Although the coexistence of heavy metals and environmental hormones always occur in aquatic environment, the information of the combined impacts remains unclear. To explore the multi-generational toxicity of cadmium (Cd) and tributyltin (TBT), adult zebrafish (Danio rerio) (F0) were exposed to different treated groups (100 ng/l Cd, 100 ng/l TBT and their mixture) for 90 d, with their offspring (F1 and F2) subsequently reared in the same exposure solutions corresponding to their parents. Both developmental neurotoxicity and thyroid disturbances were examined in the three (F0, F1, and F2) generations. Our results showed that co-exposure to Cd and TBT induced the developmental neurotoxicity in F1 and F2 generations, reflected by the significant lower levels of neurotransmitters (dopamine and serotonin) and the inhibited acetylcholinesterase (AChE) activities. And the thyroid endocrine disruption were observed in the two-generations larval offspring by parental exposure to Cd and/or TBT, including the significantly decreasing levels of thyroid hormones and the down-regulated the expression of genes involved in the hypothalamus-pituitary-thyroid axis, compared to the control. Additional, the embryonic toxicity and growth inhibition were also determined in the fish larvae. Overall, this study examined the impacts of parental co-exposure to Cd and TBT, with regard to developmental inhibition, nervous system damage and endocrine disruption, which highlighted that co-exposure influences are complicated and need to be considered for accurate environmental risk assessment.

Butylated hydroxytoluene (BHT) is one of the most frequently used synthetic phenolic antioxidants added to food and consumer products such as plastics as a preservative. Due to its high production volume, BHT has been detected in aquatic environments, raising concerns about sub-lethal toxicity. However, there are limited toxicological data for BHT, especially in fish. In this study, zebrafish embryos were exposed to BHT at concentrations ranging 0.01–100 μM for up to 6 days post fertilization (dpf). Acute toxicity was assessed, and experiments revealed that BHT had a 96 h LC50 value of 57.61 μM. At sub-lethal doses (0.1–60 μM), BHT markedly decreased heart rates of zebrafish embryos at 48 h and 72 h by ∼25–30%. Basal and maximal respiration of zebrafish embryos at 24 hpf were decreased by 59.3% and 41.4% respectively following exposure to 100 μM BHT. Behavior in zebrafish was measured at 6 dpf following exposures to 0.01–10 μM BHT. Locomotor behaviors (e.g. total distance moved and velocity) were significantly increased in larvae at doses higher than 0.1 μM BHT. In addition, dark-avoidance behavior was decreased following exposure to 0.01 μM BHT, while conversely, it was increased in zebrafish exposed to 0.1 μM BHT. To investigate potential underlying mechanisms that could explain behavioral changes, transcripts involved in dopamine signaling were measured. Relative expression of dat mRNA was increased in larval fish from the 0.01 μM BHT treatment, while there were no effects on dat mRNA levels at higher concentrations. The mRNA levels of drd3 were decreased in zebrafish from the 1 μM BHT treatment. Taken together, BHT can affect the expression of the dopamine system, which is hypothesized to be related to the abnormal anxiety-associated behavior of larval zebrafish.

Because oil spills frequently occur in coastal regions that serve as spawning habitat, characterizing the effects of oil in estuarine fish carries both economic and environmental importance. There is a breadth of research investigating the effects of crude oil on fish, however few studies have addressed how transcriptional responses to oil change throughout development or how these responses might be conserved across taxa. To investigate these effects, we performed RNA-seq and pathway analysis following oil exposure 1) in a single estuarine species (Cyprinodon variegatus) at three developmental time points (embryos, yolk-sack larvae, free-feeding larvae), and 2) in two ecologically similar species (C. variegatus and Fundulus grandis), immediately post-hatch (yolk-sack stage). Our results indicate that C. variegatus embryos mount a diminished transcriptional response to oil compared to later stages, and that few transcriptional responses are conserved throughout development. Pathway analysis of larval C. variegatus revealed dysregulation of similar biological processes at later larval stages, including alteration of cholesterol biosynthesis pathways, cardiac development processes, and immune functions. Our cross-species comparison showed that F. grandis exhibited a reduced transcriptional response compared to C. variegatus. Pathway analysis revealed that the two species shared similar immune and cardiac responses, however pathways related to cholesterol biosynthesis exhibited a divergent response as they were activated in C. variegatus but inhibited in F. grandis. Our results suggest that examination of larval stages may provide a more sensitive estimate of oil-impacts than examination of embryos, and challenge assumptions that ecologically comparable species respond to oil similarly.

The presence of microplastics in aquatic ecosystems has recently received increased attention. Small plastic particles may resemble natural food items of larval fish and other aquatic organisms, and create strong selective pressures on the feeding traits in exposed populations. Here, we examined if larval ingestion of 90 μm polystyrene microspheres, in the presence of zooplankton (Artemia nauplii, mean length = 433 μm), shows adaptive variation in the European whitefish (Coregonus lavaretus). A full-factorial experimental breeding design allowed us to estimate the relative contributions of male (sire) and female (dam) parents and full-sib family variance in early feeding traits, and also genetic (co)variation between these traits. We also monitored the magnitude of intake and elimination of microplastics from the alimentary tracts of the larvae. In general, larval whitefish ingested small numbers of microplastics (mean = 1.8, range = 0–26 particles per larva), but ingestion was marginally affected by the dam, and more strongly by the full-sib family variation. Microsphere ingestion showed no statistically significant additive genetic variation, and thus, no heritability. Moreover, microsphere ingestion rate covaried positively with the ingestion of Artemia, further suggesting that larvae cannot adaptively avoid microsphere ingestion. Together with the detected strong genetic correlation between food intake and microplastic intake, the results suggest that larval fish do not readily possess additive genetic variation that would help them to adapt to the increasing pollution by microplastics. The conflict between feeding on natural food and avoiding microplastics deserves further attention.

