Integrated QuEChERS combined with LC-MS/MS for high-throughput analysis of per- and polyfluoroalkyl substances in milk.

In this study, an integrated QuEChERS method was developed for the rapid determination of 22 per- and polyfluoroalkyl substances (PFASs) in milk by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The extraction and purification processes were combined into one step with this method. Meanwhile, the solid-liquid separation was carried out by magnetic suction (Fe3O4-SiO2) instead of the centrifugal process. The primary experimental parameters were optimized, including the type of extraction solvent, the amounts of magnetic nanomaterials (Fe3O4-SiO2), and the purification materials (ZrO2 and C18). The developed method exhibits high precision (RSDs < 9.9%), low limits of detection (0.004-0.079 µg/kg) and limits of quantitation (0.01-0.26 µg/kg), and acceptable recovery (71.7-116%) under optimized conditions. The developed integrated QuEChERS method had clear superiority in terms of sample pretreatment time, operating procedures, reagent amount, and recovery. This makes it an excellent alternative analytical technique for PFAS residue measurement at low micrograms-per-kilogram ranges with desirable sensitivity.

Stereoselective behaviors and enantiomeric effects of paclobutrazol on microorganisms during Chinese cabbage pickling process.

Studies on the differences between chiral pesticide enantiomers have caused widespread concern in the last decade. In the current work, the selective behaviors and different biological activities of paclobutrazol enantiomers during Chinese cabbage pickling process were evaluated. Results of degradation kinetics indicated that when paclobutrazol reside in raw material (Chinese cabbage) and was introduced into the pickling process, the degradation rates of the two paclobutrazol enantiomers were significantly different, the half-lives of (2R, 3R)-paclobutrazol (R-paclobutrazol) and (2S, 3S)-paclobutrazol (S-paclobutrazol) were 18.24 and 6.19 d, respectively. Besides, the conversion between the two enantiomers could also be observed, and the conversion rate of R-paclobutrazol to S-paclobutrazol was slower than that of reverse process. In addition, from the analysis of 16S rRNA and ITS sequencing, we inferred that the degradation of paclobutrazol was probably due to the presence of Pseudomonas and Serratia. Moreover, there has a significant difference in biological activity between R-paclobutrazol and S-paclobutrazol and shown an obviously enantiomeric effects on microbial community composition of pickling system. Besides, the analysis of microbial community displayed R-paclobutrazol might inhibit the growth of Erwinia (a sort of plant pathogens). Results from this study served to enhance our understanding of chiral pesticide residues on food safety and the potential risks to human health.

Comprehensive evaluation of chiral penflufen metabolite (penflufen-3-hydroxy-butyl): Identification, synthesis, enantioseparation, toxicity and enantioselective metabolism.

Identification and evaluations of pesticide metabolites are necessary for risk assessment and toxicological research. In this study, the metabolites of penflufen (a widely used chiral pesticide) in rat liver microsomes were identified using liquid chromatography Q-Exactive Plus mass spectrometry. In total, 17 penflufen metabolites were identified, and most of them were hydroxylation products, which were generated by oxygenation at different candidate sites of penflufen. The relative abundance of metabolite M12 (penflufen-3-hydroxy-butyl, 32 %) was the largest, followed by M8 (15.6 %) and M2 (12.8 %). The major metabolite penflufen-3-hydroxy-butyl was first synthesized by 11 reactions with a 99.73 % purity. The absolute configuration of M12 enantiomers were confirmed after preparing enantiomers, and establishing the enantioseparation method. The M12 enantiomers toxicity to Danio rerio (LC50, >10 mg/L) and four kinds of phytopathogens (EC50, 148-34969 mg/L) were significantly lower than parents (LC50, 0.449-24.3 mg/L; EC50, 0.027-92.0 mg/L). In rat liver microsomes, approximately 40-47 % of the penflufen enantiomers were metabolized to M12 enantiomers, and R-penflufen was preferentially metabolized. The generation concentrations of S-M12 were higher than R-M12 after 10 min, and the metabolic half-lives of R-M12 (29.0-32.5 min) were shorter than S-M12 (35.2-38.1 min), and were approximately 4 times longer than parent penflufen enantiomers (4.5-9.5 min). Simultaneously, the generated contents (relative contents) of M8 (27.1-57 %) and M10 (2.22-8.36 %) from S-penflufen were lower than those from R-penflufen (M8, 24.7-92.4 %; M10, 27.4-69.5 %). The enantioselective evaluations of M12, M10 and M8 deserve further study. These findings were helpful in understanding the fate and risks of chiral penflufen.

