Time is of the essence: The importance of considering biological rhythms in an increasingly polluted world.

Biological rhythms have a crucial role in shaping the biology and ecology of organisms. Light pollution is known to disrupt these rhythms, and evidence is emerging that chemical pollutants can cause similar disruption. Conversely, biological rhythms can influence the effects and toxicity of chemicals. Thus, by drawing insights from the extensive study of biological rhythms in biomedical and light pollution research, we can greatly improve our understanding of chemical pollution. This Essay advocates for the integration of biological rhythmicity into chemical pollution research to gain a more comprehensive understanding of how chemical pollutants affect wildlife and ecosystems. Despite historical barriers, recent experimental and technological advancements now facilitate the integration of biological rhythms into ecotoxicology, offering unprecedented, high-resolution data across spatiotemporal scales. Recognizing the importance of biological rhythms will be essential for understanding, predicting, and mitigating the complex ecological repercussions of chemical pollution.

Substrate colour guides turquoise killifish's (Nothobranchius furzeri) choice of preferred spawning habitat.

Turquoise killifish (Nothobranchius furzeri) is a promising new model species used in biomedical and ecological laboratory experiments, and should be kept under optimal conditions to ensure fish welfare and the quality of science. While the popularity of this model species is rapidly increasing, we need to improve our understanding of how the species interacts with its environment to optimize its husbandry. Specifically, turquoise killifish are substrate spawners that bury their eggs in the sediment, which can be accommodated under captive conditions, but it is not yet known whether or not turquoise killifish have a preference for a specific sediment colour. Here, we performed a laboratory experiment in which fish could choose between white, orange and black sand for spawning, colours which are relevant in both laboratory and field conditions. We assessed their preference in the context of single breeding pairs, as well as in a social group setting. Additionally, we also assessed the preference of individuals for a white versus black background in a nonmating context. Single breeding pairs deposited over 3.5 times more eggs in black compared to orange or white sand. Similarly, fish in social groups deposited over 3.5 times more eggs in black compared to orange sand, which in turn was over two times higher than that in white sand. Fish showed a slight preference for the black compared to the white zone in a nonmating context, but this did not correlate with substrate choice during the spawning tests. The results suggest that turquoise killifish select their preferred spawning location based on the colour of the substrate. These findings contribute to our understanding of the species' biology and can help to guide good welfare and scientific practice.

Combined effects of global warming and chlorpyrifos exposure on the annual fish Nothobranchius furzeri.

Global warming and environmental pollution threaten aquatic ecosystems. While interactive effects between both stressors can have more than additive consequences, these remain poorly studied for most taxa. Especially chronic exposure trials with vertebrates are scarce due to the high time- and monetary costs of such studies. We use the recently-established fish model Nothobranchius furzeri to assess the separate and combined effects of exposure to the pesticide chlorpyrifos (at 2 µg/L and 4 µg/L) and a 2 °C temperature increase. We performed a full life-cycle assessment to evaluate fitness-related endpoints including survival, total body length, maturation time, fecundity, critical thermal maximum (CTmax) and locomotor activity. Exposure to 4 µg/L chlorpyrifos slowed down male maturation, reduced fecundity and impaired growth of the fish. While the temperature increase did not affect any of the measured endpoints on its own, the combination of exposure to 2 µg/L CPF with an increase of 2 °C reduced growth and severely reduced fecundity, with almost no offspring production. Together, these findings suggest that climate change may exacerbate the impact of environmental pollution, and that interactive effects of chronic exposure to multiple stressors should be considered to predict how populations will be affected by ongoing global change.

Conspecific density and environmental complexity impact behaviour of turquoise killifish (Nothobranchius furzeri).

Fish models are essential for research in many biological and medical disciplines. With a typical lifespan of only 6 months, the Turquoise killifish (Nothobranchius furzeri) was recently established as a time- and cost-efficient model to facilitate whole-life and multigenerational studies in several research fields, including behavioural ecotoxicology. Essential information on the behavioural norm and on how laboratory conditions affect behaviour, however, is deficient. In the current study, we examined the impact of the social and structural environment on a broad spectrum of behavioural endpoints in N. furzeri. While structural enrichment affected only fish boldness and exploratory behaviour, fish rearing density affected the total body length, locomotor activity, boldness, aggressiveness and feeding behaviour of N. furzeri individuals. Overall, these results contribute to compiling a behavioural baseline for N. furzeri that increases the applicability of this new model species. Furthermore, our findings will fuel the development of improved husbandry protocols to maximize the welfare of N. furzeri in a laboratory setting.

