Integrated spectroscopic and computational analyses unravel the molecular interaction of pesticide azinphos-methyl with bovine beta-lactoglobulin.

Organophosphorus are typically hazardous chemicals used in the pharmaceutical, agricultural, and other industries. They pose a serious risk to human life and can be fatal upon direct exposure. Hence, studying the interaction between such compounds with proteins is crucial for environmental, health, and food safety. In this study, we investigated the interaction mechanism between azinphos-methyl (AZM) and ß-lactoglobulin (BLG) at pH 7.4 using a combination of biophysical techniques. Intrinsic fluorescence investigations revealed that BLG fluorescence was quenched in the presence of increasing AZM concentrations. The quenching mechanism was identified as static, as evidenced by a decrease in the fluorescence quenching constant (1.25 × 104, 1.18 × 104, and 0.86 × 104 M-1) with an increase in temperatures. Thermodynamic calculations (ΔH > 0; ΔS > 0) affirmed the formation of a complex between AZM and BLG through hydrophobic interactions. The BLG's secondary structure was found to be increased due to AZM interaction. Ultraviolet -visible spectroscopy data showed alterations in BLG conformation in the presence of AZM. Molecular docking highlighted the significant role of hydrophobic interactions involving residues such as Val43, Ile56, Ile71, Val92, Phe105, and Met107 in the binding between BLG and AZM. A docking energy of -6.9 kcal mol-1, and binding affinity of 1.15 × 105 M-1 suggest spontaneous interaction between AZM and BLG with moderate to high affinity. These findings underscore the potential health risks associated with the entry of AZM into the food chain, emphasizing the need for further consideration of its impact on human health.

A high-performance Calix@ZnO based bifunctional nanomaterial for selective detection and degradation of toxic azinphos methyl in environmental samples.

One of the key tenets of sustainable agriculture and food safety is the removal of toxic pesticides from the environment. However, developing reliable, affordable, and efficient methods for detecting and degrading pesticides into non-toxic degradable products remains an immediate matter of concern. Herein, we attempt to develop a strategy for the detection as well as degradation of highly toxic phosphorodithioate pesticide, Azinphos methyl (AZM), using hybrid zinc oxide nanoparticles (ZnO NPs). Considering the non-selectivity of bare ZnO and receptor R1, we have fabricated the heterocalixarene-based Calix (R1) over zinc oxide (ZnO) surface in situ via the sol-gel process. The synthesized heterocaliaxrene-modified ZnO (R1@ZnO) NPs show an excellent affinity for the selective and sensitive detection of AZM with a tremendously low limit of detection (68 mg L-1) and no interference from other pesticides. Degradation of AZM was fully supported by fluorescence spectroscopy, scanning electron microscopy (SEM), 1H NMR titrations, FTIR spectroscopy, cyclic voltammetry, and mass spectroscopy, which unequivocally confirmed the formation of non-toxic products. According to our findings, R1@ZnO NPs are sustainable nanomaterials that can be employed for environmental remediation since they operate in an aqueous medium.

Insights into the interaction of azinphos-methyl with bovine serum albumin: experimental and molecular docking studies.

In the present study, combining spectroscopic and molecular modeling techniques has been used to analyze azinphos-methyl binding properties, as an organophosphorus pesticide, to bovine serum albumin. The quenching interaction of azinphos-methyl with bovine serum albumin was investigated in an appropriate physiological state (pH = 7.4). Fluorescence spectroscopy, UV-visible spectroscopy, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). Findings showed differences in the secondary protein structure microenvironment following interaction with azinphos-methyl. The results from spectroscopic experiments suggest that azinphos-methyl binds to bovine serum albumin residues with a binding constant in the range of 0.099 × 105-0.209 × 105 M-1 in one binding site (Tyr 160). The experimental results are supported by computational techniques such as docking using a bovine serum albumin crystal model. The results show that azinphos-methyl is linked to the site I of bovine serum albumin (in subdomain IB), and the result was in accordance with the experimental result. Based on the negative ΔG°, ΔH° and ΔS° values, the binding between azinphos-methyl and bovine serum albumin was spontaneous, and docking studies confirmed hydrogen bonding and van der Waals forces between them.Communicated by Ramaswamy H. Sarma.

