[Determination of 14 perfluoroalkyl substances in Chinese mitten crab by multi-plug filtration cleanup coupled with ultra-performance liquid chromatography-tandem mass spectrometry].

Perfluoroalkyl substances (PFASs) have become a new food-safety problem. Dietary intake is a major pathway of human exposure to PFASs. Chinese mitten crab (Eriocheir sinensis) is a high-end aquaculture product popular among consumers in China. Conventional extraction methods for PFASs are cumbersome and time consuming, and result in incomplete purification; thus, this technique does not meet the requirements for PFAS detection. Herein, an analytical strategy was established for the rapid detection of 14 PFASs in Chinese mitten crab based on multi-plug filtration cleanup (m-PFC) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The carbon-chain length of the 14 PFASs analyzed in this study ranged from 4 to 14, and they are perfluorobutanoic acid (PFBA), perfluoro-n-pentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), perfluorotetradecanoic acid (PFTeDA), perfluoro-1-butane sulfonic acid (PFBS), perfluoro-1-hexane sulfonic acid (PFHxS), perfluoro-1-octane sulfonic acid (PFOS), and perfluoro-1-decanesulfonate (PFDS). The m-PFC column was prepared using carboxy-based multiwalled carbon nanotubes, and used to reduce the interference of sample impurities. The samples were extracted with 5 mL of 0.1% formic acid aqueous solution, 15 mL of acetonitrile and extraction salt (2 g Na2SO4 and 2 g NaCl). The supernatant (10 mL) was purified using the m-PFC column, concentrated to near dryness under nitrogen, and then redissolved in 1 mL of methanol. Finally, the sample solution was filtered through a 0.22 µm polypropylene syringe filter for UPLC-MS/MS analysis. The target analytes were separated using a Shimadzu Shim-pack G1ST-C18 chromatographic column (100 mm×2.1 mm, 2 µm) using methanol (A) and 5 mmol/L ammonium acetate aqueous solution (B) as the mobile phases via gradient elution. The linear gradient program were as follows: 0-0.5 min, 10%A-35%A; 0.5-3 min, 35%A-60%A; 3-5 min, 60%A-100%A; 5-6.5 min, 100%A; 6.5-7 min, 100%A-10%A. The target analytes were analyzed using negative electrospray ionization in multiple-reaction monitoring mode, and quantitative analysis was performed using the internal standard method. In this study, we optimized the mobile-phase system as well as the extraction solvent, time, volume, and salt. The 14 PFASs exhibited good peak shapes and sensitivities when the 5 mmol/L ammonium acetate solution-methanol system was used as the mobile phase. Compared with acetonitrile or methanol alone, the extraction efficiencies of the 14 PFASs were significantly improved when 5 mL of 0.1% formic acid aqueous solution was added, followed by 15 mL of acetonitrile. The extraction efficiencies of the 14 PFASs did not differ significantly when the extraction time was within 3-15 min. The extraction salt (MgSO4, Na2SO4, NaCl, (NH4)2SO4, and Na2SO4+NaCl) significantly affected the extraction efficiencies of the 14 PFASs. The highest extraction efficiencies of the 14 PFASs, which ranged from 47.9% to 121.9%, were obtained when Na2SO4+NaCl was used as the extraction salt. Under the optimal experimental conditions, good linearities (R2=0.998-0.999) were obtained for seven PFASs (PFBS, PFHxA, PFHpA, PFHxS, PFDA, PFDoDA, PFTeDA) at 0.10-100 µg/L, and seven PFASs (PFBA, PFPeA, PFOA, PFOS, PFNA, PFUnDA, PFDS) at 0.50-100 µg/L. The average spiked recoveries for the 14 PFASs in Chinese mitten crabs at three levels ranged from 73.1% to 120%, with relative standard deviations (RSDs) in the range of 1.68%-19.5%(n=6). The limits of detection (LODs) and quantification (LOQs) of the 14 PFASs were in the range of 0.03-0.15 and 0.10-0.50 µg/kg, respectively. The developed method was applied to the analysis of crab samples collected from three farms in Shanghai, and PFASs with total concentrations of 3.52-37.77 µg/kg were detected in all samples. The detection frequencies for PFDA, PFUnDA, PFDoDA, PFTeDA, and PFOS were 100%. PFDA, PFUnDA, PFOS, and PFDoDA were the most abundant congeners, accounting for 31.2%, 30.6%, 15.0%, and 10.9%, respectively, of the 14 PFASs detected. The proposed method is simple, efficient, accurate, and suitable for the rapid analysis of 14 PFASs in Chinese mitten crabs.

