Food dyes, or color additives, are chemicals added to industrial food products and in domestic cooking to improve the perceived flavor and attractiveness. Of natural and synthetic origin, their safety has been long discussed, and concern for human safety is now clearly manifested by warnings added on products labels. Limited attention, however, has been dedicated to the effects of these compounds on aquatic flora and fauna. For this reason, the toxicity of four different commercially available food dyes (cochineal red E120, Ponceau red E124, tartrazine yellow E102 and blue Patent E131) was assessed on three different model organisms, namely Cucumis sativus, Artemia salina and Danio rerio that occupy diverse positions in the trophic pyramid. The evidence collected indicates that food dyes may target several organs and functions, depending on the species. C. sativus rate of germination was increased by E102, while root/shoot ratio was ∼20% reduced by E102, E120 and E124, seed total chlorophylls and carotenoids were 15–20% increased by E120 and 131, and total antioxidant activity was ∼25% reduced by all dyes. Mortality and low mobility of A. salina nauplii were increased by up to 50% in presence of E124, E102 and E131, while the nauplii phototactic response was significantly altered by E102, E120 and E124. Two to four-fold increases in the hatching percentages at 48 h were induced by E124, E102 and E131 on D. rerio, associated with the occurrence of 20% of embryos showing developmental defects. These results demonstrated that the food dyes examined are far from being safe for the aquatic organisms as well as land organisms exposed during watering with contaminated water. The overall information obtained gives a realistic snapshot of the potential pollution risk exerted by food dyes and of the different organism' ability to overcome the stress induced by contamination.

Climate change and pharmaceuticals contamination constitute two of the most relevant stressors on the aquatic ecosystems, however, there is a huge lack of information regarding the interactive effects of both stressors. For that, a mesocosm experiment was implemented where adult zebrafish were exposed to combined temperature and the progestin levonorgestrel (LNG) for 21 days. Considering that the liver is one of the organs where there is a greater metabolization and accumulation of toxicants, the main objective of this work was to assess the effects of both stressors on the female zebrafish hepatocytes morphology and functioning, through stereological and immunohistochemical techniques.Our results revealed an increase of coefficient of variation of the number distribution of hepatocytes volume (CVN(υ)) for individuals exposed to LNG, which denotes an increase of the hepatocytes size variability and is suggestive of functional impacts. This was corroborated by the signs of increased glycogen content with the exposure to increased LNG concentrations and temperature, indicating modified hepatocyte glycogen metabolism. Such disturbances can be considered indicators that the fish had to deal with impacts caused by the stress factors.Regarding the immunoreactivity, from the four proteins selected (catalase, CYP1A, HSP90 and Vtg), just in two of them (catalase and Vtg) were observed some responses to both stressors. For catalase there was a hormetic response, in which exposure to lower LNG concentrations caused a significant higher positive immunostaining than under higher LNG concentrations. While, for Vtg, significant effects of temperature and LNG existed, in which a decline in Vtg immunostaining was observed with exposure to higher temperature and lower LNG concentrations. These results should be seen as a warning sign about fine impacts of multiple stressors, such as temperature and progestogens, on the structure and functioning of zebrafish liver and potentially in other aquatic organisms, and on their health implications.

β-Blockers (BB) are one of the most commonly prescribed pharmaceuticals used for treating cardiovascular and acute anxiety-related disorders. This class of drugs inhibit β-adrenoceptor signalling and given their growing, widespread use, BB are routinely detected in surface waters at nM concentrations. This is concerning as trace levels of BB impart developmental and reproductive dysfunction in non-target aquatic organisms, with potential for ecological risks. To date, environmental pharmaceutical risks to non-target animals are not part of the monitoring framework due to the lack of bioassays for assessing their biological effects. Behavioral endpoints have the advantage of a systems-level integration of multiple sensory signals and motor responses for toxicity screening; however, they are not currently used for risk assessment of environmental contaminants. The zebrafish (Danio rerio) embryo photomotor response (zfPMR) has been used in high-throughput behavioral screenings for neuroactive drug effects at high, therapeutic concentrations. Our objective here was to examine if we could utilize the zfPMR for screening environmental levels of BB. Embryos were placed into 96-well plates, exposed to chemicals and/or municipal wastewater effluent (MWWE), and their zfPMRs were measured with video-analysis. To specifically target BB, embryos were co-treated with isoproterenol, a β-adrenergic agonist that stimulates the zfPMR, and the inhibition of isoproterenol-induced response was used as a biomarker of BB exposure. Our results reveal that the inhibition of isoproterenol-stimulated zfPMRs can be used as a biosensor capable of detecting BB in the parts-per-billion to parts-per-trillion in water samples, including diluted MWWE. The method developed detects BB in spite of the presence of other neuroactive compounds in water samples. This systems level approach of rapid screening for BB effects provides the most promising evidence to date that behavioral neuromodulation can be potentially applied for environmental effects monitoring of pharmaceuticals.

