Sensitive colorimetric and fluorescence dual-mode detection of thiophanate-methyl based on spherical Fe3O4/GONRs composite nanozyme.

Pesticide detection based on nanozyme is largely limited in terms of the variety of pesticides. Herein, a spherical and well-dispersed Fe3O4/graphene oxide nanoribbons (Fe3O4/GONRs) composite nanozyme was applied to firstly develop an enzyme-free and sensitive colorimetric and fluorescence dual-mode detection of thiophanate-methyl (TM). The synthesized Fe3O4/GONRs possess excellent dual enzyme-like activities (peroxidase and catalase) and can catalyze H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) into oxidized TMB (oxTMB). We found that Fe3O4/GONRs can adsorb TM through the synergistic effect of multiple forces, thereby inhibiting the catalytic activities of nanozyme. This inhibition can modulate the transformation of TMB to oxTMB, producing dual responses of absorbance decrease (oxTMB) and fluorescence enhancement (TMB). The limits of detection (LODs) of TM were 28.1 ng/mL (colorimetric) and 8.81 ng/mL (fluorescence), respectively. Moreover, the developed method with the recoveries of 94.8-100.8% also exhibited a good potential application in the detection of pesticides residues in water and food samples.

Determination, dissipation dynamics, terminal residues and dietary risk assessment of thiophanate-methyl and its metabolite carbendazim in cowpeas collected from different locations in China under field conditions.

BACKGROUND: Thiophanate-methyl and its metabolite carbendazim are broad-spectrum fungicides used on many crops. The residues of these chemicals could result in potential environmental and human health problems. Therefore, investigations of the dissipation and residue behaviors of thiophanate-methyl and its metabolite carbendazim on cowpeas and associated dietary risk assessments are essential for the safety of agricultural products. RESULTS: A simple analytical approach using liquid chromatography with tandem mass spectrometry was developed and validated for the determination of thiophanate-methyl and carbendazim concentrations in cowpeas. Good linearity (R2 > 0.998) was obtained, and the recoveries and relative standard deviations were 80.0-104.7% and 1.4-5.2%, respectively. The dissipation rates of thiophanate-methyl, carbendazim and total carbendazim were high (half-lives of 1.61-2.46 days) and varied in the field cowpea samples because of the different weather conditions and planting patterns. Based on the definition of thiophanate-methyl, the terminal residues of total carbendazim in cowpea samples were below the maximum residue limits set by Japan for other legumes. The acute and chronic risk quotients of three analytes were 0.0-27.6% in cowpea samples gathered from all terminal residue treatments, which were below 100%. CONCLUSION: An optimized approach for detecting thiophanate-methyl and carbendazim in cowpeas was applied for the investigation of field-trial samples. The potential acute and chronic dietary risks of thiophanate-methyl, carbendazim and total carbendazim to the health of Chinese consumers were low. These results could guide the safe and proper use of thiophanate-methyl in cowpeas and offer data for the dietary risk assessment of thiophanate-methyl in cowpeas. © 2021 Society of Chemical Industry.

Substrate sterilization with thiophanate-methyl and its biodegradation to carbendazim in oyster mushroom (Pleurotus ostreatus var. florida).

Residue analysis to detect thiophanate-methyl and its primary metabolite (carbendazim) during oyster mushroom (Pleurotus ostreatus var. florida) cultivation was done for two consecutive years 2017 and 2018. Wheat straw substrate was chemically treated with different treatments of thiophate-methyl, viz, thiophanate-methyl 30 ppm + formalin 500 ppm (T1), thiophanate-methyl 40 ppm + formalin 500 ppm (T2), thiophanate-methyl 50 ppm + formalin 500 ppm (T3), thiophanate-methyl 60 ppm + formalin 500 ppm (T4), and formalin 500 ppm (T5 as control and recommended concentration), and utilized for cultivation of oyster mushroom. Treatments T3 and T4 exhibited significant difference in pH levels during both the trials. Minimum spawn run, pinhead formation, and fruit body formation time were recorded in treatments T3 and T4. Significantly higher biological efficiency (%) was recorded in treatments T3 and T4 as compared with all other treatments. No incidence of competitor molds was recorded in T3 and T4. Pesticide residue analysis for detection of thiophanate-methyl and its metabolite (carbendazim) was done in the fruit body produced in T3 and T4 treatments using liquid chromatography with tandem mass spectrometry method. No residue of thiophanate-methyl and carbendazim was detected at 50 ppm concentration of thiophanate-methyl during both the trials. However, in trial II, residue of carbendazim (5.39 µg/kg) was detected at 60 ppm. Based on the findings of the trials I and II, T3 (thiophanate-methyl 50 ppm + formalin 500 ppm) may be utilized for substrate sterilization for oyster mushroom cultivation and Pleurotus ostreatus var. florida could be recognized as microorganism which could play a role in degradation of thiophanate-methyl.

