Combined toxic effects of fluxapyroxad and multi-walled carbon nanotubes in Xenopus laevis larvae.

Carbon nanomaterials rarely exist in isolation in the natural environment, and their combined effects cannot be ignored. Multi-walled carbon nanotubes (MWCNTs) have shown tremendous potential applications in diverse fields, including pollution remediation, biomedicine, energy, and smart agriculture. However, the combined toxicities of MWCNTs and pesticides on non-target organisms, particularly amphibians, are often overlooked. Fluxapyroxad (FLX), a significant succinate dehydrogenase inhibitor fungicide, has been extensively utilized for the protection of food and cash crops and control of fungi. This raises the possibility of coexistence of MWCNTs and FLX. The objective of this study was to explore the individual and combined toxic effects of FLX and MWCNTs on the early life stages of Xenopus laevis. Embryos were exposed to varying concentrations of FLX (0, 5, and 50 µg/L) either alone or in combination with MWCNTs (100 µg/L) for a duration of 17 days. The findings indicated that co-exposure to FLX and MWCNTs worsened the inhibition of growth, liver damage, and dysregulation of enzymatic activity in tadpoles. Liver transcriptomic analysis further revealed that the presence of MWCNTs exacerbated the disturbances in glucose and lipid metabolism caused by FLX. Additionally, the combined exposure groups exhibited amplified alterations in the composition and function of the gut microflora. Our study suggests that it is imperative to pay greater attention to the agricultural applications, management and ecological risks of MWCNTs in the future, considering MWCNTs may significantly enhance the toxicity of FLX.

Uncovering hidden dangers: The combined toxicity of abamectin and lambda-cyhalothrin on honey bees.

Studying the toxic effects of pesticides on bees has consistently been a prominent area of interest for researchers. Nonetheless, existing research has predominantly concentrated on individual toxicity assessments, leaving a gap in our understanding of mixed toxicity. This study delves into the individual and combined toxic effects of abamectin (ABA) and lambda-cyhalothrin (LCY) on honey bees (Apis mellifera) in laboratory settings. We discovered that ABA (96 h-LC50 value of 0.079 mg/L) exhibited greater acute toxicity to honey bees compared to LCY (96 h-LC50 value of 9.177 mg/L). Moreover, the mixture of ABA and LCY presented an acute antagonistic effect on honey bees. Additionally, our results indicated that exposure to LCY, at medium concentration, led to a reduction in the abundance of gut core bacterium Snodgrassella. However, an increase in the abundance of Bifidobacterium was noted when exposed to a medium concentration of LCY and its mixture with ABA. Transcriptomic analysis revealed significant regulation of certain genes in the medium concentration of all three treatments compared to the control group, primarily enriching in metabolism and immune-related pathways. Following chronic exposure to field-relevant concentrations of ABA, LCY, and their mixture, there were significant alterations in the activities of immunity-related enzyme polyphenol oxidase (PPO) and detoxification enzymes glutathione S-transferase (GST) and carboxylesterase (CarE). Additionally, the expression of four genes (abaecin, cyp9e2, cyp302a1, and GstD1) associated with immune and detoxification metabolism was significantly altered. These findings suggest a potential health risk posed by the insecticides ABA and LCY to honey bees. Despite exhibiting acute antagonistic effect, mixed exposure still induced damage to bees at all levels. This study advances our knowledge of the potential adverse effects of individual or combined exposure to these two pesticides on non-target pollinators and offers crucial guidance for the use of insecticides in agricultural production.

Metabolic perturbations in zebrafish (Danio rerio) larvae exposed to sulfentrazone and imidacloprid.