Bluebirds Experience Impaired Hatching Success in Conventionally Sprayed Apple Orchard Habitats: A 31-Year Study.

We analyzed the reproductive success of eastern bluebirds (Sialia sialis) nesting in apple orchards and non-orchard nest-boxes in southern Ontario, Canada, from 1988 to 2018. Using data from 2397 nest-boxes monitored at 20 orchard sites and 52 non-orchard sites, we first modeled phenological parameters typically linked to climate change across both site types. We found that the first egg of each brood was laid significantly earlier in the season each year over our 31-year study. Clutch initiation occurred 4 days earlier in the spring in 2018 compared to 1988. Average clutch size in the first or second brood did not change significantly during our 31-year study; however, clutches were significantly smaller in orchards compared to non-orchards (0.10 ± 0.03 fewer eggs between sites). Nests built in orchards were also at 6.1-fold greater risk of parasitism and 2.1-fold greater risk of depredation than nests in non-orchards. After accounting for depredation and nest parasitism, hatching success was still significantly lower in orchards than in non-orchards. Overall, hatching success was 4%-5% lower in orchards. The probability of successfully fledging did not differ significantly between site types. In 2012, a ban on use of the organophosphate insecticide azinphos-methyl in orchards was enacted in Canada. We did not find a difference in hatching or fledging success in orchards after the ban. In our assessment of available data, we conclude that any pesticide effect on hatching success of eastern bluebirds in sprayed orchards is most likely the consequence of long-term exposure to dichlorodiphenyltrichloroethane (DDT) compounds in orchard soils and bioaccumulation in eggs rather than pesticides in use since regulation of DDT in the 1970s. Environ Toxicol Chem 2021;40:3369-3378. © 2021 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

Experimental and Established Oximes as Pretreatment before Acute Exposure to Azinphos-Methyl.

Poisoning with organophosphorus compounds (OPCs) represents an ongoing threat to civilians and rescue personal. We have previously shown that oximes, when administered prophylactically before exposure to the OPC paraoxon, are able to protect from its toxic effects. In the present study, we have assessed to what degree experimental (K-27; K-48; K-53; K-74; K-75) or established oximes (pralidoxime, obidoxime), when given as pretreatment at an equitoxic dosage of 25% of LD01, are able to reduce mortality induced by the OPC azinphos-methyl. Their efficacy was compared with that of pyridostigmine, the only FDA-approved substance for such prophylaxis. Efficacy was quantified in rats by Cox analysis, calculating the relative risk of death (RR), with RR=1 for the reference group given only azinphos-methyl, but no prophylaxis. All tested compounds significantly (p ≤ 0.05) reduced azinphos-methyl-induced mortality. In addition, the efficacy of all tested experimental and established oximes except K-53 was significantly superior to the FDA-approved compound pyridostigmine. Best protection was observed for the oximes K-48 (RR = 0.20), K-27 (RR = 0.23), and obidoxime (RR = 0.21), which were significantly more efficacious than pralidoxime and pyridostigmine. The second-best group of prophylactic compounds consisted of K-74 (RR = 0.26), K-75 (RR = 0.35) and pralidoxime (RR = 0.37), which were significantly more efficacious than pyridostigmine. Pretreatment with K-53 (RR = 0.37) and pyridostigmine (RR = 0.52) was the least efficacious. Our present data, together with previous results on other OPCs, indicate that the experimental oximes K-27 and K-48 are very promising pretreatment compounds. When penetration into the brain is undesirable, obidoxime is the most efficacious prophylactic agent already approved for clinical use.

