excessive repetitive behaviors in FXS: CaMKII, NMDA receptor subunits 2A and 2B, NLGN3, and SHANK3. Together, the fly-mutant phenotypes and mechanistic insights provide starting points for drug discovery to identify compounds that reduce dysfunctional repetitive behaviors.
... The 10-s data demonstrated a better resolution in behavioral phenotype with reaction to the light stimulus clearly visible with both sexes but lost when pooling the datasets into 2 min bins. This highlights the importance of how the data is processed and analyzed in forming conclusions as pointed out by Melvin et al. (2017) and Kohler et al. (2018a). ...
Behavior is a useful endpoint in ecotoxicological research; it links the biochemical effects of contamination with physiology of individuals, which can be applied to higher levels of organization with relevance to ecology. Animals exhibit species-specific and sex specific behaviors. Previous experiments within ecotoxicology using amphipods as models have either not separated by sex or have on the assumption that they may create more variability in the results. Therefore, the objectives of this study were to investigate the effect of time (which controlled light conditions), sex, and the interaction of time and sex on the swimming velocity in males and females of the marine amphipod Echinogammarus marinus. E. marinus exhibited a phototactic response to light, as is consistent with previous findings. It was determined that females swim significantly faster than males and that this response was enhanced during periods when the lights were switched off. This suggests that a greater understanding of the baseline unconditioned behaviors of experimental organisms is needed to avoid impacts of unknown variables on results. The results of this study reveal significant sexual differences in the baseline behavior of E. marinus which has implications for future research in ecotoxicology. It is recommended that in future research specimens be separated by sex prior to experimentation, in order to account for possible behavioral differences such as those observed in the present studies. This study also highlights the need for thorough observation of behavior over shorter time intervals as larger intervals may miss short-term variations.
... Certain limitations clearly exist in behavioral tests for acute toxicity. A recent study revealed that shorter toxic agent exposure timeframes are more more likely to elicit higher temporal variance in behavioral toxicity results with fish [69]. Currently, an improved methodology is being developed to maintain the rapidity of the acute toxicity test, and at the same time, perform sufficient acclimation. ...
The purpose of this study was to determine whether behavioral tests and metabolic profiling of organisms can be promising alternatives for assessing the health of aquatic systems. Water samples from four potential pollution sources in South Korea were collected for toxicity evaluation. First, conventional acute toxicity test in Daphnia magna and behavioral test in zebrafish was conducted to assess water quality. Second, metabolomic analysis was performed on zebrafish exposed to water samples and on environmental fish collected from the same source. Acute toxicity test in D. magna showed that none of the water samples exerted significant adverse effects. However, activity of zebrafish larvae exposed to samples from the zinc smelter (ZS) and industrial complex (IND) sites decreased compared to those exposed to samples from the reference site (RS). Metabolomic analysis using the Manhattan plot and Partial Least Square (PLS)/Orthogonal PLS Discriminant Analysis (OPLS-DA) showed differences in metabolic profiles between RS and ZS, and between IND and abandoned mine site (M). Interestingly, applying the same metabolomic analysis to environmental fish revealed patterns similar to those for zebrafish, despite the uncontrollable variables involved in environmental sampling. This study shows that metabolomics is a promising tool in assessing the health of aquatic environments.
... In addition to examining the data on swimming speed and the proportion of their time the fish spent in the top, middle, and bottom areas of the novel tank, the number of entries fish made into the top and middle areas was also included in the analysis. Further, based on the suggestion of Melvin et al. (2017) that different lengths of observation time can impact on basic swimming parameters, we separated the behavioural data into two periods (2-10 min and 10-18 min) and analysed each time period separately. There is logic in doing so because fish might feel anxious when first placed in a novel tank, but after a while become less anxious and explore their surroundings more. ...
There is concern that psychoactive drugs present in the aquatic environment could affect the behaviour of fish, and other organisms, adversely. There is considerable experimental support for this concern, although the literature is not consistent. To investigate why, fish were exposed to three concentrations of the synthetic opiate tramadol for 23–24 days, and their anxiolytic behaviour in a novel tank diving test was assessed both before and after exposure. The results were difficult to interpret. The positive control drug, the anti-depressant fluoxetine, produced the expected results: exposed fish explored the novel tank more, and swam more slowly while doing so. An initial statistical analysis of the results provided relatively weak support for the conclusion that both the low and high concentrations of tramadol affected fish behaviour, but no evidence that the intermediate concentration did. To gain further insight, UK and Japanese experts in ecotoxicology were asked for their independent opinions on the data for tramadol. These were highly valuable. For example, about half the experts replied that a low concentration of a chemical can cause effects that higher concentrations do not, although a similar number did not believe this was possible. Based both on the inconclusive effects of tramadol on the behaviour of the fish and the very varied opinions of experts on the correct interpretation of those inconclusive data, it is obvious that more research on the behavioural effects of tramadol, and probably all other psychoactive drugs, on aquatic organisms is required before any meaningful risk assessments can be conducted. The relevance of these findings may apply much more widely than just the environmental risk assessment of psychoactive drugs. They suggest that much more rigorous training of research scientists and regulators is probably required if consensus decisions are to be reached that adequately protect the environment from chemicals.
... The eastern mosquitofish is a small freshwater fish native to south-eastern North America (Pyke, 2008) that is receiving growing research interest as a model for investigating behavioural effects of chemical pollutants (e.g., Saaristo et al., 2013;Magellan et al., 2014;Martin et al., 2017;Melvin et al., 2017;Bertram et al., 2018). Mosquitofish are one of the most prolific and widely distributed freshwater fish in the world (García-Berthou et al., 2005;Pyke, 2008), and are among the world's 100 most invasive species (Lowe et al., 2000). ...
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.
... The eastern mosquitofish is a small, internally fertilising poeciliid fish with a widespread geographic distribution (Pyke, 2005(Pyke, , 2008 that is attracting increased interest as a model for investigating the impacts of chemical pollutants (e.g., Saaristo et al., 2013Saaristo et al., , 2014Magellan et al., 2014;Martin et al., 2017;Melvin et al., 2017). Mosquitofish have a coercive mating system, where males copulate with females by 'sneaking' from behind and thrusting the tip of their gonopodiumda modified anal fin used for internal fertilisationdinto the female's genital pore (Bisazza et al., 2001). ...