The intensive and widespread application of pesticides in agroecosystems can lead to the simultaneous exposure of non-target aquatic organisms to insecticides and herbicides. However, the underlying mechanisms through which aquatic organisms undergo metabolic reprogramming to withstand the combined effects of the insecticide imidacloprid (IMI) and herbicide sulfentrazone (SUL) remain poorly elucidated. This study employs metabolomics to investigate the effects of individual and combined exposures to IMI and SUL on zebrafish (Danio rerio), aiming to simulate complex environmental conditions. Metabolomics analysis revealed extensive metabolic reprogramming in larvae induced by the selected agrochemicals. Both individual and combined exposures disrupted nucleotide metabolism, inhibited glycolysis, and led to the accumulation of acetylcholine through the shared modulation of differential metabolites. Notably, individual exposure exhibited a unique mode of action. Larvae exposed to IMI alone showed mitochondrial dysfunction, potentially stemming from interference with the electron transport chain, while SUL-induced disruptions were associated with glycerophospholipid accumulation, marking it as a critical target. Additionally, calculations of the metabolic effect level index indicated antagonistic interactions between SUL and IMI mixtures at an overall metabolic level. The results obtained through investigating the lethal and sub-lethal effects also revealed that the simultaneous application of SUL and IMI may have the potential to diminish acute and developmental toxicity in zebrafish. This study underscores the significance of metabolomics as a valuable and effective strategy for deciphering the toxicity and interactions of agrochemical mixtures.

Co-exposure to deltamethrin and cyazofamid: variations in enzyme activity and gene transcription in the earthworm (Eisenia fetida).

Pesticide formulations are typically applied as mixtures, and their synergistic effects can increase toxicity to the organisms in the environment. Despite pesticide mixtures being the leading cause of pesticide exposure incidents, little attention has been given to assessing their combined toxicity and interactions. This survey purposed to reveal the cumulative toxic effects of deltamethrin (DEL) and cyazofamid (CYA) on earthworms (Eisenia fetida) by examining multiple endpoints. Our findings revealed that the LC50 values of DEL for E. fetida, following 7- and 14-day exposures, ranged from 887.7 (728-1095) to 1552 (1226-2298) mg kg-1, while those of CYA ranged from 316.8 (246.2-489.4) to 483.2 (326.1-1202) mg kg-1. The combinations of DEL and CYA induced synergistic influences on the organisms. The contents of Cu/Zn-SOD and CarE showed significant variations when exposed to DEL, CYA, and their combinations compared to the untreated group. Furthermore, the mixture administration resulted in more pronounced alterations in the expression of five genes (hsp70, tctp, gst, mt, and crt) associated with cellular stress, carcinogenesis, detoxification, and endoplasmic reticulum compared to single exposures. In conclusion, our comprehensive findings provided detailed insights into the cumulative toxic effects of chemical mixtures across miscellaneous endpoints and concentration ranges. These results underscored the importance of considering mixture administration during ecological risk evaluations of chemicals.

Enzymatic and transcriptional level changes induced by the co-presence of lead and procymidone in hook snout carp (Opsariichthys bidens).

Understanding the interactions between different environmental pollutants is necessary in ecotoxicology since environmental contaminants never appear as single components but rather in combination with other substances. Heavy metals and pesticides are commonly detected in the environment, but the characterization of their mixture toxicity has been inadequately explored. This research aimed to elucidate the mixture impacts of the heavy metal lead (Pb) and the pesticide procymidone (PCM) on the hook snout carp (Opsariichthys bidens) using an array of biomarkers. The data showed that Pb and PCM possessed almost equivalent acute toxicity to the animals, with 4-days LC50 values of 120.9 and 85.15 mg L-1, respectively. Combinations of Pb and PCM generated acute synergistic effects on O. bidens. The contents of malondialdehyde (MDA), antioxidative (SOD), apoptotic (caspase-9), and detoxifying enzymes glutathione S-transferase (GST) and cytochrome P450 (CYP450) significantly changed after most of the mixture exposures compared with the baseline level and the corresponding individual exposures. This suggests the induction of oxidative stress, cell damage, and detoxification dysfunction. The expressions of eight genes (mn-sod, cu-sod, p53, cas3, erß1, esr, ap, and klf2α) associated with oxidative stress, cell apoptosis, immune response, and hormonal functions exhibited pronounced changes when challenged with the mixture compared to the individual treatments. This indicates the occurrence of immune dysregulation and endocrine disorder. These findings provide an overall understanding of fish upon the challenge of sublethal toxicity between Pb and PCM and can be adopted to evaluate the complicated toxic mechanisms in aquatic vertebrates when exposed to heavy metal and pesticide mixtures. Additionally, these results might guide environmental regulation tactics to protect the population of aquatic vertebrates in natural ecosystems.

