Integrative time series of cellular, humoral and molecular response revealed immunotoxicity of bifenthrin to Chinese rare minnow (Gobiocypris rarus) following Pseudomonas fluorescens challenge.

Bifenthrin is a common pesticide that is widespread in aquatic environments. Although it has been shown to be toxic to aquatic organisms, its immunotoxicity and mechanism are unclear. Herein, we reported the immunotoxicity of bifenthrin on adult Chinese rare minnow (Gobiocypris rarus) after 28 days of exposure to different concentrations of bifenthrin (0.1, 0.3, and 1.0 µg/L) and 36-h Pseudomonas fluorescens challenge. Bifenthrin inhibited the fish humoral immune response to bacteria by altering the lymphocyte and neutrophil ratios and decreasing the production of lysozyme, complement component 3, immunoglobulin M, and C-reactive protein, particularly were 1.0 µg/L. Bifenthrin caused intestinal damage and significantly reduced the volume of intestinal mucus at 12 and 36 hours postinjection (hpi) (p < 0.05). Moreover, 1.0 µg/L bifenthrin significantly increased the fish mortality and bacterial loads at 12 and 36 hpi (p < 0.05). RNA-seq analysis revealed several enriched genes involved in pathogen attachment and recognition, inflammatory responses, and complement system at the early-to-mid stage of infection (4-12 hpi). Overall, our results corroborated that bifenthrin induced immunotoxicity in Gobiocypris rarus, resulting in immune dysfunction of fish and increasing their sensitivity to bacterial infection and accelerating mortality. Moreover, 4-12 hpi was better than 36 hpi for analyzing immune responses against pathogen infection in fish exposed to bifenthrin.

Long-Term Exposure to SSRI Citalopram Induces Neurotoxic Effects in Zebrafish.

Residual antidepressants are of increasing concern worldwide, yet critical information on their long-term neurotoxic impacts on nontarget aquatic animals is lacking. Here, we investigated the long-term effects (from 0 to 150 days postfertilization) of the selective serotonin reuptake inhibitor citalopram (0.1-100 µg/L) on motor function, learning, and memory in zebrafish over two generations and explored the reversibility of the effect in F1 larvae. Unlike F0+ larvae, we found that F1+ larvae displayed decreased sensorimotor performance when continuously exposed to citalopram at 100 µg/L. No adverse effects were found in F1- larvae after they were transferred to a clean medium. Whole-mount immunofluorescence assays suggested that the motor impairments were related to axonal projections of the spinal motor neurons (MNs). For F0+ adults, long-term citalopram exposure mainly caused male-specific declines in motor, learning, and memory performance. Analysis of serotonergic and cholinergic MNs revealed no significant changes in the male zebrafish spinal cord. In contrast, the number of glutamatergic spinal MNs decreased, likely associated with the impairment of motor function. Additionally, treatment with 100 µg/L citalopram significantly reduced the number of dopaminergic neurons, but no significant neuronal apoptosis was observed in the adult telencephalon. Overall, this study provides neurobehavioral evidence and novel insights into the neurotoxic mechanisms of long-term citalopram exposure and may facilitate the assessment of the environmental and health risks posed by citalopram-containing antidepressant drugs.

Environmentally relevant concentrations of fenvalerate induces immunotoxicity and reduces pathogen resistance in Chinese rare minnow (Gobiocypris rarus).