Gut microbiota is of critical importance to host health. Aryl hydrocarbon receptor (AhR) is found to be closely involved in the regulation of gut microbial dynamics. However, it is still not clear how AhR signaling shapes the gut microbiota. In the present study, adult zebrafish were acutely exposed to an AhR antagonist (CH223191), an AhR agonist (polychlorinated biphenyl 126; PCB126) or their combination for 7 d. Overall intestinal health and gut microbial community were temporally monitored (1 d, 3 d and 7 d) and inter-compared among different groups. The results showed that single exposure to PCB126 significantly disrupted the overall health of intestines (i.e., neural signaling, inflammation, epithelial barrier integrity, oxidative stress). However, CH223191 failed to inhibit but enhanced the physiological toxicities of PCB126, implying the involvement of extra mechanisms rather than AhR in the regulation of intestinal physiological activities. Dysbiosis of gut microbiota was also caused by PCB126 over time as a function of sex. It is intriguing that CH223191 successfully abolished the holistic effects of dioxin on gut microbiota, which inferred that growth of gut microbes was directly controlled by AhR activation without the involvement of host feedback modulation. When coming to detailed alterations at certain taxon, both antagonistic and synergistic interactions existed between CH223191 and dioxin, depending on fish sex, exposure duration and bacterial species. Correlation analysis found that gut inflammation was positively associated with pathogenic Legionella bacteria, but was negatively associated with epithelial barrier integrity, suggesting that integral intestinal epithelial barrier can prevent the influx of pathogenic bacteria to induce inflammatory response. Overall, this study has deciphered, for the first time, the direct regulative effects of AhR activity on gut microbiota. Future research is warranted to elucidate the specific mechanisms of AhR action on certain bacterial population.

As a widely used organotin acaricide nowadays, azocyclotin (ACT) could induce thyroidal endocrine disruption in fishes and amphibians, but its dominant disrupting mode remains unknown. In this study, zebrafish were firstly exposed to ACT (0.18–0.36 ng/mL) from 2 hpf (hours post fertilization) to 30 dpf (days post fertilization), and a series of developmental toxicological endpoints and thyroid hormones were measured. Result showed that no developmental toxicity to zebrafish was found in 0.18 and 0.24 ng/mL groups except decreased body weight (30 dpf, 0.24 ng/mL). However, exposed to 0.36 ng/mL ACT led to reductions in heartbeat (48 hpf), hatching rate (72 hpf) and bodyweight (30 dpf). General tendencies of decreases in free T3 but increases in free T4 and reductions in ratio of free T3/T4 were also found, inferring that type II deiodinase (Dio2) was repressed. This inference was confirmed by Western analysis that Dio2 expression reduced by 42.7% after 0.36 ng/mL ACT treatment. Moreover, RNA-Seq analysis implied that exposed to 0.36 ng/mL ACT altered the genome-wide gene expression profiles of zebrafish. Totally 5660 genes (involving 3154 down-regulated and 2596 up-regulated genes) were differentially expressed, and 13 deferentially expressed genes including down-regulated dio2 were significantly enriched in thyroid hormone signaling pathway. Subsequently, an in vitro thyroid receptor-reporter gene assay using GH3 cells was performed to further explore the potential disrupting mechanism. Result showed that luciferase activity slightly increased after exposure to ACT alone or ACT combined with low level T3, but was suppressed when combined with high level T3. It indicted there probably existed a competitive relationship in some extent between ACT and T3 in vivo. Overall, the present study provided preliminary evidences that long-term exposure to trace ACT repressed Dio2 expression, declined T3 and then activated thyroid receptor-mediated signaling, thereby leading to integrated thyroid endocrine disruption in zebrafish larvae.