Current-use pesticides monitoring and ecological risk assessment in vegetable soils at the provincial scale.

Pesticides represent one of the largest intentional inputs of potentially hazardous compounds into agricultural soils. However, as an important vegetable producing country, surveys on pesticide residues in soils of vegetable production areas are scarce in China. This study presented the occurrence, spatial distribution, correlation between vegetable types and pesticides, and ecological risk evaluation of 94 current-use pesticides in 184 soil samples from vegetable production areas of Zhejiang province (China). The ecological risks of pesticides to soil biota were evaluated with toxicity exposure ratios (TERs) and risk quotient (RQ). The pesticide concentrations varied largely from below the limit of quantification to 20703.06 µg/kg (chlorpyrifos). The situation of pesticide residues in Jiaxing is more serious than in other cities. Soils in the vegetable areas are highly diverse in pesticide combinations. Eisenia fetida suffered exposure risk from multiple pesticides. The risk posed by chlorpyrifos, which exhibited the highest RQs at all scenarios, was worrisome. Only a few pesticides accounted for the overall risk of a city, while the other pesticides make little or zero contribution. This work will guide the appropriate use of pesticides and manage soil ecological risks, achieving green agricultural production.

Spatio-temporal distribution of organochlorine pesticides in agricultural soils of southeast China during 2014-2019.

Organochlorine pesticides (OCPs) are organic pollutants that are persistent and undegradable in the environment. To investigate their residual concentrations, spatial and temporal distributions, and the relationship with the crops planted, 12 individual OCPs in 687 soil samples from Jiangsu, Zhejiang and Jiangxi provinces of southeast China were examined. The detection frequencies of OCPs in the studied areas were 1.89%-64.9%. The concentrations of dichloro-diphenyl-trichloroethanes (DDTs), hexachlorocyclohexanes (HCHs), and endosulfans ranged from 0.01 to 5659 µg/kg, 0.03-3.58 µg/kg, and 0.05-3235 µg/kg, respectively. Jiangsu was mainly contaminated by p,p'-DDT, p,p'-DDD and endosulfan sulfate, Zhejiang was more polluted by OCPs except δ-HCH, and Jiangxi was more vulnerable to the contamination of OCPs except o,p'-DDE. The partial least-squares discrimination analysis (PLS-DA) model with RX2 36.3-36.8% revealed that compounds with similar chemical properties tended to appear in the same year and month. All crop lands were polluted by DDTs and Endosulfans. The highest concentrations of DDTs and Endosulfans were found in citrus and vegetable fields, respectively. This study offers new insight into the layout and partitioning of OCPs in agricultural land and into insecticide management on public health and ecological safety.

Adverse effects and potential mechanisms of fluxapyroxad in Xenopus laevis on carbohydrate and lipid metabolism.

Fungicides are one of significant contributing factors to the rapid decline of amphibian species worldwide. Fluxapyroxad (FLX), an effective and broad-spectrum succinate dehydrogenase inhibitor fungicide, has attracted major concerns due to its long-lasting in the environment. However, the potential toxicity of FLX in the development of amphibians remains mostly unknown. In this research, the potential toxic effects and mechanisms of FLX on Xenopus laevis were investigated. In the acute toxicity test, the 96 h median lethal concentration (LC50) of FLX in X. laevis tadpoles was 1.645 mg/L. Based on the acute toxicity result, tadpoles at the stage 51 were exposed to 0, 0.00822, 0.0822, and 0.822 mg/L FLX during 21 days. Results demonstrated that FLX exposure led to an apparent delay in the growth and development of tadpoles and associated with severe liver injury. Additionally, FLX induced glycogen depletion and lipid accumulation in the liver of X. laevis. The biochemical analysis of plasma and liver indicated that FLX exposure could perturb liver glucose and lipid homeostasis by altering enzyme activity related to glycolysis, gluconeogenesis, fatty acid synthesis, and oxidation. Consistent with the biochemical result, FLX exposure altered the liver transcriptome profile, and the enrichment analysis of differential expression genes highlighted the adverse effects of FLX exposure on steroid biosynthesis, PPAR signaling pathway, glycolysis/gluconeogenesis, and fatty acid metabolism in the tadpole liver. Overall, our study was the first to reveal that sub-lethal concentrations of FLX could induce liver damage and produce obvious interference effects on carbohydrate and lipid metabolism of Xenopus, providing new insight into the potential chronic hazards of FLX for amphibians.