Fenvalerate is a broadly used type II pyrethroid with a potential toxic effect in fish. However, information on the immunotoxicity of fenvalerate in fish is scarce. Here, to discover the immunotoxicity of fenvalerate and its underlying mechanism in fish, adult Chinese rare minnow was exposed to fenvalerate at 0, 0.3, 1, and 3 µg/L for 28 days and then subjected to Pseudomonas fluorescens (P. fluorescens) challenge. Fenvalerate induced significant pathological changes, with disintegration of cell boundaries in the intestine, epithelial hyperplasia in gills, and vacuolation of hepatocytes at 3 µg/L treatment. Additionally, the pathological characteristics were more serious during P. fluorescens infection after fenvalerate exposure. A significant increase in neutrophil counts was observed after 3 µg/L fenvalerate exposure for 28 days (p < 0.05), whereas significantly increased monocyte and neutrophil counts and greatly decreased lymphocyte counts were detected at 24 h post-injection (hpi) with P. fluorescens (p < 0.05). Furthermore, obvious decreases in LYS, IgM, ALP, and C3 levels were detected in plasma after 3 µg/L fenvalerate exposure for 28 days, which was consistent with the results at 24 and 48 hpi. Notably, fish exposed to fenvalerate suppressed the transcription of TLR-NF-κB signaling pathway-relevant genes in response to P. fluorescens, accompanied by high mortalities and bacterial loads. Therefore, our results demonstrate that fenvalerate at environmentally relevant concentrations caused immunotoxicity in fish. This study highlights the importance of considering the combined effects of chemicals and pathogens to refine our ability to predict the effects of environmental contaminants on aquatic organisms.

Low doses and lifecycle exposure of waterborne antidepressants in zebrafish model: A survey on sperm traits, reproductive behaviours, and transcriptome responses.

Venlafaxine and citalopram have been commonly found in surface water and may disrupt fish reproduction, yet the long-term impact and the underlying mechanism are largely unknown. Here, zebrafish were exposed to 0.1-100 µg/L venlafaxine and citalopram for their entire life cycle from embryo to adult, respectively. After exposure for 180 days, the lowest observable effective concentration (LOEC) of venlafaxine and citalopram to significantly reduce the mean number of egg production in adults were 10 and 1 µg/L, respectively, whereas the fertilization rate displayed no significant changes. Further, we investigated the impacts of venlafaxine and citalopram in a reproductive context, including sperm quality and reproductive behaviour. In contrast, venlafaxine and citalopram exposure did not affect sperm quality but caused a reduction of reproductive behaviour (e.g., mating duration and mating interval) of adults exposed to 1-10 µg/L of the antidepressant. Transcriptomic profiling of the whole ovary revealed that lifecycle venlafaxine and citalopram exposure significantly affected the Na+/Cl- dependent neurotransmitter transporters signaling. Moreover, immune system-mediated ovarian regeneration and creatine metabolism regulated energy metabolism were proposed as the novel mechanism in the observed effects. Taken together, our results highlight the risk of lifecycle venlafaxine and citalopram exposure to fish reproduction and provide novel perspectives for unveiling the mechanism of female reproductive dysfunction.

Toxicity of waterborne vortioxetine, a new antidepressant, in non-target aquatic organisms: From wonder to concern drugs?

Vortioxetine is increasing in popularity as a treatment for major depressive disorder and has been detected in wastewater effluent. However, information on the toxicity and environmental risk of vortioxetine in non-target organisms is scarce. Here, embryonic and juvenile zebrafish (Danio rerio) were used to assess the toxicity of vortioxetine (0, 1, 10, 30, 100, 300, and 1000 µg/L) after 120 h and 7 d of exposure, respectively. Vortioxetine induced significant toxicity during embryonic development, including effects on survival, hatching, basal heart rate, spontaneous tail coiling and developmental abnormalities, and inhibited larval locomotor activity at concentrations higher than 30 µg/L. Additionally, vortioxetine evoked anxiolytic-like behavior and caused histopathological changes to multiple organs (gills, heart, liver and intestine) in juvenile zebrafish. Significant increase in 5-HT content was observed in whole zebrafish larvae and juvenile brain tissues from animals treated with 1 or 100 µg/L vortioxetine. Notably, the lowest effective concentrations of vortioxetine for zebrafish were mainly in the range of 10-30 µg/L, which were slightly lower than the vortioxetine therapeutic concentrations. Risk quotients assuming conservative exposure assessments were above one in European countries indicating moderate risk for the behavioral endpoints assessed. We believe that these results highlight the adverse effects of vortioxetine on non-target organisms and that further investigations will be required to provide a higher confidence.

Environmentally relevant concentrations of clozapine induced lipotoxicity and gut microbiota dysbiosis in Chinese rare minnow (Gobiocypris rarus).

