Different developmental insecticide exposure windows trigger distinct locomotor phenotypes in the early life stages of zebrafish.

Due to their extensive use and high biological activity, insecticides largely contribute to loss of biodiversity and environmental pollution. The regulation of insecticides by authorities is mainly focused on lethal concentrations. However, sub-lethal effects such as alterations in behavior and neurodevelopment can significantly affect the fitness of individual fish and their population dynamics and therefore deserve consideration. Moreover, it is important to understand the impact of exposure timing during development, about which there is currently a lack of relevant knowledge. Here, we investigated whether there are periods during neurodevelopment of fish, which are particularly vulnerable to insecticide exposure. Therefore, we exposed zebrafish embryos to six different insecticides with cholinergic mode of action for 24 h during different periods of neurodevelopment and measured locomotor output using an age-matched behavior assay. We used the organophosphates diazinon and dimethoate, the carbamates pirimicarb and methomyl as well as the neonicotinoids thiacloprid and imidacloprid because they are abundant in the environment and cholinergic signaling plays a major role during key processes of neurodevelopment. We found that early embryonic motor behaviors, as measured by spontaneous tail coiling, increased upon exposure to most insecticides, while later movements, measured through touch-evoked response and a light-dark transition assay, rather decreased for the same insecticides and exposure duration. Moreover, the observed effects were more pronounced when exposure windows were temporally closer to the performing of the respective behavioral assay. However, the measured behavioral effects recovered after a short period, indicating that none of the exposure windows chosen here are particularly critical, but rather that insecticides acutely interfere with neuronal function at all stages as long as they are present. Overall, our results contribute to a better understanding of risks posed by cholinergic insecticides to fish and provide an important basis for the development of safe regulations to improve environmental health.

Zebrafish Larvae Rapidly Recover from Locomotor Effects and Neuromuscular Alterations Induced by Cholinergic Insecticides.

Owing to the importance of acetylcholine as a neurotransmitter, many insecticides target the cholinergic system. Across phyla, cholinergic signaling is essential for many neuro-developmental processes including axonal pathfinding and synaptogenesis. Consequently, early-life exposure to such insecticides can disturb these processes, resulting in an impaired nervous system. One test frequently used to assess developmental neurotoxicity is the zebrafish light-dark transition test, which measures larval locomotion as a response to light changes. However, it is only poorly understood which structural alterations cause insecticide-induced locomotion defects and how persistent these alterations are. Therefore, this study aimed to link locomotion defects with effects on neuromuscular structures, including motorneurons, synapses, and muscles, and to investigate the longevity of the effects. The cholinergic insecticides diazinon and dimethoate (organophosphates), methomyl and pirimicarb (carbamates), and imidacloprid and thiacloprid (neonicotinoids) were used to induce hypoactivity. Our analyses revealed that some insecticides did not alter any of the structures assessed, while others affected axon branching (methomyl, imidacloprid) or muscle integrity (methomyl, thiacloprid). The majority of effects, even structural, were reversible within 24 to 72 h. Overall, we find that both neurodevelopmental and non-neurodevelopmental effects of different longevity can account for the reduced locomotion. These findings provide unprecedented insights into the underpinnings of insecticide-induced hypoactivity.

Implementation of Zebrafish Ontologies for Toxicology Screening.