Prostaglandin Metabolome Profiles in Zebrafish (Danio rerio) Exposed to Acetochlor and Butachlor.

Prostaglandins (PGs) are critically important signaling molecules that play key roles in normal and pathophysiological processes. Many endocrine-disrupting chemicals have been found to suppress PG synthesis; however, studies about the effects of pesticides on PGs are limited. The effects of two known endocrine disrupting herbicides, acetochlor (AC) and butachlor (BC), on PG metabolites in zebrafish (Danio rerio) females and males were studied using widely targeted metabolomics analysis based on ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In total, 40 PG metabolites were detected in 24 zebrafish samples, including female and male samples, with and without exposure to AC or BC at the sub-lethal concentration of 100 µg/L for 96 h. Among them, 19 PGs significantly responded to AC or BC treatment, including 18 PGs that were upregulated. The enzyme-linked immunosorbent assay (ELISA) test in zebrafish showed BC could cause significant upregulation of an isoprostane metabolite, 5-iPF2a-VI, which is positively related to the elevated level of reactive oxygen species (ROS). The present study guides us to conduct a further study to determine whether PG metabolites, including isoprostanes, could be potential biomarkers for chloracetamide herbicides.

The acute toxicity, mechanism, bioconcentration and elimination of fluxametamide on zebrafish (Danio rerio).

Fluxametamide is a completely novel and the first isoxazoline insecticide used to control agricultural pests and has high insecticidal properties. To expand its usage in the paddy field, its potential toxicological effects on fish are necessary to make clear. In this study, the acute toxicity, bioconcentration and elimination of fluxametamide to zebrafish Danio rerio, and the action mode of it on the heteromeric Drα1ß2Sγ2 and Drα1ß2S GABA receptor was respectively determined by HPLC and two-electrode voltage clamp technique. Fluxametamide exhibited high toxicity to D. rerio, whereas slightly inhibited the GABA-stimulated current of Drα1ß2Sγ2 or Drα1ß2S. It showed high bioconcentration level in D. rerio at 0.0314 mg L-1 and 0.157 mg L-1, with bioconcentration factors at steady state of 1491.55 and 2875.28, respectively. The concentration of fluxametamide in D. rerio rapidly decreased from 47.84 ± 0.12 to 9.77 ± 1.13 mg kg-1 in 0.0314 mg L-1 or from 393.19 ± 0.46 to 46.93 ± 2.88 mg kg-1 in 0.157 mg L-1 within 10 days, and steadily kept at a low level after 18 days. In conclusion, fluxametamide has highly acute toxicity to D. rerio, and might induce high bioconcentration in a short time. As we know, this is the first report to provide a theoretical basis for evaluating the potential risk of fluxametamide on fish, and guidance for the application of fluxametamide.

Analysis method development and health risk assessment of pesticide and heavy metal residues in Dendrobium Candidum.