Clozapine (CLZ) is a neuroactive pharmaceutical that is frequently detected in aquatic environments. Although the cardiotoxicity, developmental toxicity, and neurotoxicity of CLZ in aquatic non-target organisms have been reported, its lipotoxicity and underlying mechanism are unknown. Therefore, in this study, 2-month-old Chinese rare minnows were exposed to 0, 0.1, 1, and 10 µg/L CLZ for 90 days. Overt dyslipidemia was observed after CLZ exposure, whereas the body weights of females significantly increased after CLZ exposure (p < 0.05). In addition, obvious hepatocyte vacuolization and hepatic lipid droplet accumulation were observed at all treatment groups (p < 0.05). The activities of sterol regulatory element binding proteins 1 (SREBP1) and fatty acid synthase (FAS) were significantly upregulated at the 1 and 10 µg/L CLZ treatment groups (p < 0.05). Moreover, evident cell boundary disintegration of the intestinal villi and increasing mucus secretion were observed at all treatment groups (p < 0.05). Furthermore, the diversity of the gut microbiota increased, whereas the relative abundances of Proteobacteria, Firmicutes and Bacteroidetes significantly increased after CLZ exposure (p < 0.05). Furthermore, significantly increased bacterial secondary bile acid biosynthesis activity in Chinese rare minnows was observed after 1 µg/L CLZ exposure (p < 0.05). Therefore, our findings confirmed that CLZ induced lipotoxicity by stimulating SREBP1 and affecting the bacterial secondary bile acid biosynthesis activity in Chinese rare minnows.

Risks to aquatic environments posed by 14 pharmaceuticals as illustrated by their effects on zebrafish behaviour.

The presence of pharmaceutical residues in aquatic ecosystems is a worldwide problem that may pose serious threats and challenges to the environment, especially to the safety of aquatic biota. In the present study, we investigated the effects of 14 environmentally relevant pharmaceutical compounds on individual and collective-related behaviours in juvenile zebrafish (Danio rerio) for 21 days. The tested concentrations of the compounds spanned three orders of magnitude. This study also compared the potential risks of these compounds in Chinese surface waters based on the data on their toxic effects or only on behavioural effects. In the case of individual behaviours, most antidepressants, but not anti-inflammatory agents or blood lipid-lowering agents, decreased fish locomotor activity (LMA) and individual social activity (IDS); however, all three classes of compounds induced significant disruptions in the light/dark transition locomotor response (LMR-L/D) performance, even at lower treatment levels (0.1-1 µg/L). Furthermore, collective behaviour (CLB) analysis suggested that most of the compounds significantly altered the group sociability of fish and frequently occurred at environmentally relevant concentrations. Finally, a risk assessment suggested that the presence of ibuprofen, fluoxetine, and venlafaxine in the surface waters of China poses a relatively high risk to fish, regardless of the risk ranking based on the data of the toxic or behavioural effects.

Evaluating environmental impact of STP effluents on receiving water in Beijing by the joint use of chemical analysis and biomonitoring.

To evaluate the environmental impact of receiving water from the Qinghe River sewage treatment plant (STP) effluents in Beijing, we collected sediments and Bellamya aeruginosa (Up-site, Discharge-site, and Down-site) both in 2017 and 2018 and analyzed the samples via chemical analysis, biological responses and transcriptomics. In two years of data, our biological results showed that AChE activities presented different degrees of influence on B. aeruginosa captured at sampling points of the STP compared to control sites (P < 0.05). Additionally, indicators of the antioxidant system (e.g., SOD, CAT, GST, EROD activity) and MDA content were significantly increased in the whole tissue at the Up-site of the STP. Integration of the assessed biomarkers using the integrated biomarker response (IBR) index ranked the environmental impact at sites as Up-site > Discharge-site > Down-site. In terms of the transcriptome data, B. aeruginosa collected from the Discharge-site of the STP showed greater transcriptomic response than it did from all other sites. KEGG pathway analysis revealed that sewage significantly altered the expression of genes involved in xenobiotics by cytochrome P450, drug metabolism-cytochrome P450, glutathione metabolism, oxidative phosphorylation, citrate (TCA) cycle, glycolysis/gluconeogenesis, apoptotic and Parkinson's disease. The concentrations of 34 organic pollutants (17 PAHs, 10 PAEs, 7 EDCs) were measured. The chemical concentrations of pollutants decreased from Up-site to Down-site and were well correlated with enzyme activity, IBR, and transcriptomic results. Our results demonstrated that the combined use of chemical analysis, biological responses and transcriptome data is necessary to validate the efficacy of a battery of biomarkers chosen to detect environmental stress due to pollution.