Chiral pesticides levels in peri-urban area near Yangtze River and their correlations with water quality and microbial communities.

Pesticides are considered to be the second-largest non-point source pollution in water. Our research assayed the river network of typical agricultural areas in the middle and lower Yangtze River as the study area. Pesticides residues in aquatic environment were determined by QuEChERS, combined with high-performance liquid chromatography tandem mass spectrometry, or gas chromatograph-mass spectrometer. At chiral pesticides' levels, we detected pesticides contents in water, classified and counted the types of pesticides, and analyzed their environmental risk assessment. Furthermore, potential correlations between chiral pesticides concentrations and water quality indicators were assayed. Additionally, we explored their relations with microbial communities at species levels. Enantiomers of Diclofop-methyl, Ethiprole, Difenoconazole and Epoxiconazole were enantioselectively distributed. More interestingly, due to various chiral environment of the sampling site, the enantiomers of Tebuconazole Acetochlor, Glufosinate ammonium and Bifenthrin had completely different distributions at different sites. Based on that, the chiral pesticides Diclofop-methyl, Bifenthrin, Ethiprole, Tebuconazole and Difenoconazole are enantioselective to the risk of aquatic environment. Generally, enantiomeric selectivity had high positive correlations with total nitrogen and phosphorus. Then we found that chiral fate behavior of Tebuconazole and Paichongding in water might be affected by prokaryotes. In addition, the chiral behavior of Diclofop-methyl, Propiconazole, Difenoconazole, and Tebuconazole isomers in water might be negatively affected by eukaryotes. That research helped us to comprehensively understand the impact of non-point source pollution of chiral pesticides in aquatic environment and provided basic data support for developing biological and water quality indicators for monitoring pollution in aquatic environment.

Zeolitic imidazolate framework-8 as a dispersive solid phase extraction sorbent for simultaneous determination of 145 pesticide residues in polyphenol-rich plants.

Many plants have a high polyphenol content, which causes the matrix effect and makes the analysis of trace pesticide residues highly challenging. A common approach to improving matrix effects is to purify pesticides through the use of sorbents, but this requires a combination of multiple sorbents and extensive use. Zeolitic imidazolate framework-8 is widely used for pesticide analysis due to its high porosity, large specific surface area, and versatility. Here, we established and validated a modified quick, easy, cheap, effective, rugged, and safe method based on a zeolitic imidazolate framework-8 that was used to test the removal ability for polyphenols. To this end, 145 pesticide residues in peppermint, perilla, fennel, and mulberry leaves were analyzed by the modified method coupled with liquid chromatography with tandem mass spectrometry. The mean recoveries of all pesticide residues were in the range of 74.3-103.7%, with mean relative standard deviations ≤9.1% at spiked concentrations of 1, 10, 50, and 100 µg/kg for mulberry leaves. The limits of quantitation of the method ranged from 1 to 50 µg/kg. This study offers a reliable approach for the accurate quantitative analysis of various trace substances in the polyphenol-rich plants.

Low-dose cadmium affects the enantioselective bioaccumulation and dissipation of chiral penflufen in zebrafish (Danio rerio).

Pesticides are currently extensively used in agriculture, forestry, animal husbandry, and environmental hygiene, and their residues have become a global environmental problem, which can easily form combined pollution with heavy metals. The present study examined the effects of chronic (28 days) aqueous exposure of chiral penflufen (rac-penflufen, R-(-)-penflufen and S-(+)-penflufen), a widely used fungicide, with/without cadmium (Cd), a highly toxic heavy metal in zebrafish (Danio rerio). After rac-penflufen individual or combined exposure with Cd, the bioaccumulation and residual levels of S-(+)-penflufen were significantly higher than R-(-)-penflufen, and the effects of Cd were insignificantly. But for penflufen enantiomer, the effects of Cd were more serious for R-(-)-penflufen, which could increase the bioaccumulation (up to1.73 times), inhibit the dissipation (up to 32.3%) and enhance the residue (up to 5.35 times) of R-(-)-penflufen in zebrafish, decreasing the enantioselectivity. However, significant increase of S-(+)-penflufen concentrations was only found in viscera under co-exposure of Cd. The tissue distribution of penflufen enantiomers were not affected by the presence of Cd, and no interconversion of the two enantiomers occurred regardless of the presence of Cd. These findings indicated that co-contamination with Cd could increase the persistence of R-(-)-penflufen in zebrafish, thus increasing the environmental risks. The significant differences of Cd effects on chiral pesticide enantiomer and racemate indicated that the combined pollution of heavy metal and chiral pesticide might have enantiomer-specific, which should raise concern, and the enantioselective mechanism deserve further study.