Herbal medicines that are widely used worldwide are easily contaminated by pesticides and heavy metals, threatening human health. In this study, a modified QuEChERS pre-treatment method combined with HPLC/GC-MS/MS was established for the determination of 24 pesticide residues in Dendrobium candidum. The average recoveries of 24 pesticides in D. candidum were 76.9-110.0% with the relative standard deviation (RSD) of 0.28-11.40%, and their limits of detection (LOD) and limits of quantitation (LOQ) were 0.005-10 and 0.011-22 µg kg-1, respectively. The results showed that 83.33% of all samples had detected pesticide residues with the concentrations of 0.06-312.83 µg kg-1. Meanwhile, microwave digestion combined with ICP-MS was used to detect the residues of 8 heavy metals in D. candidum. The average recoveries of 8 heavy metals were 82.7-108.1% with an RSD of 1.4-8.0%, and their LOD and LOQ were 0.0001-0.05 mg kg-1 and 0.0003-0.2 mg kg-1, respectively. The results indicated that 8 heavy metals were all detected in all samples, and the highest concentration of Zn was 11.97 mg kg-1. Furthermore, the health risk assessment showed that the risk of the detected pesticides and heavy metals in samples to humans, specifically to the general population including adults and children, was acceptable.

Insights into the combined effects of environmental concentration of difenoconazole and tebuconazole on zebrafish early life stage.

Limited literature had focused on the combined effect of triazole fungicides on aquatic organisms at environmental concentrations. In this research, difenoconazole (DIF) and tebuconazole (TEB) mixture exhibited additive effect on the acute toxicity to zebrafish embryos. The transcriptomics and metabolomics demonstrated DIF and TEB mixtures at aquatic life benchmark and environmental concentration simultaneously influenced the lipid metabolism, arachidonic acid metabolism, steroid hormone biosynthesis and tryptophan metabolism, but showed diverse response patterns mediating the combined effects on zebrafish embryos after 120 h exposure. The DIF and TEB mixture at aquatic life benchmark caused combined effect on yolk sac resorption and metabolites, was less than the additive effect of individual DIF and TEB. It was found environmental concentration of DIF and TEB caused much lower levels of IFN and IL6, induced higher levels of PGE2, l-kynurenine and formylanthranilate in zebrafish larvae, and their binary mixture caused synergistic effect on the accumulation of metabolites in metabolic pathways, which might cause more negative effect and risk on growth in zebrafish later life stages. Results further demonstrated that adding arachidonic acid (AA) increased the transcripts of Pla2, Ptgs1, Cyp19a and Cxcl8b, allayed the accumulation of PLA2 and 17ß-E2, and induced more PGF2α, IFN and IL6 levels in zebrafish larvae, indicated AA metabolism might play important regulatory roles on hormone synthesis and immune response caused by DIF and TEB mixtures. Current results indicated the risk assessment of mixtures based on single concentration may not precisely estimate the environmental risk and health effect, it is crucially important to consider the multi-concentration combinations, and more attention should be paid to the environmental concentration.

Old pesticide, new use: Smart and safe enantiomer of isocarbophos in locust control.

Locust plagues are still worldwide problems. Selecting active enantiomers from current chiral insecticides is necessary for controlling locusts and mitigating the pesticide pollution in agricultural lands. Herein, two enantiomers of isocarbophos (ICP) were separated and the enantioselectivity in insecticidal activity against the pest Locusta migratoria manilensis (L. migratoria) and mechanisms were investigated. The significant difference of LD50 between (+)-ICP (0.609 mg/kg bw) and (-)-ICP (79.412 mg/kg bw) demonstrated that (+)-ICP was a more effective enantiomer. The enantioselectivity in insecticidal activity of ICP enantiomers could be attributed to the selective affinity to acetylcholinesterase (AChE). Results of in vivo and in vitro assays suggested that AChE was more sensitive to (+)-ICP. In addition, molecular docking showed that the -CDOKER energies of (+)-ICP and (-)-ICP were 25.6652 and 24.4169, respectively, which suggested a stronger affinity between (+)-ICP and AChE. Significant selectivity also occurred in detoxifying enzymes activities (carboxylesterases (CarEs) and glutathione S-transferases (GSTs)) and related gene expressions. Suppression of detoxifying enzymes activities with (+)-ICP treatment suggested that (-)-ICP may induce the detoxifying enzyme-mediated ICP resistance. A more comprehensive understanding of the enantioselectivity of ICP is necessary for improving regulation and risk assessment of ICP.