... The behavioural endpoints such as average, maximum or angular velocity are very informative parameters for behavioural pattern assessment. Velocity is a derivative ratio between the time spend moving and the distance travel (Melvin et al. 2017). Moreover, when determining the baseline behaviour of fish, the average velocity is more stable in relation to time then other endpoints (Makaras et al. 2018). ...
The aim of this research was to assess interactions between metals at low exposure concentrations (Maximum-Permissible-Concentrations accepted for the inland waters in EU) and to assess possible influence of background exposure (10-times reduced concentration of a single metal) on toxicological significance of selected biomarkers in Salmo salar after treatment with metal mixture (Zn – 0.1, Cu – 0.01, Ni – 0.01, Cr – 0.01, Pb – 0.005 and Cd – 0.005 mg/L). The tissue-specific bioaccumulation, genotoxicity and cytotoxicity responses (erythrocytic nuclear abnormalities assay) in peripheral blood, kidneys, gills and liver erythrocytes of fish to metal mixtures were assessed after 14 days treatment. Treatment with primary mixture (MIX) or two variants of this mixture (Cr↓ (10 times reduced Cr6+ concentration) and Cu↓ (10 times reduced Cu2+ concentration)) induced the strongest responses in genotoxicity and cytotoxicity endpoints. Exposure to these mixtures highly affected Zn, Cu and Cd bioaccumulation in liver tissue. The highest amount of Ni accumulated was measured after Cd↓ treatment in all tissues. Treatments with reduced concentration of non-essential metal resulted in an increased accumulation of Pb, Ni, or Cd; treatments with reduced concentration of essential metal resulted in a reduced accumulation of certain metals (especially Cd and Pb) in tissues compared between treatments. Glucose content in blood and behavioural endpoints were evaluated after short-term exposure to metal mixtures (MIX, Cr↓, Cu↓). Significant increase in blood glucose concentration was measured after all treatments. These metal mixtures elicit significant behavioural alterations in fish. Consequently, this research revealed a significant influence of background exposure considering mixture toxicity.
... The eastern mosquitofish is a small, internally fertilising poeciliid fish with a widespread geographic distribution (Pyke, 2005(Pyke, , 2008 that is attracting increased interest as a model for investigating the impacts of chemical pollutants (e.g., Saaristo et al., 2013Saaristo et al., , 2014Magellan et al., 2014;Martin et al., 2017;Melvin et al., 2017). Mosquitofish have a coercive mating system, where males copulate with females by 'sneaking' from behind and thrusting the tip of their gonopodiumda modified anal fin used for internal fertilisationdinto the female's genital pore (Bisazza et al., 2001). ...
... For example, Gao et al. (2017) evaluated the effect of early life exposure to benzo[a]pyrene on adult behavioral performance on a NTT and a T-maze and was able to link a phenotype of neurodegeneration to changes in DNA methyltransferase expression, decreased neurotransmitter levels, and decreased dopaminergic neurons in brain sections. Although adult zebrafish behavior testing is well described, factors such as biological replicate, test day, and experimental protocols can have a strong effect on behavioral results (Kalueff et al., 2016), highlighting the need for reporting and standardization of information, such as acclimation period, to help improve reproducibility of results (Melvin et al., 2017). ...
The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect. Additionally, advances in behavioral characterization and experimental methodology allow for observation of recognizable phenotypic changes after xenobiotic exposure. Future directions in zebrafish research are predicted to take advantage of CRISPR-Cas9 genome editing methods in creating models of disease and interrogating mechanisms of action with fluorescent reporters or tagged proteins. Zebrafish can also model developmental origins of health and disease and multi- and transgenerational toxicity. The zebrafish has many advantages as a toxicologic model and new methodologies and areas of study continue to expand the usefulness and application of the zebrafish.
... Also, for many benthic species, including those at bathyal and abyssal depths, the speed at which a behavioral response can be elicited may be too slow to represent a viable means to avoid toxicant exposure (Ward et al., 2013). In addition, the potential variability in the "typical" behavioral response to toxicant exposure, which may impede their application as biomarker responses, has been identified by other research teams (e.g., Garcia-March et al., 2008;Melvin et al., 2017). Behavioral avoidance is also not possible for sedentary or attached species. ...
In January 2017, the International Seabed Authority released a discussion paper on the development of Environmental Regulations for deep-sea mining (DSM) within the Area Beyond National Jurisdiction (the “Area”). With the release of this paper, the prospect for commercial mining in the Area within the next decade has become very real. Moreover, within nations' Exclusive Economic Zones, the exploitation of deep-sea mineral ore resources could take place on very much shorter time scales and, indeed, may have already started. However, potentially toxic metal mixtures may be released at sea during different stages of the mining process and in different physical phases (dissolved or particulate). As toxicants, metals can disrupt organism physiology and performance, and therefore may impact whole populations, leading to ecosystem scale effects. A challenge to the prediction of toxicity is that deep-sea ore deposits include complex mixtures of minerals, including potentially toxic metals such as copper, cadmium, zinc, and lead, as well as rare earth elements. Whereas the individual toxicity of some of these dissolved metals has been established in laboratory studies, the complex and variable mineral composition of seabed resources makes the a priori prediction of the toxic risk of DSM extremely challenging. Furthermore, although extensive data quantify the toxicity of metals in solution in shallow-water organisms, these may not be representative of the toxicity in deep-sea organisms, which may differ biochemically and physiologically and which will experience those toxicants under conditions of low temperature, high hydrostatic pressure, and potentially altered pH. In this synthesis, we present a summation of recent advances in our understanding of the potential toxic impacts of metal exposure to deep-sea meio- to megafauna at low temperature and high pressure, and consider the limitation of deriving lethal limits based on the paradigm of exposure to single metals in solution. We consider the potential for long-term and far-field impacts to key benthic invertebrates, including the very real prospect of sub-lethal impacts and behavioral perturbation of exposed species. In conclusion, we advocate the adoption of an existing practical framework for characterizing bulk resource toxicity in advance of exploitation.
