Analysis of polar metabolites of organophosphorus insecticides on an anion-exchange chromatographic column

A simple and rapid procedure is described for the qualitative and quantitative analysis of metabolites of malathion and other insecticides. The omission of an extraction at low pH, and mild conditions of anion-exchange chromatography on QAE-Sephadex prevent the degradation of a malathion metabolite, S-(1-carboxy-2-ethoxycarbonyl)-ethyl O,O-dimethyl phosphorothioate (malaoxon α-carboxylic acid), which takes place under strongly acid conditions. Disadvantages of the commonly used fractionation of malathion and malaoxon metabolites based on partitioning are discussed.     

Toxicokinetics of Malathion in Larval Stages of the Toad Bufo arenarum (Hensel): Effect of Exogenous Spermidine

The aim of this work was to study the absorption, biotransformation, and excretion of malathion (¹⁴C-methoxy) and its metabolites in larval stages of the toad Bufo arenarum (Hensel). Also, changes in malathion metabolization by the action of the exogenous polyamine spermidine were studied. Malathion clearance from the media was uniexponential, and spermidine reduced the uptake in the larvae, causing an increase in the apparent half-life of the toxicant. Concomitant with this effect, spermidine increased the level of induction of mixed-function oxidases due to malathion and caused a progressively higher malaoxon/malathion ratio. As a consequence of the higher conversion to the active metabolite malaoxon, spermidine also provoked a significant enhancement in the inhibitory effect of Malathion on acetylcholinesterase activity. [methoxy¹⁴C]malathion metabolites, such as carboxylesterase and glutathione S-transferase products, were detected in the toad larvae and in the media. The excreted products of carboxylesterase activity were about 70% of the total radioactivity, and the glutathione S-transferase products (methyl glutathione) were 20–30% of the total radioactivity. No significant variations in the levels of excreted products due to the action of exogenous spermidine were detected. Malathion inhibited carboxylesterase activity, independent of the presence of spermidine in the media. In turn, glutathione S-transferase activity was induced by spermidine, but was not affected by the exposure to low concentrations of malathion for 48 h. We conclude that the presence of spermidine in the medium modifies malathion toxicokinetics, increasing its toxicity in B. arenarum larvae.     

Oxidative cleavage of a carboxyester bond as a mechanism of resistance to malaoxon in houseflies

The synergistic effect of triphenyl phosphate (a carboxyesterase inhibitor), sesamex (inhibitor of microsomal oxidation) and O,O-diethyl O-phenyl phosphorothioate on the toxicity of malathion and malaoxon for one susceptible and two resistant strains of housefly was studies. It was found that in the resistant strain G (characterized by high carboxyesterase activity) both malathion and malaoxon were synergized by triphenyl phosphate, but only malaoxon (and not malathion) by sesamex. The other resistant strain E 1, moderately tolerant for malathion but highly resistant to malaoxon, differed from strain G in that triphenyl phosphate had no effect; its response to sesamex was similar to that of strain G. The third synergist, O,O-diethyl O-phenyl phosphorothioate, combined the properties of triphenyl phosphate and sesamex. It was found to be the best of the three compounds used.Biochemical in vitro studies showed that both resistant strains could degrade malaoxon oxidatively at a rate at least 10 × higher than that of the susceptible strain. This oxidation could be inhibited by very low concentrations of the thiono analogue; a malaoxon to malathion ratio of 10:1 gave an inhibition of about 70% at a malaoxon concentration of 5 × 10^?6M. The product of this oxidation is malaoxon β-monocarboxylic acid. This metabolite was also found 1 hr after application of malaoxon in vivo.The results mentioned in this paper indicate that houseflies may become resistant to malaoxon by an increased rate of oxidative carboxyester bond cleavage.     