Analysis of the dissipation kinetics of thiophanate-methyl and its metabolite carbendazim in apple leaves using a modified QuEChERS-UPLC-MS/MS method.

As one of the main fungicides for the apple leaf disease control, thiophanate-methyl (TM) mainly exerts its fungicidal activity in the form of its metabolite carbendazim (MBC), whose dissipation kinetics is very distinct from that of its parent but has been paid little attention. The aim of this work was to investigate the dissipation kinetics of TM and its active metabolite MBC in apple leaves using a modified QuEChERS-UPLC-MS/MS method. The results showed that TM and MBC could be quickly extracted by this modified QuEChERS procedure with recoveries of 81.7-96.5%. The method linearity was in the range of 0.01-50.0 mg kg-1 with the quantification limit of 0.01 mg kg-1 . Then this method was applied to the analysis of fungicide dissipation kinetics in apple leaves. The results showed that the dissipation kinetics of TM for the test in 3 months can be described by a first-order kinetics model with a DT50 (dissipation half-life) range of 5.23-6.03 days and the kinetics for MBC can be described by a first-order absorption-dissipation model with the Tmax (time needed to reach peak concentration) range of 4.78-7.09 days. These models can scientifically describe the behavior of TM and MBC in apple leaves, which provides necessary data for scientific application.

Guar gum-crosslinked-Soya lecithin nanohydrogel sheets as effective adsorbent for the removal of thiophanate methyl fungicide.

Rapid increase in use of fungicides for the agricultural and industrial purposes has marked the deterioration of water resources which ultimately affects the human life. Accordingly, various attempts have been made in the removal of these noxious compounds. In the same context, we are presenting biopolymers based nanohydrogel sheets; guar gum-crosslinked-Soya lecithin nanohydrogel sheets (GG-crosslinked-SY NHS) used for the effective removal of a fungicide; thiophanate methyl from aqueous solution. Guar gum and soya lecithin were employed as the biopolymers in the fabrication of nanohydrogel sheets due to their non- toxic nature, easy availability, cheapness and significant properties. Due to the presence of highly reactive functional groups onto the surface of GG-crosslinked-SY NHS, good adsorption results have been obtained. Maximum adsorption capacity of 59.205mg/g was observed with 20mg GG-crosslinked-SY NHS and 25ppm thiophanate methyl solution concentration as calculated from the Langmuir isotherm. Results showed that neutral pH favoured the adsorption process. Kinetics results were indicative of the physical interactions between the thiophanate methyl and GG-crosslinked-SY NHS surface. Thermodynamic results have shown the spontaneous and endothermic adsorption process.

Oxidative stress and histopathological changes induced by methylthiophanate, a systemic fungicide, in blood, liver and kidney of adult rats.

BACKGROUND: Methyl-thiophanate (MT), a fungicide largely used in agriculture throughout the world including Tunisia, protects many vegetables, fruits and field crops against a wide spectrum of fungal diseases. Oxidative stress has been proposed as a possible mechanism involved in MT toxicity on non-target organism. METHODS: In the present study, the effect of MT injected intraperitoneally to adult rats at 300 or 500 mg/kg of body weight was studied on blood, liver and kidney. RESULTS: Our results showed 3 days after MT injection, a significant decrease in hemoglobin and hematocrit values. A disruption in total white blood cells and platelets also occurred. Accordingly, an increased in malondialdehyde, H2O2 and advanced oxidation protein levels in liver and kidney were noted with the two doses. A significant change in plasma biomarkers and organ enzymatic and non-enzymatic activities were observed after MT treatment. The modifications in biochemical parameters were substantiated by histopathological data. CONCLUSION: These data confirmed the pro-oxidant effects of this fungicide. Accordingly, care must be taken to avoid mammalian and human exposure to MT.

Characterization of the ultraviolet-visible photoproducts of thiophanate-methyl using high performance liquid chromatography coupled with high resolution tandem mass spectrometry-Detection in grapes and tomatoes.

UV-visible irradiation of thiophanate-methyl (TM) led to the formation of nine photoproducts that were characterized by high performance liquid chromatography coupled with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Although carbendazime has been reported in the literature to be the major metabolite and photoproduct of thiophanate-methyl, it was not detected in this study. However, an isomer of carbendazime referred as PP2, which was unambiguously characterized owing to CID experiments, was found in great abundance. Grape berries and cherry tomatoes treated with aqueous solutions of thiophanate-methyl were submitted to irradiation under laboratory conditions. TM and PP2 were detected in both peel and flesh of berries. The ability of TM and PP2 to pass through the fruit skin has been shown to be highly compound and matrix dependent. In vitro bioassays on Vibrio fischeri bacteria showed that the global ecotoxicity of the TM solution increases significantly with the irradiation time. PP2 should likely contribute to this ecotoxicity enhancement since in silico estimations for Daphnia magna provide a LC50 value seven times lower for PP2 than for the parent molecule.

Methyl-thiophanate increases reactive oxygen species production and induces genotoxicity in rat peripheral blood.

Methylthiophanate is one of the widely used fungicides to control important fungal diseases of crops. The aim of this study was to elucidate the short-term hematoxicity and genotoxicity effects of methylthiophanate administered by intraperitoneal way at three doses (300, 500 and 700 mg/kg of body weight) after 24, 48 and 72 h. Our results showed, 24 h after methylthiophanate injection, a hematological perturbation such as red blood cells (p < 0.05, p < 0.05 and p < 0.01) and hemoglobin content (p < 0.05), respectively, and a noticeable genotoxic effect in WBC evidenced by a significant increase in the frequency of the micronuclei and a decrease in cell viability. An increase in erythrocyte osmotic fragility was also noted after 24 and 48 h of methylthiophanate treatment at graded doses. A significant increase in hydrogen peroxide, advanced oxidation of protein products and malondialdehyde levels, in erythrocytes of methylthiophanate-treated rats with 300, 500 and 700 mg/kg of body weight, was also observed after 24 h of treatment (p < 0.05, p < 0.01 and p < 0.001, respectively), suggesting the implication of oxidative stress in its toxicity. Antioxidants activities of superoxide dismutase and glutathione peroxidase in erythrocytes significantly increased (p < 0.001) 24 h after the highest dose injected. While all these parameters were improved after 72 h of methylthiophanate injection (300, 500 and 700 mg/kg body weight). In conclusion, these data showed that the exposure of adult rats to methylthiophanate resulted in oxidative stress leading to hematotoxicity and the impairment of defence system, confirming the pro-oxidant and genotoxic effects of this fungicide.

Spinach or amaranth may represent highest residue of thiophanate-methyl with open field application on six leaf vegetables.

To select representative crop among leaf vegetables which may contain the highest residue after fungicide uses, open field applications with thiophanate-methyl on six crops including pakchoi, rape, crown daisy, amaranth, spinach and lettuce were designed and conducted. In this study, a high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry with selected reaction monitoring was used to simultaneously determine thiophanate methyl and its metabolite carbendazim residue in various samples. The limit of quantification for thiophanate methyl and carbendazim were established in the range of 0.005-0.01 mg kg(-1) for all samples. It was shown that recoveries ranged from 67.8 % to 102.3 % for thiophanate methyl, and 72.0 %-112.6 % for carbendazim at spiked levels of 0.01-0.1 mg kg(-1). It's found that thiophanate methyl converts to carbendazim very quickly. In supervised field trials, the half-lives of thiophanate methyl in six leaf vegetables were in the range of 1.26-2.65 days, and the half-lives of carbendazim were in the range of 2.53-4.28 days. It was also found that thiophanate methyl residue in spinach and amaranth was higher than others after application. It's recommended that spinach or amaranth can be selected as representative crop in leaf vegetables in study of systemic fungicides with similarity as thiophanate methyl.

Rational coformer or solvent selection for pharmaceutical cocrystallization or desolvation.

It is demonstrated that the fluid-phase thermodynamics theory conductor-like screening model for real solvents (COSMO-RS) as implemented in the COSMOtherm software can be used for accurate and efficient screening of coformers for active pharmaceutical ingredient (API) cocrystallization. The excess enthalpy, H(ex) , between an API-coformer mixture relative to the pure components reflects the tendency of those two compounds to cocrystallize. Thus, predictive calculations may be performed with decent effort on a large set of molecular data in order to identify potentially new cocrystal systems. In addition, it is demonstrated that COSMO-RS theory allows reasonable ranking of coformers for API solubility improvement. As a result, experiments may be focused on those coformers, which have an increased probability of cocrystallization, leading to the largest improvement of the API solubility. In a similar way as potential coformers are identified for cocrystallization, solvents that do not tend to form solvates may be determined based on the highest H(ex) s with the API. The approach was successfully tested on tyrosine kinase inhibitor axitinib, which has a propensity to form relatively stable solvated structures with the majority of common solvents, as well as on thiophanate-methyl and thiophanate-ethyl benzimidazole fungicides, which form channel solvates.

Optical spectroscopy studies of the interaction between thiophanate methyl and human serum albumin for biosensor applications.

Optical properties of the interaction between thiophanate methyl and human serum albumin have been investigated for biosensor applications. The interaction between human serum albumin (HSA) and thiophanate methyl (MT) was investigated by UV-Vis absorption spectra and atomic force microscopy. The optical constants (refractive index, absorption index, band gap and dielectric properties) of HSA, MT and MT+HSA films were determined using absorbance, transmittance and reflectance spectra. The refractive index dispersion curve (>530 nm) exhibits the normal dispersion. The refractive index of the MT+HSA is higher than both HSA and MT alone due to the highest reflectance of the mixture of MT and HSA. This behavior is indicative of the complex formation between the MT and HSA.

Biodegradation kinetics of the benzimidazole fungicide thiophanate-methyl by bacteria isolated from loamy sand soil.

Degradation of the fungicide thiophanate-methyl (TM) by Enterobacter sp. TDS-1 and Bacillus sp. TDS-2 isolated from sandy soil previously treated with TM was studied in mineral salt medium (MSM) and soil. Both strains were able to grow in MSM supplemented with TM (50 mg l(-1)) as the sole carbon source. Over a 16 days incubation period, 60 and 77% of the initial dose of TM were degraded by strains TDS-1 and TDS-2, respectively, and disappearance of TM was described by first-order kinetics. Medium supplementation with glucose markedly stimulated bacterial growth; while the final rate of TM degradation was reduced by 21 and 27% for strains TDS-1 and TDS-2, respectively as compared to medium with TM only. Moreover, this additional carbon source changed the TM degradation kinetics, which proceeded according to a zero-order model. This effect was linked to substrate competition and/or a strong decrease of medium pH. Isolates degraded TM (100 mg kg(-1)) in soil with rate constants of 0.186 and 0.210 day(-1), following first-order rate kinetics, and the time in which the initial TM concentration was reduced by 50% (DT50) in soils inoculated with strains TDS-1 and TDS-2 were 6.3 and 5.1 days, respectively. Analysis of TM degradation products in soil showed that the tested strains may have the potential to transform carbendazim (MBC) to 2-aminobenzimidazole (2-AB), and may be useful for a bioremediation of MBC-polluted soils.

Fungicide methyl thiophanate binding at sub-domain IIA of human serum albumin triggers conformational change and protein damage.

Fluorescence quenching data on interaction of a fungicide methyl thiophanate (MT) with human serum albumin (HSA) elucidated a primary binding site at sub-domain IIA. Stern-Volmer algorithm and double log plot revealed the binding affinity (K(a)) and capacity (n) of HSA as 1.65 x 10(4)M(-1) and 1.0 (r(2)=0.99), respectively. Cyclic voltammetric and circular dichroism (CD) studies reaffirmed MT-HSA binding and demonstrated reduction in alpha-helical content of HSA. Substantial release of the carbonyl and acid-soluble amino groups from MT treated HSA suggested protein damage. The plausible mechanism of methyl ((+)CH(3)) group transfer from MT to side chain NH group of tryptophan and HSA degradation elucidates the toxicological and clinical implications of this fungicide.