Determination of 25 polybrominated diphenyl ethers in Chinese mitten crab ecosystems by gas chromatography-mass spectrometry.

A sensitive and reliable method for determining 25 polybrominated diphenyl ethers (PBDEs) in Chinese mitten crabs and their ecosystems ranging from the growing environment to edible feed by gas chromatography coupled to triple quadrupole mass spectrometry with advanced electron ionization (GC-AEI-MS/MS) was developed and validated. Accelerated solvent extraction (ASE) and liquid-liquid extraction were used to extract solid and water samples, respectively. On the basis of a traditional acid-base silica column, deactivated silica was added and n-hexane elution was used to increase the effect of separation and purification. Two oven temperature programs were applied to achieve good separation of low brominated congeners and increase the sensitivity of high brominated congeners. The method provided good linearity (>0.9996). The recoveries of four matrices were in the range of 82-115% and the method quantification limits (MQLs) in crabs, feed, sediment and water ranged from 0.36-6 pg per g wet weight, 0.69-22.29 pg per g dry weight, 1.02-25.26 pg per g dry weight, and 2.43-40.14 pg L-1, respectively. The proposed method was used for ten samples from two aquatic sites and PBDEs were detected in Chinese mitten crabs, commercial feed and sediment, with the highest in crabs. This analytical technique can be used to monitor the content and the accumulation behavior of PBDEs in Chinese mitten crab ecosystems or other aquaculture systems.

Effects of Thiamethoxam and Fenvalerate Residue Levels on Light-Stable Isotopes of Leafy Vegetables.

Accurate identification of the rational and standardized use of pesticides is important for the sustainable development of agriculture while maintaining a high quality. The insecticides thiamethoxam and fenvalerate and the vegetables spinach, cabbage, and lettuce were used here as study objects. Descriptive analysis and primary reaction kinetic equations were used to analyze the changes in metabolic residues of the two insecticides after different numbers of application in three vegetables. The effects of pesticide residue levels on the δ13C, δ15N, δ2H, and δ18O values of vegetables were analyzed by one-way analysis of variance and correlation analysis. Partial least squares discriminant analysis (PLS-DA) was applied to build discrimination models of the vegetables with different pesticide residues based on stable isotopes. The results showed that the first degradation residues of thiamethoxam and fenvalerate in spinach, cabbage, and lettuce conformed to primary reaction kinetic equations, but the degradation half-lives were long, and accumulation occurred in the second application. The differences in the four stable isotope ratios in the control group of the three vegetables were statistically significant, and two-thirds of the stable isotope ratios in the three vegetables with different numbers of pesticide applications were significantly different. The δ13C and δ15N values of spinach, the δ13C, δ15N, and δ2H values of cabbage, and the δ13C, δ15N, δ2H, and δ18O values of lettuce were significantly correlated with different residues of thiamethoxam and/or fenvalerate applications. The control groups of the three vegetables, spinach-thiamethoxam-first, spinach-thiamethoxam-second, cabbage-thiamethoxam-second, cabbage-fenvalerate-first, and lettuce-thiamethoxam-first, were fully identified by PLS-DA models, while the identification models of other vegetables containing pesticide residues still need to be further improved. The results provide technical support for identifying the rational use of pesticides in vegetables and provide a reference method for guaranteeing the authenticity of green and organic vegetables.

Dissipation, Bioconcentration and Dietary Risk Assessment of Thiamethoxam and Its Metabolites in Agaricus bisporus and Substrates under Different Application Methods.