Can Indirect Herbicide Exposure Modify the Response of the Colorado Potato Beetle to an Organophosphate Insecticide?

Organisms live in complex multivariate environments. In agroecosystems, this complexity is often human-induced as pest individuals can be exposed to many xenobiotics simultaneously. Predicting the effects of multiple stressors can be problematic, as two or more stressors can have interactive effects. Our objective was to investigate whether indirect glyphosate-based herbicide (GBH) exposure of the host plant has interactive effects in combination with an insecticide (azinphos-methyl) on an invasive pest Colorado potato beetle (Leptinotarsa decemlineata Say). We tested the effects of GBH and insecticide on the survival, insecticide target genes expression (acetylcholinesterase genes) and oxidative status biomarkers (glutathione S-transferase [GST], glucose-6-phosphate dehydrogenase [G6PDH], glutathione reductase homolog [GR], glutathione peroxidase homolog [GPx], total glutathione [totGSH], glutathione reduced-oxidized [GSH: GSSG], catalase [CAT], superoxide dismutase [SOD], lipid hydroperoxides). We found that exposure to indirect GBH has no single or interactive effects in combination with the insecticide on larval survival. However, prior exposure to GBH inhibits Ldace1 gene expression by 0.55-fold, which is the target site for the organophosphate and carbamate insecticides. This difference disappears when individuals are exposed to both GBH and insecticide, suggesting an antagonistic effect. On the other hand, oxidative status biomarker scores (PCAs of GPx, GR, and CAT) were decreased when exposed to both stressors, indicating a synergistic effect. Overall, we found that indirect GBH exposure can have both antagonistic and synergistic effects in combination with an insecticide, which should be considered when aiming for an ecologically relevant risk assessment of multiple human-induced stressors.

Human Oral Buccal Microbiomes Are Associated with Farmworker Status and Azinphos-Methyl Agricultural Pesticide Exposure.

In a longitudinal agricultural community cohort sampling of 65 adult farmworkers and 52 adult nonfarmworkers, we investigated agricultural pesticide exposure-associated changes in the oral buccal microbiota. We found a seasonally persistent association between the detected blood concentration of the insecticide azinphos-methyl and the taxonomic composition of the buccal swab oral microbiome. Blood and buccal samples were collected concurrently from individual subjects in two seasons, spring/summer 2005 and winter 2006. Mass spectrometry quantified blood concentrations of the organophosphate insecticide azinphos-methyl. Buccal oral microbiome samples were 16S rRNA gene DNA sequenced, assigned to the bacterial taxonomy, and analyzed after "centered-log-ratio" transformation to handle the compositional nature of the proportional abundances of bacteria per sample. Nonparametric analysis of the transformed microbiome data for individuals with and without azinphos-methyl blood detection showed significant perturbations in seven common bacterial taxa (>0.5% of sample mean read depth), including significant reductions in members of the common oral bacterial genus Streptococcus Diversity in centered-log-ratio composition between individuals' microbiomes was also investigated using principal-component analysis (PCA) to reveal two primary PCA clusters of microbiome types. The spring/summer "exposed" microbiome cluster with significantly less bacterial diversity was enriched for farmworkers and contained 27 of the 30 individuals who also had azinphos-methyl agricultural pesticide exposure detected in the blood. IMPORTANCE: In this study, we show in human subjects that organophosphate pesticide exposure is associated with large-scale significant alterations of the oral buccal microbiota composition, with extinctions of whole taxa suggested in some individuals. The persistence of this association from the spring/summer to the winter also suggests that long-lasting effects on the commensal microbiota have occurred. The important health-related outcomes of these agricultural community individuals' pesticide-associated microbiome perturbations are not understood at this time. Future investigations should index medical and dental records for common and chronic diseases that may be interactively caused by this association between pesticide exposure and microbiome alteration.

In vitro and in vivo studies of cholinesterases and carboxylesterases in Planorbarius corneus exposed to a phosphorodithioate insecticide: Finding the most sensitive combination of enzymes, substrates, tissues and recovery capacity.