The aim of this study was to investigate the long-term effects of a short exposure to natural sediments within the Athabasca oil sand formation to critical stages of embryo-larval development in fathead minnows (Pimephales promelas). Three different sediments were used: Ref sediment from the upper Steepbank River tested at 3 g/L (containing 12.2 ng/g ∑PAHs), and two bitumen-rich sediments tested at 1 and 3 g/L; one from the Ells River (Ells downstream, 6480 ng/g ∑PAHs) and one from the Steepbank River (Stp downstream, 4660 ng/g ∑PAHs). Eggs and larvae were exposed to sediments for 21 days, then transferred to clean water for a 5-month grow-out and recovery period. Larval fish had significantly decreased survival after exposure to 3 g/L sediment from Stp downstream, and decreased growth (length and weight at 16 days post hatch) in Ells and Stp downstream sediments at both 1 and 3 g/L. Decreased tail length was a sensitive endpoint in larval fish exposed to Ells and Stp downstream sediments for 21 days compared to Ref sediment. After the grow-out in clean water, all growth effects from the bitumen-containing sediments recovered, but adult fish from Stp downstream 3 g/L sediment had significant increases in jaw deformities. The study shows the potential for fish to recover from the decreased growth effects caused by sediments containing oil sands-related compounds, but that some effects of the early-life sediment exposure occur later on in adult fish.

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.

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.

Perfluorododecanoic acid (PFDoA), a kind of perfluorinated carboxylic acid (PFCA) with 12 carbon atoms, has an extensive industrial utilization and is widespread in both wildlife and the water environment, and was reported to have the potential to cause a disruption in the thyroid hormone system homeostasis. In this study, zebrafish embryos/larvae were exposed to different concentrations of PFDoA (0, 0.24, 1.2, 6 mg/L) for 96 h post-fertilization (hpf). PFDoA exposure caused obvious growth restriction connected with the reduced thyroid hormones (THs) contents in zebrafish larvae, strengthening the interference effect on the growth of fish larvae. The transcriptional level of genes within the hypothalamic-pituitary-thyroid (HPT) axis was analyzed. The gene expression levels of thyrotropin-releasing hormone (trh) and corticotrophin-releasing hormone (crh) were upregulated upon exposure to 6 mg/L of PFDoA, and iodothyronine deiodinases (dio2) was upregulated in the 1.2 mg/L PFDoA group. The transcription of thyroglobulin (tg) and thyroid receptor (trβ) were significantly downregulated upon exposure to 1.2 mg/L and 6 mg/L of PFDoA. PFDoA could also decrease the levels of sodium/iodide symporter (nis) and transthyretin (ttr) gene expression in a concentration-dependent manner after exposure. A significant decrease in thyroid-stimulating hormoneβ (tshβ), uridinediphosphate-glucuronosyltransferase (ugt1ab) and thyroid receptor (trα) gene expression were observed at 6 mg/L PFDoA exposure. Upregulation and downregulation of iodothyronine deiodinases (dio1) gene expression were observed upon the treatment of 1.2 mg/L and 6 mg/L PFDoA, respectively. All the data demonstrated that gene expression in the HPT axis altered after different PFDoA treatment and the potential mechanisms of the disruption of thyroid status could occur at several steps in the process of synthesis, regulation, and action of thyroid hormones.

To determine whether natural recovery was occurring in a depositional area of the St. Marys River (Ontario, Canada) known as East Bellevue Marine Park (EBMP), sediment was collected from two depth ranges, 0–5 cm and 0–10 cm, and subjected to a series of laboratory toxicity tests and chemical analysis. Toxicological responses (survival, growth, reproduction, development) of four benthic invertebrates and the fathead minnow were compared at test vs. reference sites using univariate and multivariate (ordination) techniques. Temporal trends in sediment chemistry and invertebrate toxicity were examined with time series data from 2008 through to 2018. Polycyclic aromatic hydrocarbons (PAHs; ≤ 37 mg/kg) and petroleum hydrocarbons (PHCs; ≤ 6266 mg/kg) were elevated in EBMP compared to reference sites (PAHs, ≤ 1.6 mg/kg; PHCs ≤ 180 mg/kg). Comparatively, the 0–5 cm sediment layer had lower concentrations of all contaminants than the 0–10 cm layer at three of four test sites. Over time, contaminant concentrations have mostly remained stable or have decreased. There were no significant differences in survival, growth, or development of the larval fish in EBMP compared to the upstream reference sites, and no differences between sampling depths. However, most EBMP sediments were toxic to invertebrates, driven by reduced reproduction by the worm Tubifex and reduced survival by the amphipod Hyalella. Among habitat variables, a combination of different classes of compounds based on ordination scores (PHCs, oil and grease, metals) was most strongly correlated to toxicological response. There was little to no difference in toxicity between sampling depths based on integrated endpoint response; however, individual endpoints showed mostly greater toxicity from exposure to the 0–10 cm layer. Over time, toxicity has mostly remained stable or showed improvement. These results provided some positive indications that gradual natural recovery is occurring in EBMP.

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.