Environmentally relevant concentrations of carbamazepine induced lipid metabolism disorder of Chinese rare minnow (Gobiocypris rarus) in a gender-specific pattern.

Carbamazepine (CBZ), an anticonvulsant and mood stabilizer, is ubiquitous distributed in aquatic environment. Though the toxicity and endocrine disrupting effect of CBZ on non-target organisms have been studied, its lipotoxity are scarcely known. To assess the lipotoxicity of CBZ, 2-month-old Chinese rare minnow were exposed to 0, 1, 10, and 100 µg/L CBZ for 90 d. Obvious dyslipidemia was observed after 30 d and 90 d exposure, whereas overt hyperlipidemia was observed in males at 100 µg/L treatments. Severe lipid droplet accumulation in livers was observed at 10 and 100 µg/L treatments for 30 d and in females, whereas those was observed at all treatments in males. In addition, serious mitochondria damage was observed in males at 100 µg/L treatments. After 90 d exposure, the enzyme activities of FAS and ACCα were significantly increased at 10 and 100 µg/L treatments, whereas HMGCR were markedly increased at 100 µg/L treatments (p < 0.05). However, ACCß were markedly decreased in females at 10 and 100 µg/L treatments and in males at all treatments (p < 0.05). The transcription levels of fasn, accα, hmgcrα, fdft1, idi1, plin1, plin2, caveolin1, and caveolin2 were significantly increased at 100 µg/L treatments (p < 0.05). Moreover, the body weight was obviously increased at 10 and 100 µg/L treatments in males (p < 0.05). Our results confirmed that environmental relevant concentrations CBZ induced lipid metabolism disorder and mitochondria damage of Chinese rare minnow in a gender-specific pattern, which provided a new insight into the lipotoxicity mechanism of CBZ.

Environmentally relevant concentrations of bifenthrin induce changes in behaviour, biomarkers, histological characteristics, and the transcriptome in Corbicula fluminea.

Bifenthrin (BF) is an insecticide that is commonly used to control agricultural and domestic pests and is widespread in aquatic environments. Although previous studies have found that BF is toxic to aquatic organisms, such a comprehensive study of the mechanism of toxic effects in bivalves is not common. In this study, to assess the toxic effects of BF on bivalves, adult Corbicula fluminea (C. fluminea) were exposed to 0, 1, 5, and 25 µg/L BF for 15 days. Transcriptome analysis revealed that BF exposure significantly altered the expression of genes involved in detoxification, antioxidation, and metabolism. Moreover, the ROS content and GST activity at 25 µg/L treatments were significantly increased (p < 0.05), and significant increases of MDA concentration and CAT activity were observed at 5 and 25 µg/L treatments (p < 0.05). However, AChE activity was markedly inhibited at 25 µg/L treatments (p < 0.05). In addition, vacuolation in the digestive tubules and the hemolytic infiltration of connective tissue were observed at all treatments, and the degeneration of the digestive tubule was observed at 5 and 25 µg/L treatments. In the behavioural assay, the siphoning behaviour of C. fluminea was significantly inhibited at 25 µg/L treatments (p < 0.05), whereas no significant change in burrowing behaviour was observed. Our findings suggested that BF exposure caused changes in detoxification, antioxidation, and metabolism pathways, biomarker activity or concentrations and histopathological characteristics, resulting in changes in behaviour. Therefore, our findings provide a basis for further evaluation of the toxicity of pyrethroid insecticides in bivalves.

Neonicotinoids caused oxidative stress and DNA damage in juvenile Chinese rare minnows (Gobiocypris rarus).