Magnetic polymer particles as a highly efficient and facile cleanup adsorbent for multi-pesticide residues analysis in aquatic products.

A novel and efficient sample pretreatment procedure using magnetic particles was exploited for the determination of multi-pesticide residues in aquatic products. The magnetic adsorbent was prepared using divinyl benzene and N-vinyl pyrrolidone as functional monomers modified on the Fe3O4 @SiO2. The obtained magnetic adsorbent, octadecylsilane sorbents, and graphitized carbon black were employed as effective adsorbents to remove matrix interferences in aquatic products, and their dosages were optimized. Satisfactory levels of accuracy and precision were procured under optimum conditions. The method limits of quantification ranged from 0.1 to 2.0 µg/kg. The analytical accuracy of the developed method for the analysis of multi-pesticide residues in freshwater and seafood products was validated. It was found to be suitable for the analysis of multi-pesticide residues in different types of aquatic products. Additionally, the method was successfully applied for the analysis of pesticide residues in fish samples obtained from aquaculture plants located in Zhejiang Province, China. The detected concentrations of pesticides ranged from 0.14 to 0.95 µg/kg. In general, this method shows promising application prospects for the rapid determination of multi-pesticide residues in aquatic products.

Toxicological Study on Chiral Fluoxetine Exposure to Adult Zebrafish (Danio rerio): Enantioselective and Sexual Mechanism on Disruption of the Brain Serotonergic System.

The increasing number of people with depression worldwide has led to concerns regarding antidepressant contamination in aquatic environments, which could have the risk of negative effects on aquatic organisms. Chirality increases its toxicity potentials. Accordingly, we investigated the negative effects of racemic (rac-), R-, and S-FX at environmental levels (100 ng/L) on the brain serotonergic system in zebrafish (Danio rerio) for 42 days. Additionally, we measured the whole-body concentrations of FX and norfluoxetine (NFX). We found that S-FX exposure disrupted the brain serotonergic system more severely than rac- and R-FX exposure. The mechanism underlying this disruption induced by S-FX was sex-specific, with female zebrafish showing disruption of the serotonin (5-HT) release process but male zebrafish showing disruption of the 5-HT synthesis process. In addition, enantioselective enrichment and biotransformation (R-FX to R-NFX and S-FX to S-NFX) occurred in zebrafish. Sex-specific accumulation was also observed, with higher concentrations in females. Our study provides evidence for enantiomer- and sex-specific effects of FX exposure at biologically relevant concentrations. More broadly, our study demonstrated that SSRI antidepressants, such as FX, can affect aquatic life by causing important shifts in not only their active sites of the serotonin transporter.

Enantioselective toxicity and mechanism of chiral fungicide penflufen based on experiments and computational chemistry.

Penflufen fungicide is widely used as a racemate, which has potential ecological risks to aquatic organisms, while its enantioselective toxicity data is limited. This study aimed to differentiate the enantioselective toxicity difference of penflufen enantiomers, and illuminate the enantioselective mechanism from the insight of enantiomer-protein specific binding. The semipreparative separation and absolute configuration of penflufen enantiomers were conducted. The acute toxicity of S-(+)-penflufen was 54 times higher than R-(-)-penflufen to Danio rerio, and the coexistence of R-(-)-penflufen could increase the exposure risk of S-(+)-penflufen. For chronic toxicity, after low-dose long-term exposure, rac-penflufen and S-(+)-penflufen inducted more serious oxidative stress than R-(-)-penflufen in D. rerio, and inhibited the succinate dehydrogenase (SDH) activity significantly. For target phytopathogen, the toxicity difference of S-(+)-penflufen and R-(-)-penflufen was up to 148 times for Rhizoctonia solani. Based on the toxic unit analysis, the toxic interactions of antagonistic effect and concentration addition were found between penflufen enantiomers, indicating the coexistence of R-(-)-penflufen could increase overuse and environmental risks. Computational chemistry was used to illuminate the enantioselectivity mechanism, and the lower binding energy between the active site of SDH and S-(+)-penflufen contributed to the higher toxicity. The higher target toxicity might be due to the hydrophobic pocket of CybL in R. solani was more benefited to S-(+)-penflufen binding SDH than Botrytis cinerea. These results could be helpful for further understanding the potential risk of chiral penflufen in the environment, demonstrating the importance of understanding the enantioselective difference of chiral pesticides, and providing a new insight for analyzing the enantioselective mechanism.