In order to acquire scientific evidence for the application of thiamethoxam (TMX) in Agaricus bisporus cultivation, residue and dissipation experiments for field trials were performed with the application of TMX in compost and casing soil, respectively. An effective QuEChERS method was established to analyze TMX and its two metabolites, clothianidin (CLO) and thiamethoxam-urea (TMX-urea), in compost, casing soil, and fruiting bodies. The results indicated that the TMX dissipation half-lives (t1/2) at dosages of 10 and 50 mg kg-1 were 19.74 d (day) and 28.87 d in compost and 33.54 d and 42.59 d in casing soil, individually. TMX, CLO, and TMX-urea were observed after TMX application in compost and casing soil. For TMX applied to the casing soil, only TMX residues were detected in fruiting bodies with bioconcentration factors (BCFs) of 0.0003~0.0009. In addition, both the chronic risk quotient (RQ) and acute risk quotient (HQ) values of TMX in fruiting bodies were far less than 1, which means the dietary health risks to humans were acceptable. However, in the TMX application to the compost, these analytes were not detected in the fruiting bodies. This suggested that the application of TMX in compost was safer than in casing soil during A. bisporus cultivation.

Toxic effects and transcriptional responses in zebrafish liver cells following perfluorooctanoic acid exposure.

As a typical type of persistent organic pollutant, perfluorooctanoic acid (PFOA) is pervasive in the environment. Multiple studies have found that PFOA has hepatotoxicity, but the mechanism remains poorly understood. In this study, the toxic effects of different concentrations of PFOA on zebrafish liver cells were systematically assessed by recording cell survival, ultrastructural observations, and transcriptome analyses. The results showed that the inhibition of cell viability and the massive accumulation of autophagic vacuoles were observed at 400 µM PFOA, while transcriptomic changes occurred with treatments of 1 and 400 µM PFOA. The transcription levels of 1055 (977 up- and 78 down-regulated genes) and 520 (446 up- and 74 down-regulated genes) genes were significantly changed after treatment with 1 and 400 µM PFOA, respectively. Based on Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, significant expression changes were observed in autophagy, tight junction, signal transduction, immune system, endocrine system, and metabolism-related pathways, indicating that such processes were greatly affected by PFOA exposure. The findings of this study will provide a scientific basis for the toxic effects and potential toxic mechanisms of PFOA on zebrafish, and provide information for ecological risk assessments.

Efficient Removal of Nonylphenol Contamination from Water Using Optimized Magnesium Silicate.

Nonylphenol (NP) is considered to be an environmentally toxic, endocrine-disrupting chemical that affects humans and ecosystems. Adsorption is one of the most promising approaches for the removal of nonylphenol contamination from water. Herein, in order to design an adsorbent with high adsorption capacity, magnesium silicate with different Mg/Si ratios was successfully synthesized by a sol-gel method at 60 °C. Magnesium silicate with a Mg/Si ratio of 1:6 was found to possess the best adsorption performance, with maximum 4-NP sorption 30.84 mg/g under 25 °C and 0.2 g/L adsorbent dose. The adsorption was negatively affected by increasing adsorbent dose and temperature. The kinetics and isotherm of 4-NP adsorption by Mg/Si were well described by the pseudo-second-order and Sips model, respectively, and behavior was proven to be physisorption-enhanced by a chemical effect. Detailed characterization by XRD, BET, and SEM confirmed that the magnesium silicate possesses an amorphous, mesoporous structure. The study will contribute to the applicability of cheap magnesium silicate for removal of NP contamination in water.

New insights into the transfer and accumulation of dioxins and dioxin-like PCBs in the food web of farmed Chinese mitten crabs: A typical case from the Yangtze River area.

Dioxins and dioxin-like polychlorinated biphenyls (DL-PCBs) transfer and accumulation behavior remains poorly understood in the farmed Chinese mitten crab (Eriocheir sinensis). In this study, dioxins and DL-PCBs concentration in 48 farming crabs in lower reaches of the Yangtze River was monitored and controlled field design was conducted in a typical farm to dissect the dioxins and DL-PCBs contamination in crab food web (crab, feeds, and environment). Results showed that dioxins and DL-PCBs were ubiquitous in farmed crabs with concentrations ranging from 0.390 to 37.2 pg toxic equivalents (TEQ) g-1 ww and do not present a health risk to general consumers. Of the total dioxins TEQ found in crab in treated farms, 45.6% was attributed to direct transfer from the aquaculture environment and 46.5% to the consumption of snails. Consumption of feed material accounted for nearly all of the total DL-PCBs TEQ, divided as 58.2% from feed and 41.8% from snails. These results demonstrated that dominant routes of dioxins accumulation in crabs were transferred for the sediment-snail-crab and sediment-crab chains, whereas DL-PCBs is mainly transferred through consumption of feeds and snails. To our knowledge, this work is the first report of snails serving as a biomagnification medium that promotes accumulation of dioxins in mitten crabs. This observation provided crucial insight to prevent and reduce contamination of crab by dioxins and DL-PCBs.