With the expansion of human settlements and the environmental changes brought on by human activity and pollutants, toxicology and risk assessment of piscine species is becoming increasingly of interest to scientists involved in environmental research and connected disciplines. This book focuses specifically on environmental risk assessment in fish species from different zoogeographical regions of the world.
Fish Species in Environmental Risk Assessment Strategies is an ideal companion to toxicologists and ecologists interested in risk assessment in the environments of ichthyic fauna, particularly those with an interest in the deleterious impact introduced by human activity. The book is also of interest to those working in conservation biology, biological invasion, biocontrol, habitat management and related disciplines.
Chemical pollution within the aquatic environment is an ongoing concern causing detrimental effects to the health of various fish populations around the world. Pharmaceuticals have become a key focus in many fish toxicological studies, causing significant changes to individual fish behaviour, with emerging interest in their ability to alter circadian rhythms. Circadian rhythms in fish are sensitive to changes to the surrounding environment, but few studies have characterised how pharmaceuticals such as antidepressants can influence these patterns. Our study investigated the circadian rhythms of male and female eastern mosquitofish (Gambusia holbrooki) collected from a freshwater pond in southeast Queensland. Mosquitofish were exposed with a control and different concentrations (1 μg/L, 10 μg/L and 100 μg/L) of the antidepressant duloxetine during 2 x 7 day and 2 x 24hr exposure periods. Behavioural endpoints such as average velocity, maximum velocity and average angular (turning) velocity were measured as the 'activity levels’ of fish between day-time and night-time through the aid of an infrared spectrum video recording camera. Our findings indicate male and female fish exhibited lower activity patterns when exposed to duloxetine. This was highlighted for the average velocity and average angular velocity as these endpoints were an apparent inverse to each other during the day and night. Additionally, female circadian rhythms were identified to be less pronounced to those in males due to the requirement of greater nutrient intake for different ecological processes. Our study indicates the need to explore further effects of other pharmaceutical contaminants, particularly antidepressants at low concentrations found within the aquatic environment to understand their potential risks to normal circadian processes in fish.
Sectional channelization of natural rivers for flood mitigation causes discontinuity of the riverine corridor, leading to unfavorable conditions for fish movement in the longitudinal direction. To minimize the negative effects, hydraulic structures (e.g. deflectors) are proposed as potential restoration measures in concrete flood channels. In this study, indoor flume experiments were carried out to assess the variation of flow field (velocity magnitude, turbulent kinetic energy, and Reynolds stress) and bed morphology with the inclusion of deflectors. The flow and sediment regimes were then compared in the presence or absence of deflectors. The experimental results indicated that deflectors triggered a non-uniform flow pattern and stimulated riffle-pool sequence formation through local scouring and subsequent deposition. To better understand the response of predaceous chub (Parazacco spilurus) and mud carp (Cirrhinus molitorella) to deflectors, fish trajectories, distribution, and number of transits were analyzed using a video monitoring system. The results suggested that fish responded differently to the baseline (without deflector) and deflector scenarios: (1) fish struggled against the oncoming current when crossing the physical barrier in the channelized section; (2) fish moved and rested in the diverse habitats created by deflectors. Additionally, the presence of sediments in the channel bed was essential in attracting fish to the channelized section. Therefore, it was concluded that using deflectors at appropriate intervals and allowing the existence of sediments in flood channel sections can benefit fish movement and enhance channel naturalization.
Fish batches are often transported from aquacultures to laboratory facilities, and this fact triggers a pre-stressed condition that can cause alterations in both physiological and behavioural status of animals. The acclimation before fish trials is highly recommended to ideally ensure basal levels for the species, but there is still limited information concerning the period of time and parameters to be included for the evaluation. Facing those facts, brown trout (Salmo trutta f. fario) juveniles subjected to an acute transport (around 2 h) were here tested during 42 days post-stress (at 12 h, 7, 14, 28 and 42 days), in order to: 1) define a multi-parameter approach, based on biochemical, physical, morphological, nutritional and behavioural parameters that best characterize the fish acclimation and welfare status; and (2) propose an adequate acclimation period for brown trout juveniles after transportation to new housing conditions.
Behavioural changes were noted with an overall stabilization at the 17th day. Fish tended to occupy preferentially the bottom of the tank, a smaller number of tank sections, decreased the swimming activity and the number of abnormal movements. Reaction time to food decreased until it was instantaneous, and the reaction to food gradually changed over time, so that on the 1st day there was no reaction and, from the 30th day, most of the fish moved continuously to reach the food. Feed intake did not occur on the 1st day, and then increased gradually. Despite that, there were no differences in fish condition and hepatosomatic indices over time.
In general, glucose was relatively stable along the assay, and cholesterol decreased at the 7th and 14th days, compared to 12 h, possibly as a consequence of the high metabolic demand induced by stress. Cortisol varied from 21,841.9 pg/mL (12 h) to 2162.2 pg/mL (42 day). As for cortisol, the volume-weighted nuclear volume of interrenal cells was not significantly distinct between sampling days, but a minimum of 96.33 μm³ was reached at 28 days. Higher haemoglobin values were obtained at 28th and 42th days, in relation to 12 h and the 14th day. Triglycerides, potassium and haematocrit did not change. The quantitative skin analyses (fragments and histological sections) showed a reduction in the pigmented area, with stabilization at 14th day.
Acclimation of brown trout juveniles should be monitored using a multi-parameter evaluation including behavioural, biochemical, nutritional and morphological analyses. Overall, it is recommended an acclimation period of at least 28 days.