High-throughput determination of fungicides in grapes using thin-film microextraction coupled with liquid chromatography-tandem mass spectrometry.

A high-throughput and environmentally friendly method based on 96-well plate thin-film microextraction was established to determine 14 fungicides in grapes and grape juice using liquid chromatography-tandem mass spectrometry. The thin-film microextraction optimized method consisted of 60 min of extraction at pH 6.0 with the addition of sodium chloride (2-5%). Acetonitrile/water in the ratio of 8:2 was used for desorption analytes for 60 min. Evaluation of different extractive phases showed that polyacrylonitrile-polystyrene-divinylbenzene was the optimum coating. The linearity of the method was good in the range of 0.01-0.5 µg/mL for 14 fungicides with determination coefficients (R2 ) from 0.990 to 0.999, which indicated good linearity for both the grape juice and grape matrixes. The limit of detection was in the range of 0.002-0.01 µg/mL. The limit of quantitation was in the range of 0.01 mg/kg according to the minimum fortified level. The average absolute recoveries of the 14 fungicides ranged from 75.0 to 118.3%. The intraday relative standard deviation (n = 4) and interday relative standard deviation (n = 4) were 5.6-13.0% and 1.6-6.4%, respectively. This study showed that this method can be used for analyzing 96 samples in parallel, and the sample preparation time was approximately 2.0 min per sample. In addition, this approach offers a green and low-cost sample pretreatment technique for future analyses.

Enantioselective toxic effects and digestion of furalaxyl enantiomers in Scenedesmus obliquus.

Research on the enantioselective environmental behavior of chiral pesticides has been a hot spot of environmental chemistry recently. In this study, the acute toxicity and digestion of furalaxyl enantiomers were determined on the aquatic algae Scendesmus obliquus. After exposure for 96 hours, the EC50 values for (S)-furalaxyl and (R)-furalaxyl were 13.59 and 15.26 mg/L, respectively. In addition, enantioselectivity was observed from the determined chlorophyll contents and antioxidant enzyme (CAT and SOD) activities of algae cells after exposure to furalaxyl enantiomers for 96 hours. The digestion rate of (S)-furalaxyl and (R)-furalaxyl were almost the same in S. obliquus. On the basis of these data, the inactive enantiomers (S)- furalaxyl is more toxic than the active one on the non-target species S. obliquus, indicating that such enantiomeric differences should be taken into consideration in the study of pesticide risk assessment.

Comparison of triadimefon and its metabolite on acute toxicity and chronic effects during the early development of Rana nigromaculata tadpoles.

Pesticides are one of major causes for amphibian population declines and the behavior of pesticide metabolite products to amphibians has become a rising concern. In this study, the acute toxicity and the chronic effects of triadimefon and triadimenol (the metabolite of triadimefon) on Rana. nigromaculata were investigated. In the acute assay, significant differences were observed in antioxidant enzyme activities and malondialdehyde levels between the triadimefon and triadimenol. The 96 h-acute toxicity of triadimefon (25.97 mg/L) and triadimenol (34.55 mg/L) to tadpoles was low. In 28d-chronic exposure, we studied the relative expression of tadpoles genes related to thyroid hormone-dependent metamorphic development, histological examination of liver and some biological index, including wet weight, snout-to-vent length (SVL) and development stages. The results revealed that the effects of triadimefon and triadimenol on tadpole development are driven by a disruption of the hormonal pathways involved in metamorphosis. Interestingly, triadimefon was more harmful on R. nigromaculata than triadimenol at high dose, whereas the reverse result was observed at low doses. According to the relative expression of thyroid hormone-dependent genes, we also found that the two compounds may have different mechanisms of toxic action on R. nigromaculata. Our study developed a pragmatic approach for use in the risk assessment of pesticide and its metabolite，and increased the information and understanding of the impacts of fungicides and other potential endocrine disrupting environmental contaminants on amphibians.