Toxicological evaluation of chemicals using early-life stage zebrafish (Danio rerio) involves the observation and recording of altered phenotypes. Substantial variability has been observed among researchers in phenotypes reported from similar studies, as well as a lack of consistent data annotation, indicating a need for both terminological and data harmonization. When examined from a data science perspective, many of these apparent differences can be parsed into the same or similar endpoints whose measurements differ only in time, methodology, or nomenclature. Ontological knowledge structures can be leveraged to integrate diverse data sets across terminologies, scales, and modalities. Building on this premise, the National Toxicology Program's Systematic Evaluation of the Application of Zebrafish in Toxicology undertook a collaborative exercise to evaluate how the application of standardized phenotype terminology improved data consistency. To accomplish this, zebrafish researchers were asked to assess images of zebrafish larvae for morphological malformations in two surveys. In the first survey, researchers were asked to annotate observed malformations using their own terminology. In the second survey, researchers were asked to annotate the images from a list of terms and definitions from the Zebrafish Phenotype Ontology. Analysis of the results suggested that the use of ontology terms increased consistency and decreased ambiguity, but a larger study is needed to confirm. We conclude that utilizing a common data standard will not only reduce the heterogeneity of reported terms but increases agreement and repeatability between different laboratories. Thus, we advocate for the development of a zebrafish phenotype atlas to help laboratories create interoperable, computable data.

Biological effect and chemical monitoring of Watch List substances in European surface waters: Steroidal estrogens and diclofenac - Effect-based methods for monitoring frameworks.

Three steroidal estrogens, 17α-ethinylestradiol (EE2), 17ß-estradiol (E2), estrone (E1), and the non-steroidal anti-inflammatory drug (NSAID), diclofenac have been included in the first Watch List of the Water Framework Directive (WFD, EU Directive 2000/60/EC, EU Implementing Decision 2015/495). This triggered the need for more EU-wide surface water monitoring data on these micropollutants, before they can be considered for inclusion in the list of priority substances regularly monitored in aquatic ecosystems. The revision of the priority substance list of the WFD offers the opportunity to incorporate more holistic bioanalytical approaches, such as effect-based monitoring, alongside single substance chemical monitoring. Effect-based methods (EBMs) are able to measure total biological activities (e.g., estrogenic activity or cyxlooxygenase [COX]-inhibition) of specific group of substances (such as estrogens and NSAIDs) in the aquatic environment at low concentrations (pg/L). This makes them potential tools for a cost-effective and ecotoxicologically comprehensive water quality assessment. In parallel, the use of such methods could build a bridge from chemical status assessments towards ecological status assessments by adressing mixture effects for relevant modes of action. Our study aimed to assess the suitability of implementing EBMs in the WFD, by conducting a large-scale sampling and analysis campaign of more than 70 surface waters across Europe. This resulted in the generation of high-quality chemical and effect-based monitoring data for the selected Watch List substances. Overall, water samples contained low estrogenicity (0.01-1.3 ng E2-Equivalent/L) and a range of COX-inhibition activity similar to previously reported levels (12-1600 ng Diclofenac-Equivalent/L). Comparison between effect-based and conventional analytical chemical methods showed that the chemical analytical approach for steroidal estrogens resulted in more (76%) non-quantifiable data, i.e., concentrations were below detection limits, compared to the EBMs (28%). These results demonstrate the excellent and sensitive screening capability of EBMs.

Sub-Lethal Peak Exposure to Insecticides Triggers Olfaction-Mediated Avoidance in Zebrafish Larvae.

In agricultural areas, insecticides inevitably reach water bodies via leaching or run-off. While designed to be neurotoxic to insects, insecticides have adverse effects on a multitude of organisms due to the high conservation of the nervous system among phyla. To estimate the ecological effects of insecticides, it is important to investigate their impact on non-target organisms such as fish. Using zebrafish as the model, we investigated how different classes of insecticides influence fish behavior and uncovered neuronal underpinnings of the associated behavioral changes, providing an unprecedented insight into the perception of these chemicals by fish. We observed that zebrafish larvae avoid diazinon and imidacloprid while showing no response to other insecticides with the same mode of action. Moreover, ablation of olfaction abolished the aversive responses, indicating that fish smelled the insecticides. Assessment of neuronal activity in 289 brain regions showed that hypothalamic areas involved in stress response were among the regions with the largest changes, indicating that the observed behavioral response resembles reactions to stimuli that threaten homeostasis, such as changes in water chemistry. Our results contribute to the understanding of the environmental impact of insecticide exposure and can help refine acute toxicity assessment.

Approaches to Test the Neurotoxicity of Environmental Contaminants in the Zebrafish Model: From Behavior to Molecular Mechanisms.

The occurrence of neuroactive chemicals in the aquatic environment is on the rise and poses a potential threat to aquatic biota of currently unpredictable outcome. In particular, subtle changes caused by these chemicals to an organism's sensation or behavior are difficult to tackle with current test systems that focus on rodents or with in vitro test systems that omit whole-animal responses. In recent years, the zebrafish (Danio rerio) has become a popular model organism for toxicological studies and testing strategies, such as the standardized use of zebrafish early life stages in the Organisation for Economic Co-operation and Development's guideline 236. In terms of neurotoxicity, the zebrafish provides a powerful model to investigate changes to the nervous system from several different angles, offering the ability to tackle the mechanisms of action of chemicals in detail. The mechanistic understanding gained through the analysis of this model species provides a good basic knowledge of how neuroactive chemicals might interact with a teleost nervous system. Such information can help infer potential effects occurring to other species exposed to neuroactive chemicals in their aquatic environment and predicting potential risks of a chemical for the aquatic ecosystem. In the present article, we highlight approaches ranging from behavioral to structural, functional, and molecular analysis of the larval zebrafish nervous system, providing a holistic view of potential neurotoxic outcomes. Environ Toxicol Chem 2021;40:989-1006. © 2020 SETAC.

Sampling rates for passive samplers exposed to a field-relevant peak of 42 organic pesticides.

Pesticide concentrations in agricultural streams are often characterised by a low level of baseline exposure and episodic peak concentrations associated with heavy rainfall events. Traditional sampling methods such as grab sampling, which are still largely used in governmental monitoring, typically miss peak concentrations. Passive sampling represents a cost-efficient alternative but requires the additional determination of sampling rates to calculate time-weighted average (TWA) water concentrations from the accumulated pesticide mass in the sampler. To date, sampling rates have largely been determined in experiments with constant exposure, which does not necessarily reflect field situations. Using Empore styrene-divinylbenzene (SDB) passive sampler disks mounted in metal holders, we determined sampling rates for 42 organic pesticides, of which 27 sampling rates were lacking before. The SDB disks were in an artificial channel system exposed to a field-relevant pesticide peak. We used an open-source algorithm to estimate coefficients of equations for the accumulated pesticide mass in disks and to determine exposure time-dependent sampling rates. These sampling rates ranged from 0.02 to 0.98 L d-1 and corresponded to those from previous studies determined with constant exposure. The prediction of sampling rates using compound properties was unreliable. Hence, experiments are required to determine reliable sampling rates. We discuss the use of passive sampling to estimate peak concentrations. Overall, our study provides sampling rates and computer code to determine these under peak exposure designs and suggests that passive sampling is suitable to estimate peak pesticide concentrations in field studies.

Fish-gut-on-chip: development of a microfluidic bioreactor to study the role of the fish intestine in vitro.

In this study we present the first fish-gut-on-chip model. This model is based on the reconstruction of the intestinal barrier by culturing two intestinal cell lines from rainbow trout, namely epithelial RTgutGC and fibroblastic RTgutF, in an artificial microenvironment. For a realistic mimicry of the interface between the intestinal lumen and the interior of the organism we i) developed ultrathin and highly porous silicon nitride membranes that serve as basement membrane analogues and provide a culture interface for the fish cells; ii) constructed a unique micro-well plate-based microfluidic bioreactor that enables parallelization of experiments and creates realistic fluid flow exposure scenarios for the cells; iii) integrated electrodes in the reactor for non-invasive impedance sensing of cellular well-being. In a first approach, we used this reactor to investigate the response of epithelial fish cells to in vivo-like shear stress rates of 0.002-0.06 dyne per cm2, resulting from fluid flow within the intestinal lumen. Moreover, we investigated the interplay of epithelial and fibroblast cells under optimal flow conditions to carefully evaluate the benefits and drawbacks of the more complex reconstruction of the intestinal architecture. With our fish-gut-on-chip model we open up new strategies for a better understanding of basic fish physiology, for the refinement of fish feed in aquaculture and for predicting chemical uptake and bioaccumulation in fish for environmental risk assessment. The basic principles of our reactor prototype, including the use of ultrathin membranes, an open microfluidic circuit for perfusion and the micro-well plate-based format for simplified handling and avoidance of air-bubbles, will as well be of great value for other barrier-on-chip models.

