Bifenazate, a potent acaricide that targets mitochondrial complex III, exhibits selective toxicity (>280-fold) toward phytophagous mites versus predatory mites. Here, a systematic study was conducted to clarify the selective mechanism. Nontarget factors were excluded through epidermal penetration tests and assessment of detoxification enzymes\' activities. Quantification of IC50 values, ATP content, and reactive oxygen species (ROS) levels revealed that differences in drug-target binding determine the toxicity selectivity. Structural modeling and molecular docking revealed that variations in key amino acid sites within the cytochrome b (cytb) target might regulate this selectivity, which was validated through a microscale thermophoresis assay. Significant disparities were observed in the binding affinity between bifenazate and recombinant cytb proteins derived from phytophagous mites and predatory mites. Mutating isoleucine 139 to leucine notably reduced the binding affinity of bifenazate to cytb. Insights into bifenazate selectivity between phytophagous and predatory mites inform a basis for developing compounds that target cytochrome b.

Acaricides used against Tetranychus urticae Koch, 1836 (Acari: Tetranychidae) in cotton fields cause control failure over time. To determine the resistance status of T. urticae populations to tebufenpyrad and bifenazate, different populations collected from Aydın (AYD), Adana (ADA), Şanlıurfa (SAN), and Diyarbakır (DIY) provinces of Türkiye, between 2019 and 2020, were subjected to diagnostic dose bioassays. Firstly, the spider mites were eliminated with a discriminating dose. Afterwards, LC50 and LC90 of the remaining populations were determined and the ten highest resistant populations were selected. The highest phenotypic resistance to bifenazate was observed in AYD4 and DIY2 (LC50 57.14 mg L- 1 with 85.01-fold and LC50 30.15 mg L- 1with 44.86-fold, respectively), while the lowest phenotypic resistance was found in SAN6 (LC50 1.5 mg L- 1; 2.28-fold). Considering the phenotypic resistance to tebufenpyrad, the highest resistance was found in AYD4 population (LC50 96.81 mg L- 1; 12.92-fold), while the lowest - in DIY28 population (LC50 21.23 mg L- 1; 2.83-fold). In pharmacokinetic studies, the ADA16 population was compared with the sensitive German Susceptible Strain population and it was determined that carboxylesterase activity was statistically higher (1.46 ± 0.04 nmol/min/mg protein enzyme activation 2.70-fold). The highest activation of glutathione S-transferase was detected in ADA16 (1.49 ± 0.01 nmol/min/mg protein; 2.32-fold). No mutations were found in PSST (METI 1), the point mutation site for tebufenpyrad, and Cytb (METI 3), the point mutation site for bifenazate. In terms of phenotypic resistance, bifenazate was found to be moderately resistant in two populations (85.01 and 44.86-fold), while tebufenpyrad was moderately resistant in one population (12.92-fold). This study showed that both acaricides are still effective against T. urticae populations.

Bifenazate is widely recognized as an effective acaricide for citrus production in various regions. Detecting both the parent compound of bifenazate and its metabolite, bifenazate-diazene, simultaneously can be challenging owing to their tendency to undergo chemical interconversion. Current methods developed for detecting bifenazate or bifenazate-diazene residues often involve lengthy incubation periods and may not effectively separate the two compounds. In this study, we developed a convenient and fast method based on a modified QuEChERS method assisted by oxidants to concurrently detect bifenazate and bifenazate-diazene. Based on preliminary analysis, it appears that ferric chloride has the ability to react with a reducing substance present in citrus, which may prevent the reduction of bifenazate-diazene. The method was validated and applied in a field trial. This work reports a novel strategy to establish a balanced \'neutral\' condition to create a potential method for efficient determination of bifenazate acaricide residues in fruit matrices.

Bifenazate is a novel acaricide that has been widely used to control spider mites. Interestingly, we found bifenazate had a biological activity against the diamondback moth (Plutella xylostella), one of the most economically important pests on crucifer crops around the world. However, the molecular mechanisms underlying the response of P. xylostella to bifenazate treatment are not clear. In this study, we first estimated the LC30 dose of bifenazate for third-instar P. xylostella larvae. Then, in order to identify genes that respond to the treatment of this insecticide, the comparative transcriptome profiles were used to analyze the gene expression changes in P. xylostella larvae after exposure to LC30 of bifenazate. In total, 757 differentially expressed genes (DEGs) between bifenazate-treated and control P. xylostella larvae were identified, in which 526 and 231 genes were up-regulated and down-regulated, respectively. The further Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the xenobiotics metabolisms pathway was significantly enriched, with ten detoxifying enzyme genes (four P450s, five glutathione S-transferases (GSTs), and one UDP-Glucuronosyltransferase (UGT)) were up-regulated, and their expression patterns were validated by qRT-PCR as well. Interestingly, the present results showed that 17 cuticular protein (CP) genes were also remarkably up-regulated, including 15 CPR family genes. Additionally, the oxidative phosphorylation pathway was found to be activated with eight mitochondrial genes up-regulated in bifenazate-treated larvae. In contrast, we found some genes that were involved in tyrosine metabolism and purine pathways were down-regulated, indicating these two pathways of bifenazate-exposed larvae were significantly inhibited. In conclusion, the present study would help us to better understand the molecular mechanisms of sublethal doses of bifenazate detoxification and action in P. xylostella.

The dissipation, conversion and risk assessment of bifenazate and bifenazate-diazene in garlic plant were studied by a modified QuEChERS method coupled with UHPLC-MS/MS for the first time. Bifenazate dissipated rapidly in garlic chive and serpent garlic with the half-lives of 3.0-3.9 days and 6.1-6.9 days, respectively. Bifenazate residue on garlic (<0.01 mg/kg) was significantly lower than the other two matrices in the whole growing period, which meant residues in the above-ground part were not transferred to the garlic. Furthermore, garlic chive had higher residues than serpent garlic due to the differences in morphological characteristics. Bifenazate-diazene was easier to convert to bifenazate, with the conversion rates of 93%, 16% and 32% in garlic, serpent garlic and garlic chive extracts, respectively. Additionally, the dietary intake risk for bifenazate was acceptable with RQchronic < 100% according to the international and national assessments.

A rapid, sensitive, and effective multiresidue analytical method was established to investigate the degradation rate and final residues of bifenthrin, bifenazate, and its metabolite bifenazate-diazene in apples, and the dietary risk of consumers was evaluated. The residues of bifenthrin, bifenazate, and bifenazate-diazene in apple samples from 12 different apple-producing areas of China were determined by high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS). The average recoveries of the three compounds in apples were 88.4-104.6%, and the relative standard deviations (RSDs) were 1.3-10.5%. The limit of quantification (LOQ) for each compound was 0.01 mg/kg. Although the degradation half-lives of bifenthrin, bifenazate, and bifenazate-diazene were 17.8-28.9, 4.3-7.8, and 5.0-5.8 days, under good agricultural practice (GAP) conditions, the final residues of bifenthrin, bifenazate, and the sum of bifenazate and its metabolite bifenazate-diazene in apples were <0.01-0.049, < 0.01-0.027, and <0.02-0.056 mg/kg, respectively, which were lower than the maximum residue limit (MRL) in China. By comparing the deterministic model with the probabilistic model, the results of the probabilistic model at the P95 level (12.91-48.9% for bifenthrin, 17.48-52.01% for bifenazate including its metabolite) were selected as reasonable assessment criteria for chronic dietary risk, and the acute risk was at the P99.9 level (3.00-15.59% for bifenthrin). Although the exposure risk calculated by both the deterministic model and the probabilistic model was less than 100%, the risk to children is significantly higher than that of the general population. This suggests that in future research and policy making, we should pay more attention to the risk of vulnerable groups such as children.

The conclusions of the EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Sweden, and co-rapporteur Member State, Italy, for the pesticide active substance bifenazate are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative uses of bifenazate as an acaricide on strawberry, fruiting vegetables (tomatoes, peppers, aubergines, cucumbers, courgettes, melons, watermelons), flowering and ornamental plants and nursery ornamentals and updated following the request to peer review the exposure and risk assessments for bifenazate. The reliable end points, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified.

The residues of bifenazate (sum of bifenazate and bifenazate-diazene) and etoxazole in whole citrus and pulp collected from twelve regions of China were monitored and their chronic dietary risk to consumer were also evaluated. The citrus samples were extracted by a QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, and analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The average recoveries of target compounds were ranged from 83 to 100% with relative standard deviations (RSDs) of 0.59-11.8%. The limits of quantification (LOQs) for three analytes were 0.01 mg/kg. At the interval to harvest of 20 and 30 days, the residues of total bifenazate and etoxazole were from below 0.02 to 0.26 mg/kg and from below 0.01 to 0.30 mg/kg in citrus samples. The chronic risk quotients (RQs) were below 100%, indicating no unacceptable risk to consumers.

Bifenazate is a novel acaricide for selective foliar spraying and is widely used to control mites in agricultural production. However, its toxicity to aquatic organisms is unknown. Here, a zebrafish model was used to study bifenazate toxicity to aquatic organisms. Exposure to bifenazate was found to cause severe cardiotoxicity in zebrafish embryos, along with disorders in the gene expression related to heart development. Bifenazate also caused oxidative stress. Cardiotoxicity caused by bifenazate was partially rescued by astaxanthin (an antioxidant), accompanied by cardiac genes and oxidative stress-related indicators becoming normalized. Our results showed that exposure to bifenazate can significantly change the ATPase activity and gene expression levels of the calcium signaling pathway. These led to heart failure, in which the blood accumulated outside the heart without entering it, eventually leading to death. The results indicated that bifenazate exposure caused cardiotoxicity in zebrafish embryos through the induction of oxidative stress and inhibition of the calcium signaling pathway.

Bifenazate is a widely used acaricide, but its biological safety remains unknown. In the present study, the immunotoxic effects of exposure to bifenazate on zebrafish larvae were evaluated for the first time. Firstly, after exposure to bifenazate, the body length of the zebrafish larvae became shorter and the yolk sac swelled. Secondly, the number of innate immune cells and adaptive immune cells was greatly reduced. Following exposure to bifenazate, oxidative stress levels in the zebrafish increased significantly, antioxidant activity was inhibited, and the expression of genes related to antioxidants, such as those of the glutathione metabolism pathway, changed, including gclm, prdx1, serpine1, and gss. In addition, inflammatory factors such as CXCL-c1c, IFN-γ, iL-8, iL-6, and MYD88 were abnormally expressed. The use of astaxanthin was effective in rescuing the developmental toxicity caused by bifenazate exposure. In summary, bifenazate exposure is immunotoxic and can cause oxidative stress in zebrafish larvae.