Tissue distribution and toxicity effects of myclobutanil enantiomers in lizards (Eremias argus).

In recent years, serious environmental pollution has caused a decrease in the abundance of many species worldwide. Reptiles are the most diverse group of terrestrial vertebrates. There are large amounts of toxicological data available regarding myclobutanil, but the adverse effects of myclobutanil on lizards has not been widely reported. In this study, treatment groups were orally administered a single-dose of myclobutanil (20mg/kg body weight (bw)). Subsequently, it was found that there were differences in myclobutanil levels between the different tissues and concentrations also changed with degradation time. The tissue concentrations of myclobutanil decreased in the order of: stomach > liver > lung > blood > testis > kidney > heart > brain. Based on our results, the liver and testis were considered to be the main target organs in lizards, indicating that the myclobutanil could induce potential hepatic and reproductive toxicity on lizards. Meanwhile, it was also demonstrated that the toxic effects of myclobutanil was different in different species, and the distribution of different pesticides in lizards were different.

Effects of benthic organism Tubifex tubifex on hexachlorocyclohexane isomers transfer and distribution into freshwater sediment.

In this study, bioaccumulation and elimination of HCHs in tubifex, and the distribution of HCHs in overlying water and sediment, were studied during a 10-d experiment. A sensitive method was developed for the determination of HCHs in samples based on gas chromatograph (GC) equipped with a nickel-63 electron capture detector (µECD). The limit of detection (LOD) was 0.35 µg/kg for α-HCH and 0.82 µg/kg for ß-HCH. Tubifex accumulated HCHs rapidly, and the curves were approximately M-type. The highest level was reached on the 7th day, with 0.34 mg/kg(wwt) for α-HCH and 0.87 mg/kg(wwt) for ß-HCH in worms. The AFs of ß-HCH in tubifex were higher than those of α-HCH. Moreover, the existence of tubifex significantly reduced ß-HCH fluxes from the overlying water to sediment by uptake or degradation and decreased the concentrations of ß-HCH in the sediment, but it had little influence on α-HCH fluxes. Moreover, enantioselectivity of α-HCH enantiomers was not observed in tubifex, whether in the bioaccumulation or elimination experiments. At the end of the elimination experiment, approximately 80% and 70% of α-HCH and ß-HCH were eliminated, and the depuration half-lives were 4.43 and 5.39 days, respectively.

Comparison of Different Extraction Methods for Analysis of 10 Organochlorine Pesticides: Application of MAE-SPE Method in Soil from Beijing.

Four commonly applied extraction techniques for organochlorine pesticides, microwave-assisted extraction (MAE), accelerated solvent extraction (ASE), quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction and ultrasonic solvent extraction, were applied on soil samples in order to evaluate their performances. The extracts were analyzed by GC-ECD and confirmed by GC-MS/MS. The MAE and QuEChERS extraction methods generally yielded higher results compared to the ultrasonication and ASE methods, while the lowest recovery (56.8 %) for o,p'-DDD was obtained using the QuEChERS method. The MAE method was further applied to six different soils from Beijing. In the soil samples only α-endosulfan and ß-endosulfan were not detected. The ratios of α-HCH/γ-HCH and α-HCH/ß-HCH indicated HCH residues likely originated from historical use of HCHs, and that technical HCHs were not likely being currently applied in Beijing.

Stereoselective bioaccumulation and metabolite formation of triadimefon in Tubifex tubifex.