One Health.

Bertram and colleagues introduce the One Health concept, an interdisciplinary framework that aims to sustainably advance and safeguard the health of humans, animals, and the environment.

Fish predation affects invertebrate community structure of tropical temporary ponds, with downstream effects on phytoplankton that are obscured by pesticide pollution.

Aquatic biota of tropical temporary ponds typically experience a wide range of stressors that can drive the structure and dynamics of natural communities. Particularly in regions with intense agricultural activity, aquatic biota may not only experience predation pressure but also stress from pesticides that inadvertently enter the ponds. We increasingly understand how these different sources of stress affect classic model taxa under controlled laboratory conditions, but how predators and pesticides may jointly affect pond invertebrate communities is still unclear, particularly for tropical systems. Here, we conducted an outdoor mesocosm experiment to study how fish predation combined with exposure to an environmentally relevant concentration of the commonly used insecticide cypermethrin (0.8 ng/L) affects the structure of invertebrate communities, and its potential effects on leaf litter decomposition and invertebrate grazing efficiency as measures of ecosystem functioning. A total of seven invertebrate taxa were recorded in the mesocosm communities. Fish predation effectively lowered the number of invertebrate taxa, with fish mesocosms being dominated by high densities of rotifers, associated with lower phytoplankton levels, but only when communities were not simultaneously exposed to cypermethrin. In contrast, cypermethrin exposure did not affect invertebrate community structure, and neither fish predation nor cypermethrin exposure affected our measures of ecosystem functioning. These findings suggest that predation by killifish can strongly affect invertebrate community structure of tropical temporary ponds, and that downstream effects on phytoplankton biomass can be mediated by exposure to cypermethrin. More broadly, we contend that a deeper understanding of (tropical) temporary pond ecology is necessary to effectively manage these increasingly polluted systems.

Assessing Environmental Risks during the Drug Development Process for Parasitic Vector-Borne Diseases: A Critical Reflection.

Parasitic vector-borne diseases (VBDs) represent nearly 20% of the global burden of infectious diseases. Moreover, the spread of VBDs is enhanced by global travel, urbanization, and climate change. Treatment of VBDs faces challenges due to limitations of existing drugs, as the potential for side effects in nontarget species raises significant environmental concerns. Consequently, considering environmental risks early in drug development processes is critically important. Here, we examine the environmental risk assessment process for veterinary medicinal products in the European Union and identify major gaps in the ecotoxicity data of these drugs. By highlighting the scarcity of ecotoxicological data for commonly used antiparasitic drugs, we stress the urgent need for considering the One Health concept. We advocate for employing predictive tools and nonanimal methodologies such as New Approach Methodologies at early stages of antiparasitic drug research and development. Furthermore, adopting progressive approaches to mitigate ecological risks requires the integration of nonstandard tests that account for real-world complexities and use environmentally relevant exposure scenarios. Such a strategy is vital for a sustainable drug development process as it adheres to the principles of One Health, ultimately contributing to a healthier and more sustainable world.

Deciphering Host-Parasite Interplay in Leishmania Infection through a One Health View of Proteomics Studies on Drug Resistance.

Recent efforts in the study of vector-borne parasitic diseases (VBPDs) have emphasized an increased consideration for preventing drug resistance and promoting the environmental safety of drugs, from the beginning of the drug discovery pipeline. The intensive use of the few available antileishmanial drugs has led to the spreading of hyper-resistant Leishmania infantum strains, resulting in a chronic burden of the disease. In the present work, we have investigated the biochemical mechanisms of resistance to antimonials, paromomycin, and miltefosine in three drug-resistant parasitic strains from human clinical isolates, using a whole-cell mass spectrometry proteomics approach. We identified 14 differentially expressed proteins that were validated with their transcripts. Next, we employed functional association networks to identify parasite-specific proteins as potential targets for novel drug discovery studies. We used SeqAPASS analysis to predict susceptibility based on the evolutionary conservation of protein drug targets across species. MATH-domain-containing protein, adenosine triphosphate (ATP)-binding cassette B2, histone H4, calpain-like cysteine peptidase, and trypanothione reductase emerged as top candidates. Overall, this work identifies new biological targets for designing drugs to prevent the development of Leishmania drug resistance, while aligning with One Health principles that emphasize the interconnected health of people, animals, and ecosystems.

EthoCRED: a framework to guide reporting and evaluation of the relevance and reliability of behavioural ecotoxicity studies.

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.

