Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants.

We examined ingestion and retention rates of microplastics (MPs) by two freshwater (Japanese medaka and zebrafish) and two marine fish species (Indian medaka and clown anemonefish) to determine their color preferences and gastrointestinal-tract retention times. In our ingestion experiments, clown anemonefish ingested the most MP particles, followed by zebrafish, and then Japanese and Indian medaka. Next, we investigated color preferences among five MP colors. Red, yellow, and green MP were ingested at higher rates than gray and blue MPs for all tested fish species. To test whether these differences truly reflect a recognition of and preference for certain colors based on color vision, we investigated the preferences of clown anemonefish for MP colors under light and dark conditions. Under dark conditions, ingestion of MP particles was reduced, and color preferences were not observed. Finally, we assessed gastrointestinal-tract retention times for all four fish species. Some individuals retained MP particles in their gastrointestinal tracts for over 24 h after ingestion. Our results show that fish rely on color vision to recognize and express preferences for certain MP colors. In addition, MP excretion times varied widely among individuals. Our results provide new insights into accidental MP ingestion by fishes.

Once in the aquatic ecosystems, nanoplastics (NPls) can interact with other contaminants acting as vectors of transport and altering their toxicological effects towards organisms. Thus, the present study aims to investigate how polystyrene NPls (44 nm) interact with the herbicide phenmedipham (PHE) and affect its toxicity to zebrafish embryos. Single exposures to 0, 0.015, 0.15, 1.5, 15 and 150 mg/L NPls and 0.02, 0.2, 2 and 20 mg/L PHE were performed. Embryos were also exposed to the binominal combinations: 0.015 mg/L NPls + 2 mg/L PHE, 0.015 mg/L NPls + 20 mg/L PHE, 1.5 mg/L NPls + 2 mg/L PHE and 1.5 mg/L NPls + 20 mg/L PHE. Due to the low solubility of PHE in water, a solvent control was performed (0.01% acetone). PHE was quantified. Mortality, heartbeat and hatching rate, malformations appearance, locomotor behavior and biomarkers related to oxidative stress, neurotransmission and energy budgets were analyzed. During 96 h, NPls and PHE single and combined exposures did not affect embryos development. After 120 h, NPls induced hyperactivity and PHE induced hypoactivity. After 96 h, NPls increased catalase activity and PHE increased glutathione S-transferases activity. On the combination 0.015 mg/L NPls + 20 mg/L PHE, hyperactivity behavior was found, similar to 0.015 mg/L NPls, and cholinesterase activity was inhibited. Additionally, the combination 1.5 mg/L NPls + 20 mg/L PHE increased both catalase and glutathione S-transferases activities. The combination NPls with PHE affected more biochemical endpoints than the single exposures, showing the higher effect of the binominal combinations. Dissimilar interactions effects – no interaction, synergism and antagonism – between NPls and PHE were found. The current study shows that the effects of NPls on bioavailability and toxicity of other contaminants (e.g. PHE) cannot be ignored during the assessment of NPls environmental behavior and risks.

Aquatic natural colloids are closely related to the environmental behavior of pollutants, which may affect their bioavailability in aquatic organisms. This study explored the potential mechanisms of the natural colloids at environmentally relevant concentrations affecting the bioaccumulation process of benzophenone-3 (BP3) in zebrafish (Danio rerio). The results of kinetic model fitting showed that the natural colloids decreased the uptake and loss rate of BP3 by zebrafish but prolonged the time to reach the cumulative equilibrium, eventually resulting in a higher cumulative concentration in zebrafish. According to the tissue concentration at equilibrium and the results of toxicokinetic analysis, the presence of high molecular colloids could enhance the bioaccumulation of freely dissolved BP3 due to its high desorption rate with BP3 in the intestines of fish, increasing the freely dissolved BP3 concentrations to which zebrafish were exposed. Both natural colloids and BP3 could enhance the cell permeability of zebrafish, which allowed colloid-bound BP3 to directly enter the fish and accumulate in its muscle. Besides, although both natural colloids and BP3 could cause the metabolic disorders in adult zebrafish, they affected the physiological and biochemical activities of zebrafish through different pathways. The disturbance of glutathione metabolism in zebrafish induced by natural colloids may be the reason for the diminished ability of zebrafish to clear and transform BP3 in the mixture system. The carrier effect of natural colloids and reduced clearance ability of zebrafish eventually increased the bioaccumulation of BP3 in zebrafish. This study highlights the significance of natural colloids at environmentally relevant concentrations on the biological effects of emerging contaminants in actual waters, however, natural colloids are always ignored in most field investigation of pollutants, which would ultimately lead to an underestimation of the true ecological risk of pollutants.