The immunotoxicity of decabromodiphenyl ether (BDE-209) on broiler chicks by transcriptome profiling analysis.

Decabromodiphenyl ether (BDE-209) has drawn significant attention due to its suppression of immune functions in animals and even humans. In order to explore the mechanism through which BDE-209 affects the immune system, broiler chicks were fed a diet containing various concentrations of BDE-209 (0, 0.004, 0.04, 0.4, and 4 g/kg) for 42 days. Histopathological observations of immune organs found damaged and necrotic lymphocytes in the spleen and bursa, and losses of lymphoid cells in thymic gland. The activities of catalase, glutathione, glutathione peroxidase, and superoxide dismutase in both the spleen and serum were affected by BDE-209. Obvious bioaccumulation effect was found in spleen tissues (high to 1339 ± 181.9 µg/kg). Furthermore, transcriptome sequencing analyses of the spleen identified 424 upregulated and 301 downregulated DEGs, and the cytokine-cytokine receptor interaction signal pathway was most significantly enriched based on the Kyoto Encyclopedia of Genes and Genomes database. Quantitative real-time PCR affirmed the decreased expressions of interleukin IL18, IL18R1, IL18RAP, IL21, as well as interferon gamma IFNG and tumor necrosis factor superfamily members TNFSF8, indicating significant interference to immunomodulation function and possible disease progression in inflammatory effects resulting from BDE-209 exposure. The immunotoxicity of BDE-209 may cause the suppression of immune and physiological functions of spleen cells, leading to inflammation and apoptosis and ultimately spleen atrophy.

Study on the Dynamic Difference between Single and Mixed Residues of Three Neonicotinoids in Brassica chinensis L.

Multiple insecticides' residues after the mixed application of several neonicotinoids cause combined pollution and bring new challenges to food safety and pest control during agricultural production. In this study, three neonicotinoid insecticides, namely imidacloprid (IMI), acetamiprid (ACE), and thiamethoxam (TMX), were mixed and evenly sprayed on Brassica chinensis L. in the field. Then, the insecticides' residues were dynamically monitored to determine the differences in their rates of dissipation and final residues after 10 days. The results showed that the dissipation kinetics of neonicotinoids still conformed to the first-order kinetic model for binary or ternary application of neonicotinoid mixtures, with all determination coefficients (R2) being above 0.9 and the dissipation half-life (DT50) being 2.87-6.74 d. For treatment groups with five times the recommended dosages (IMI 300 g·hm-2, ACE 900 g·hm-2, and TMX 600 g·hm-2), mixed insecticides had a slower dissipation rate, and the DT50 values of mixtures were longer than those of single insecticides. Moreover, the final insecticide residues with mixed application were higher than those of single compounds at 10 d after spraying. Thus, mixed applications of neonicotinoids may increase food safety risks as they increase the final insecticide residues in Brassica chinensis L., and care should therefore be taken when considering the combined use of such compounds.

Determination of Polybrominated Diphenyl Ethers in Water Samples Using Effervescent-Assisted Dispersive Liquid-Liquid Icroextraction with Solidification of the Aqueous Phase.