Esterase-mediated malathion resistance in the human head louse, Pediculus capitis (Anoplura: Pediculidae)

Resistance in a dual malathion- and permethrin-resistant head louse strain (BR-HL) was studied. BR-HL was 3.6- and 3.7-fold more resistant to malathion and permethrin, respectively, compared to insecticide-susceptible EC-HL. S,S,S-Tributylphosphorotrithioate synergized malathion toxicity by 2.1-fold but not permethrin toxicity in BR-HL. Piperonyl butoxide did not synergize malathion or permethrin toxicity. Malathion carboxylesterase (MCE) activity was 13.3-fold and general esterase activity was 3.9-fold higher in BR-HL versus EC-HL. There were no significant differences in phosphotriesterase, glutathione S-transferase, and acetylcholinesterase activities between strains. There was no differential sensitivity in acetylcholinesterase inhibition by malaoxon. Esterases from BR-HL had higher affinities and hydrolysis efficiencies versus EC-HL using various naphthyl-substituted esters. Protein content of BR-HL females and males was 1.6- and 1.3-fold higher, respectively, versus EC-HL adults. Electrophoresis revealed two esterases with increased intensity and a unique esterase associated with BR-HL. Thus, increased MCE activity and over-expressed esterases appear to be involved in malathion resistance in the head louse.     

The in vitro degradation of [14C]malathion by enzymatic extracts from resistant and susceptible strains of Drosophila melanogaster

Enzyme preparations from Drosophila melanogaster flies degraded [¹⁴C]malathion to α- and β-malathion monoacids and, hence, were considered to contain malathion carboxylesterase (ME) activity. Although ME- activity was stable during preincubation in the absence of malathion, it decreased dramatically during the course of the reaction, and could not be completely recovered by Sephadex G-25 chromatography. Furthermore, the protein fraction after chromatography still contained ¹⁴C, suggesting that the enzyme had become inhibited by a bound, ¹⁴C-labeled derivative. Extracts from a resistant (malathion-selected), an intermediate control, and the susceptible Canton S strains of D. melanogaster differed in the lability of ME activity during the reaction. This difference was partly attributed to the production of small amounts of malaoxon (2–8%) by the extracts from the more resistant strains. No consistent strain differences were found when the rate of malathion degradation was measured during the first minute of reaction, either with or without a microsomal oxidase inhibitor (metyrapone) present. These results, together with the cross-resistance of the malathion-selected strain to other insecticides and the lack of a synergistic effect of two carboxylesterase inhibitors (triphenyl phosphate and S,S,S-tributylphosphorotrithioate) suggested that malathion carboxylesterase does not contribute significantly to the observed differences in malathion resistance between strains.     

Toxicodynamics of malaoxon in house flies

The fate of malaoxon was studied in a susceptible and a resistant strain of house fly following topical application. Sublethal doses were used: 160 pmol for the S-strain (0.17 × LD₅₀) and 1570 pmol for the R-strain (0.1 × LD₅₀). The penetration rates are dose dependent and semilog plots of the external amount vs time show that these rates are not proportional to this external amount. Internal concentrations of malaoxon rapidly increase following administration, reach maximum values between 30 min and 2 hr (depending on dose), and then slowly decrease. The rate of metabolic degradation is highest in the early stage of the intoxication process. A three-compartment pharmacokinetic model is postulated to explain the experimental data quantitatively. The first compartment represents external malaoxon, the other two represent internal parent compound. Statistical analysis shows that the penetration rate is better described with a sum of two exponentials rather than with a single exponential decay. In the model, degradation occurs in the first internal compartment and is assumed to be first order. Malaoxon is distributed between the two internal compartments slowly with first-order kinetics. Parameter estimation with curve-fitting procedures for the internal processes (degradation and exchange) shows that there is not one set of parameter values that can be used for both strains simultaneously. This prompted a study of possible interstrain differences in degradation capacities. It was found that in vitro the R-strain had a fourfold higher oxidative breakdown rate of malaoxon. Taking this difference into account it is possible to explain the two sets of data with one kinetic model, although other alternatives cannot be excluded.     

The Persistence and Fate of Malathion Residues in Stored Beans (Phaseolus vulgaris) and Maize (Zea mays)

Two experimental models simulating the traditional storage conditions prevalent in Kenya, i.e. the open basket model and the modern wooden box model, were used to study the rate of dissipation and fate of malathion residues in maize grains and beans stored for periods of up to one year at ambient temperatures averaging 23°C. The grain samples were initially treated with 10·36 mg kg⁻¹ of radiolabelled malathion dust prior to storage and portions analysed at regular intervals for malathion, malaoxon and the transformation products isomalathion, malathion α-monocarboxylic acid and malathion β-monocarboxylic acid using a combination of chromatographic, radioisotopic and mass-spectrometric techniques. The findings showed a gradual penetration of malathion into the grains in amounts which were slightly higher in maize than in beans irrespective of the method of storage. After 51 weeks of storage, 34–60% of the initial residues persisted in all the grains. The total residual levels were slightly higher in beans than in maize irrespective of the storage methods though the persistence was a little higher in the wooden box than in the open basket. The rates of dissipation of the pesticide from the grains decreased with storage time and followed a biphasic pattern. Applying first-order reaction kinetics, the following half-lives were obtained: maize grains stored in open basket: 194 days; maize grains stored in closed wooden box: 261 days; beans stored in open basket: 259 days; beans stored in closed wooden box: 405 days. Beans stored in the wooden box had higher levels of bound residues than those sampled from the open basket. This trend was similar in maize grains although the concentrations were lower. The analysis of malathion metabolites confirmed the degradation trend of the residues.     

Toxicity of ddt and malathion to various larval stages of Mamestra brassicae L.

The toxicity of DDT and malathion to the larvae of Mamestra brassicae was determined following several methods of application. The toxicity (LD₅₀), expressed as μg insecticide per g of insect, did not change significantly between larval instars (a) when either insecticide was injected into fourth to sixth instars; (b) when DDT was applied in the food of fifth and sixth instars; or (c) when malathion was applied topically to second to sixth instars. Significant changes in toxicity were found between successive instars when DDT was applied topically, but there was no clear trend. When malathion was applied in the food, the fifth instars were more susceptible than the sixth instars; it was found that the former consumed a toxic dose of malathion at a greater rate, and that probably malathion was degraded in the gut at a slower rate. In a contact test, the first to third instars were far more susceptible than the later instars to malathion; with DDT this trend was much less marked. Uptake studies with [¹⁴C]malathion showed that differences in the contact toxicity of malathion between instars could be explained, at least partly, by the decline in uptake per unit weight with increasing larval size.     

鱼类肝脏酯酶的同工酶构成及其对磷酸三苯酯和马拉氧磷的敏感性

用聚丙烯酰胺凝胶电泳法分离了麦穗鱼 (Pseudorasbora p arva)、金鱼 (Carassius auratus)、尼罗罗非鱼 (Tilapja nilotica)、食蚊鱼 (Gambusia affinis)、虹鳟 (Salmo gairdneri)等五种鱼的肝脏酯酶的同工酶 ,以乙酸α-萘酯为底物测定了它们的活性。在麦穗鱼、金鱼、尼罗罗非鱼体内发现两条酶带 ,在食蚊鱼和虹鳟体内发现一条酶带。离体抑制率实验表明 ,五种鱼肝脏内均含有对磷酸三苯酶敏感的酶带 ,而对马拉氧磷敏感的酶带只存在于麦穗鱼、金鱼、尼罗罗非鱼体内。酯酶同工酶分布型的意义及与马拉硫磷选择毒性的关系在本文中进行了讨论。     

In vitro metabolism of insecticides during midgut penetration

A study of the metabolism of ¹⁴C-labeled dieldrin, DDT, malathion, and carbaryl during penetration of the isolated midgut of two insects (Blaberus discoidalis and Manduca sexta) and a section of the intestine of a mammal (Mus musculus) is reported. There was appreciable metabolism of malathion during penetration, including differences in the activation reaction to malaoxon, between insects and mammals. Metabolism was relatively slow during penetration of carbaryl and the chlorinated hydrocarbon insecticides, and little difference in metabolic patterns was noted among the three species. The penetration studies were supported by experiments in which insecticides were incubated with intact and homogenized midgut preparations.     

Variation in the sensitivity of Callosobruchus (Coleoptera: Bruchidae) acetylcholinesterase to the organophosphate insecticide malaoxon: effect of species, geographical strain and food type

BACKGROUND: Bruchid beetles, Callosobruchus species, are serious pests of economically important grain legumes; their activity in stores is often controlled by the use of synthetic insecticides. Esterases are known to be involved in insecticide resistance in insects. However, there is a dearth of information on esterase activity in the genus Callosobruchus. In this study, the effect of species, geographical strain and food type on the variation in acetylcholinesterase (AChE) activity and its inhibition by malaoxon (malathion metabolite) was investigated using an in vitro spectrophotometric method. RESULT: AChE activity varied significantly among species and strains and also among legume type used for rearing them. Generally, irrespective of species, strain or food type, the higher the AChE activity of a population, the higher is its inhibition by malaoxon. C. chinensis had the highest AChE activity of the species studied, and in the presence of malaoxon it had the lowest remaining AChE activity, while C. rhodesianus retained the highest activity. CONCLUSION: A first‐hand knowledge of AChE activity in regional Callosobruchus in line with the prevailing food types should be of utmost importance to grain legume breeders, researchers on plant materials for bruchid control and pesticide manufacturer/applicators for a robust integrated management of these bruchids. Copyright © 2012 Society of Chemical Industry     

Increased activity and reduced sensitivity of acetylcholinesterase associated with malathion resistance in a field population of the oriental migratory locust, Locusta migratoria manilensis (Meyen)

The susceptibility to malathion, and the activity and sensitivity of acetylcholinesterase (AChE, EC 1.1.1.7) were compared between two populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen) collected from Wudi County of Shandong Province in East China and Huangliu County of Hainan Province in South China. Huangliu population showed 8.5-fold resistance to malathion compared with Wudi population. AChE from Huangliu population showed 4.8-fold higher activity than that from Wudi population toward the model substrate acetylthiocholine (ATC). Kinetic studies indicated that AChE from Huangliu population had 2.6-fold lower affinity, but 5.0-fold higher catalytic activity toward ATC than AChE from Wudi population. Significantly increased activity of AChE in Huangliu population was also confirmed by non-denaturing polyacrylamide gel electrophoresis. Inhibition kinetics revealed that AChE from Huangliu population was 9.8-, 2.4-, 8.0- and 7.7-fold less sensitive to inhibition by paraoxon, malaoxon, chlopyrifos oxon, demeton-S-methyl, respectively, than that from Wudi population. Our studies revealed that a mild resistance to malathion in Huangliu population was associated with reduced sensitivity and increased catalytic activity of AChE. Our results suggest that alterations of AChE may play an important role conferring or contribute to malathion resistance in Huangliu population of the locust.     

The biochemistry of insecticide resistance in Anopheles sacharovi: Comparative studies with a range of insecticide susceptible and resistant Anopheles and Culex species

Fourth instar larvae, the progeny from wild-caught Anopheles sacharovi females, were subjected to a number of biochemical tests and the results were compared to those from similar tests on laboratory insecticide resistant and susceptible strains of anopheline and culicine mosquitoes. DDT resistance in An. sacharovi is associated with the ability to rapidly metabolise DDT to DDE. The organophosphorus and carbamate resistance was not associated with quantitative changes in esterases, multifunction oxidases, or glutathione S-transferase. The acetylcholinesterase was less sensitive to malaoxon and propoxur than laboratory susceptible An. albimanus, and plots of inhibition suggest that the population was polymorphic for more than one form of acetylcholinesterase. Metabolism studies on malathion and pirimiphos methyl did not indicate resistance due to increased metabolism. There was no evidence of penetration barriers contributing to resistance to either DDT or malathion, and there was no indication of any resistance to pirimiphos methyl in our tests.     

Organophosphorus insecticides affect normal polyamine metabolism in amphibian embryogenesis

The objective of the present study was to evaluate the concentration- and time-dependent effects of the organophosphorus insecticides malathion and azinphos-methyl on polyamine metabolism, and relate them to normal and altered embryonic development of the common toad Rhinella arenarum. Control embryos showed that the higher polyamines spermidine and spermine acquired importance with respect to the diamine putrescine as embryonic development progressed. The activity of ornithine decarboxylase significantly decreased in complete operculum embryos. Continuous exposure to malathion caused a decrease in polyamine levels during embryonic development. However, there was an increase in putrescine levels in complete operculum embryos exposed to a sublethal concentration of the insecticide. Embryos exposed to malathion displayed a decrease in fresh weight and size, along with an increase in the number of malformed individuals. R. arenarum embryos exposed to a lethal concentration of azinphos-methyl showed an increase in putrescine levels and a decrease in spermidine and spermine levels, accompanied by an increase in ornithine decarboxylase activity. In conclusion, as the embryonic development of the toad R. arenarum progresses, polyamine metabolism shifts to higher polyamine levels with a more preponderant contribution of spermidine and spermine with respect to putrescine and involves a dramatic change in ornithine decarboxylase activity, one of the key regulatory enzymes of the pathway. Organophosphorus insecticides are capable of altering polyamine metabolism, slowing embryo development in parallel with a reduction in spermidine and spermine levels. An increase in the oxidative degradation of polyamines might be involved in the toxic action of organophosphorus insecticides and might also be related to other effects such as teratogenesis.     

Baseline toxicity of several pesticides to Hyaliodes vitripennis (Say) (Hemiptera: Miridae)

Hyaliodes vitripennis (Say) is a univoltine indigenous predacious mirid. It has been reported in several orchards where IPM programmes are used. It is a generalist, and feeds on phytophagous mites in addition to other arthropods. In Quebec, a foliar application of imidacloprid, deltamethrin or lambda‐cyhalothrin is used at least once per season to manage arthropod pests such as leafhoppers and leaf‐eating caterpillars. Meanwhile, several applications of metiram, flusilazole, myclobutanil and mancozeb are made to control apple scab [Venturia inaequalis (Cooke) Winter]. In laboratory trials, comparison of lethal concentrations of the three insecticides against H vitripennis nymphs and adults showed no significant difference. However, when lethal concentrations were compared between two growth stages for each insecticide, a significant difference was noted between adults and nymphs treated with lambda‐cyhalothrin, adults being more susceptible than nymphs. No such difference could be detected for imidacloprid or deltamethrin. When LC₅₀ values were compared with the manufacturer's label rates, deltamethrin and imidacloprid were toxic to the nymphs and adults, and lambda‐cyhalothrin was slightly toxic to the nymphs and moderately toxic to the adults. Among the fungicides evaluated in the laboratory, myclobutanil showed moderate toxicity to adults at the manufacturer's label rate. The remaining fungicides had no toxic effects to adults or nymphs, even at four times the manufacturer's label rate. © 2001 Society of Chemical Industry     

麦穗鱼脑乙酰胆碱酯酶(AChE)的纯化及其比较性研究

用PEG2000双水相萃取、DEAE-Sephadex A-50和Sephadex G-200方法分离纯化麦穗鱼Pseudorasbora parva脑中的乙酰胆碱酯酶(AChE),然后比较分析纯酶液与粗酶液中鱼脑AChE 的动力学特性和抗抑制性,以便更直接地了解底物与酶以及毒剂与酶的反应关系。研究结果表明,经过一系列步骤的纯化,最后所得的AChE是纯度较高的酶液;通过对AChE的动力学研究发现,纯化后的麦穗鱼脑AChE与底物之间的亲和力和其在粗酶状态时没有显著的差别,而且纯化后的鱼脑AChE对底物抑制作用更敏感;抗抑制性研究发现,纯化后的麦穗鱼脑AChE对经溴水氧化的马拉硫磷(malathion)和三唑磷(triazophos)的敏感性显著高于粗酶状态的AChE,酶更易受抑制。     

Differential susceptibility to abamectin and two bioactive avermectin analogs in abamectin-resistant and -susceptible strains of Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