Polyhalogenated polycyclic aromatic hydrocarbons (HPAHs) represent a major environmental concern due to their persistency and toxicity. Among them, mono-halogenated (HNs) and halomethyl naphthalenes (HMNs) are not well-studied, and the toxicity of many HNs to fishes has not been reported. In this study, we exposed zebrafish (Danio rerio) embryos to naphthalene and five HNs at concentrations ranging from 0.25 to 2.0 mg L⁻¹ to assess acute toxicities and developmental effects. Among them, 2-bromomethyl naphthalene (2-BMN) produced moderate lethal effects (96-h LC₅₀ = 1.4 mg L⁻¹) and significantly reduced hatchability. Abnormal phenotypes, including pericardial edema, spine curvature, and shortened body length, were also induced by 2-BMN (96-h EC₅₀ = 0.45 mg L⁻¹). Treatments of 0.5–2.0 mg L⁻¹ 2-BMN evoked cardiac malformations via significant down-regulation of the cacna1c gene, which codes the voltage-dependent calcium channel, at 72 hpf and up-regulation of the nppa gene, responsible for the expression of natriuretic peptides, at 96 hpf in zebrafish. One presumable toxic photo-dissociated metabolite of 2-BMN, the 2-naphthylmethyl radical, may be responsible for the toxic effect on zebrafish embryos. HPAHs must be monitored and managed due to their adverse effects on living organisms at low concentrations.

Sex determination is a complex process that can be influenced by environment in various taxa. Disturbed environments can affect population sex ratios and thus threaten their viability. Emerging evidences support a role of epigenetic mechanisms, notably DNA methylation, in environmental sex determination (ESD). In this work, using zebrafish as model and a transgenerational experiment comprising 4 successive generations, we report a strength link between the promotor methylation level of three genes in female gonads and population sex ratio. One generation of zebrafish was exposed throughout its lifetime to cadmium (Cd), a non-essential metal, at an environmentally relevant concentration. The subsequent generations were not exposed. At the first and the third generation a subset of individuals was exposed to an elevated temperature, a well-known masculinizing factor in zebrafish. While heat was associated to an increase in the methylation level of cyp19a1a gene and population masculinization, foxl2a/dmrt1 methylation levels appeared to be influenced by Cd and fish density leading to offspring feminization. Ancestral Cd exposure indeed led to a progressive feminization of the population over generations and affected the sex plastic response of zebrafish in response to heat. The effect of Cd on the methylation level of foxl2a was observed until the third generation, supporting potential transgenerational inheritance. Our results support (i) a key role of cyp19a1a methylation in SD in zebrafish in response to environmental cues and (ii) the fact that the environment experienced by parents, namely mothers in the present case, can affect their offspring sex ratio via environment-induced DNA methylation changes in gonads.

Various microplastics (MPs) are found in the environment and organisms. MP residues in organisms can affect health; however, their impacts on metabolism in aquatic organisms remain unclear. In this study, zebrafish embryos were exposed to polyethylene MPs with sizes ranging from 1 to 4 μm at concentrations of 0, 10, 100, and 1000 μg/L for 7 days. Through qPCR technology, the results indicated that zebrafish exposed to polyethylene MPs exhibited significant change in microbes of the phyla Firmicutes, Bacteroidetes, Proteobacteria, and Verrucomicrobia, etc. Moreover, 16S RNA gene sequencing revealed that there was a significant difference in alpha diversity between the control and 1000 μg/L MP-treated groups. At the genus level, the abundance of Aeromonas, Shewanella, Microbacterium, Nevskia and Methyloversatilis have increased remarkably. Conversely, the abundance of Pseudomonas, Ralstonia and Stenotrophomonas were significant reduction after MPs exposure. In addition, the levels of TG (triglyceride), TCHO (total cholesterol), NEFA (nonesterified fatty acid), TBA (total bile acid), GLU (glucose) and pyruvic acid significantly changed in MP-treated larval zebrafish, indicating that their metabolism was disturbed by MPs. Transcriptional levels of glucose and lipid metabolism-related genes showed a decreasing trend. Furthermore, LC/MS-based nontargeted metabolomics analysis demonstrated that a total of 59 phospholipid-related substances exhibited significant changes in larval fish treated with 1000 μg/L MPs. The mRNA levels of phospholipid metabolism-related genes were also obviously changed. Pearson correlation analysis indicated that the abundance of Aeromonas, Shewanella and Chitinibacter bacteria showed a negative correlation with most phospholipids, while Nevskia, Parvibacter and Lysobacter showed a positive correlation with most phospholipids. Based on these results, it is suggested that 1–4 μm PE-MPs could impact the microbiome and metabolism of larval zebrafish. All of these results indicated that the health risk of MPs cannot be ignored.