An effective and sensitive method is necessary for the determination of polybrominated diphenyl ethers (PBDEs) pollutants in water. In this study, effervescent-assisted dispersive liquid-liquid microextraction with solidification of the aqueous phase (EA-DLLME-SAP), followed by Gas Chromatography-Tandem Mass Spectrometry (GC-MS-MS) quantitative analysis, was established for the preconcentration and determination of PBDEs in real environmental water samples. 1,1,2,2-Tetrachloroethane was used as the extractant and directly dispersed into the water phase of the aqueous samples with the aid of a large number of carbon dioxide bubbles generated via the acid-base reaction of acetic acid and sodium bicarbonate, which did not require the use of a dispersant during the extraction process. The key factors affecting the extraction recovery were optimized, and an internal standard was used for quantitative analysis, which gave good linearity ranges of 1-100 ng·L-1 (BDEs 28, 47, 99, and 100), 2-200 ng·L-1 (BDEs 153, 154, and 183) and 5-500 ng·L-1 (BDE 209) with limits of quantification in the range of 1.0-5.0 ng·L-1. The accuracy was verified with relative standard deviations < 8.5% observed in tap, lake, river and reservoir water samples with relative recoveries ranging from 67.2 to 102.6%. The presented method contributes to the determination of PBDEs in environmental water samples.

Isotope chemometrics determines farming methods and geographical origin of vegetables from Yangtze River Delta Region, China.

Shanghai city has encountered possible food fraud regarding the geographical mislabeling of vegetables for economic gain. A combination of δ13C, δ15N, δ2H and δ18O values and partial least squares discrimination analysis and support vector machine (SVM) methods were used for the first time to assess farming methods and determine the origin of vegetables from Shanghai city, Anhui and Zhejiang provinces. The results showed that 65.8% of Shanghai vegetables, 38.2% of Anhui vegetables and 23.6% of Zhejiang vegetables appeared to be grown using green or organic farming methods. The optimal discriminant model was obtained using SVM with a predictive accuracy of 100% for Shanghai vegetables. Zhejiang vegetables had a predictive accuracy of 91.7%, while it was difficult to distinguish Anhui vegetables from Shanghai or Zhejiang vegetables. Therefore, this study provided a useful method to identify vegetable farming methods and discriminate vegetables from Shanghai and Zhejiang.

Assessing the combined toxicity effects of three neonicotinoid pesticide mixtures on human neuroblastoma SK-N-SH and lepidopteran Sf-9 cells.

Neonicotinoid insecticides have been widely used in plant protection against pests worldwide. Generally, more than one neonicotinoids are detected in plants and foods, and such mixtures may show combined toxicity and increase the risk for both insects and higher organisms. In this study, the combined toxicity of imidacloprid (IM), acetamiprid (AC) and thiamethoxam (TH) was investigated using human neuroblastoma cell line (SK-N-SH) and lepidopteran cell line (Sf-9). Results showed that binary and ternary mixtures could enhance the inhibition of growth of both SK-N-SH and Sf-9 cells at low doses. In SK-N-SH cells, based on CompuSyn software analysis, all the mixtures of IM+AC, IM+TH, AC+TH and IM+AC+TH showed synergistic effects at concentrations < 50 mg/L, but IM+AC, IM+TH showed antagonistic effects at higher concentrations. For Sf-9 cells, all mixtures revealed synergistic effects at low concentrations (< 0.1 mg/L) except IM+AC, showing antagonism at higher concentrations (> 0.5 mg/L). The toxicity thresholds of mixtures denoted by BMDL10 values were all lower than those for single pesticides and the combined BMDL10 value of AC+TH was the lowest one. It is concluded that the co-occurrence of several neonicotinoid insecticides may enhance their toxicity and aggravate the health risk for both insects and human.

Imidacloprid dissipation, metabolism and accumulation in Agaricus bisporus fruits, casing soil and compost and dietary risk assessment.