Organophosphate insecticides (OPs) continue to be an important class of agrochemicals used in modern agriculture worldwide. Even though these pesticides persist in the environment for a relatively short time, they show a high acute toxicity that may represent a serious hazard for wildlife. Sub-lethal effects on non-target species are a focus in pest management programs and should be used as biomarkers. Cholinesterases (ChEs) are the most used biomarker of OP exposure in vertebrate and invertebrate species. However, the combined monitoring of ChE and carboxylesterase (CE) activities may provide a more useful indication of exposure and effect of the organisms. The objective of the present work was to find the most sensitive combination of enzyme, substrate, tissue and capacity to recovery of B-esterases in the freshwater gastropod Planorbarius corneus exposed to the OP azinphos-methyl. For this purpose, ChE and CE activities in different tissues of P. corneus (head-foot, pulmonary region, digestive gland, gonads and whole organism soft tissue) were studied. Measurements of ChE activity were performed using three substrates: acetylthiocholine, propionylthiocholine and butyrylthiocholine and CE activity using four different substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1-naphthyl acetate, and 2-naphthyl acetate in control and exposed organisms. Finally, the recovery rates of ChE and CE activities following 48h exposure to azinphos-methyl were analyzed. Our results show a preference for acetylthiocholine as substrate, a high inhibition with eserine (a selective ChE inhibitor) and inhibition with excess of substrate in all the analyzed tissues. The highest ChE and CE activity was found in the pulmonary region and in the digestive gland, respectively. The highest CE Vmax was obtained with 1 and 2-naphthyl acetate in all the tissues. CEs were more sensitive than ChE to azinphos-methyl exposure. The highest sensitivity was found using p-nitrophenyl acetate and butyrate as substrates. On the other hand, CEs of the digestive gland and the pulmonary region were more sensitive than CEs of the whole organism soft tissue. Regarding the recovery of enzyme activities after 48h exposure, ChE and CEs with p-nitrophenyl butyrate reached control values after 14days in the digestive gland and after 21days in the pulmonary region. Our results show marked differences in P. corneus basal ChE and CE activities depending on substrates and the tissue. Also, both tissue-dependent and substrate-dependent variations in sensitivity to azinphos-methyl exposure and recovery were obtained. CEs measured with p-nitrophenyl butyrate in the pulmonary region were the best combination to be used as biomarker of exposure to azinphos-methyl due to their sensitivity and low recovery capacity. Environmental concentrations of azinphos-methyl inhibited CE activity so they could be used as effective biomarkers of aquatic contamination.

Genomic and Transcriptomic Associations Identify a New Insecticide Resistance Phenotype for the Selective Sweep at the Cyp6g1 Locus of Drosophila melanogaster.

Scans of the Drosophila melanogaster genome have identified organophosphate resistance loci among those with the most pronounced signature of positive selection. In this study, the molecular basis of resistance to the organophosphate insecticide azinphos-methyl was investigated using the Drosophila Genetic Reference Panel, and genome-wide association. Recently released full transcriptome data were used to extend the utility of the Drosophila Genetic Reference Panel resource beyond traditional genome-wide association studies to allow systems genetics analyses of phenotypes. We found that both genomic and transcriptomic associations independently identified Cyp6g1, a gene involved in resistance to DDT and neonicotinoid insecticides, as the top candidate for azinphos-methyl resistance. This was verified by transgenically overexpressing Cyp6g1 using natural regulatory elements from a resistant allele, resulting in a 6.5-fold increase in resistance. We also identified four novel candidate genes associated with azinphos-methyl resistance, all of which are involved in either regulation of fat storage, or nervous system development. In Cyp6g1, we find a demonstrable resistance locus, a verification that transcriptome data can be used to identify variants associated with insecticide resistance, and an overlap between peaks of a genome-wide association study, and a genome-wide selective sweep analysis.