The traditional ecotoxicity assays (forced exposure) tend to use organisms that are cultured under controlled conditions or that come from undisturbed ecosystems, with no (or negligible) previous contact with contamination. The same occurs in the non-forced approach, in which organisms are exposed to a contamination gradient and can move between different concentrations choosing the less toxic one. Considering that organisms inhabiting contaminated ecosystems tend to be gradually exposed to contamination, an abrupt exposure from uncontaminated conditions to a contaminated environment might present two problems: lack of ecological relevance to a scenario where the contamination occurs gradually and a magnification of the toxicity due to the sudden change in the environmental conditions. Therefore, a key question should be addressed: might a previous exposure to contamination reduce the organisms' perception of the danger of a contaminant (hypothesis of time-delayed avoidance due to pre-acclimation-TDADP), altering their avoidance response pattern? We tested the avoidance of zebrafish (Danio rerio: ±2 months old) populations when exposed to a copper gradient (0-400 μg/L). The populations differed according to the period (24 h and 7 and 30 days) in which they were acclimated to copper (ca. 400 μg/L). The avoidance in the 2 h experiments changed as a consequence of the acclimation period. In the population that was not previously acclimated, 40% of the fish moved to the less contaminated compartment and only 6.7% stayed in the most contaminated one; for the other populations those values were, respectively, 31 and 11% (24 h-acclimation), 28 and 26% (7 day-acclimation) and 19 and 27% (30 day-acclimation). An abrupt exposure to a contaminant might overestimate the response if this is analyzed in the short-term. When the avoidance tests were prolonged to 24 h, the avoidance tended to reach similar values to those of the non-acclimated population, thus supporting our TDADP hypothesis.
Environmental contamination contributes to the threatened status of many amphibian populations. Many contaminants alter behaviour at concentrations commonly experienced in the environment, with negative consequences for individual fitness, populations and communities. A comprehensive, quantitative evaluation of the behavioural sensitivity of amphibians is warranted to better understand the population-level and resultant ecological impacts of contaminants. We conducted a systematic review and meta-analysis evaluating behavioural changes following exposure to contaminants. Most studies were conducted in North America and Europe on larval stages, and 64% of the 116 studies focussed on the effects of insecticides. We found that a suite of contaminants influence a wide range of behaviours in amphibians, with insecticides typically invoking the strongest responses. In particular, insecticides increased rates of abnormal swimming, and reduced escape responses to simulated predator attacks. Our analysis identified five key needs for future research, in particular the need: (1) for researchers to provide more details of experimental protocols and results (2) to develop a strong research base for future quantitative reviews, (3) to broaden the suite of contaminants tested, (4) to better study and thus understand the effects of multiple stressors, and (5) to establish the ecological importance of behavioural alterations. Behavioural endpoints provide useful sub-lethal indicators of how contaminants influence amphibians, and coupled with standard ecotoxicological endpoints, can provide valuable information for population models assessing the broader ecological consequences of environmental contamination.
This study examined changes in locomotor activity of the rainbow trout (Oncorhynchus mykiss) juveniles exposed to sublethal concentrations of hexavalent chromium (Cr⁶⁺) (as a single pollutant) and landfill leachate (as an complex mixture of mainly organic compounds, including trace amounts of metals). Fish were first examined for baseline behavior patterns for 3 h to determine the appropriate duration of acclimation and the control level. The average velocity of fish was found to be the most informative among other endpoints throughout the 3 h long acclimation. Under the effect of both test substances, fish locomotor activity significantly increased after 5 min reaching maximum values after 10 min of exposure. The juveniles exposed to leachate were more responsive than those exposed to Cr⁶⁺. However, in this study we did not succeed in identifying pollutants from the elicited fish behavioral response patterns.
Recently there has been a growing concern that many published research findings do not hold up in attempts to replicate them. We argue that this problem may originate from a culture of ‘you can publish if you found a significant effect’. This culture creates a systematic bias against the null hypothesis which renders meta-analyses questionable and may even lead to a situation where hypotheses become difficult to falsify. In order to pinpoint the sources of error and possible solutions, we review current scientific practices with regard to their effect on the probability of drawing a false-positive conclusion. We explain why the proportion of published false-positive findings is expected to increase with (i) decreasing sample size, (ii) increasing pursuit of novelty, (iii) various forms of multiple testing and researcher flexibility, and (iv) incorrect P-values, especially due to unaccounted pseudoreplication, i.e. the non-independence of data points (clustered data). We provide examples showing how statistical pitfalls and psychological traps lead to conclusions that are biased and unreliable, and we show how these mistakes can be avoided. Ultimately, we hope to contribute to a culture of ‘you can publish if your study is rigorous’. To this end, we highlight promising strategies towards making science more objective. Specifically, we enthusiastically encourage scientists to preregister their studies (including a priori hypotheses and complete analysis plans), to blind observers to treatment groups during data collection and analysis, and unconditionally to report all results. Also, we advocate reallocating some efforts away from seeking novelty and discovery and towards replicating important research findings of one's own and of others for the benefit of the scientific community as a whole. We believe these efforts will be aided by a shift in evaluation criteria away from the current system which values metrics of ‘impact’ almost exclusively and towards a system which explicitly values indices of scientific rigour.
Psychoactive drugs are frequently detected in the aquatic environment. The evolutionary conservation of the molecular targets of these drugs in fish suggest that they may elicit mode-of-action mediated effects in fish as they do in humans, and one the key open question is at what exposure concentrations these effects might occur. In the present study, we investigated the uptake and tissue distribution of the benzodiazepine oxazepam in the fathead minnow (Pimephales promelas) after 28 days of waterborne exposure to 0.8, 4.7, and 30.6 µg L(-1) . Successively, we explored the relationship between the internal concentrations of oxazepam and the effects on fish exploratory behaviour quantified by performing two types of behavioural tests, the Novel Tank Diving Test and the Shelter-seeking Test. The highest internal concentrations of oxazepam were found in brain, followed by plasma and liver, whereas muscle presented the lowest values. Average concentrations measured in the plasma of fish from the three exposure groups were, respectively, 8.7 ± 5.7, 30.3 ± 16.1, and 98.8 ± 72.9 µg L(-1) .Significant correlations between plasma and tissue concentrations of oxazepam were found in all three groups. Exposure of fish to 30.6µg L(-1) in water produced plasma concentrations within or just below the Human Therapeutic Plasma Concentration (HT PC) range in many individuals. Statistically significant behavioural effects in the Novel Tank Diving test were observed in fish exposed to 4.7µg L(-1) . In this group, plasma concentrations of oxazepam were approximately one third of the lowest HT PC value. No significant effects were observed in fish exposed to the lowest and highest concentrations. The significance of these results is discussed in the context of the species-specific behaviour of fathead minnow and existing knowledge of oxazepam pharmacology. This article is protected by copyright. All rights reserved.