Insufficient evidence exists regarding the visible physiological toxic endpoints of MPs exposures on zebrafish larvae due to their small sizes. Herein, the impacts of micro-polystyrene particles (μ-PS) and 100 nm polystyrene particles (n-PS) on the toxicity of cadmium (Cd) through altering neutrophil expressions were identified and quantified in the transgenic zebrafish (Danio rerio) larvae Tg(lyz:DsRed2), and the effects were size-dependent. When exposed together with μ-PS, the amount of neutrophils in Cd treated zebrafish larvae decreased by 25.56% through reducing Cd content in the larvae. By contrast, although n-PS exposure caused lower Cd content in the larvae, the expression of neutrophils under their combined exposure remained high. The mechanism of immune toxicity was analyzed based on the results of metabonomics. n-PS induced high oxidative stress in the larvae, which promoted taurine metabolism and unsaturated fatty biosynthesis in n-PS + Cd treatment. This observation was accordance with the significant inhibition of the activities of superoxide dismutase and catalase enzymes detected in their combined treatment. Moreover, n-PS promoted the metabolic pathways of catabolic processes, amino acid metabolism, purine metabolism, and steroid hormone biosynthesis in Cd treated zebrafish larvae. Nanoplasctis widely coexist with other pollutants in the environment at relatively low concentrations. We conclude that more bio-markers of immune impact should be explored to identify their toxicological mechanisms and mitigate the effects on the environment.

Strobilurins are popular fungicides used in agriculture on a global scale. Due to their widespread use as agrochemicals, they can enter aquatic environments at concentrations that can elicit adverse effects in organisms. This review synthesizes the current state of knowledge regarding the toxic effects of strobilurin fungicides on aquatic species, including algal species, Daphnia magna, and fish species, to determine risk to aquatic organisms and ecosystems. Data show that the toxicities of strobilurins vary widely across aquatic species. Strobilurins bind cytochrome bc1 in mitochondrial complex III in fungi, and as such, research in aquatic species has focused on mitochondria-related endpoints following exposures to strobilurins. In fish, studies into the activities of mitochondrial complexes and the expression of genes involved in the electron transfer chain have been conducted, converging on the theme that mitochondrial complexes and their enzymes are impaired by strobilurins. In general, the order of toxicity of strobilurins for fish species are pyraoxystrobin > pyraclostrobin ≈ trifloxystrobin > picoxystrobin > kresoxim-methyl > fluoxastrobin > azoxystrobin. In addition to mitochondrial toxicity, studies also report genotoxicity, immunotoxicity, cardiotoxicity, neurotoxicity, and endocrine disruption, and each of these events can potentially impact whole organism-level processes such as development, reproduction, and behavior. Screening data from the US Environmental Protection Agency ToxCast database supports the hypothesis that these fungicides may act as endocrine disruptors, and high throughput data suggest estrogen receptor alpha and thyroid hormone receptor beta can be activated by some strobilurins. It is recommended that studies investigate the potential for endocrine disruption by strobilurins more thoroughly in aquatic species. Based on molecular, physiological, and developmental outcomes, a proposed adverse outcome pathway is presented with complex III inhibition in the electron transfer chain as a molecular initiating event. This review comprehensively addresses sub-lethal toxicity mechanisms of strobilurin fungicides, important as the detection of strobilurins in aquatic environments suggests exposure risks in wildlife.

Azoxystrobin (AZ) and pyraclostrobin (PY) are strobilurin fungicides that inhibit fungal mitochondrial respiration. In this study, a representative model, zebrafish (Danio rerio), was used as a test species for acute and developmental toxicity. Survival and malformation rates were observed only PY-treated embryos, with an LC₅₀ value of 77.75 ppb accompanied by a dramatic decrease in hatching rate, while AZ did not show great mortality. Morphological changes were observed in PY-treated embryos with the occurrence of pericadial edema at 25 ppb. A delay in growth was observed after treatment with pyraclostrobin at 50 ppb. Use of genetically engineered Tg(cmlc:EGFP) allowed fluorescence observation during heart development. PY interfered with normal heart development via upregulation of the nppa gene responsible for the expression of natriuretic peptides. Heart function was dramatically reduced as indicated by reduced heart rates. Increased expression of the nppa gene was also seen in AZ-treated embryos. The expression level of cyp24a1 was also up-regulated, while ugt1a1 and sult1st6 were down-regulated after treatment of zebrafish embryos with AZ or PY. Overall, strobilurin fungicides might inhibit normal heart formation and function within the range of concentrations tested.