Conifer flavonoid compounds inhibit detoxification enzymes and synergize insecticides.

Detoxification by glutathione S-transferases (GSTs) and esterases are important mechanisms associated with insecticide resistance. Discovery of novel GST and esterase inhibitors from phytochemicals could provide potential new insecticide synergists. Conifer tree species contain flavonoids, such as taxifolin, that inhibit in vitro GST activity. The objectives were to test the relative effectiveness of taxifolin as an enzyme inhibitor and as an insecticide synergist in combination with the organophosphorous insecticide, Guthion (50% azinphos-methyl), and the botanical insecticide, pyrethrum, using an insecticide-resistant Colorado potato beetle (CPB) Leptinotarsa decemlineata (Say) strain. Both taxifolin and its isomer, quercetin, increased the mortality of 1(st) instar CPB larvae after 48h when combined with Guthion, but not pyrethrum. Taxifolin had greater in vitro esterase inhibition compared with the commonly used esterase inhibitor, S, S, S-tributyl phosphorotrithioate (DEF). An in vivo esterase and GST inhibition effect after ingestion of taxifolin was measured, however DEF caused a greater suppression of esterase activity. This study demonstrated that flavonoid compounds have both in vitro and in vivo esterase inhibition, which is likely responsible for the insecticide synergism observed in insecticide-resistant CPB.

B-esterase determination and organophosphate insecticide inhibitory effects in JEG-3 trophoblasts.

The placenta and trophoblasts express several B-esterases. This family includes acethylcholinesterase (AChE), carboxylesterase (CES) and butyrylcholinesterase (BChE), which are important targets of organophosphate insecticide (OP) toxicity. To better understand OP effects on trophoblasts, B-esterase basal activity and kinetic behavior were studied in JEG-3 choriocarcinoma cell cultures. Effects of the OP azinphos-methyl (Am) and chlorpyrifos (Cp) on cellular enzyme activity were also evaluated. JEG-3 cells showed measurable activity levels of AChE and CES, while BChE was undetected. Recorded Km for AChE and CES were 0.33 and 0.26 mM respectively. Native gel electrophoresis and RT-PCR analysis demonstrated CES1 and CES2 isoform expression. Cells exposed for 4 and 24 h to the OP Am or Cp, showed a differential CES and AChE inhibition profiles. Am inhibited CES and AChE at 4 h treatment while Cp showed the highest inhibition profile at 24 h. Interestingly, both insecticides differentially affected CES1 and CES2 activities. Results demonstrated that JEG-3 trophoblasts express AChE, CES1 and CES2. B-esterase enzymes were inhibited by in vitro OP exposure, indicating that JEG-3 cells metabolization capabilities include phase I enzymes, able to bioactivate OP. In addition, since CES enzymes are important for medicinal drug activation/deactivation, OP exposure may interfere with trophoblast CES metabolization, probably being relevant in a co-exposure scenario during pregnancy.

Organophosphate Resistance and its Main Mechanism in Populations of Codling Moth (Lepidoptera: Tortricidae) from Central Chile.

The codling moth, Cydia pomonella (L.), is the key pest of apple production worldwide. Insecticide resistance has been reported in all producing countries, based on five different mechanisms. Codling moth in Chile has resistance to azinphos-methyl and tebufenozide in post-diapausing larvae. However, there are no studies about the susceptibility of these populations to insecticides from other chemical groups. Therefore, the efficacy of azinphos-methyl, chlorpyrifos-ethyl, esfenvalerate, methoxyfenozide, tebufenozide, and thiacloprid on neonate and post-diapausing larvae from six field populations was investigated, and identified resistance mechanisms in this species were evaluated. Neonate larvae were susceptible to all insecticides studied, but post-diapausing larvae from four populations were resistant to chlorpyrifos, one of them was also resistant to azinphos-methyl, and another one was resistant to tebufenozide. The acetylcholinesterase insensitivity mutation was not detected, and the sodium channel knockdown resistance mutation was present in a low frequency in one population. Detoxifying enzymatic activity of glutathione S-transferases, esterases, and cytochrome P-450 monooxygenases in adults differed among populations, but chlorpyrifos resistance was associated only with a decreased esterase activity as shown by a significant negative correlation between chlorpyrifos mortality and esterase activity.