Fish are increasingly popular subjects in behavioural and neurobiological research. It is therefore important that they are housed and handled appropriately to ensure good welfare and reliable scientific findings, and that species-appropriate behavioural tests (e.g. of cognitive/affective states) are developed. Routine handling of captive animals may cause physiological stress responses that lead to anxiety-like states (e.g. increased perception of danger). In fish, these may be particularly pronounced when handling during tank-to-tank transfer involves removal from water into air. Here we develop and use a new combined scototaxis (preference for dark over light areas) and novel-tank-diving test, alongside conventional open-field and novel-object tests, to measure the effects of transferring three-spined sticklebacks (Gasterosteus aculeatus) between tanks using a box or net (in and out of water respectively). Preference tests for dark over light areas confirmed the presence of scototaxis in this species. Open-field and novel-object tests failed to detect any significant differences between net and box-handled fish. However, the combined diving and scototaxis detected consistent differences between the treatments. Net-handled fish spent less time on the dark side of the tank, less time in the bottom third, and kept a greater distance from the ‘safe’ bottom dark area than box-handled fish. Possible explanations for this reduction in anxiety-like behaviour in net-handled fish are discussed. The combined diving and scototaxis test may be a sensitive and taxon-appropriate method for measuring anxiety-like states in fish.
Sub-lethal toxicological responses are common occurrences in aquatic animals exposed to sewage wastewater and organic wastewater contaminants. Behavioural alterations are particularly sensitive indicators of sub-lethal toxicological stress in animals exposed to various pollutants, and often correlate with higher-level outcomes. Diurnal activity patterns in many fish species are sensitive to changes in natural biotic factors, but few studies have explored how environmental pollutants influence such rhythms. We investigated diurnal activity patterns in the mosquitofish (Gambusia holbrooki), after exposure to UV-treated sewage and a mixture of key contaminants identified through chemical analysis and subsequent risk-based prioritization of the wastewater. Exposure to 50% and 100% wastewater abolished daytime activity levels in male, but not female fish. Chemical analysis identified fluoxetine, diazinon and triclosan above their reported predicted-no-effect-concentrations (PNECs), and fish were thus exposed to a mixture of these compounds at 1, 10 and 100 μg l–1. Behavioural responses were highly consistent between fish exposed to wastewater and the contaminant mixture, indicating that these prioritized contaminants are indeed likely contributing to the observed effects. Effective concentrations of the mixture were considerably lower than those reported as eliciting behavioural effects in previous studies exploring each of these compounds alone. Results warn of the potential for negative higher-level consequences associated with exposures of fish to common organic wastewater contaminants, as altered diurnal activity patterns could conceivably scale-up to influence performance including foraging success and predator avoidance. Further research is necessary to increase our understanding of linkages between alterations to diurnal activities and effects at the population level.
The question of how hunger affects locomotory behaviour, in particular how it affects the kinematics of movement and an animal’s interaction with the physical structures in its environment is of broad relevance in behavioural ecology. We experimentally manipulated the hunger levels of individual mosquitofish ( Gambusia holbrooki) and recorded their swimming behaviour in shoals of 4 fish. We found that hungry individuals in shoals moved at greater speeds and had higher turning speeds than satiated individuals in shoals, as well as a greater variance in speed and turning speeds. We also found that hungry individuals explored more of the arena and used more of its internal space, away from the square arena’s walls and displayed less wall-following behaviour than satiated individuals. A functional explanation for this change in swimming behaviour and interaction with environmental heterogeneity is discussed in the context of social foraging, as is the consequence of these results for models of search patterns and collective movement.
The basic conclusions in almost all reports on new drug applications and in all publications in toxicology are based on statistical methods. However, serious contradictions exist in practice: designs with small samples sizes but use of asymptotic methods (i.e. constructed for larger sample sizes), statistically significant findings without biological relevance (and vice versa), proof of hazard vs. proof of safety, testing (e.g. no observed effect level) vs. estimation (e.g. benchmark dose), available statistical theory vs. related user-friendly software. In this review the biostatistical developments since about the year 2000 onwards are discussed, mainly structured for repeated-dose studies, mutagenicity, carcinogenicity, reproductive and ecotoxicological assays. A critical discussion is included on the unnecessarily conservative evaluation proposed in guidelines, the inadequate but almost always used proof of hazard approach, and the limitation of data-dependent decision-tree approaches.
Eastern and Western Gambusia (i.e., Gambusia holbrooki and G. affinis, respectively) are considered together here because these two fish species are very closely related, similar in appearance,
similar in biology and often confused. Widely divergent attitudes have developed with regard to these fish with some viewing
them as being highly beneficial to humans through controlling mosquitoes and the diseases they harbor, and others expressing
concern about the negative impacts that these fish may have on other species with which they interact. Because of the widespread
distribution, high levels of abundance, ease of capture and captive maintenance, and divergent attitudes, a very large and
diffuse literature has developed with regard to these species. In fact, few fish species have been studied as much as or more
than these two species combined. There has, however, been no comprehensive review of their biology published to date. As it
is not possible to provide a comprehensive review of Gambusia biology in one reasonably sized document, I provide here a review of aspects of their biology at the level of species and
individual. In another review I focused instead on the levels of population and species communities and consider the impacts
that these fish have on mosquitoes and other organisms (Pyke, unpublished). As would be expected of such widespread and abundant
species, Gambusia affinis and G. holbrooki are clearly very tolerant, adaptable and variable in their biology, at both an individual and population level. Both individuals
and populations can tolerate, and often thrive within, a wide range of conditions and the abilities of individuals to do this
are enhanced if they have time to acclimate to any changes. Populations can adapt through genetic or evolutionary changes
in response to conditions that vary in space or time, and there is significant genetic variation within and between populations.
Gambusia affinis and G. holbrooki, introduced worldwide from eastern North America, are collectively the most abundant, widespread freshwater fish in the world, which is not surprising because they tolerate, and sometimes thrive under, an exceptional range of environmental conditions and have high reproductive potential. Some know them as mosquitofish because of a legendary ability to control mosquitoes, and diseases they carry, while others doubt this ability or argue that indigenous fish are equally or more effective.
However, rigorous evidence to support these views remains scant, so the legend persists. Some know them as plague minnow because of negative impacts on many native animal species, and abundant evidence exists to support this view. Despite such polarized attitudes toward them, their high abundance and wide distribution, and a large scientific literature devoted to them, many important aspects of their biology remain poorly known.
The anti-depressant fluoxetine is widely present in the aquatic environment. Typical river concentrations are in the low ng/L range. Many ecotoxicity studies have assessed the effects of this pharmaceutical on a range of aquatic species. Some studies report that ng, or even pg, per litre concentrations cause effects, whereas other studies report that effects only occur when the water concentration is in the μg/L range. It seems unlikely that all reported effects will be repeatable. Many of the studies have considerable limitations. Currently it is impossible to ascertain what environmental concentrations of fluoxetine pose a risk to aquatic organisms. The key question can be answered only by high quality, reproducible research.
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.
C. Glenn Begley explains how to recognize the preclinical papers in
which the data won't stand up.
Reviews the literature which examined the effects of exposing organisms to aversive events which they cannot control. Motivational, cognitive, and emotional effects of uncontrollability are examined. It is hypothesized that when events are uncontrollable the organism learns that its behavior and outcomes are independent, and this learning produces the motivational, cognitive, and emotional effects of uncontrollability. Research which supports this learned helplessness hypothesis is described along with alternative hypotheses which have been offered as explanations of the learned helplessness effect. The application of this hypothesis to rats and man is examined. (114 ref) (PsycINFO Database Record (c) 2012 APA, all rights reserved)
Behavioural studies comparing hatchery and wild-caught fish are useful to improve selec-tion for aquaculture and restocking programmes. We examined swimming behaviour characteristics in wild captured and domesticated sea bass juveniles before and after elic-iting a startle response at 8 different ages and always on naive individuals. We specifically investigated whether domestication impacts juvenile sea bass behaviour and whether the first months of captivity induce behavioural modifications in wild juveniles. An appara-tus was designed to mimic a predator attack by presenting a sudden visual and mechanical stimuli simultaneously in 8 arenas where single individuals were placed and video recorded. The reactivity response was evaluated and different swimming variables including angu-lar velocity, total distance travelled, mean velocity, immobility and distance from stimulus point were analysed from videos taken 5 min before stimulus actuation, 5 and 15 min after. Otolith readings showed that wild and domesticated juveniles were of similar age (∼55 days at the start of the experiment and ∼125 at the end of experiment). There were consistent behavioural differences (e.g. higher angular velocity and distance from stimulus point in wild fish) demonstrating that domestication reduces flight response behaviour. There were also similarities between both fish origins (similar response to stimulus actuation: decrease of total distance travelled and mean velocity, increase of angular velocity and immobility). A decrease over time in reactivity and variability in swimming responses among fish of both origins showed that captivity only does not fully explain wild fish behaviour changes and ontogenic modifications are likely interplaying.
The effects of wall color stimuli on diving, and the effects of depth stimuli on scototaxis, were assessed in zebrafish. Three groups of fish were confined to a black, a white, or a transparent tank, and tested for depth preference. Two groups of fish were confined to a deep or a shallow tank, and tested for black-white preference. As predicted, fish preferred the deep half of a split-tank over the shallow half, and preferred the black half of a black/white tank over the white half. Results indicated that the tank wall color significantly affected depth preference, with the transparent tank producing the strongest depth preference and the black tank producing the weakest preference. Tank depth, however, did not significantly affect color preference. Additionally, wall color significantly affected shuttling and immobility, while depth significantly affected shuttling and thigmotaxis. These results are consistent with previous indications that the diving response and scototaxis may reflect dissociable mechanisms of behavior. We conclude that the two tests are complementary rather than interchangeable, and that further research on the motivational systems underlying behavior in each of the two tests is needed.
How should ecologists and evolutionary biologists analyze nonnormal data that involve random effects? Nonnormal data such as counts or proportions often defy classical statistical procedures. Generalized linear mixed models (GLMMs) provide a more flexible approach for analyzing nonnormal data when random effects are present. The explosion of research on GLMMs in the last decade has generated considerable uncertainty for practitioners in ecology and evolution. Despite the availability of accurate techniques for estimating GLMM parameters in simple cases, complex GLMMs are challenging to fit and statistical inference such as hypothesis testing remains difficult. We review the use (and misuse) of GLMMs in ecology and evolution, discuss estimation and inference and summarize 'best-practice' data analysis procedures for scientists facing this challenge.
Changes in animal movement (frequency or speed of locomotion) following exposure to a toxicant are frequently considered a biomarker of contaminant exposure and are some of the most widely reported behavioral results in toxicological literature. However, the ecological consequences of such behavioral changes, such as effects on toxicant transfer in foodwebs, are far less well understood, complicated in part by the short-term nature of laboratory experiments and the lack of complementary field studies where the nature of toxicant exposure is more complex. Here we examine whether naturally exposed individuals of the round goby, a benthic, site-loyal fish, move in a manner similar to conspecifics from less contaminated habitats. In the laboratory, round goby from a relatively cleaner site showed greater activity and exploration than goby from two highly contaminated sites. Male fish were more active than females but the site effects were similar in both sexes. In contrast to laboratory findings, a field mark-recapture study of 881 round goby showed that fish from the cleaner site did not move greater distances or exhibit shorter residence times within the site than round goby from highly contaminated sites. Our results indicate that while behavioral changes in the laboratory may be one of several useful diagnostics of toxicant exposure of wild-exposed animals, they do not necessarily translate readily into measurable differences in a natural context. Thus, the potential fitness consequences of toxicant exposure based on behavioral changes need to be assessed carefully.
The zebrafish is an important model organism for neuro-anatomy and developmental genetics. It also offers opportunities for investigating the functional and evolutionary genetics of behaviour but these have yet to be exploited. The ecology of anti-predator behaviour has been widely studied in fish and has been shown to vary among populations and between wild and domesticated (laboratory) fish. Here, we utilise the strong behavioural differences present between a wild-derived strain of fish from Bangladesh and the laboratory strain AB. In total, 184 F2 fish were generated and tested for shoaling tendency and willingness to approach an unfamiliar object ('boldness'). Our results indicate the existence of QTL for boldness on chromosomes 9 and 16 and suggest another genomic region that influences anti-predator behaviour on chromosome 21. QTL for growth rate, weight and fat content, all of which are elevated in laboratory fish, were detected on chromosome 23. These initial results confirm the potential for QTL mapping of behavioural traits in zebrafish and also for dissecting the consequences of selection during domestication.