To assess the effects of neonicotinoid insecticides on fish, juvenile Chinese rare minnows (Gobiocypris rarus) were exposed to 0.1, 0.5, or 2.0 mg/L neonicotinoid insecticides (imidacloprid, nitenpyram, and dinotefuran) for 60 days. The endpoints, including oxidative stress and DNA damage, were determined. The results of oxidative stress assays showed that SOD activities were significantly increased in the 2.0 mg/L imidacloprid and 0.5 mg/L nitenpyram and dinotefuran treatments (p < 0.05). CAT activity was significantly increased with 0.1 mg/L nitenpyram (p < 0.05), whereas it was significantly decreased in the 0.1 and 2.0 mg/L dinotefuran treatment groups (p < 0.05). Moreover, MDA content was significantly decreased in all imidacloprid treatments and in the 0.5 and 2.0 mg/L dinotefuran treatments (p < 0.05); however, it was significantly increased in the 0.1 mg/L nitenpyram treatment (p < 0.05). GSH content was significantly increased at all treatments except for the 0.5 mg/L dinotefuran treatment (p < 0.05). The transcript expression results showed that gstm mRNA expression was significantly inhibited by 0.5 and 2.0 mg/L imidacloprid, and gstp1 mRNA expression was significantly inhibited by all nitenpyram treatments (p < 0.05). In addition, ugt1a mRNA expression was significantly inhibited in the 0.5 mg/L nitenpyram treatment (p < 0.05). The results of the DNA damage assay showed that tail moments were significantly increased by the 2.0 mg/L imidacloprid treatment (p < 0.01), while tail DNA was significantly increased by 0.5 and 2.0 mg/L imidacloprid, 2.0 mg/L nitenpyram and all dinotefuran treatments (p < 0.01). Moreover, olive tail moments were significantly increased by the 0.5 and 2.0 mg/L imidacloprid and 2.0 mg/L dinotefuran treatments (p < 0.01). Therefore, our oxidative stress and DNA damage findings demonstrated that imidacloprid and nitenpyram could cause adverse effects on juvenile rare minnows.

Three organophosphate flame retardants (OPFRs) reduce sperm quality in Chinese rare minnows (Gobiocypris rarus).

Organophosphate flame retardants (OPFRs) are widespread in the aquatic environment, but the effects of these chemicals on reproductive toxicity are far from clear. In this study, sperm quality in adult male Chinese rare minnows after exposure to tris-(2-butoxyethyl) phosphate (TBOEP), tris-(1,3-dichloro-2-propyl) phosphate (TDCIPP), and triphenyl phosphate (TPHP) was investigated. No obvious change in sperm concentration and vitality was observed after treatments, whereas significant changes in sperm velocity and morphology were found following all treatments (P < 0.05). Moreover, OPFR exposure significantly increased the apoptosis ratios in testis cells. Analysis of the transcriptomic data revealed that Na+/K+ ATPase (NKA) related genes were significantly downregulated, and the NKA enzyme activities after all treatments were significantly inhibited (P < 0.05). However, no obvious change in hormone levels in the groups exposed to TBOEP and TDCIPP was observed. These findings indicate that the OPFR-induced reduction of sperm quality might be due to the effects of OPFRs on NKA enzyme instead of changes in hormone levels.

3-(4-Methylbenzylidene) camphor induced reproduction toxicity and antiandrogenicity in Japanese medaka (Oryzias latipes).

To assess the toxic effects of 3-(4-Methylbenzylidene) camphor (4-MBC) at environmentally relevant concentrations on the reproduction and development of Japanese medaka (Oryzias latipes), adult paired medaka (F0) were exposed to 5, 50, and 500 µg/L 4-MBC for 28 d in the current study. The fecundity and fertility were significantly decreased at 500 µg/L 4-MBC (p < 0.05). Histological observations showed that spermatogenesis in F0 males was significantly inhibited at 50 and 500 µg/L 4-MBC, similar to the effects obtained with all treatments of plasma 11-ketotestosterone (p < 0.05). Moreover, the plasma vitellogenin and estradiol levels in F0 females were significantly increased at 5 µg/L 4-MBC (p < 0.05). All the transcripts of hypothalamic-pituitary-gonadal (HPG) axis-related genes tested in the brains and gonads of males were significantly increased at all treatments, similar to the effects obtained for erα, erß and vtg in the livers and in contrast to those found for arα in the livers (p < 0.05). Equal numbers of embryos were exposed to tap water and 4-MBC solutions. Significantly increased times to hatching, decreased hatching rates and decreased body lengths at 14-day post-hatching (dph) were obtained at 500 µg/L 4-MBC treatment (p < 0.05). The cumulative death rates at 14 dph were significantly increased with all the treatments (p < 0.05). Therefore, our results showed that long-term exposure to 50 and 500 µg/L 4-MBC causes reproductive and developmental toxicity and thus provide new insight into antiandrogenicity and the mechanism of 4-MBC in Japanese medaka.