Microalgae.

Microalgae, in the strictest definition, are eukaryotic, unicellular microorganisms that are photosynthetic and typically have an aquatic lifestyle. Despite the fact that cyanobacteria (or 'blue-green algae') are prokaryotic, and are therefore not true algae, we have included them in this overview because they have a similar physiology and ecology to eukaryotic microalgae, and share many biotechnological applications. In this Primer, we discuss the diversity of microalgae, their evolutionary origin and ecological importance, the role they have played in human affairs so far, and how they can help to accelerate the transition to a more sustainable society.

Long-term exposure to a pharmaceutical pollutant affects geotaxic behaviour in the adult but not juvenile life stage of killifish.

Ecosystems around the world are increasingly polluted with pharmaceutical compounds that may perturb wildlife behaviour. Because many pharmaceuticals are continuously present in the aquatic environment, animals are often exposed to them across several life stages or even their entire life. Despite a large body of literature showing various impacts of exposure to pharmaceuticals on fish, hardly any long-term studies across different life stages have been conducted which makes it hard to accurately estimate the ecological outcomes of pharmaceutical pollution. Here, we performed a laboratory experiment in which we exposed hatchlings of the fish model Nothobranchius furzeri to an environmentally relevant concentration (0.5 µg/L) of the antidepressant fluoxetine until well into adulthood. We monitored total body length and geotaxic behaviour (i.e. gravity-mediated activity) of each fish as two traits that are ecologically relevant and naturally differ between juvenile and adult killifish. Fish exposed to fluoxetine were smaller compared to control fish, an effect that became more apparent as fish aged. Even though fluoxetine did not affect average swimming depth of either juveniles or adults, nor the time spent at the surface or bottom of the water column, exposed fish changed their position in the water column (depth) more frequently in the adult but not juvenile phase. These results suggest that important morphological and behavioural responses to pharmaceutical exposure-and their potential ecological consequences-may only emerge later in time and/or during specific life stages. Therefore, our results highlight the importance of considering ecologically relevant timescales across developmental stages when studying the ecotoxicology of pharmaceuticals.

Environmental risks of a commonly used pyrethroid: Insights from temporary pond species of the Lake Manyara Basin, Tanzania.

Environmental risks posed by widespread pesticide application have attracted global attention. Currently, chemical risk assessments in aquatic environments rely on extrapolation of toxicity data from classic model species. However, similar assessments based on local species could be complementary, particularly for unusual living environments such as temporary ponds. Here, we carried out an environmental risk assessment (ERA) of a pyrethroid model compound, cypermethrin, based on local temporary pond species. First, we measured cypermethrin residue concentrations in rivers, irrigation canals and temporary ponds in the Lake Manyara Basin (LMB). Then, we estimated the environmental risks of cypermethrin by combining these data with acute toxicity data of three resident species across three trophic levels: primary producers (Arthrospira platensis), invertebrate grazers (Streptocephalus lamellifer) and fish (Nothobranchius neumanni). Furthermore, we compared the derived ERA to that obtained using toxicity data from literature of classic model species. Cypermethrin residue concentrations in contaminated systems of the LMB ranged from 0.01 to 57.9 ng/L. For temporary pond species, S. lamellifer was the most sensitive one with a 96 h-LC50 of 0.14 ng/L. Regardless of the assumed exposure concentration (0.01 and 57.9 ng/L), the estimated risks were low for primary producers and high for invertebrate grazers, both for local species as well as for classic model species. The highest detected cypermethrin concentration resulted in a moderate risk estimation for local fish species, while the estimated risk was high when considering classic fish models. Our results confirm that, at least for pyrethroids, ERAs with classic model species are useful to estimate chemical risks in temporary pond ecosystems, and suggest that complementary ERAs based on local species could help to fine-tune environmental regulations to specific local conditions and conservation targets.

Open science.

The term 'open science' refers to a range of methods, tools, platforms and practices that aim to make scientific research more accessible, transparent, reproducible and reliable. This includes, for example, sharing code, data and research materials, embracing new publishing formats such as registered reports and preprints, pursuing replication studies and reanalyses, optimising statistical approaches to improve evidence assessment and re-evaluating institutional incentives. The ongoing shift towards open science practices is partly due to mounting evidence that studies across disciplines suffer from biases, underpowered designs and irreproducible or non-replicable results. It also stems from a general desire amongst many researchers to reduce hyper-competitivity in science and instead promote collaborative research that benefits science and society.