Locomotor behavior is commonly affected by contaminants, and the pattern of fish swimming is a highly organized species-specific response. In the current study, we examined the locomotor behavioral response of the mosquitofish, Gambusia affinis, which was exposed to a sublethal concentration (LC(5), 20 microg/L) of mercuric chloride (HgCl2) for 28 days and monitored using a computer vision system. The EthoVision video tracking system for automation of behavioral studies at regular intervals revealed abnormal locomotor behavior such as reduction in swimming speed (cm/s) and distance traveled per unit time. The effects of this metal on the gill morphology and bioaccumulation in different body parts were also investigated. High-resolution microscopy studies revealed abnormal gill morphology, with fusion of primary lamellae along with deep lesions and erosions in the secondary lamellae. The bioaccumulation concentrations in head, body, and viscera were determined by cold vapor atomic absorption spectrometric technique at regular intervals. The results indicated that the accumulation of mercury was the highest in viscera followed by head and body, with bioconcentration factors (BCFs) of 3.99, 2.18, and 1.57 and uptake rate constants (k1) of 17.91, 11.02, and 8.13, respectively. These observations indicate that alterations in fish behavior under subacute stress can provide important information useful in predicting the stress.
The zebra fish has been a preferred subject of genetic analysis. It produces a large number of offspring that can be kept in small aquaria, it can be easily mutagenized using chemical mutagens (e.g., ethyl nitrosourea [ENU]), and high-resolution genetic maps exist that aid identification of novel genes. Libraries containing large numbers of mutant fish have been generated, and the genetic mechanisms of the development of zebra fish, whose embryo is transparent, have been extensively studied. Given the extensive homology of its genome with that of other vertebrate species including our own and given the available genetic tools, zebra fish has become a popular model organism. Despite this popularity, however, surprisingly little is known about its behavior. It is argued that behavioral analysis is a powerful tool with which the function of the brain may be studied, and the zebra fish will represent an excellent subject of such analysis. The present paper is a proof of concept study that uses pharmacological manipulation (exposure to alcohol) to show that the zebra fish is amenable to the behavioral genetic analysis of aggression and thus may allow us to reveal molecular mechanisms of this behavioral phenomenon relevant to vertebrates.
Measurement error adds noise to predictions, increases uncertainty in parameter estimates, and makes it more difficult to discover new phenomena or to distinguish among competing theories. A common view is that any study finding an effect under noisy conditions provides evidence that the underlying effect is particularly strong and robust. Yet, statistical significance conveys very little information when measurements are noisy. In noisy research settings, poor measurement can contribute to exaggerated estimates of effect size. This problem and related misunderstandings are key components in a feedback loop that perpetuates the replication crisis in science.
Current advances the ability to assay adult aquatic vertebrate behaviour are potentially very useful to aquatic toxicologists wishing to characterise the effects of pollutants on behaviour, cognition or neurodevelopment. This review considers two specific challenges faced by researchers wishing to exploit these technologies: maximising reliability and validity. It will suggest two behavioural procedures, with the potential for automation and high-throughput implementation, which can be used to measure social cohesion and anxiety, two areas of interest in behavioural aquatic toxicology. In addition, the review will make recommendations about how these procedures (and others) could be carried out to maximise reliability and validity.
Chemical pollution is a pervasive and insidious agent of environmental change. One class of chemical pollutant threatening ecosystems globally are 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 17β-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 17β-trenbolone was also associated with greater male mass. However, no effect of female exposure to 17β-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 17β-trenbolone, demonstrating the possibility of widespread disruption of mating systems of aquatic organisms by common agricultural contaminants.
Copyright © 2015. Published by Elsevier Inc.
Freshwater environments are common repositories for the discharge of large volumes of domestic and industrial waste, particularly through wastewater effluent. One common group of chemical pollutants present in wastewater are endocrine disrupting chemicals (EDCs), which can induce morphological and behavioural changes in aquatic organisms. The aim of this study was to compare the reproductive behaviour and morphology of a freshwater fish, the mosquitofish (Gambusia holbrooki), collected from two sites (wastewater treatment plant (WWTP) and a putative pristine site). The mosquitofish is a sexually dimorphic livebearer with a coercive mating system. Males inseminate females using their modified anal fin as an intromittent organ. Despite this, females are able to exert some control over the success of male mating attempts by selectively associating with, or avoiding, certain males over others. Using standard laboratory assays of reproductive behaviour, we found that mosquitofish males living in close proximity to WWTP showed increased mating activity compared to those inhabiting a pristine site. More specifically, during behavioural trials in which males were allowed to interact with females separated by a transparent divider, we found that WWTP-males spent more time associating with females. Concordant with this, when males and females were subsequently allowed to interact freely, WWTP-males also spent more time chasing and orienting towards the females. As a result, females from both sites showed more interest towards the WWTP-site males. Male anal fin morphology, however, did not differ between sites. Our study illustrates that lifetime exposure to WWTP-effluents can greatly affect male behaviour. The results underscore the importance of behaviour as a potential tool for investigating unknown contaminants in the environment.
We have become progressively more concerned about the quality of some published ecotoxicology research. Others have also expressed concern. It is not uncommon for basic, but extremely important, factors to apparently be ignored. For example, exposure concentrations in laboratory experiments are sometimes not measured, and hence there is no evidence that the test organisms were actually exposed to the test substance, let alone at the stated concentrations. To try to improve the quality of ecotoxicology research, we suggest twelve basic principles that should be considered, not at the point of publication of the results, but during the experimental design. These principles range from carefully considering essential aspects of experimental design through to accurately defining the exposure, as well as unbiased analysis and reporting of the results. Although not all principles will apply to all studies, we offer these principles in the hope that they will improve the quality of the science that is available to regulators. Science is an evidence-based discipline and it is important that we and the regulators can trust the evidence presented to us. Significant resources often have to be devoted to refuting the results of poor research when those resources could be utilised more effectively.
