Combined effects of chlorpyrifos and cyfluthrin on neurobehavior and neurotransmitter levels in larval zebrafish.

Chlorpyrifos and cyfluthrin are insecticides commonly used in agriculture. The mixed residues of chlorpyrifos and cyfluthrin in the aquatic environment may have combined effects on nontarget species. Therefore, studying the combined toxic effects and mechanisms of pesticide mixtures is of great significance to environmental risk assessment. To evaluate the risk of combined exposure, we examined the effects of both compounds, separately and together, on motor activity, acetylcholinesterase (AChE) activity, and neurotransmitter levels in larval zebrafish. Chlorpyrifos exposure significantly reduced functional motor capacity (swim distance and velocity) and enhanced meandering, while cyfluthrin exposure alone had no significant effects on swim parameters. However, combined exposure significantly reduced total swimming distance and mean velocity and increased meandering. Both compounds alone and the combination significantly reduced AChE activity, and the combined effect was antagonistic. Combined exposure also significantly altered the concentrations of serotonin, serotonin precursors, and dopamine precursors, as well as concentrations of the amino acid neurotransmitters glycine, alanine, and aspartic acid. Combined exposure to chlorpyrifos and cyfluthrin exhibited distinct joint action modes in terms of neurobehavior, AChE activity, and neurotransmitter levels, thereby providing an experimental basis for assessing the combined exposure to chlorpyrifos and cyfluthrin's environmental risk.

Duplex Surface Enhanced Raman Scattering-Based Lateral Flow Immunosensor for the Low-Level Detection of Antibiotic Residues in Milk.

A duplex surface enhanced Raman scattering (SERS)-based lateral flow immunosensor was established for the simultaneous detection of two common antibiotic residues including tetracycline and penicillin in milk. The newly synthesized Au@Ag nanoparticles were labeled with different Raman molecules including 5,5-dithiobis-2-nitrobenzoic acid (DTNB) or 4-mercaptobenzoic acid (MBA), followed by the conjugation of anti-tetracycline monoclonal antibody or anti-penicillin receptor, forming two kinds of SERS nanoprobes. The two nanoprobes can recognize tetracycline-BSA and ampicillin-BSA, respectively, which facilitates the simultaneous detection of the two types of antibiotics on a single test line. After optimization, detection limits of tetracycline and penicillin as low as 0.015 ng/mL and 0.010 ng/mL, respectively, were achieved. These values were far below those of most of other documented bio-analytical approaches. Moreover, the spiking test demonstrates an excellent assay accuracy with recoveries of 88.8% to 111.3%, and satisfactory assay precision with relative standard deviation below 16%. Consequently, the results demonstrate that the SERS-based lateral flow immunosensor developed in this study has the advantages of excellent assay sensitivity and remarkable multiplexing capability, thus it will have great application potential in food safety monitoring.

Dietary Bacillus velezensis KNF-209 supplementation improves growth performance, enhances immunity, and promotes gut health in broilers.