Recovery study of cholinesterases and neurotoxic signs in the non-target freshwater invertebrate Chilina gibbosa after an acute exposure to an environmental concentration of azinphos-methyl.

Azinphos-methyl belongs to the class of organophosphate insecticides which are recognized for their anticholinesterase action. It is one of the most frequently used insecticides in the Upper Valley of Río Negro and Río Neuquén in Argentina, where agriculture represents the second most important economic activity. It has been detected in water from this North Patagonian region throughout the year and the maximum concentration found was 22.48 µg L(-1) during the application period. Chilina gibbosa is a freshwater gastropod widely distributed in South America, particularly in Patagonia, Argentina and in Southern Chile. Toxicological studies performed with C. gibbosa in our laboratory have reported neurotoxicity signs and cholinesterase inhibition after exposure to azinphos-methyl for 48 h. Recovery studies together with characterization of the enzyme and sensitivity of the enzyme to pesticides can improve the toxicological evaluation. However, little is known about recovery patterns in organisms exposed to organophosphates. The aim of the present work was to evaluate the recovery capacity (during 21 days in pesticide-free water) of cholinesterase activity and neurotoxicity in C. gibbosa after 48 h of exposure to azinphos-methyl. Also, lethality and carboxylesterase activity were registered during the recovery period. Regarding enzyme activities, after a 48-h exposure to 20 µg L(-1) of azinphos-methyl, cholinesterases showed an inhibition of 85% with respect to control, while carboxylesterases were not affected. After 21 days in pesticide-free water, cholinesterases continued to be inhibited (70%). Severe neurotoxicity signs were observed after exposure: 82% of the snails presented lack of adherence to vessels, 11% showed weak adherence, and 96% exhibited an abnormal protrusion of the head-foot region from shell. After 21 days in pesticide-free water, only 15% of the snails presented severe signs of neurotoxicity. However, during the recovery period significant lethality (30%) was registered in treated snails. C. gibbosa is a very sensitive organism to azinphos-methyl. These snails play an important role in the structure and function of aquatic food webs in this region. Thus, a decline of this species' population would probably have an impact on aquatic and non-aquatic communities. Our results show that C. gibbosa is a relevant sentinel species for studying exposure and effects of azinphos-methyl using behavioral and biochemical biomarkers. Neurotoxic behavioral signs are very sensitive, non-destructive biomarkers, which can be easily detected for about one week after acute exposure. Cholinesterse activity is a very useful biomarker showing a high sensitivity and a slow recovery capacity increasing the possibility to indirectly detect organophosphates for long periods after a contaminant event.

Differential sensitivity in embryonic stages of the zebrafish (Danio rerio): The role of toxicokinetics for stage-specific susceptibility for azinphos-methyl lethal effects.

The occasionally observed differential chemical sensitivity in embryonic life stages of fish is still poorly understood and could represent an important issue for understanding the time course of toxicity and the toxic modes of action of chemicals. In this study we analyzed the toxicity of the acetylcholinesterase inhibitor azinphos-methyl (APM) in different life-stages of zebrafish embryos. To this end, the LC50 of three 48h-exposure windows were determined (12µM for 0-48, no mortality observed for 24-72 and 72-120hpf up to a concentration of 79µM). We hypothesized that the differential sensitivity of the stage-specific embryos may be related to differences in uptake of the compound and/or internal concentrations. Therefore, internal concentrations were determined using HPLC. Similar levels and time courses of internal concentrations for all three exposure windows were observed. Bioconcentration amounted to a factor of about 30. Short-term exposure windows for a concentration 4-fold above the calculated LC50 (47µM) identified the period of 0-4hpf as the most sensitive time window for APM toxicity. Our results indicate that the differential sensitivity of APM in the embryos is not related to differences in internal concentrations but related to a stage specific mechanisms of toxicity.