Triadimefon, a chiral fungicide, could be metabolized to triadimenol which has two chiral centers. In this work, Tubifex tubifex was exposed to triadimefon through the aqueous and soil phase to explore the relative importance of the routes of uptake. Bioaccumulation of triadimefon in tubifex was detected in both treatments, and the kinetics of the accumulation processes were significantly different in these two experiments. In spiked water treatment, (S)-triadimefon was preferentially accumulated over the (R)-triadimefon, whereas the enantioselective bioaccumulation was not detected in the spiked soil microenvironment. Simultaneously, four stereoisomers of triadimenol were also found in the tubifex tissue. Although the amount of these stereoisomers were different from each other with relatively more accumulation of the most fungi-toxic stereoisomer (1S,2R), the abundance ratios in the two exposure treatments were similar at the same sampling, following the order (1S,2S) > (1R,2S) > (1R,2R) > (1S,2R). The bioaccumulation factor was calculated for parent compound triadimefon and metabolite enrichment factor for metabolite. The results showed that both uptake routes, epidermal contact in the aqueous phase and ingestion of solid particles in soil, were important to the bioaccumulation of the triadimefon and triadimenol in tubifex.

The chiral separation and enantioselective degradation of the chiral herbicide napropamide.

The chiral pesticide enantiomers often have different toxic effects and environmental behaviors, which suggests that the risk assessments should be on an enantiomeric level. In this work, the chiral separation of the napropamide enantiomers and the stereoselective degradation in tomato, cucumber, rape, cabbage, and soil were investigated. Napropamide enantiomers could be separated absolutely by high-performance liquid chromatography (HPLC) using a Chiralpak IC column with a resolution factor of 11.75 under the optimized condition. Solid phase extraction (SPE) was used for cleanup of the enantiomers in the vegetable samples. The residue analysis method was validated. Good linearities (R(2) = 0.9997) and recoveries (71.43% -97.64%) were obtained. The limits of detection (LOD) were 0.05 mg/kg in soil and 0.20 mg/kg in vegetables. The results of degradation showed that napropamide dissipated rapidly in vegetables with half-lives of only 1.13-2.21 days, but much more slowly in soil, with a half-life of 11.95 d. Slight stereoselective degradation of the two enantiomers was only observed in cabbage, with enantiomeric fraction (EF) = 0.46, and there was no enantioselectivity in the other vegetables. The degradation of napropamide in the five matrixes was fast, and there was no enantioselectivity.

Enantioselective bioaccumulation and dissipation of soil-associated metalaxyl enantiomers in tubifex.

Many pesticides are chiral compounds and stereochemistry is an important factor for any reaction of chiral structures in biological systems. In this study, experiment about bioaccumulation of the two metalaxyl enantiomers in Tubifex (Oligochaeta, Tubificida) was conducted in laboratory aquatic ecosystems. Terrestrial soil spiked with two dose levels of metalaxyl was employed as the artificial bottom substrate. A method of determination of metalaxyl enantiomers in tubifex tissue, soil and overlying water were developed by HPLC. During a 14-day exposure, concentrations of metalaxyl in tubifex increased with the of soil concentration, however, the enantioselective bioaccumulation was only detected at high-dose exposure group, with the preferential accumulation of (-)-(R)-metalaxyl. The bioturbation activity of tubifex decreased water clarity and released soil-associated metalaxyl to overlying water. In those experiments where tubifex was exposed to metalaxyl from soil, pore water and overlying water, each route contributed to the total body burden, and our results indicated the pore water and soil are the primary exposure routes for high-dose exposure concentration treatment.

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.

Toxic mechanisms of imazalil, azoxystrobin and their mixture to hook snout carp (Opsariichthys bidens).

While combinations of pesticides better represent actual conditions within aquatic ecosystems, the specific toxic effects of these combinations have not been determined yet. The objective of this research was to assess the combined impact of imazalil and azoxystrobin on the hook snout carp (Opsariichthys bidens) and delve into the underlying causes. Our findings indicated that the 4-day LC50 value for imazalil (1.85 mg L-1) was greater than that for azoxystrobin (0.90 mg L-1). When imazalil and azoxystrobin were combined, they presented a heightened effect on the species. Enzyme activities like SOD, CAT, GST, and CarE, along with androgen and estrogen levels, displayed marked differences in most single and combined treatments in comparison to the baseline group. Moreover, four genes (mn-sod, cu-sod, il-1, and esr) related to oxidative stress, immunity, and the endocrine system exhibited more pronounced expression changes when exposed to combined pesticides rather than individual ones. Our tests revealed that the combined use of imazalil and azoxystrobin had more detrimental effect on aquatic vertebrates than when evaluated individually. This finding suggested that future ecological hazard analyses based only on individual tests might not sufficiently safeguard our aquatic ecosystems.