Selenium (Se) is a metalloid of potential interest from both a toxicological and nutritional perspective, having a range of safe intake. The adverse neuro-behavioural effects of Se have been investigated in both humans and fishes, but little is known about its effects on social behaviours or the serotonergic signaling pathway in the brain. In the present study, we investigated the effects of chorionic dietary exposure to Se (as selenomethionine) at different concentrations (control, 2.1, 11.6 or 31.5 μg/g dry wt.) on antipredator avoidance, shoaling behaviour, and social group preferences in adult zebrafish (Danio rerio). In addition, we also measured the expression of important genes in the serotonergic pathway that influence social behaviours. After 60 days of exposure, the highest dose (31.5 μg/g dry wt.) caused the highest level of baseline fear behaviour, with fish swimming lower in the water column and in tighter shoals compared to fish in the other treatments. With high levels of baseline fear, these fish did not significantly intensify fear behaviours in response to predation risk in the form of exposure to chemical alarm cues. When individual fish were given an opportunity to shoal with groups of differing sizes (3 vs. 4 individuals), fish exposed to the high dose spent less time with groups in general, and only control fish showed a significant preference for the larger group. In the zebrafish brain, we found significant upregulation in the mRNA expression of serotonin receptors (htr1aa and htr1b), a transporter (slc6a4a), and tryptophan hydroxylase-2 (tph2), whereas there was a downregulation of the monoamine oxidase (mao) gene. The results of this study suggest that disruption of serotonergic neurotransmission might have been responsible for Se-induced impairment of antipredator and social behaviour in zebrafish.

Spirocyclic tetramic acids are widely used in controlling phytophagous mite species throughout the world. the data set is incomplete and provides insufficient evidence for drawing the same conclusion for fish. To fill the gap whether these acaricides alter lipid metabolism on vertebrates, zebrafish embryos exposed to a series concentration of pesticides, the developmental effects, enzyme activities and levels of gene expression were assessed, battery of biomarker utilized by the integrated biomarker response (IBRv2) model. The 96 h-LC₅₀ of spirodiclofen, spiromesifen and spirotetramat were 0.14, 0.12 and 5.94 mg/L, respectively. Yolk sac deformity, pericardial edema, spinal curvature and tail malformation were observed. Three spirocyclic acids were unfavouring the lipid accumulation of by inhibited the acetyl-CoA carboxylase (ACC), fatty acid synthesis (FAS), fatty acid binding proteins (FABP2) and lipoprotein lipase (LPL) activity. The total cholesterol (TCHO) level significantly decreased in the 0.072 mg/L spirodiclofen group and 0.015 and 0.030 mg/L in the spiromesifen groups. No expected change in spirotetramat group on the TCHO and triglycerides (TGs) levels for any of the treatments. The mRNA levels of the genes related to lipid metabolism also significantly altered. In both spirodiclofen and spiromesifen, ACC achieved the highest scores among a battery of biomarkers using integrated biomarker response (IBRv2). The results suggest that spiromesifen was the most toxic for embryos development and spirodiclofen was the most toxic for lipid metabolism in embryos. The 0.07 mg/L of spirodiclofen, 0.05 mg/L of spiromesifen and 2.00 mg/L would cause malformation on zebrafish embryos. This study will provide new insight that fatty acid metabolism may be a suitable biomarker for the spirocyclic tetramic acids in fish species.