Reversible cholinesterase inhibitors as pre-treatment for exposure to organophosphates: assessment using azinphos-methyl.

Pre-treatment with reversible acetylcholinesterase (AChE) inhibitors before organophosphorous compound (OPC) exposure can reduce OPC-induced mortality. However, pyridostigmine, the only substance employed for such prophylaxis, is merely efficacious against a limited number of OPCs. In search of more efficacious and broad-range alternatives, we have compared in vivo the ability of five reversible AChE inhibitors (pyridostigmine, physostigmine, ranitidine, tacrine and K-27) to reduce mortality induced by the OPC azinphos-methyl. Protection was quantified using Cox analysis by determining the relative risk (RR) of death in rats that were administered these AChE inhibitors in equitoxic dosage (25% of LD01) 30 min before azinphos-methyl exposure. Azinphos-methyl-induced mortality was significantly reduced by all five tested compounds as compared with the reference group that was only exposed to azinphos-methyl without prior pre-treatment (RR = 1). The most efficacious prophylactic agents were K-27 (RR = 0.15) and physostigmine (RR = 0.21), being significantly more efficacious than ranitidine (RR = 0.62) and pyridostigmine (RR = 0.37). Pre-treatment with tacrine (RR = 0.29) was significantly more efficacious than pre-treatment with ranitidine, but the difference between tacrine and pyridostigmine was not significant. Our results indicate that prophylactic administration of the oxime K-27 may be a promising alternative in cases of imminent OPC exposure.

Susceptibility in field populations of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), in Ontario and Quebec apple orchards to a selection of insecticides.

BACKGROUND: Codling moth is a major pest of pome fruit worldwide. Insecticide resistance has become a widespread pest management issue. However, the current status of insecticide resistance in Ontario and Quebec codling moth populations is unknown. RESULTS: Codling moth populations were collected from 27 orchards in Ontario and Quebec from 2008 to 2010. A series of laboratory bioassays were performed to establish baseline susceptibility of adults and larvae to azinphos-methyl, thiacloprid, chlorantraniliprole and methoxyfenozide. Adult codling moth percentage mortality ranged from 22 to 97% and from 21 to 85% when exposed to topical bioassays using azinphos-methyl and thiacloprid respectively. Azinphos-methyl LC50 values from three selected orchards were ca fivefold greater than those from an insecticide-susceptible population. Neonate larva percentage mortality ranged from 5 to 50%, from 15 to 65%, from 90 to 100% and from 10 to 40% when exposed to diet bioassays using azinphos-methyl, thiacloprid, chlorantraniliprole and methoxyfenozide respectively. CONCLUSION: Based on the response of the field-collected populations, resistance development to some registered insecticides was evident in some Ontario and Quebec populations. With the present status of insecticide resistance documented in these regions, modifications to codling moth management strategies should be initiated before changes in field efficacy occur.

Comparative assessment of in vitro and in vivo toxicity of azinphos methyl and its commercial formulation.