Methyl thiophanate as a DNA minor groove binder produces MT-Cu(II)-DNA ternary complex preferably with AT rich region for initiation of DNA damage.

Interaction of a genotoxic fungicide methyl thiophanate (MT) has been studied in vitro with calf thymus DNA. Fluorescence quenching data revealed the binding constant (K(a)=3.23 x 10(4)M(-1)) and binding capacity (n=1.1) of MT with ctDNA. Ligand displacement studies using specific probes suggested the MT binding at DNA minor groove. The docking analysis further substantiated MT interaction with at least three AT base pairs within the DNA groove. A discernable change in E(0)' value with decreased peak currents in cyclic voltammogram, and peak shifts in CD spectra reflected the formation of MT-ctDNA and MT-ctDNA-Cu(II) complexes. The results elucidate the significance of specific MT-DNA interactions as an initiating event in MT-induced DNA damage.

Assessment of methyl thiophanate-Cu (II) induced DNA damage in human lymphocytes.

Dimethyl 4,4'-(O-phenylene)bis(3-thioallophanate), commonly known as methyl thiophanate (MT), is a category-III acute toxicant and suspected carcinogen to humans. Hence, the ability of this benzimidazole class of fungicide to engender DNA strand breaks was investigated using alkaline single cell gel electrophoresis (SCGE), alkaline unwinding and cytokinesis-blocked micronucleus (CBMN) assays. The SCGE of human lymphocytes treated with 1mM MT for 3h at 37 degrees C showed much higher Olive tail moment (OTM) value of 40.3+/-2.6 (p<0.001) vis-à-vis 3.3+/-0.09 in DMSO control. Treatment of cultured lymphocytes for 24h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). Stoichiometric data revealed the intrinsic property of MT to bind with Cu (II) and its reduction to Cu (I), which is known to form reactive oxygen species (ROS). We have detected the intracellular ROS generation in MT treated lymphocytes and observed an elevated level of MT-induced strand breaks per unit of calf thymus DNA in presence of Cu (II). Overall the data suggested that the formation of MT-Cu (II)-DNA ternary complex and consequent ROS generation, owing to Cu (II)/Cu (I) redox cycling in DNA proximity, is responsible for MT-induced DNA damage.

In vitro study on the interaction between thiophanate methyl and human serum albumin.

Thiophanate methyl (MT) is one of the widely used fungicides to control important fungal diseases of crops, which has led to potential toxicological risk to public health. Several different transport proteins exist in blood plasma, but albumin only is bound by a wide diversity of xenobiotics reversibly with high affinity. We studied the interaction of MT with human serum albumin by using spectroscopic methods including fluorescence quenching technology, UV and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The result of fluorescence titration revealed that MT could quench the intrinsic fluorescence of HSA. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses. In addition, the studies of FT-IR spectroscopy showed that the binding of MT to HSA changed molecular conformation of HSA. The results obtained from molecular modeling showed that the interaction between MT and HSA was dominated by hydrophobic force, and there was also hydrogen bond interaction between the pesticide and the residues of HSA, which was in good agreement with the result of binding mode.

Determination of thiophanate methyl and carbendazim residues in vegetable samples using microwave-assisted extraction.

Microwave-assisted extraction (MAE) was carried out for the determination of the fungicides thiophanate methyl [1.2-alpha-(3-methoxycarbonyl-2-thioureido)benzene] and carbendazim (methyl benzimidazol-2-ylcarbamate) in vegetable samples. Two vegetable samples, cabbage and tomatoes, were fortified with the two pesticides and subjected to MAE followed by cleanup to remove co-extractives prior to analysis by high-performance liquid chromatography (HPLC). Using the selected microwave exposure time and power setting, the recoveries of carbendazim ranged from 69 to 75%. But thiophanate methyl could not be recovered as the parent compound. It was converted and recovered as carbendazim. The conversion was quantitative as confirmed by high-performance liquid chromatography-mass spectrometry (HPLC-MS).