Exposure to environmentally relevant concentrations of deltamethrin renders the Chinese rare minnow (Gobiocypris rarus) vulnerable to Pseudomonas fluorescens infection.

In this study, to assess the immunotoxicity of deltamethrin on fish, adult Chinese rare minnows (Gobiocypris rarus) were exposed to 0.1, 0.3, and 1 µg/L deltamethrin for 28 d. Many immunological parameters and histopathological alterations were determined. The results showed that lymphocyte number was markedly decreased at 0.3 and 1 µg/L treatments, whereas the neutrophil number was strongly increased at 1 µg/L treatments (p < 0.05). Furthermore, lysozyme (LYS), immunoglobulin M (IgM), and complement component 3 (C3) levels at 0.3 and 1 µg/L treatments were markedly reduced, whereas alkaline phosphatase (ALP) activity were marked increased at 1 µg/L treatments (p < 0.05). The transcripts of almost all TLR (Toll-like receptor) signaling pathway-related genes were up-regulated. Histological lesions in the livers, intestines, and gills were observed at all treatments. Then, all remaining fish from controls and deltamethrin-exposed groups were injected with Pseudomonas fluorescens (P. fluorescens) for 48 h. At 24 and 48 h post-injection with P. fluorescens (hpi), the lymphocyte numbers were strongly reduced at 0.3 and 1 µg/L deltamethrin-exposed groups, whereas LYS and C3 levels were strongly reduced at 0.3 and 1 µg/L deltamethrin-exposed groups (p < 0.05). Obvious reduces in IgM levels were also detected at 0.3 and 1 µg/L deltamethrin-exposed groups at 48 hpi (p < 0.05). The transcripts of almost all TLR signaling pathway-related genes were significantly down-regulated, whereas the levels of related microRNAs (miRNAs) were markedly increased at all deltamethrin-exposed groups at 24 and 48 hpi. Moreover, the bacterial load in the liver and the mortality of fish were significantly increased at 1 µg/L deltamethrin-exposed groups at 24 and 48 hpi (p < 0.05). Furthermore, obvious histological damage in the livers, intestines, and gills were observed at all deltamethrin-exposed fish at 48 hpi. Overall, our results demonstrated that environmentally relevant concentration deltamethrin suppressed immunity and rendered the fish vulnerable to P. fluorescens infection, subsequently inducing mortality.

Reproductive toxicity and estrogen activity in Japanese medaka (Oryzias latipes) exposed to environmentally relevant concentrations of octocrylene.

The growing use of octocrylene (OC) in sunscreens has posed a great threat to aquatic organisms. In the present study, to assess its reproductive toxicity and mechanism, paired Japanese medaka (Oryzias latipes) (F0) were exposed to OC at nominal concentrations of 5, 50, and 500 µg/L for 28 d. Significant increases were observed in the gonadosomatic index (GSI) and hepatosomatic index (HSI) of F0 medaka at 500 µg/L OC (p < 0.05) without significant differences in fecundity. The fertility was significantly decreased at all treatments (p < 0.05). Significant increases in the percent of mature oocytes were observed at 5 and 500 µg/L OC, in which contrary to the percent of spermatozoa (p < 0.05). The plasma sex hormones and vitellogenin levels significantly increased in males at all treatments and in females at 50 and 500 µg/L OC (p < 0.05). In addition, the levels of fshß and lhß in the brains and the levels of fshr, lhr and cyp17α in the gonads were significantly upregulated in males at all treatments (p < 0.05), in line with those of ar, erα, erß and cyp19ß in the brains of male and female. The upregulation of vtg in male and female livers was observed only at 500 µg/L OC and upregulation of star and hsd3ß was observed in testis at all treatments (p < 0.05). Continued exposure to OC significantly induced increases in the time to hatching, morphological abnormality rates, and cumulative death rates of F1 embryos, inconsistent with body length of F1 larvae (p < 0.05). Therefore, the responses of the exposed fish at the biochemical and molecular levels indicated reproductive toxicity and estrogenic activity of OC, providing insights into the mechanism of OC.