The toxic effects of Gusathion (GUS), which is a commercial organophosphate (OP) pesticide, and also its active ingredient, azinphos methyl (AzM), are evaluated comparatively with in vitro and in vivo studies. Initially, the 96-h LC50 values of AzM and GUS were estimated for two different life stages of Xenopus laevis, embryos, and tadpoles. The actual AzM concentrations in exposure media were monitored by high-performance liquid chromatography. Also, the sub-lethal effects of these compounds to tadpoles were determined 24 h later at exposure concentrations of 0.1 and 1 mg/L using selected biomarker enzymes such as acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione S-transferase (GST), glutathione reductase, lactate dehydrogenase, and aspartate aminotrasferase. Differences in AChE inhibition capacities of AzM and GUS were evaluated under in vitro conditions between frogs and fish in the second part of this study. The AChE activities in a pure electrical eel AChE solution and in brain homogenates of adult Cyprinus carpio, Pelophylax ridibundus, and X. laevis were assayed after in vitro exposure to 0.05, 0.5, 5, and 50 mg/L concentrations of AzM and GUS. According to in vivo studies AChE, CaE and GST are important biomarkers of the effect of OP exposure while CaE may be more effective in short-term, low-concentration exposures. The results of in vitro studies showed that amphibian brain AChEs were relatively more resistant to OP exposure than fish AChEs. The resistance may be the cause of the lower toxicity/lethality of OP compounds to amphibians than to fish.

Development of a passive air sampler to measure airborne organophosphorus pesticides and oxygen analogs in an agricultural community.

Organophosphorus pesticides are some of the most widely used insecticides in the US, and spray drift may result in human exposures. We investigate sampling methodologies using the polyurethane foam passive air sampling device to measure cumulative monthly airborne concentrations of OP pesticides chlorpyrifos, azinphos-methyl, and oxygen analogs. Passive sampling rates (m(3)d(-1)) were determined using calculations using chemical properties, loss of depuration compounds, and calibration with side-by-side active air sampling in a dynamic laboratory exposure chamber and in the field. The effects of temperature, relative humidity, and wind velocity on outdoor sampling rates were examined at 23 sites in Yakima Valley, Washington. Indoor sampling rates were significantly lower than outdoors. Outdoor rates significantly increased with average wind velocity, with high rates (>4m(3)d(-1)) observed above 8ms(-1). In exposure chamber studies, very little oxygen analog was observed on the PUF-PAS, yet substantial amounts chlorpyrifos-oxon and azinphos methyl oxon were measured in outdoor samples. PUF-PAS is a practical and useful alternative to AAS because it results in little artificial transformation to the oxygen analog during sampling, it provides cumulative exposure estimates, and the measured sampling rates were comparable to rates for other SVOCs. It is ideal for community based participatory research due to low subject burden and simple deployment in remote areas.

Biomarker responses and morphological effects in juvenile tilapia Oreochromis mossambicus following sequential exposure to the organophosphate azinphos-methyl.

Pesticides are contaminants of aquatic environments. Such ecosystems in the Western Cape, South Africa are at risk as most organophosphates are highly toxic to fish and other aquatic organisms. The objective of this experimental study was firstly to determine the acute toxicity of azinphos-methyl (AZP) to juvenile fish (Oreochromis mossambicus) and, secondly, to investigate the effects of repeated exposure of fish to an array of sublethal concentrations on morphological parameters such as growth, condition factor and organ-somatic indices. Food consumption and feeding response time were investigated as ecologically relevant behavioral endpoints which could affect growth, reproduction and survival and subsequently causes impacts at the population and/or community level. Finally, acetylcholinesterase (AChE) was used as biomarker to investigate effects at sub-organismal level following sequential exposure to AZP. The aim was to determine how sequential spraying procedures, using different exposure concentrations and intervals, affected fish as reflected by their responses at different organizational levels. A dose-dependent effect on feeding impairment was observed in the feeding response experiment. The correlation found between growth impairment, feeding activity and AChE inhibition therefore indicates that frequency of exposure can play an important role regarding the severity of impacts to non-target organisms. This study provides evidence that AZP has harmful effects on non-target aquatic organisms, such as fish which can be manifested in the early developmental stages. Sequential exposures showed that dosage and frequency of spraying and spraying interval could exacerbate harmful effects. AChE inhibition and organosomatic indices can be used effectively to measure effects.

Cholinesterases and neurotoxicity as highly sensitive biomarkers for an organophosphate insecticide in a freshwater gastropod (Chilina gibbosa) with low sensitivity carboxylesterases.

In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 µg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 µg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 µg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 µg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 µg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile.