Phthalimide Fungicides Research Articles

Revealing the mechanistic interactions of profenofos and captan pesticides with serum protein via biophysical and computational investigations

Profenofos (PF) and captan (CT) are among the most utilized organophosphorus insecticides and phthalimide fungicides, respectively. To elucidate the physicochemical and influential toxicokinetic factors, the mechanistic interactions of serum albumin and either PF or CT were carried out in the current study using a series of spectroscopy and computational analyses. Both PF and CT could bind to bovine serum albumin (BSA), a representative serum protein, with moderate binding constants in a range of 103–104 M−1. The bindings of PF and CT did not induce noticeable BSA’s structural changes. Both pesticides bound preferentially to the site I pocket of BSA, where the hydrophobic interaction was the main binding mode of PF, and the electrostatic interaction drove the binding of CT. As a result, PF and CT may not only induce direct toxicity by themselves, but also compete with therapeutic drugs and essential substances to sit in the Sudlow site I of serum albumin, which may interfere with the pharmacokinetics and equilibrium of drugs and other substances causing consequent adverse effects.

Folpet promotes apoptosis of bovine mammary epithelial cells via disruption of redox homeostasis and activation of MAPK cascades.

Folpet, a phthalimide fungicide, is an agrochemical used to prevent fungal diseases in several crops. The toxicity of folpet has been demonstrated in Cyprinus carpio, pigs, and the human respiratory system. However, despite the possibilities of ingestion of folpet through feed, detrimental influences of folpet on dairy cattle have not been documented. Thus, this study aimed to record the harmful effects of folpet on the bovine mammary system and milk production using mammary epithelial cells (MAC-T cells), which play an essential role in the maintenance of yield and quality of milk production. In this study, we first confirmed that folpet exhibited cytotoxicity against MAC-T cells in both 2D and 3D cultures. Folpet treatment caused apoptosis, dysregulated intracellular calcium levels, and mitochondrial membrane potential, leading to cell death. We further demonstrated the induction of oxidative stress upon folpet treatment by assessing reactive oxygen species (ROS) content and lipid peroxidation in MAC-T cells. ROS generation following folpet treatment induced activation of MAPK cascades, including ERK1/2, JNK, and p38 signaling. This is the first report highlighting the detrimental impacts of folpet on bovine mammary glands and, consequently, the dairy industry by elucidating intracellular mechanisms using MAC-T cells.

Captan-induced increase in the concentrations of intracellular Ca2+ and Zn2+ and its correlation with oxidative stress in rat thymic lymphocytes

Captan, a phthalimide fungicide, is considered to be relatively nontoxic to mammals. There is a possibility that captan affects membrane and cellular parameters of mammalian cells, resulting in adverse effects, because of high residue levels. To test the possibility, we examined the effects of captan on rat thymic lymphocytes using flow-cytometry with appropriate fluorescent probes. Treatment with 10 and 30 μM captan induced apoptotic and necrotic cell death. Before cell death occurred, captan elevated the intracellular concentrations of Ca2+ and Zn2+ and decreased the concentration of cellular thiol compounds. These captan-induced phenomena are shown to cause cell death and are similar to those caused by oxidative stress. Captan also elevated the cytotoxicity of hydrogen peroxide. Results indicate that 10 and 30 μM captan cause cytotoxic effects on mammalian cells. Despite no report on the significant environmental toxicity hazard of captan in humans, it may exhibit adverse effects, described above, on wild organisms.

Necrotic and apoptotic cell death induced by Captan on Saccharomyces cerevisiae

Captan is one of the most widely used broad-spectrum fungicide applied to control several early and late diseases of grapes, apples, and other fruits and vegetables, and as other phthalimide fungicides is defined as a multisite compound with thiol-reactivity. Captan can affect non-target organisms as yeasts, modifying microbial populations and fermentation processes. In this study, we asked whether Captan thiol-reactivity and other mechanisms are involved in acute Captan-induced cell death on aerobic growing Saccharomyces cerevisiae. Thus for, we analyze cellular protein and non-protein thiols, cell membrane integrity, reactive oxygen species accumulation, phosphatidylserine externalization, and apoptotic mutants behavior. The results showed that when submitted to acute Captan treatment most cells lost their membrane integrity and died by necrosis due to Captan reaction with thiols. However, part of the cells, even maintaining their membrane integrity, lost their culture ability. These cells showed an apoptotic behavior that may be the result of non-protein thiol depletion and consequent increase of reactive oxygen species (ROS). ROS accumulation triggers a metacaspase-dependent apoptotic cascade, as shown by the higher viability of the yca1-deleted mutant. Together, necrosis and apoptosis are responsible for the high mortality detected after acute Captan treatment of aerobically growing cells of S. cerevisiae.

Captan (Pesticides).

Food Safety Commission of Japan (FSCJ) conducted a risk assessment of captan (CAS No. 133-06-2), a phthalimide fungicide, based on results from various studies. Major adverse effects of captan were observed in suppressed body weight, and also in duodenal mucosal hyperplasia in mice. No adverse effect on fertility was detected. Increases in incidence of duodenal adenoma and adenocarcinoma were identified in mice. Negative results were however obtained from a gene mutation assay of the target in transgenic mice. No genotoxicity relevant to human health of captan was recognized in spite of the positive results in vitro. Therefore, a genotoxic mechanism was unlikely involved in the tumor development, and it enabled us to establish a threshold in the assessment. In developmental toxicity studies, captan, at the doses causing maternal toxicity, increased external alterations as well as skeletal and soft tissue alterations in the fetus of rabbits and hamsters. No captan-induced teratogenicity was detected in rats. Captan (parent compound only) was identified as the residue definition for dietary risk assessment in agricultural and livestock products. The lowest no-observed-adverse-effect level (NOAEL) obtained from all the studies was 10 mg/kg bw/day. FSCJ specified an acceptable daily intake (ADI) of 0.1 mg/kg bw/day by applying a safety factor of 100 to the NOAEL. The lowest NOAEL for potential adverse effects of a single oral administration of captan was 30 mg/kg bw/day in a developmental toxicity study in rabbits. FSCJ specified an acute reference dose (ARfD) of 0.3 mg/kg bw, for women who are or may be pregnant, by applying a safety factor of 100 to the NOAEL. In addition, FSCJ specified an ARfD of 3 mg/kg bw, for general population, by applying a safety factor of 100 to the no-observed-effect level (NOEL) of 300 mg/kg bw obtained from a general pharmacology study in mice.

Folpet (Pesticides).

Food Safety Commission of Japan (FSCJ) conducted a risk assessment of folpet (CAS No. 133-07-3), a phthalimide fungicide, based on results from various studies. Major adverse effects of folpet were observed in hyper-keratosis in rats and in duodenal mucosal hyperplasia in mice. No neurotoxicity and adverse effect on fertility were detected. Increases in the incidence of duodenal adenoma and adenocarcinoma were identified in carcinogenicity studies in mice. FSCJ recognized no genotoxicity relevant to human health of folpet in spite of the positive results in vitro. Therefore, a genotoxic mechanism was unlikely involved in the tumor development, and it enabled us to establish a threshold in the assessment. In developmental toxicity studies, no adverse effects observed in fetus at the dose without maternal toxicity. No folpet-induced teratogenicity was detected in rats. Folpet (parent compound only) was identified as the residue definition for dietary risk assessment in agricultural products. The lowest no-observed-adverse-effect level (NOAEL) obtained in all the studies was 10 mg/kg bw/day in several studies in dogs, rats and rabbits. FSCJ specified an acceptable daily intake (ADI) of 0.1 mg/kg bw/day by applying a safety factor of 100 to the NOAEL. The lowest NOAEL for potential adverse effects of a single oral administration of folpet was 10 mg/kg bw/day in a developmental toxicity study in rabbits. FSCJ specified an acute reference dose (ARfD) of 0.1 mg/kg bw/day, for women who are or may be pregnant, by applying a safety factor of 100 to the NOAEL. FSCJ considered it unnecessary to specify ARfD for general population.

Impact of Fungicide Residues on Polymerase Chain Reaction and on Yeast Metabolism

ABSTRACT The indiscriminate use of pesticides on grape crops is harmful for consumers´ healthin “in natura” consumption and in the ingestion of wine and grape juice. During winemaking, a rapid and efficient fermentation stage is critical to avoid proliferation of contaminating microorganisms and to guarantee the product´s quality. Polymerase chain reaction (PCR) has the advantage of detecting these contaminants in the early stages of fermentation. However,this enzymatic reaction may also be susceptible to specific problems, reducing its efficiency. Agricultural practices, such as fungicide treatments, may be a source of PCR inhibiting factors and may also interfere in the normal course of fermentation.The action of the pesticides captan and folpet on PCR and on yeast metabolism was evaluated, once these phthalimide compounds are widely employed in Brazilian vineyards. DNA amplification was only observed at 75 and 37.5 µg/mL of captan concentrations, whereas with folpet, amplification was observed only in the two lowest concentrations tested (42.2 and 21.1µg/mL).Besides the strong inhibition on Taq polymerase activity, phthalimides also inhibited yeast metabolism at all concentrations analyzed.Grape must containing captan and folpet residues could not be transformed into wine due to stuck fermentation caused by the inhibition of yeast metabolism. Non-compliance with the waiting period for phthalimide fungicides may result in financial liabilities to the viticulture sector.The use of yeasts with high fungicide sensitivity should be selected for must fermentation as a strategy for sustainable wine production and to assure that products comply with health and food safety standards.