Effect of imidacloprid on the behavior, antioxidant system, multixenobiotic resistance, and histopathology of Asian freshwater clams (Corbicula fluminea).

In the current study, to investigate the effect of imidacloprid on benthic bivalves, adult Asian clams (Corbicula fluminea) were exposed to 0, 20, 200, and 2000 µg/L imidacloprid for 30 d. Imidacloprid significantly inhibited the siphoning and burrowing behaviour (p < 0.05) of the clams. Significant histopathological changes were associated with degeneration of the cilium, the contraction and adhesion of the lymphocyte, and the swelling of epithelium cells in gills, and there was notable degeneration in the digestive tubules, haemolytic infiltration in the connective tissue and epithelial cell necrosis in the digestive glands in the 2000 µg/L treatment group. The activity of AChE in the digestive glands was significantly inhibited at all treatment levels, whereas this inhibition was observed in gills only in the 2000 µg/L treatment (p < 0.05). Additionally, indicators of the antioxidant system (e.g., SOD, CAT, and GST activity) and MDA content were significantly increased in the gills and digestive glands with all treatments (p < 0.05). Moreover, the mRNA expression levels of Hsp genes (hsp 22, hsp 40, hsp 60, hsp 70, hsp 90) and multixenobiotic resistance (MXR) system-related genes (abcb1, abcc1) were significantly downregulated (p < 0.05). Therefore, our results suggest that imidacloprid changes the oxidative stress, cellular detoxification, and MXR system of C. fluminea. Our findings provide new insights into the effects of neonicotinoids on benthic bivalves such as C. fluminea.

Organophosphate flame retardants (OPFRs) induce genotoxicity in vivo: A survey on apoptosis, DNA methylation, DNA oxidative damage, liver metabolites, and transcriptomics.

BACKGROUND: As potential substitutes for polybrominated diphenyl ethers (PBDEs), organophosphate flame retardants (OPFRs) have been frequently detected in the environment. However, the genotoxicity induced by these OPFRs has rarely been described, and the results reported in previous studies are conflicting and inconsistent. OBJECTIVES: The present study aimed to determine how OPFRs induced genetic toxicity in vivo. METHODS: Using Chinese rare minnow as a model, the toxicity of three OPFRs was screened with RNA-seq. To verify the OPFR-induced genotoxicity, alkaline comet assay, cell apoptosis analysis, HPLC-based DNA methylation assay, 8-OHdG assay, bioconcentration and biotransformation investigation were performed. RESULTS: According to transcriptomic data, TDCIPP exposure substantially altered the pathways related to DNA damage, including the cell cycle, DNA replication, Fanconi anemia pathway, p53 signaling pathway, and various DNA repair pathways. Although TBOEP and TPHP did not affect DNA damage, TDCIPP induced DNA damage in a dose-dependent manner. TDCIPP also induced apoptosis, altered the activities of caspase-3 and -9, and increased the 8-OHdG levels, while a significant difference in the levels of DNA methylation induced by OPFRs was not observed. CONCLUSIONS: Based on these results, TDCIPP induced DNA oxidative damage, eventually leading to genotoxicity in vivo.

Fish behavior: A promising model for aquatic toxicology research.

