Integrated toxicity assessment of DEHP and DBP toward aquatic ecosystem based on multiple trophic model assays.

To comprehensively understand the toxic risks of phthalates to aquatic ecosystems, we examined the acute toxicity of di-(2-ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) on multiple trophic models, including algae (Chlorella vulgaris), Daphnia magna and fish (Danio rerio, Pseudorasbora parva). Thus, a 15-day zebrafish exposure was conducted to trace the dynamic changes of phthalate-induced toxic effects. Among the four species, D. magna exhibited the strongest sensitivity to both DEHP and DBP, followed by D. rerio and P. parva. C. vulgaris exhibited the lowest sensitivity to phthalates. The sub-chronic zebrafish assay demonstrated that 1000 µg/L DBP induced significant mortality at 15 days post-exposure (dpe), and DEHP exhibited no lethality at the tested concentrations (10-5000 µg/L). Zebrafish hepatic SOD activity and sod transcription levels were inhibited by DBP from 3 dpe, which was accompanied by increased malondialdehyde level, while zebrafish exposed to DEHP exhibited less oxidative damage. Both DEHP and DBP induced time-dependent alterations on Ache activity in zebrafish brains, thus indicating the potential neurotoxicity toward aquatic organisms. Additionally, 1000 µg/L and higher concentration of DBP caused hepatic DNA damage in zebrafish from 7 dpe. These results provide a better understanding of the health risks of phthalate to water environment.

A multi-omics approach reveals molecular mechanisms by which phthalates induce cardiac defects in zebrafish (Danio rerio).

The potential risks of phthalates affecting human and animal health as well as the environment are emerging as serious concerns worldwide. However, the mechanism by which phthalates induce developmental effects is under debate. Herein, we found that embryonic exposure of zebrafish to di-(2-ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) increased the rate of heart defects including abnormal heart rate and pericardial edema. Changes in the transcriptional profile demonstrated that genes involved in the development of the heart, such as tbx5b, nppa, ctnt, my17, cmlc1, were significantly altered by DEHP and DBP at 50 µg/L, which agreed with the abnormal cardiac outcomes. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) further showed that significant hypomethylation of nppa and ctnt was identified after DEHP and DBP exposure, which was consistent with the up-regulation of these genes. Notably, hypermethylation on the promoter region (<1 kb) of tbx5b was found after DEHP and DBP exposure, which might be responsible for its decrease in transcription. In conclusion, phthalates have the potential to induce cardiac birth defects, which might be associated with the transcriptional regulation of the involved developmental factors such as tbx5b. These findings would contribute to understand the molecular pathways that mediated the cardiac defects caused by phthalates.

Evaluation of the spinal effects of phthalates in a zebrafish embryo assay.

Phthalates (phthalate esters, PAEs) are commonly used as plasticizers and are emerging concerns worldwide for their potential influence on the environment and general public health. Thus, identification of the negative effects and involved mechanisms of PAEs is necessary. Herein, we found that embryonic exposure of zebrafish to di-(2-ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) significantly induced spinal defects, such as inhibited spontaneous movement at 24 h post-fertilization (hpf), spine curvature and body length decrease at 96 hpf. The transcriptional level of the genes that are related to the development of the notochord (col8a1a and ngs), muscle (stac3, klhl41a and smyd2b) and skeleton (bmp2, spp1) were significantly altered by DEHP and DBP at 50 and 250 µg/L, which might be associated with the observed morphological changes. Notably, DBP and DEHP altered the locomotor activity of zebrafish larvae at 144 hpf, which might be due to the abnormal development of the spine and skeletal system. In conclusion, phthalates caused spinal birth defects in zebrafish embryos, induced transcriptional alterations of the spinal developmental genes, and led to abnormal behavior.

The mechanisms underlying the developmental effects of bisphenol F on zebrafish.

With the increasing use of bisphenol F (BPF) as BPA alternative, BPF are widely distributed in multiple environment media. Our previous study demonstrated that BPF possess equivalent toxicity towards zebrafish as BPA, while its toxic mechanism remains largely unknown. To investigate the mechanisms mediating the developmental effects of BPF, zebrafish embryos were exposed to 0.0005, 0.5, and 5.0 mg/L BPF. Morphological examination indicated that BPF exposure led to depigmentation, decreased heart rate, inhibited spontaneous movement, hatch inhibition, and spinal deformation. Motor neuron-green fluorescence zebrafish assay indicated that exposure to 0.5 or 5.0 mg/L BPF affected embryonic motor neuron development, which is consistent with the spinal defect and spontaneous movement inhibition. Transcriptomic analysis showed that genes associated with the observed symptoms, including neuron development (ngln2a, socs3a, fosb), cardiac development (klf2a), and spinal deformation (ngs, col8a1a, egr2a), were down-regulated after exposure to either 0.0005 (environmental relevant concentration) or 0.5 mg/L BPF. This partially explained the mechanisms underlying the effects of BPF. In conclusion, BPF had the potential to affect zebrafish development even at environmental level through down-regulating associated genes.

0# Diesel water-accommodated fraction induced lipid homeostasis alteration in zebrafish embryos.

To investigate the developmental effects and corresponding molecular mechanism of diesel in freshwater organisms, zebrafish embryos were exposed to 0# diesel water-accommodated fraction (WAF) at different concentrations. Mortality, embryonic morphological endpoints, transcriptional profile and lipid profile were evaluated after exposure. Exposure to 0# diesel WAF had no significant effect on the survival of zebrafish embryos from 1.5 to 96 hpf. However, a significant increase in mortality was observed at 144 and 196 hpf in the groups of 20 and 40 mg/L 0# diesel WAF. RNA-Seq results demonstrated that 0# diesel WAF could induce significant alterations in transcription profile at concentrations of 0.05 mg/L (the limit for petroleum hydrocarbon concentration in surface water in China) and 5 mg/L. Gene Ontology enrichment and similarity analysis indicated that lipid metabolism, lipid synthesis, biological transport, drug metabolism and homeostatic processes were the most altered biological processes after exposure to 0# diesel WAF. Further, transcription levels of genes involved in cholesterol and fatty acid synthesis were significantly inhibited by diesel WAF according to qPCR results. Lipidomics results also indicated that several lipid species (cholesterol ester, fatty acid, diglyceride and triglyceride) decreased after 0# diesel WAF exposure. These results reflect the potential risk of diesel pollution in freshwater ecosystems especially on the alteration of lipid homeostasis and enable a better understanding of the molecular pathways underlying the action of diesel WAF in zebrafish embryos.

New insights into the mechanism of phthalate-induced developmental effects.

To investigate the biological pathways involved in phthalate-induced developmental effects, zebrafish embryos were exposed to different concentrations of di-(2-ethylhexyl) (DEHP) and di-butyl phthalate (DBP) for 96 h. Embryonic exposure to DEHP and DBP induced body length decrease, yolk sac abnormities, and immune responses (up-regulation of immune proteins and genes). The lipidomic results showed that at a concentration of 50 µg/L, DEHP and DBP significantly reduced the levels of fatty acids, triglycerides, diacylglycerol, and cholesterol. These effects are partly explained by biological pathway enrichment based on data from the transcriptional and proteomic profiles. Co-exposure to DBP and ER antagonist did not significantly relieve the toxic symptoms compared with exposure to DBP alone. This indicates that phthalate-induced developmental abnormities in zebrafish might not be mediated by the ER pathway. In conclusion, we identified the possible biological pathways that mediate phthalate-induced developmental effects and found that these effects may not be driven by estrogenic activation.

The enantioselective toxicity and oxidative stress of beta-cypermethrin on zebrafish.

Although the toxicity of beta-cypermethrin (beta-CYP) to aquatic organisms has become a significant concern in recent years, its enantioselective effects on non-target organisms is poorly understood. To investigate the enantioselective toxicity of beta-CYP on zebrafish, adult zebrafish were exposed to a series of isometric concentrations of four beta-CYP enantiomers and the beta-CYP racemate for 96 h. In addition, the activities of four antioxidant enzymes and the malondialdehyde (MDA) content in zebrafish liver and brain were tested after 15 and 30 days beta-CYP enantiomers and racemate exposure under environmentally relevant dosages (0.01 and 0.1 µg/L). According to the acute toxicity results, the 1R-cis-αS and 1R-trans-αS enantiomers were more lethal than 1S-cis-αR and 1S-trans-αR. At 0.1 µg/L, the 1R-cis-αS and 1R-trans-αS enantiomers, and the beta-CYP racemate could significantly induce a hepatic MDA content at 30 days post exposure (dpe), while only 1R-cis-αS caused brain lipid peroxidation. An apparent regulation of antioxidase levels was observed in zebrafish liver and brain after exposure to the 1R-cis-αS and 1R-trans-αS enantiomers, and the beta-CYP racemate. In contrast, no significant oxidative stress was observed in zebrafish exposed to 1S-cis-αR and 1S-trans-αR enantiomers under the test concentrations. This work demonstrated the occurrence of enantioselectivity in toxicity and oxidative stress of beta-CYP to adult zebrafish, which should be considered in environmental risk assessments.

Oxidative Stress in Shellfish Sinonovacula constricta Exposed to the Water Accommodated Fraction of Zero Sulfur Diesel Oil and Pinghu Crude Oil.

This study investigated the oxidative stress of the water accommodated fraction (WAF) of zero sulfur diesel oil and Pinghu crude oil on Sinonovacula constricta, respectively. The oxidative stress of oil pollutants on organisms was measured by typical antioxidases, such as SOD, CAT, GST, and POD. Toxicity was quantitatively evaluated by combining IBR (integrated biomarker responses). Results demonstrated that different concentrations of oil caused different degrees of induction to the four antioxidases. Compared with the control group, all test groups presented enzyme induction or inhibition during exposure period. Combined with the calculated IBR values, the high-concentration group of the zero sulfur diesel oil could cause the largest biological effect changes, which reflected its high oxidative stress. The zero sulfur diesel oil had stronger toxicity than Pinghu crude oil.

The developmental effect of difenoconazole on zebrafish embryos: A mechanism research.

Difenoconazole is a widely used triazole fungicide and has been reported to have negative impacts on zebrafish embryos. To investigate the mechanism of its developmental toxicity, zebrafish embryos were exposed to 0.5 and 2.0 mg/L difenoconazole for 96 h. The morphological and physiological indicators of embryo development were tested. The total cholesterol (TCHO) level, triglyceride (TG) level and malondialdehyde (MDA) content were measured at 96 hpf (hours post-fertilization). In addition, the transcription of genes related to embryo development, the antioxidant system, lipid synthesis and metabolism was quantified. Our results showed that a large suite of symptoms were induced by difenoconazole, including hatching regression, heart rate decrease, growth inhibition and teratogenic effects. 0.5 mg/L difenoconazole could significantly increase the TG content of zebrafish embryos at 96 hpf, while no apparent change in the TCHO and MDA level was observed post 96 h exposure. Q-PCR (quantitative real-time polymerase chain reaction) results showed that the transcription of genes related to embryonic development was decreased after exposure. Genes related to hatching, retinoic acid metabolism and lipid homeostasis were up-regulated by difenoconazole.