Low-dose cadmium stress increases the bioaccumulation and toxicity of dinotefuran enantiomers in zebrafish (Danio rerio)?

Co-occurrence of pesticides and heavy metals has attracted extensive attention. The enantioselective behaviors of dinotefuran to aquatic organisms have not been reported, and the effects of cadmium (Cd) was absent, which were investigated in this study at environmentally relevant concentrations. The enantioselective accumulation and elimination of dinotefuran enantiomers were observed in zebrafish, and it had tissue specificity. The S-dinotefuran concentrations were higher than R-dinotefuran in heads and viscera, but it was opposite in muscles. There existed competition between S-dinotefuran and R-dinotefuran, and the existence of S-dinotefuran might decrease the accumulation and elimination of the R-dinotefuran in zebrafish. When co-exposure to Cd and dinotefuran, the accumulation concentrations of dinotefuran enantiomers increased in zebrafish at the initial stage, which were opposite latterly. The accumulation concentrations of R-dinotefuran in R + Cd treatment in fish were 3.4 times higher than those in R-dinotefuran treatment, and the enantiomer fraction (EF) values changed from 0.484 to 0.195. The oxidative stress of S-dinotefuran on zebrafish was highest, followed by rac- and R-dinotefuran. Co-exposure to Cd led to toxicity increase for R-dinotefuran, the malonaldehyde (MDA) content decreased significantly in R + Cd treatment during 7-28 days, while obvious declination of MDA contents was found on the 28th day in R-dinotefuran treatment. Furthermore, compared to R-dinotefuran treatment, Cd increased the relative expression of cz-sod (3.4 times), cas3 (1.6 times) and p53 (5.7 times) in R + Cd treatment. The co-exposure of Cd might alter the environmental behaviors and toxicity effects of dinotefuran enantiomers in zebrafish, including the enantioselectivity. The effects of Cd on accumulation and toxicity of R-dinotefuran were greater than those on S-dinotefuran. Thus, it is necessary to consider the effects of coexistent metals to chiral pesticides in ecological risk. SUMMARIZES: The enantioselective accumulation and elimination of dinotefuran enantiomers had tissue specificity. Cd increased the accumulation and toxicity of R-dinotefuran in zebrafish.

New insights into cardiotoxicity induced by chiral fluoxetine at environmental-level: Enantioselective arrhythmia in developmental zebrafish (Danio rerio).

Fluoxetine is frequently detected in aquatic environment, and chronic FLX exposure exhibits adverse effects on aquatic communities. Its chirality makes the adverse effects more complicated. This study aimed at the enantioselective cardiotoxicity in developmental zebrafish induced by racemic (rac-)/S-/R-fluoxetine. The accumulation profiles demonstrated that biotransformation of fluoxetine to norfluoxetine occurred during rac-fluoxetine exposure, with a higher enrichment of S-norfluoxetine than R-norfluoxetine. Heart malformations including pericardial edema, circulation abnormalities, and thrombosis were observed, and enantioselective changes also occurred. According to H&E staining and Masson's trichrome staining, the loose severity of cardiac structure and cardiac fibrosis in rac-norfluoxetine treated group was worse than that in fluoxetine treated groups. Results of toxicity-associated parameters in our homochiral enantiomers' exposure also indicated that the toxicity induced by S-fluoxetine was more severe than R-fluoxetine. Enantioselective arrhythmia in developmental zebrafish after chiral fluoxetine exposure could be caused by myocardial fibrosis, abnormal developmental processes, and the biotransformation of fluoxetine to norfluoxetine could make that worse. Our findings can be used to assess the environmental risk of the two enantiomers of fluoxetine that induce cardiotoxicity in aquatic organisms.

Pesticide multi-residues in Dendrobium officinale Kimura et Migo: Method validation, residue levels and dietary exposure risk assessment.

Dendrobium officinale Kimura et Migo (D. officinale) is a dual-use plant with both botanical medicine and food applications, drawing increasing attentions. Pesticides are inevitably applied on D. officinale in commercial artificial-sheltered cultivation, yet little is known about pesticide residue levels in D. officinale. A modified high through-put QuEChERS method coupled with HPLC-MS/MS was developed and validated to detect 76 pesticides in D. officinale. Graphitized multi-wall carbon nanotubes (g-MWCNTs) was selected as the clean-up sorbent, showing relative weak affinity to triazole fungicide having planar structure in their molecular and low matrix effects of pesticides in D. officinale samples compared to MWCNTs and pesticarb. The validated method was applied to analyze pesticide residues in 86 real D. officinale samples collected from three main producing provinces. 43 different pesticides were detected with highest residue of 6.11 mg/kg for dimethomorph. Given possible health risks related to pesticide residues, accordingly, risk assessment of human exposure to pesticides via intake of D. officinale was thus performed, indicating that the pesticide residue in fresh or dry D. officinale would not cause potential risk to human health either in the long-term or short-term. This work improved our understanding of potential exposure risk of pesticide multi-residues in D. officinale.

Effects of difenoconazole on hepatotoxicity, lipid metabolism and gut microbiota in zebrafish (Danio rerio).

In current study, larvae and adult zebrafish were exposed to difenoconazole to assess its effect on hepatotoxicity, lipid metabolism and gut microbiota. Results demonstrated that difenoconazole could induce hepatotoxicity in zebrafish larvae and adult, 0.400, 1.00, 2.00 mg/L difenoconazole caused yolk retention, yolk sac edema or liver degeneration after embryos exposure for 120 h, hepatocyte vacuolization and neoplasm necrosis were observed in adult liver after 0.400 mg/L difenoconazole exposure for 21 d. RNA sequencing showed that the 41 and 567 differentially expressed genes in zebrafish larvae and liver induced by 0.400 mg/L difenoconazole, were concentrated in pathways related to protein digestion and absorption, pancreatic secretion, steroid biosynthesis, and different metabolic pathways including galactose or sugar metabolism. Difenoconazole exposure caused lipid accumulation in larval yolk sac, and the elevated triglyceride (TG), malondialdehyde (MDA) and reactive oxygen species (ROS) levels in larvae and liver, which further confirmed the lipid metabolism disorders induced by difenoconazole. The results further showed that difenoconazole increased the abundance of gut microbiota such as Firmicutes, Aeromonas, Enterobacteriaceae and Bacteroides, further suggested that gut microbiota might participate in lipid metabolism and hepatotoxicity during zebrafish development. These findings advanced the field of the difenoconazole-induced developmental toxicity in larvae and adult zebrafish, and the imbalance of gut microbiota provided the plausible mode of action for the liver damage and disordered lipid metabolism in zebrafish.

Changes in thyroid hormone levels and related gene expressions in embryo-larval zebrafish exposed to binary combinations of bifenthrin and acetochlor.

Bifenthrin (BF) and acetochlor (AT) are widely used as an insecticide and herbicide, respectively, which are introduced to the aquatic environment as a natural result. Although the thyroid active substances may coexist in the environment, their joint effects on fish have not been identified. We examined the joint toxicity of BF and AT in zebrafish (Danio rerio) in this study. An acute lethal toxicity test indicated that the median lethal concentration (LC50) values of BF and AT under 96 h treatment were 0.40 and 4.56 µmol L-1, respectively. The binary mixture of BF + AT displayed an antagonistic effect on the acute lethal toxicity. After 14 days post fertilization (dpf) with exposure to individual pesticides at sub-lethal concentrations of, no effects were observed on the catalase (CAT) and peroxidase (POD) activities, while the binary mixtures (except for the 7.2 × 10-3 µmol L-1 BF + 1.2 × 10-2 µmol L-1 AT exposure group) significantly induced the CAT activity. The superoxide dismutase (SOD) activity and triiodothyronine (T3) level were significantly increased in all exposure groups. The thyroxine (T4) level remained unchanged after exposure to individual pesticides, but significantly increased in the 7.2 × 10-3 µmol L-1 BF + 1.2 × 10-2 µmol L-1 AT group. The expressions of the genes Dio2, TRa, TSHß and CRH in the thyroid hormone (TH) axis were significantly up-regulated in the 7.2 × 10-3 µmol L-1 BF + 0.4 × 10-2 µmol L-1 AT group. Our data indicated that the binary mixture of BF + AT significantly altered the antioxidant enzyme activities and gene expressions in the hypothalamic-pituitary-thyroid (HPT) axis and changed the TH levels.

Mitochondrial dysfunction, apoptosis and transcriptomic alterations induced by four strobilurins in zebrafish (Danio rerio) early life stages.

Though the toxicity of strobilurins on non-target aquatic organisms has been characterized, the associated toxic mechanisms have not been fully explored. The present study showed that the larval stage was the most sensitive developmental stage in zebrafish, and pyraclostrobin (PY) had the highest acute toxicity to embryos, larvae, juvenile and adult with 96 h-LC50 at 0.048 mg/L, 0.029 mg/L, 0.039 mg/L, 0.031 mg/L respectively, when compared with the toxicity of trifloxystrobin (TR), kresoxim-methyl (KM) and azoxystrobin (AZ) at corresponding developmental stage. Then we investigated the transcriptomics and developmental toxicity of TR, KM, AZ and PY on zebrafish embryos after 72 h exposure. RNA-seq revealed that the pathways related to cell apoptosis and cancer, and cellular components organelle membrane and mitochondrion, were markedly affected after TR, KM, AZ and PY exposure during zebrafish early life stages. The results were further confirmed by the induction of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) activities, the elevation of H2O2, malondialdehyde (MDA) and reactive oxygen species (ROS) level, as well as the reduction of intracellular calcium ions (Ca2+) and mitochondrial membrane potential (MMP), which indicated that strobilurins could cause mitochondrial dysfunction and cell apoptosis. The present study was performed a systematic analysis of strobilurins to zebrafish at multi-levels, which provided suggestions for further investigation of molecular mechanisms underlying the toxicity induced by strobilurins on aquatic organisms.

Significant role of supercritical fluid chromatography - mass spectrometry in improving the matrix effect and analytical efficiency during multi-pesticides residue analysis of complex chrysanthemum samples.

As an important "food and drug dual-use" product, chrysanthemums are widely used in both botanical medicine and food applications. However, the misuse of pesticides during chrysanthemum cultivation makes pesticide residue monitoring crucial. The aim of the present work was to address this practical demand for the simultaneous determination of multiple pesticide residues in various species of chrysanthemums. Both the sample pre-treatment and instrumental methods were systematically investigated. Seven chrysanthemum samples were extracted using acetonitrile and purified by dispersive solid-phase extraction with amino-modified multi-walled carbon nanotubes (MWCNTs-NH2) and C18 as the cleanup co-adsorbents. After optimizing the amounts of MWCNTs-NH2 and C18, matrix effects could not be avoided during LC-MS/MS analysis of 112 pesticides, although satisfactory recoveries were obtained. The use of SFC-MS/MS was evaluated, which demonstrated the significant positive role of SFC-MS/MS in reducing the matrix effects during pesticide residue analysis. In addition, the use of SFC-MS/MS permitted a shorter run time and afforded greater analytical efficiency. Method validation was further performed to evaluate the linearity, sensitivity, recovery, and precision of the developed method. Good linearity was observed for 92% of the analytes in the concentration range of 2-250 µg L-1 for all seven of the chrysanthemum samples. The LODs of the 112 pesticides ranged from 0.01 to 31.41 µg L-1, depending on the sample, while the mean recoveries of all of the spiked pesticides ranged from 81.8% to 102% for concentrations of 20, 50, and 200 µg kg-1. These results clearly demonstrate the applicability of the developed method for the simultaneous determination of multi-pesticides in various chrysanthemum samples.