Fish behaviors have great potential as models for the study of pharmacology, genetics, and neuroscience. Zebrafish (Danio rerio), Japanese medaka (Oryzias latipes) and Chinese rare minnow (Gobiocypris rarus) are popular freshwater animal models. However, their behavioral use in aquatic toxicology research is generally hampered by oversimplified behavioral tasks and the fact that they are not well-developed animal models for toxicology. Here, this study presented a comparative analysis of multiple behavioral traits (i.e., anxiety-like behavior, novel object recognition, social preferences, habituation to light-dark stimulus and noise stimulus, and spatial learning and memory). We found that only medaka (d-rR) presented a weak or no response to repeated light-dark stimulus and noise stimulus. In addition, no significant behavioral changes were observed for the three species of juvenile fish models after 7 days of exposure to 0.01% v/v carrier solvents (i.e., ethanol, acetone, and DMSO). In contrast to zebrafish and Chinese rare minnow, medaka showed no significant changes in spatial memory after subacute exposure to 1 mg/L imidacloprid or 2.5 µg/L chlorpyrifos (cpf); instead, a hyperactivity response in the open field test and reduced social time were induced by cpf and imidacloprid, respectively. Our results suggest that: (1) behavioral effects are negligible when using <0.01% v/v carrier solvents for behavioral assessment; (2) given the differences in sensitivities of behavioral responses, a single behavior used alone as an endpoint may be insufficient for estimating the toxic impacts of pesticides or other environmental contaminants. In conclusion, these results could have major implications for aquatic toxicology research and water quality monitoring and ecotoxicological risk assessment.

Global microRNA and isomiR expression associated with liver metabolism is induced by organophosphorus flame retardant exposure in male Chinese rare minnow (Gobiocypris rarus).

To reveal the adverse effects of organophosphorus flame retardants (OPFRs) on aquatic organisms at the epigenetic level, male Chinese rare minnows were exposed to 0.24 mg/L tris(2­butoxyethyl) phosphate (TBOEP), 0.04 mg/L tris(1,3­dichloro­2­propyl) phosphate (TDCIPP), or 0.012 mg/L triphenyl phosphate (TPHP) for 14 days. The effects of sub-acute OPFR exposure on liver miRNA and the 3' isomiR expression profiles of Chinese rare minnows were investigated. Through small RNA sequencing and bioinformatics analysis, a total of 32, 84, and 19 differentially expressed miRNAs were detected for TBOEP, TDCIPP, and TPHP exposure, respectively (p < 0.05). Target prediction of the differentially expressed miRNAs and pathway enrichment analysis indicated that predicted altered mRNAs for all three OPFRs were associated with metabolic pathways, whereas base excision repair was only predicted to be perturbed by the TPHP treatment. In addition, 3' isomiR-Us were unexpectedly abundant in all groups (e.g., miR-143), and TDCIPP strongly increased the ratio of 3' isomiR-U expression. Finally, histological examination and metabolic enzyme activity analyses werein agreement with the predicted metabolic pathways. As such, our study indicates that the investigation of epigenetics changes in miRNA gene transcription is a considerable method for the assessment of aquatic toxicity.

Triphenyl Phosphate (TPHP)-Induced Neurotoxicity in Adult Male Chinese Rare Minnows ( Gobiocypris rarus).

The neurotoxicity of triphenyl phosphate (TPHP) in exposed humans and laboratory animals is under debate. The rapid crossing of the blood-brain barrier (BBB) and high distribution of TPHP in fish brains have raised widespread concerns about potential neurotoxicity. Adult male Chinese rare minnows ( Gobiocypris rarus) were used as a model and exposed to 0, 20, or 100 µg/L TPHP for 28 days. We evaluated the BBB permeability, neuroinflammatory response, cell proliferation and apoptosis, synaptic plasticity and synapse loss in fish brains via the learning/memory performance of fish following 28 days of TPHP exposure. TPHP significantly increased the BBB permeability, activated the neuroinflammatory response, and decreased the tight junction-related mRNA levels of claudin-5α and occludin in the fish brain. In addition, cell proliferation was inhibited by treatment with 100 µg/L TPHP, but no significant apoptosis was observed in the brain. Fish exposed to 100 µg/L TPHP exhibited significantly decreased dendritic arborization in pyramidal neurons in the cerebellum (Ce), and the maze test indicated impaired learning/memory performance. Taken together, these findings provide scientific evidence that TPHP is neurotoxic to fish and further suggest that TPHP may not be a safe alternative for aquatic organisms.