Pesticide sensitivity of Nothobranchius neumanni, a temporary pond predator with a non-generic life-history.

Pesticides are crucial to improve agricultural productivity, but often adversely affect surrounding aquatic systems and their fauna. To determine the environmental risk of pesticides, routine ecotoxicological tests are performed on several organisms, including standard fish models. However, these typically do not include fish species from variable habitats and with non-generic life-histories. In particular, inhabitants from temporary ponds such as annual killifish are conventionally understood to be resilient to natural stressors which could translate to higher pesticide resistance or, alternatively, trade-off with their resistance to pesticides and render them more sensitive than classic fish models. Using standard exposure tests, we assessed short-term toxicity effects of two commonly used pesticides, Roundup and cypermethrin, on the annual killifish Nothobranchius neumanni, and compared its sensitivity with that of classic fish models. For Roundup, we found a 72 h-LC50 of 1.79 ± 0.11 mg/L, which is lower than the values reported for zebrafish, medaka, fathead minnow and rainbow trout, suggesting that N. neumanni is more sensitive to the compound. The opposite was true for cypermethrin, with a 72 h-LC50 of 0.27 ± 0.03 mg/L. However, these LC50-values do not deviate strongly from those reported for other fish species, supporting earlier findings in the congeneric N. furzeri that the sensitivity of annual killifish to pollutants is similar to that of classic fish models despite their assumed robustness to environmental stress.

Neurochemical exposure disrupts sex-specific trade-offs between body length and behaviour in a freshwater crustacean.

Ongoing pollution of aquatic ecosystems with neurochemical compounds warrants an improved understanding of how this affects key organisms. Neurochemicals are shown to alter the behaviour of common study species but it remains difficult to translate these results to biologically meaningful predictions across taxa. This is partly because studies on species with non-generic life-history strategies such as many freshwater crustaceans are currently underrepresented. Here, we use a laboratory experiment to assess baseline behavioural variation (spontaneous activity level and geotaxic behaviour) in the freshwater fairy shrimp Branchipodopsis wolfi and how this is affected by chronic exposure to an environmentally-relevant concentration of the anxiolytic pharmaceutical fluoxetine. The more conspicuously coloured and larger females of the species were overall less active and more benthic than males. Moreover, amongst females, vertical activity was negatively associated with size, while an opposite relationship was found for males. These trade-offs are likely part of an antipredator strategy to reduce the probability of being detected by visual hunters, but disappeared after exposure to fluoxetine. This is of particular interest since it is an effective proof of principle that neurochemicals may impact ecologically-relevant trade-offs between conspicuous morphology and antipredator behaviour. In natural ecosystems, such disturbed antipredator behavioural responses could have far-reaching fitness consequences.

Natural daily patterns in fish behaviour may confound results of ecotoxicological testing.

Low doses of neuroactive chemicals end up in the environment and disrupt behaviour of non-target organisms. Although a whole range of studies have documented pollutant-induced changes in behaviour, natural daily variability in behaviour is rarely taken into account. This is surprising because biological rhythms may affect the outcome of experiments, are adaptive and are expected to be sensitive to neurochemical exposure. Here, we exploit daily behavioural variation in the fish model Nothobranchius furzeri to examine if behavioural effects of chronic exposure (74 days) to an environmentally relevant level (28 ng/L) of the neurochemical fluoxetine depend on the time of day. Fluoxetine exposure induced an increase in anxiety-related behaviour that was slightly more pronounced in the evening compared to the morning. Moreover, open-field locomotor activity was disrupted and daily patterns in activity lifted upon exposure to the compound. These results imply that short-term behavioural variability should be considered both to standardise ecological risk assessment of neuroactive chemicals as well as to better understand the environmental impact of such compounds in aquatic ecosystems.

Towards improved fish tests in ecotoxicology - Efficient chronic and multi-generational testing with the killifish Nothobranchius furzeri.

As many freshwaters are chemically polluted, one of the challenges for policy makers is to determine the potential impact of these pollutants on ecosystems and to define safe concentrations. Common practice is the use of ecotoxicological assays to assess the response of model organisms from different trophic levels such as algae, invertebrates and fish during exposure to dilutions of a specific compound. Ideally, ecotoxicological assessments of (pseudo-)persistent chemicals should be performed across the life-cycle or even multiple generations for an accurate risk assessment. Multigenerational tests with fish are, however, impractical and costly given the long lifespan and generation time of classic model species. Here, we suggest a framework for more relevant, time- and cost-efficient fish-based testing in ecotoxicology and align it with accredited test guidelines. Next, we introduce an upcoming fish model, the turquoise killifish Nothobranchius furzeri, and show how it facilitates such research agendas due to a short lifespan and generation time. Through a review of fish-based exposure studies with a set of reference toxicants, we position N. furzeri as a sensitive species, suitable for screening effects of different pollutant types. Ultimately, we perform a cost-benefit analysis and propose a plan of action for the introduction of N. furzeri into accredited test guidelines.