Current understanding of how environmental aging of microplastics contributes to their ecotoxicity is low. We investigated whether incubation of microplastics in waters with different organic load and toxic potential alters the toxicity of microplastics to crustacean Daphnia magna, fish embryos Danio rerio and plant Lemna minor. Polyethylene primary microplastics; specifically microbeads from facial scrub; were subjected to 3-weeks incubation in low affected spring water, river water, effluent from the municipal wastewater treatment plant (WWTP) and municipal landfill leachate. Primary microplastics had no acute effect on D. magna mobility and D. rerio embryos development. While high organic load wastewaters; WWTP effluent and landfill leachate; showed evident toxicity for D. magna and D. rerio embryos, microplastics aged in these wastewaters had no effect. This suggests that adsorption of pollutants from wastewaters to microplastic particles was not high enough to induce acute toxicity to D. magna and D. rerio. On the contrary, primary microplastics affected the root growth of L. minor. Interestingly, aging of microplastics in low organic-load waters mitigated the toxicity of microplastics for L. minor, while microplastics aged in high-organic load waters had the same adverse effect as primary microplastics. Partly, these effects can be explained by different extent of coating on microplastics in different water samples. This study suggests that aging of microplastics in wastewaters and natural waters did not significantly enhance the toxicity to selected test species, but further studies on plants may be of interest.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been detected in various environmental media and has been implicated as a weak mutagen or carcinogen, but whether TDCIPP can promote the progression of liver tumor remains unclear. In this study, krasⱽ¹² genetically modified zebrafish, Tg(fabp10:rtTA2s-M2; TRE2:EGFP-krasᴳ¹²ⱽ), a model system in which liver tumors can be induced by doxycycline (DOX), was used to evaluate the liver tumor promotion potential of TDCIPP. Briefly, krasⱽ¹² transgenic females were exposed to 0.3 mg/L TDCIPP, 20 mg/L DOX or a binary mixture of 0.3 mg/L TDCIPP with 20 mg/L DOX, and liver size, histopathology, and transcriptional profiles of liver were determined. Treatment with TDCIPP resulted in increased liver size and caused more aggressive hepatocellular carcinoma (HCC). Compared with the exposure to DOX, TDCIPP in the presence of DOX up-regulated the expression of genes relevant with salmonella infection and the toll-like receptor signaling pathway. These results implied an occurrence of inflammatory reaction, which was sustained by the increase in the amount of infiltrated neutrophils in the liver of Tg(lyz:DsRed2) transgenic zebrafish larvae whose neutrophils were labelled by red fluorescent protein under the lysozyme C promoter. Furthermore, compared with the binary exposure of DOX and TDCIPP, treatment with a ternary mixture of TDCIPP, DOX and inflammatory response inhibitor (ketoprofen) significantly decrease the liver size and the amounts of neutrophils in the livers of kras and lyz double transgenic zebrafish larvae. Collectively, our results suggested that TDCIPP could promote the liver tumor progression by induction of hepatic inflammatory responses.

The growing production and extensive use of organophosphate flame retardants (OPFRs) have led to an increase in their environmental distribution and human exposure. Developmental toxicity is a major concern of OPFRs' adverse health effects. However, the impact of OPFRs exposure on vascular development and the toxicity pathway for developmental defects are poorly understood. In this study, we investigated the effects of exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCPP), a frequently detected OPFR, on early vascular development, and the possible role of nuclear factor erythroid 2-related factor (Nrf2)-dependent angiogenic pathway in TDCPP's vascular toxicity. TDCPP exposure at 300 and 500 μg/L impeded the growth of intersegmental vessels (ISV), a type of microvessels, as early as 30 hpf. Consistently, a similar pattern of decreased extension and remodeling of common cardinal vein (CCV), a typical macrovessel, was observed in zebrafish at 48 hpf and 72 hpf. Developing vasculature in zebrafish was more sensitive than general developmental parameters to TDCPP exposure. The expression of genes related to VEGF signaling pathway dose-dependently decreased in TDCPP-treated larvae. In in vitro experiments using human umbilical vein endothelial cells (HUVECs), the increased cell proliferation induced by VEGF was suppressed by TDCPP exposure in a dose-dependent fashion. In addition, we found a repression of Nrf2 expression and activity in TDCPP-treated larvae and HUVECs. Strikingly, the application of CDDO-Im, a potent Nrf2 activator, enhanced VEGF and protected against defective vascular development in zebrafish. Our results reveal that vascular impairment is a sensitive index for early exposure to TDCPP, which could be considered in the environmental risk assessment of OPFRs. The identification of Nrf2-mediating VEGF pathway provides new insight into the adverse outcome pathway (AOP) of OPFRs.