G*Power (Erdfelder, Faul, & Buchner, 1996) was designed as a general stand-alone power analysis program for statistical tests commonly used in social and behavioral research. G*Power 3 is a major extension of, and improvement over, the previous versions. It runs on widely used computer platforms (i.e., Windows XP, Windows Vista, and Mac OS X 10.4) and covers many different statistical tests of the t, F, and chi2 test families. In addition, it includes power analyses for z tests and some exact tests. G*Power 3 provides improved effect size calculators and graphic options, supports both distribution-based and design-based input modes, and offers all types of power analyses in which users might be interested. Like its predecessors, G*Power 3 is free.
Aquatic ecosystems are subject to a number of anthropogenic disturbances, including environmental toxicants. The efficient monitoring of water resources is fundamental for effective management of water quality and aquatic ecosystems. Spot sampling and continuous water quality monitoring based on physicochemical factors are conducted to assess water quality. However, not all contaminants or synergistic and antagonistic toxic effects can be determined by solely analyzing the physicochemical factors. Thus, various biotests have been developed using long-term and automatic observation studies based on the ability of the aquatic organisms to continuously sense a wide range of pollutants. In addition, a biological early warning system (BEWS) has been developed based on the response behaviors of organisms to continuously detect a wide range of pollutants for effective water quality monitoring and management. However, large amounts of data exhibiting non-linearity and individual behavioral variation are continuously accumulated over long-term and continuous behavioral monitoring studies. Thus, appropriate mathematical and computational data analyses are necessary to manage and interpret such large datasets. Here, we review the development and application of BEWS by using various groups of organisms and the computational methods used to process the behavioral monitoring data.
The present study was conducted to investigate whether individual rainbow trout Oncorhynchus mykiss can simultaneously anticipate two daily meals, and to evaluate the influence of time-restricted feeding schedules and light-dark (L:D) cycles on the timing of self-feeding activity. Individual rainbow trout were capable of anticipating not only a single meal but also two daily meals scheduled at different times; the duration of anticipation was short and precise. This seems to be the first convincing evidence to show that a single fish can simultaneously anticipate two daily meals. Change in the onset and cessation of light did not affect the duration of food-anticipatory activity, although it led to shift the phases of the light-related self-feeding activity. Moreover, under constant light and ad lib feeding conditions, only one band of rhythmic activity was observed in individual rainbow trout that had previously developed food-anticipatory activity to each of the two daily meals.
The feeding and swimming behaviors of Pseudorasboraparva and Rasboradaniconius (Cyprinidae) with two different prey types (Daphniapulex and Artemiasalina) at different densities (0.5, 1, 2, 5, 10, or 25perl) were studied after 36h of food deprivation. Full satiation was defined
as the cumulative number of attacks performed until fish attain a constant attack rate which for P.parva was 425 and R.daniconius was 390 attacks. Initial feeding rates showed marked variation with prey availability. Feeding rates of fish in high prey
concentrations were higher at the beginning of the experiment and decreased faster than in low prey densities. Decreases in
the feeding rate at high prey densities were due to faster attainment of satiation. Feeding rates of fish across high prey
densities reached a steady level after satiation. Swimming speeds of fish were inversely proportional to prey density. Moreover,
the change in swimming speeds was directly related to the level of satiation. The ratios of the attack rate and the encounter
rate against prey density of both fish reveal that the search for prey triggered swimming and thereby feeding during the transition
from hungry to satiation. The findings of this study demonstrate that satiation plays an important role in fish foraging that
should be considered a significant factor in foraging analysis.
Laboratory experiments were performed to determine behavioral preferences of Gambusia affinis for habitat variables in partitioned aquaria. The fish preferred calm water, dark-colored substrates and subsurface vegetation providing lateral concealment. Floating cover, which obstructed access to the surface, was avoided. Lack of preference for real over plastic plant cover indicates that visual rather than chemical cues are involved. These laboratory preferences correspond to the reported microhabitat distribution of G. affinis in nature and indicate the presence of an active habitat preference, as opposed to passive habitat correlation, in this species. Species-specific habitat preferences, which may be narrower where a species occurs sympatrically with its congeners, probably form part of a reproductive isolating mechanism.
Behavioral toxicity tests, if properly designed, can be used in conjunction with standard acute lethality tests, chronic full or partial life cycle tests, and early life stage toxicity tests to add ecological realism to toxicant assessments and the regulations made as an outgrowth of these assessments. Changes in certain fish behaviors, especially cough rate and avoidance reactions, are very sensitive indicators of sublethal exposure to metals. Other tests involving predator avoidance, feeding behavior, learning, social interactions, and a variety of locomotor behaviors show promise but have been insufficiently studied to judge their sensitivity or utility. No behavioral tests have been standardized and few have been verified in the field. We discuss the behavioral tests that have been used with metals, examine their sensitivity compared with standard laboratory toxicity tests, and assess the potential ecological significance of the behavioral changes observed.
Escape responses of fishes have long been studied as a model locomotor behavior in which hypothesized maximal or near-maximal muscle power output is used to generate rapid body bending. In this paper we present the results of experiments that challenged zebrafish (Danio rerio) to perform escape responses in water of altered viscosity, to better understand the effects that the fluid mechanical environment exerts on kinematics. We quantified escape kinematics using 1000 frames s(-1) high-speed video, and compared escape response kinematics of fish in three media that differed in viscosity: 1 mPa s (normal water), 10 mPa s and 20 mPa s (20 times normal water viscosity). We hypothesized that because viscosity is increased but not density there will be a different effect on kinematic variables resulting from unsteady (acceleration-dependent) hydrodynamic forces and steady (velocity-dependent) ones. Similarly, we hypothesized that the kinematics of stage 1 will be less affected by viscosity than those of stage 2, as higher angular velocities are reached during stage 1 resulting in higher Reynolds numbers. Our results showed a significant overall effect of viscosity on escape response kinematics but the effect was not in accordance with our predictions. Statistical tests showed that increasing viscosity significantly decreased displacement of the center of mass during stage 1 and after 30 ms, and decreased maximum velocity of the center of mass, maximum angular velocity and acceleration during stage 1, but increased time to maximum angular acceleration and time to maximum linear velocity of the center of mass. Remarkably, increasing water viscosity 20 times did not significantly affect the duration of stage 1 or stage 2.