Generation-specific and interactive effects of pesticide and antidepressant exposure in a fish model call for multi-stressor and multigenerational testing.

Ecological risks of a pollutant are typically assessed via short-term exposure of model organisms to that single compound. Such tests are informative, but cannot ascertain effects of long-term and multigenerational mixed-stressor exposure with which organisms are often confronted in their natural environment. Therefore, full life-cycle and multigenerational tests are needed. Yet, these are hampered due to long lifespans and generation times of many standard laboratory species, in particular for vertebrates such as fish. With a typical lifespan of 6 months and a generation time of about 3 months, the turquoise killifish (Nothobranchius furzeri) may be an ideal model for multigenerational testing. In this study, we assessed the impact of full life-cycle exposure to the emerging pollutant fluoxetine (0, 0.5 µg/L) in combination with chronic exposure during adulthood to the pesticide 3,4-dichloroaniline (0, 50, 100 µg/L) over two successive generations of N. furzeri. Overall, both life-history and behaviour were affected by exposure to fluoxetine and 3,4-DCA. Inhibitory effects of single chemical exposure on growth and fecundity were generation-dependent, while enhanced swimming acceleration and feeding in response to fluoxetine were dependent on the presence of 3,4-DCA. Together, these findings show the relevance of a multi-stressor approach across successive generations. Although full life-cycle and multigenerational tests are typically assumed to be impractical and costly for fish, we deliver an effective demonstration that such studies are possible within a timespan of less than 6 months with the killifish N. furzeri as a model organism.

Antidepressant exposure reduces body size, increases fecundity and alters social behavior in the short-lived killifish Nothobranchius furzeri.

Social and mating behavior are fundamental fitness determinants in fish. Although fish are increasingly exposed to pharmaceutical compounds that may alter expression of such behavior, potential effects are understudied. Here, we examine the impact of lifelong exposure to two concentrations (0.7 and 5.3 µg/L) of the antidepressant fluoxetine on fecundity and social behavior (i.e. sociability and male-male aggression) in the turquoise killifish, Nothobranchius furzeri. When exposed to the highest concentration of fluoxetine (5.3 µg/L), fish were smaller at maturation but they more frequently engaged in mating. In addition, in both fluoxetine treatments females roughly doubled their overall fecundity while egg fertilization rates were the same for exposed and unexposed fish. Although aggression of male fish was not impacted by fluoxetine exposure, exposed male fish (5.3 µg/L) spent more time in the proximity of a group of conspecifics, which implies an increased sociability in these individuals. Overall, the results of this study indicate that exposure to fluoxetine may result in disrupted male sociability, increased mating frequency and an increased reproductive output in fish populations.

Improving the reliability and ecological validity of pharmaceutical risk assessment: Turquoise killifish (Nothobranchius furzeri) as a model in behavioral ecotoxicology.

Pharmaceuticals are essential for human well-being, but their increasing and continuous use pollutes the environment. Although behavioral ecotoxicology is increasingly advocated to assess the effects of pharmaceutical pollution on wildlife and ecosystems, a consensus on the actual environmental risks is lacking for most compounds. The main limitation is the lack of standardized reproducible tests that are based on sensitive behavioral endpoints and that accommodate a high ecological relevance. In the present study, we assessed the impact of a 3-wk exposure to the antidepressant fluoxetine on multiple behavioral traits in the promising new model organism Nothobranchius furzeri (turquoise killifish). Overall, our study shows that fluoxetine can impact feeding behavior, habitat choice in a novel environment, and antipredator response of N. furzeri individuals; effects on spontaneous activity and exploration tendency were less pronounced. However, effects became only apparent when individuals were exposed to fluoxetine concentrations that were 10 times higher than typical concentrations in natural aquatic environments. Ecotoxicologists are challenged to maximize both the reliability and ecological validity of risk assessments of pollutants. Our study contributes to the development of a time- and cost-efficient, standardized ecotoxicological test based on sensitive, ecologically relevant behavioral endpoints in N. furzeri. Environ Toxicol Chem 2019;38:262-270. © 2018 SETAC.