Quantitation of phenyl-amide and phthalimide fungicide formulations in solid-state via UV–MALDI mass spectrometry—matrix effects in soils

Purpose Quantitative analysis of fungicides metalaxyl-M, rac-metalaxyl (1), captafol (2), captan (3), folpet (4), metalaxyl acid (5), cis-1,2,3,6-tetrahydrophthalimide (6), cis-1,2,3,6-tetrahydrophthalamid acid (7), o-phthalic acid (8), and phthalamide (9), in sterilized and nonsterilized soil fractions.

A review of methods for the analysis of orphan and difficult pesticides: glyphosate, glufosinate, quaternary ammonium and phenoxy acid herbicides, and dithiocarbamate and phthalimide fungicides.

This article reviews the chromatography/MS methodologies for analysis of pesticide residues of orphan and difficult chemical classes in a variety of sample matrixes including water, urine, blood, and food. The review focuses on pesticide classes that are not commonly included in multiresidue analysis methods such as highly polar or ionic herbicides including glyphosate, glufosinate, quaternary ammonium, and phenoxy acid herbicides, and some of their major degradation or metabolite products. In addition, dithiocarbamate and phthalimide fungicides, which are thermally unstable and have stability issues in some solvents or sample matrixes, are also examined due to their special needs in residue analysis.

Bacterial Degradation of Fungicide Captan

The phthalimide fungicide captan has been widely used to control plant pathogenic fungi. A strain of Bacillus circulans utilized the fungicide captan as sole source of carbon and energy. The organism degraded captan by a pathway involving its initial hydrolysis to yield cis-1,2,3,6-tetrahydrophthalimide, a compound without fungicidal activity. The formation of this compound was confirmed by HPLC, IR, NMR, and mass spectral analysis. The results also revealed that cis-1,2,3,6-tetrahydrophthalimide was further degraded to o-phthalic acid by a protocatechuate pathway. These findings indicated that there was a complete mineralization of fungicide captan by B. circulans.

Determination of Captan in Water, Peaches, and Apple Juice by a Magnetic Particle-Based Immunoassay

Abstract A magnetic particle-based enzyme-linked immunosorbent assay (EUSA) was used to determine captan residues in water, peaches, and apple juice. Precision studies of captan-spiked water samples showed coefficients of variation (CVs) of 24% at 0.1 ppm and 15% at 2.0 ppm. Average recoveries from water were 110–118%, with an estimated sensitivity of 0.01 ppm for captan. Low cross-reactivity was seen with the related phthalimide fungicides capta-fol and folpet. A least-detectable dose (LDD) of 1 ppm was estimated for captafol in the immunoassay; an LDD of 8.6 ppm was estimated for folpet. Apple juice samples were analyzed by a 3-step procedure: the sample was concentrated on a Cis bonded silica solid-phase column, the eluate was diluted in buffer, and the sample was assayed by ELISA. The detection limit in apple juice was estimated to be 0.1 ppm on the basis of the detection limit observed in water and the required calculation factors; the average recovery was 115%. Acetone extraction, Cis bonded silica solid-phase extraction column, and the immunoassay were used consecutively to analyze peach samples. The detection limit in peaches was estimated to be 0.15 ppm on the basis of the detection limit observed in water and the required calculation factors. Captan residues were found in all but one of the peach samples and ranged from 0.15 to 1.12 ppm (|ig/g). Recovery of added captan from peaches averaged 98% for both spike levels tested. Results of analysis of captan in peaches by immunoassay and gas chromatography with electron capture detection (GC/ECD) were compared. GC/ECD gave an average recovery of 75%; immunoassay gave an average recovery of 112%.

Genetic effects of phthalimide fungicides on diploid Aspergillus nidulans

AbstractA few specimens of ‘Ridomil 25 WP’, a commercial formulation of metalaxyl, showed high genetic activity on a heterozygous diploid strain of Aspergillus nidulans. This activity was due to an impurity which was identified by mass and n.m.r. spectroscopy as captafol. This and the related fungicides captan, dichlofluanid, and tolyfluanid greatly increased the yield of euploid colour segregants from the diploid strain. At low captafol concentrations, most of the induced segregants resulted from mitotic crossing over but chromosome non‐disjunction or loss was also caused at high concentrations. Mitotic crossing over could be induced also by vapour‐phase action of captafol and captan. The mechanism by which these phthalimides may exert their genetic effects is discussed.

Evaluation of the genetic activity profiles of 65 pesticides

We have previously reported the qualitative results of a major study on 65 pesticides (Waters et al., 1982). Dose information from this investigation (either lowest effective or highest ineffective dose tested) has now been incorporated into a computerized data management system. This report focuses on the qualitative profiles of genetic activity produced by these pesticides and our efforts to classify them according to their genotoxic effects and chemical structures. Three main categories may be distinguished based on the qualitative results: Category 1 pesticides were active in most of the in vitro and in vivo assays employed. These 9 compounds include the structurally similar organophosphate insecticides, acephate, demeton, monocrotophos and trichlorfon; the phthalimide fungicide analogues, captan and folpet; and the thiocarbamate herbicide analogues, diallate, sulfallate and triallate. The 26 Category 2 compounds demonstrated fewer positive results and may be subdivided into two parts, one of which contains 12 halogenated aromatic or heterocyclic ring compounds, including the phenoxy herbicides, 2,4-D, 2,4-DB and 2,4,5-T. The remaining part of Category 2 (14 compounds) consists of structurally similar organophosphate insecticides, azinphos-methyl, crotoxyphos, disulfoton, methyl parathion; three similar ethylenebisdithiocarbamate fungicides, maneb, mancozeb, and zineb; three similar pyrethroid insecticides, allethrin, chrysanthemic acid, and ethyl chrysanthemate; and four structurally diverse compounds, cacodylic acid, dinoseb, sec.-butylamine and benomyl. The third category of 30 pesticides gave negative results in all tests and represents structurally diverse compounds. Using the computerized profile matching methodology, from 2080 possible pairwise chemical combinations of the 65 pesticides, 20 statistically significant pairs were selected. 6 groups of pesticides were identified which were substantially similar to groups of pesticides we had formed previously (Waters et al., 1982) based on genetic activity and chemical structure. The matches showed excellent qualitative and, in most cases, excellent quantitative agreement. Hence it appears that specific patterns of test results present in the genetic activity profiles are related directly to chemical structure. Conversely, the data suggests that certain groups of compounds may be recognized by a well defined series of concordant tests results. As additional data is added, comparison of test results for new chemicals with existing data for known genotoxicants should aid in the evaluation of potential genetic health hazards.

Effects of phthalimide fungicides on the accumulation of fenarimol byAspergillus nidulans

Phthalimide fungicides (captafol, captan and folpet) enhanced the accumulation of fenarimol by the mycelium of a wild-type strain and a fenarimol-resistant strain of Aspergillus nidulans. The accumulation is ascribed to inhibition of active efflux of fenarimol from the mycelium. It is assumed that the synergistic action observed between the phthalimide fungicides and fenarimol with respect to fungitoxicity, was caused by the increased accumulation of fenarimol.

Establishment of Bioassay Methods for the Evaluation of Acute Toxicity of Pesticides to Aquatic Organisms

“The standard method for the evaluation of acute toxicity of pesticides to fish” and “A method for the evaluation of acute toxicity of pesticides to daphnids” were established to give 48h TLm values for carp and 3h TLm values for Daphnia pulex or Moina macrocopa. With these and some modified methods, acute toxicity of all the already registered pesticides to carp and daphnids were evaluated, and factors influencing the toxicity of pesticides or sensitivity of test organisms to pesticides were investigated. The results are summarized as follows: (1) Rotenone and organochlorine insecticides, organomercurial, organotin, dimethyl dithiocarbamate and phthalimide fungicides, dinitrophenol and pentachlorphenol herbicides have high toxicity to carp, while organophosphorus and phenylcarbamate insecticides are extremely toxic to daphnids. (2) In general, emulsifiable concentrates, technical products, wettable powders, dust and granules are toxic to carp, in this order. (3) The majority of pesticides are more toxic to carp and daphnids at higher temperatures, but some, including folpet and DDT, are less toxic to carp and daphnids, respectively, at higher temperatures. (4) The majority of pesticides are less toxic at higher pH. (5) No apparent synergism or antagonism is observed to occur between 6 pairs of organophosphorus and phenyl carbamate insecticides with carp. (6) No remarkable change in sensitivity of carp is observed with growth to 6 pesticides, including organophosphorus insecticides and pentachlorphenol herbicide, although sensitivity to endosulfan insecticide is extremely high at earlier developmental stages after floating. (7) Dietary pesticides are in general less toxic to carp than pesticides in bath exposure systems. In addition, sensitivity of other aquatic organisms to pesticides was evaluated by the recently developed method. Results are summarized as follows: (1) Gold fish, tanago; Rhodeus moriokae, dojo; Misgurnus anguillicaudatus, and medata; Oryzias latipes, have sensitivity similar to carp. (2) Larvae of dragonfly; Orthetrum albistylum speciosum, and mayfly; Cloëon dipterum, are found to have lower sensitivity than daphnids, but the correlations of TLm values among them are comparatively high. (3) The molluscean animals, Indoplanorbis exustus, Cipangopaludina chinensis malleata, Semisulcospira libertine and Physa acuta have low sensitivity to pesticides in general. (4) Tadpoles of Bufo bufo japonicas, Rana brevipoda and Rana catesbeiana have low sensitivity to pesticides in general.