Monitoring estrogenic activities of waste and surface waters using a novel in vivo zebrafish embryonic (EASZY) assay: Comparison with in vitro cell-based assays and determination of effect-based trigger values.

This study reports the use of the recently developed EASZY assay that uses transgenic cyp19a1b-GFP zebrafish (Danio rerio) embryos to assess in vivo estrogenic activity of 33 surface (SW) and waste water (WW) samples collected across Europe that were previously well-characterized for estrogen hormones and in vitro estrogenic activity. We showed that 18 out of the 33 SW and WW samples induced estrogenic responses in the EASZY assay leading to a significant and concentration-dependent up-regulation of the ER-regulated cyp19a1b gene expression in the developing brain. The in vivo 17ß-estradiol-equivalents (EEQs) were highly correlated with, both, the chemical analytical risk quotient (RQ) based on steroidal estrogen concentrations and EEQs reported from five different in vitro reporter gene assays. Regression analyses between the vitro and in vivo effect concentrations allowed us to determine an optimal cut-off value for each in vitro assay, above which in vivo responses were observed. These in vitro assay-specific effect-based trigger values (EBTs), ranging from 0.28 to 0.58 ng EEQ/L define the sensitivity and specificity of the individual in vitro assays for predicting a risk associated with substances acting through the same mode of action in water samples. Altogether, this study demonstrates the toxicological relevance of in vitro-based assessment of estrogenic activity and recommends the use of such in vitro/in vivo comparative approach to refine and validate EBTs for mechanism-based bioassays.

Combination of In Situ Feeding Rate Experiments and Chemical Body Burden Analysis to Assess the Influence of Micropollutants in Wastewater on Gammarus pulex.

Wastewater discharge is one of the main sources of micropollutants within the aquatic environment. To reduce the risks for the aquatic environment, the reduction of the chemical load of wastewater treatment plant effluent is critical. Based on this need, additional treatment methods, such as ozonation, are currently being tested in several wastewater treatment plants (WWTPs). In the present study, effects were investigated using in situ feeding experiments with Gammarus pulex and body burden analyses of frequently detected micropollutants which used a Quick Easy Cheap Effective Rugged and Safe (QuEChERS) multi-residue method to quantify internal concentrations in collected gammarids. Information obtained from these experiments complemented data from the chemical analysis of water samples and bioassays, which predominantly cover hydrophilic substances. When comparing up- and downstream feeding rates of Gammarus pulex for seven days, relative to the WWTPs, no significant acute effects were detected, although a slight trend of increased feeding rate downstream of the WWTP Aachen-Soers was observed. The chemical load released by the WWTP or at other points, or by diffuse sources, might be too low to lead to clear acute effects on G. pulex. However, some compounds found in wastewater are able to alter the microbial community on its leaves, leading to an increase in the feeding rate of G. pulex. Chemical analysis of internal concentrations of pollutants in the tissues of collected gammarids suggests a potential risk for chronic effects with the chemicals imidacloprid, thiacloprid, carbendazim, and 1H-benzotriazole when exceeding the critical toxic unit value of -3. This study has demonstrated that a combination of acute testing and measurement of the internal concentration of micropollutants that might lead to chronic effects is an efficient tool for investigating river systems, assuming all relevant factors (e.g., species or season) are taken into account.