As resistance to currently used insecticides increases in the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), abamectin and its 4″-epi and 8,9-oxide analogs may serve as likely replacements if proven effective. We previously selected an abamectin-resistant strain of CPB (AB-F) that is suitable for the determination of cross-resistance to these two bioactive avermectin analogs. Using bioassay and logit analysis, the present work shows that, on average and following normalization by weight, the larval stages of the insecticide-susceptible SS strain are equally sensitive to the toxic action of abamectin and its 4″-epi and 8,9-oxide analogs, indicating that all three compounds retain high bioactivity towards the susceptible larval stages. Abamectin and the two analogs also are similar in toxicity to the larval stages of the AB-F strain. However, the AB-F larval stages are significantly less sensitive on average to these insecticides than the SS larval stages, indicating resistance to abamectin and cross-resistance to both the two analogs tested. Likewise, adults of the AB-F strain are significantly less sensitive to the toxic action of abamectin and the two analogs compared to SS adults, also indicative of resistance and cross-resistance. Abamectin is significantly more toxic, however, to both SS and AB-F adults, respectively, than either of the two analogs. The reduction in sensitivity was particularly evident in SS adults to both the 4″-epi and 8,9-oxide analogs. Additionally, adults of the SS strain are significantly less sensitive to the toxic action of abamectin and the two analogs when compared to SS larval stages. There is no significant differences, however, in the toxicity of these three insecticides, respectively, between larval and adults stages of the AB-F strain. This phenomenon results in lower resistance ratio (RR) values calculated for the two avermectin analogs compared to those calculated for abamectin regardless of the insect stage examined but is particularly evident and significant in the adult stage. This relative decrease in resistance levels is primarily associated with SS adults that are less sensitive to the toxic action of these insecticides. The decrease in abamectin toxicity is apparently due to significantly increased levels of P450 associated with SS adults versus forth instars and to similar levels in adults of the SS and AB-F strains. Because abamectin resistance in CPB is due in large part to enhanced oxidative metabolism of abamectin, it is likely that the SS adults are more tolerant to abamectin due to an enhanced level of oxidative detoxification. Finally, neither abamectin nor the two analogs are structurally protected at the specific molecular locations, C₃″, C₂₄, and C₂₆ carbons, which leads to enhanced oxidative metabolism, resistance and cross-resistance. Structurally-protected avermectin analogs at these vulnerable intramolecular sites are likely to be more effective insecticides in suppressing the development of oxidative detoxification-based resistance to abamectin, as would the use of oxidative synergists.     

The effects on resistant mosquitoes of interrupted exposure to insecticides

In paired tests on batches of malathion- and dieldrin-resistant Anopheles arabiensis, one of each pair received a continuous exposure to either malathion or dieldrin for 16 h or more, while the other received a non-exposed rest phase in the middle of the exposure period. Among dieldrin-resistant mosquitoes, the mortality rate was decreased in the group that experienced the interrupted, as opposed to the continuous exposures; however, the survival rate of malathion-resistant adults was unaffected by interrupted exposure. There was a significant positive correlation between the mortality rate and the adult age for malathion, but the responses to dieldrin were independent of age. Movement of adult mosquitoes to and from malathion deposits in sprayed houses should not impair the efficacy of this insecticide, whereas prolonged absence from contact with dieldrin would require even longer exposures to achieve the same mortality in the insects.     

Non-target-site-based resistance to ALS-inhibiting herbicides in six Bromus rigidus populations from Western Australian cropping fields

BACKGROUND: Bromus rigidus is a common weed species that has increased in cropping fields owing to limited control options. During a random field survey in Western Australia, six B. rigidus populations that had survived in‐crop weed control programmes were collected. The study aimed to determine the resistance profile of these six populations. RESULTS: Based on dose–response studies, all six B. rigidus populations had a low‐level resistance to sulfosulfuron and sulfometuron (both sulfonylurea herbicides) while remaining susceptible to herbicides with other modes of action. ALS in vitro activity assays revealed no differences in enzyme sensitivity between susceptible and resistant populations, while the use of malathion (a cytochrome P450 inhibitor) in combination with sulfosulfuron caused the resistant populations to behave like the susceptible population. CONCLUSION: This study established that these six B. rigidus populations have a low‐level resistance to the ALS‐inhibiting sulfonylurea herbicides, but are able to be controlled by other herbicide modes of action. The low‐level, malathion‐reversible resistance, together with a sensitive ALS, strongly suggest that a non‐target‐site enhanced metabolism is the mechanism of resistance. Copyright © 2012 Society of Chemical Industry     

Potentiation of malathion by other insecticides against Musca domestica

Twenty-seven insecticides, including organochlorine, organophosphorus compounds and carbamates were tried for potentiation of the toxic effect of malathion. Carbaryl, diazinon, fenthion, gamma-BHC, parathion and tetrachlorvinphos potentiated the action of malathion. Fourteen insecticides gave additive effects while seven proved antagonistic. Insecticides which showed potentiation, were further tested by mixing with malathion in the ratios of 1:9, 3:7, 5:5, 7:3 and 9:1. All the five combinations in the six mixtures gave potentiating effects. Maximum potentiation of 17.6 times was observed in 1:9 combination of malathion and gamma-BHC. The proportions in which the insecticides were mixed had a pronounced effect on the toxic effect of malathion.