An improved understanding of imidacloprid (IMI) metabolism and accumulation in casing soil or compost-mushroom systems will help to optimise the safe use of IMI for agricultural pest control in Agaricus bisporus cultivation. In this study, the dissipation, metabolites and accumulation of IMI in casing soil or compost-A. bisporus systems were investigated. The results show that the IMI dissipation half-lives at doses of 10 and 50 mg kg-1 were 65 d and 59 d in casing soil and 6.6 d and 6.2 d in compost, respectively during the cultivation period. Three IMI metabolites were observed in casing soil during cultivation of mushrooms. Urea-imidacloprid (IMI-urea) was a major metabolite, accounting for more than 59%. In compost, the main metabolite in the first 5 days was IMI-urea, from 5 to 10 days olefin-imidacloprid (IMI-ole) and 6-chloronicotinic acid (6-CNA) were both the most prominent compounds, and after 10 days 6-CNA alone. At 50 mg kg-1 dose, IMI and the metabolites (IMI-urea, IMI-ole) were detected in the fruiting body with IMI applied to casing soil and only the metabolite 6-CNA was detected in fruiting body with IMI applied to compost. The bio concentration factors (BCFs) of IMI-ole and IMI-urea were higher than of IMI and 6-CNA, and the BCFs had no obvious relationship with log Kow. Furthermore, the potential dietary risk of IMI in A. bisporus was acceptable when application rates in casing soil or compost was up to 50 mg kg-1. Our study supports a safe use of IMI as agricultural pest control in A. bisporus cultivation.

Efficacy of different dosages of ivermectin injectable against the Hypoderma spp. in yaks.

A chemotherapy trial was conducted to determine the lowest dosage of injectable preparation of ivermectin against Hypoderma spp. infestation in yaks in Tibetan areas in Tianzhu county, Gansu province, in northwest of China. One hundred and sixty yaks were randomly divided into four groups of 40 yaks for the trial. The first three groups were treated by subcutaneous injection in the neck with 0.1% ivermectin (respectively, 1, 5, 10 microg/kg body weight). The fourth group was not treated and considered as control group. All the experiments were performed in November 2000 and the animals were examined for the presence of warbles in the next March and May. The results indicated that there was no warbles found on the back of treated animal while third stage larvae were palpated on back of some of the yaks in control group. It is concluded that dosage of 1 microg/kg ivermectin injectable was sufficient to kill or stop development of larvae of Hypoderma spp. in naturally infected yaks if administrated in November.

Experiments on transmission of an unidentified Theileria sp. to small ruminants with Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum.

Experiments on the transmission of an unidentified Theileria sp. infective for small ruminants by Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum were carried out. Three Theileria-free batches of adult, larvae, and nymphs of laboratory reared H. qinghaiensis and Hy. a. anatolicum ticks were infected by feeding them on sheep infected with Theileria sp. The Theileria sp. was originally isolated from adult ticks of H. qinghaiensis, by inoculation of blood stabilates or tick transmission. H. qinghaiensis has been shown to be capable of transmitting the Theileria sp. infective for small ruminants transstadially to sheep and goats. The nymphs developed from the larvae engorged on the sheep infected with the parasite transmitted the pathogen to splenectomized sheep with prepatent periods of 30, 31 days, respectively; but the subsequent adult ticks of H. qinghaiensis derived from the nymphs did not transmit the pathogen to sheep. However, adults developed from the nymphs engorged on the sheep infected with the parasite transmitted the pathogen to sheep with prepatent periods of 24-27 days. The larvae, nymphs and adult ticks derived from female H. qinghaiensis ticks engorged on infected sheep were not able to transmit the parasite transovarially. The same experiments were done with Hy. a. anatolicum, but examination for presence of piroplasma of Theileria sp. from all animals were negative, demonstrating that Hy. a. anatolicum could not transmit the organism to sheep or goats.

Description of a new Babesia sp. infective for cattle in China.

A Babesia species has been identified and shown to be transmitted by Hyalomma anatolicum anatolicum in China. When larvae, nymphs and adults developed from engorged females H. a. anatolicum collected from cattle in Xinjiang province were infested onto the Babesia-free calves, piroplasms of Babesia sp. were seen in blood smears from cattle infested with nymphs (2 calves), but not from the calves infested with larvae (1 calf or adult ticks (2 calves). This Babesia sp. proved to be of low virulence, causing 3% parasites which lasted 3-4 days.

Transmission of an unidentified Theileria species to small ruminants by Haemaphysalis qinghaiensis ticks collected in the field.

The transmission of a recently identified Theileria species pathogenic for sheep and goats in northern China is described. Haemaphysalis qinghaiensis nymphs which had been collected from grass in epidemic areas were able to transmit this Theileria species to sheep. The pathogen was also transmitted to sheep and goats by three batches of adult ticks collected from grass, ticks collected when moving about on sheep and ticks which were found partially engorged on sheep or goats.