This study aimed to investigate the effects of dietary Bacillus velezensis KNF-209 (BV-KNF-209) on the growth performance, immunity, and gut health of broilers. A total of 540 one-day-old male Cobb-500 broilers were randomly divided into 5 groups of 6 replicates with 18 broilers per replicate. Dietary treatments were corn-soybean meal basal diets supplemented with 0, 50, 100, 200, and 400 mg/kg BV-KNF-209 (CON, BV 50, BV 100, BV 200, and BV 400 groups, respectively) for 42 d. Compared with the CON group, the average daily gains (ADG) at 0 to 42 d in the BV 100 and BV 200 groups were significantly increased (P < 0.01), and the feed-to-gain (F:G) ratios were significantly decreased at 0 to 21 d (P < 0.01) and 0 to 42 d (P < 0.05). The BV 200 and BV 400 groups had higher serum immunoglobulin M (IgM) levels at d 21 and 42 (P < 0.05). The serum levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were significantly decreased in the BV 50, BV 100, and BV 200 groups at d 21 (P < 0.05), and serum IL-1ß and IL-6 levels were also reduced in the BV 100 and BV 200 groups at d 42 (P < 0.05). Meanwhile, increased interleukin-10 (IL-10) levels in the jejunal and ileal mucosa at d 42 were observed in the BV 100, BV 200, and BV 400 groups (P < 0.05), while the IL-1ß and IL-6 levels (P < 0.01) were decreased. The BV 200 and BV 400 groups showed significantly higher activities of lipase and trypsin (P < 0.05) in jejunal digesta as well as higher activities of amylase and trypsin (P < 0.01) in ileal digesta at d 42. The cecal acetic acid and propionic acid levels in the BV groups and lactic acid levels in the BV 50, BV 100, and BV 200 groups (P < 0.05) were significantly higher compared to those in the CON group. Overall, dietary BV-KNF-209 supplementation significantly improved broiler growth performance, an effect that may have been achieved by heightening immunity, increasing digestive enzyme activity, and raising intestinal short-chain fatty acids and lactic acid levels.

Determination of Zearalenone and Its Derivatives in Feed by Gas Chromatography-Mass Spectrometry with Immunoaffinity Column Cleanup and Isotope Dilution.

In this study, a gas chromatography-mass spectrometry (GC-MS) method was established for the determination of zearalenone and its five derivatives in feed, including zearalanone, α-zearalanol, ß-zearalanol, α-zearalenol, and ß-zearalenol. An effective immunoaffinity column was prepared for sample purification, which was followed by the silane derivatization of the eluate after an immunoaffinity chromatography analysis for target compounds by GC-MS. Matrix effects were corrected by an isotope internal standard of zearalenone in this method. The six analytes had a good linear relationship in the range of 2-500 ng/mL, and the correlation coefficients were all greater than 0.99. The limits of detection (LODs) and limits of quantification (LOQs) were less than 1.5 µg/kg and 5.0 µg/kg, respectively. The average spike recoveries for the six feed matrices ranged from 89.6% to 112.3% with relative standard deviations (RSDs) less than 12.6%. Twenty actual feed samples were analyzed using the established method, and at least one target was detected. The established GC-MS method was proven to be reliable and suitable for the determination of zearalenone and its derivatives in feed.

Immunoaffinity Cleanup and Isotope Dilution-Based Liquid Chromatography Tandem Mass Spectrometry for the Determination of Six Major Mycotoxins in Feed and Feedstuff.

In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of deoxynivalenol, aflatoxin B1, zearalenone, ochratoxin A, T-2 toxin and fumonisin B1 in feed and feedstuff was established. The sample was extracted with an acetonitrile-water mixture (60:40, v/v), purified by an immunoaffinity column, eluted with a methanol-acetic acid mixture (98:2, v/v), and reconstituted with a methanol-water mixture (50:50, v/v) after drying with nitrogen. Finally, the reconstituted solution was detected by LC-MS/MS and quantified by isotope internal standard method. The six mycotoxins had a good linear relationship in a certain concentration range, the correlation coefficients were all greater than 0.99, the limits of detection were between 0.075 and 1.5 µg·kg-1, and the limits of quantification were between 0.5 and 5 µg·kg-1. The average spike recoveries in the four feed matrices ranged from 84.2% to 117.1% with relative standard deviations less than 11.6%. Thirty-six actual feed samples were analyzed for mycotoxins, and at least one mycotoxin was detected in each sample. The proposed method is reliable and suitable for detecting common mycotoxins in feed samples.

Antagonistic effects and mechanisms of carbendazim and chlorpyrifos on the neurobehavior of larval zebrafish.

Residues from multiple pesticides are frequently detected on vegetables, which may produce combined toxicity not predicted by individual toxicity data. As these combined effects present additional dangers to food safety, we have compared individual to combined effects for a variety of pesticides. Carbendazim and chlorpyrifos are the two most commonly detected pesticides in vegetables, and previous studies reported that combined exposure results in synergistic developmental toxicity to zebrafish embryos. In this study, individual and combined effects on zebrafish motor activity were examined following individual and combined exposure to assess nervous system toxicity. Further, transcriptomics methods were used to identify potential molecular mechanisms for individual and combined toxicity. Carbendazim alone induced a disorganized swim pattern characterized by increased angular velocity, turn angle, meander, and acceleration during light-dark transition, while chlorpyrifos alone reduced average swim speed and light-dark acceleration. Combined treatment significantly reduced average swim velocity and total distance traveled. Combination indices indicated strong antagonism between compounds for average speed and light-dark acceleration. Transcriptomics (RNA-seq) showed that carbendazim significantly altered the expression of genes involved in antigen processing and presentation, apoptosis, autophagy, and metabolism, including ctslb, cyp7a1, hsp70l, and ugt1a1. Alternatively, chlorpyrifos significantly altered genes involved in various nervous system-related pathways, including glutamatergic, GABAergic, dopaminergic, and calcium signaling. Protein-protein interaction (PPI) network analysis suggested that chlorpyrifos significantly downregulated genes related to light transduction, resulting in decreased sensitivity to light-dark transitions, while antagonism mainly reflected divergent effects on phototransduction and retinol metabolism. Carbendazim had no significant effects on vision-related genes such as gnat1 and gngt1, while chlorpyrifos downregulated expression, an effect reversed by the combination. Comprehensive toxicity analyses must include joint effects of co-applied pesticides for enhanced food safety.

Antagonistic Effects of Enrofloxacin on Carbendazim-Induced Developmental Toxicity in Zebrafish Embryos.

Carbendazim (CAR) and enrofloxacin (ENF) are frequently detected in fruits and meat products, respectively. Since most people consume fruits, vegetables, and meat products, combined exposure is possible, necessitating further evaluation of toxic interactions. In this study, the developmental toxicity of separate and combined exposure was examined in zebrafish embryos. Carbendazim exposure at 0.79 mg/L and above significantly affected developmental parameters, while enrofloxacin alone had no substantial effects on these developmental parameters within the selected concentration range (0.10-0.40 mg/L). Surprisingly, ENF antagonized the CAR-evoked reduction in the 48 hpf (hours post-fertilization) hatching rate and the increases in the 96 hpf malformation and lethality rates. The results revealed that the antagonism might be associated with reciprocal effects of these compounds on metabolism-related genes, such as cyp7a1 and apoa1a. These results reveal a complex interaction between ENF and CAR on metabolic regulation during development and highlight the importance of combined assessment for agents with the potential for simultaneous exposure.

Dual near-infrared fluorescence-based lateral flow immunosensor for the detection of zearalenone and deoxynivalenol in maize.

A novel dual near-infrared fluorescence-based lateral flow immunosensor was developed to determine zearalenone and deoxynivalenol in maize. Two near-infrared dyes with distinct fluorescence characteristics were utilized to separately label the anti-zearalenone and anti-deoxynivalenol antibodies as detection reagents. The capture antigens zearalenone-BSA and deoxynivalenol-BSA were mixed and immobilized on the same test line of nitrocellulose membrane. This assay format facilitates simultaneous detection of the two mycotoxins on a single test line. After optimizing experimental parameters, the limits of detection for zearalenone and deoxynivalenol were as low as 0.55 µg/kg and 3.8 µg/kg in maize, respectively. The spiking experiment yielded recovery ratios ranging from 81.7% to 107.3% with coefficients of variation less than 14% demonstrating high assay accuracy and precision. Moreover, the actual sample analysis produced consistent results between this method and instrumental method. Therefore, the developed immunosensor can serve as an accurate and efficient approach for monitoring mycotoxins in agricultural products.