OP Pesticides Research Articles

Biochemical mechanisms of resistance in Daphnia magna exposed to the insecticide fenitrothion

Resistance to fenitrothion and enzyme activities associated with the toxicity and metabolism of organophosphorus insecticides were measured in three genetically unique Daphnia magna clones collected from rice fields of Delta del Ebro (NE Spain) during the growing season and a lab sensitive clone. The studied clones showed up to sixfold differences in resistance to fenitrothion. The lack of correlation between in vitro sensitivity of acetylcholinesterase (AChE) to fenitrooxon and resistance to fenitrothion indicated that insensitivity of AChE to the most active oxon metabolite was not involved in the observed differences in resistance. Inhibition of mixed- function oxidases (MFOs) by piperonyl butoxide (PBO) increased the tolerance to fenitrothion by almost 20-fold in all clones without altering their relative ranking of resistance. Conversely, when exposed to fenitrooxon, the studied clones showed similar levels of tolerance, thus indicating that clonal differences in the conversion of fenitrothion to fenitrooxon by MFOs were involved in the observed resistance patterns. Despite that resistant clones showed over 1.5 higher activities of carboxilesterase (CbE) than sensitive ones, toxicity tests with 2-( O-cresyl)-4H-1,3,2-benzodioxaphosphorin-2 oxide, which is a specific inhibitor of these enzymes, evidenced that this system only contributed marginally to the observed clonal differences in tolerance. Glutathione- S-transferases activity (GST) varied across clones but not under exposure to fenitrothion, and was only related with tolerance levels in the field clones. In summary, our results indicate that MFO mediated differences on the bio-activation of the phosphorotionate OP pesticide to its active oxon metabolite contributed mostly in explaining the observed moderate levels of resistance, whereas the activities of CbE and GST had only a marginal role.

Genetically engineered resistance to organophosphate herbicides provides a new scoreable and selectable marker system for transgenic plants

Organophosphate hydrolase (OPH, E.C. 3.1.8.1; encoded by the bacterial opd gene) provides a new scoreable and selectable genetic marker system for use in plant cell culture and regenerated plant tissue. OPH hydrolyzes a wide range of substrates that produce visually detectable products, which can be readily quantified in biological tissues. A variety of different OP compounds, both herbicides and pesticides, have been identified as acceptable enzymatic substrates, which can be used to generate transgenic markers for various types of plant tissues. For example, transgenic leaf tissue was easily differentiated from non-transgenic tissue by a simple fluorescent assay utilizing the OP insecticide coroxon. Transformed callus and intact whole seed could be easily distinguished from non-transformed tissue using novel non-destructive methods which allowed callus or seeds to grow and/or to germinate after phenotypic scoring with non-herbicidal OP insecticides such as paraoxon. In addition to being used as a scoreable phenotypic markers with various OP pesticides, the OP compounds Haloxon and Bensulide (Bensumec-4LF) were effective as positive selection agents for callus and germinating seeds.

Impairment in Clinical Indices in Acute Organophosphate Insecticide Poisoning Patients in India

This report defines with the impairment in clinical indices and laboratory investigations in acute organophosphate poisoning patients admitted to Outdoor Patient Department of B.J. Medical College and Civil Hospital, Ahmedabad – 380016, India. Out of 121 OP poisoning patients (65.3% males and 35.7% females), about 70% cases were educated (matriculate and higher secondary passed) and remaining illiterate. It was noticed that suicidal and intentional poisoning being a major cause as recorded in 80.2% cases followed by occupational (9.1%), accidental (6.6%), homicidal (1.6%) and unknown (2.5%). The major signs and symptoms reported by the poisoning cases were muscarinic and nicotinic manifestations. ECG abnormalities were recorded in 46 cases (38%); the sinus tachycardia (24%), depression of ST segment and inversion of T wave (7.4%) and sinus bradycardia (6.6%). Plasma and RBCs ChE activities in OP poisoning cases were significantly decreased (p≤0.01). The activities of serum LDH and CK activities were significantly elevated (p≤0.01) in poisoning cases indicating muscular functional impairment due to OP toxicity. In a sub-set of samples, the serum levels of IgA, IgG and circulating immune complexes (C3 and C4) were also found significantly increased (p≤0.01). The findings indicated that administration of atropine with 2-PAM increases the probability of survival although the results showed no significant variation. Logistic regression analysis showed that the survival rate of the cases was about 6 times more when number of days (indirectly the quantum of atropine and 2-PAM received by the cases) increased from 3 days. When duration of atropine administration was divided into 4 quartiles, it was observed that there is significant trend of survival of the cases (63% survival in the 1st quartile, 80% survival for 2nd quartile and 100% for 3rd and 4th quartile). The findings of this study reflect the usefulness of few clinical indices in management of OP pesticide poisoning and also support to impose the restrictions on the access of very toxic pesticides.

In vivo chlorpyrifos induced oxidative stress: Attenuation by antioxidant vitamins

Chlorpyrifos ( O, O′-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothionate, CPF) exposure in rats causes elevation in the levels of thiobarbituric acid reactive substances (TBARS) and inhibition of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), and glucose-6-phosphate dehydrogenase (G6PDH) activities in the liver, kidney, spleen, and brain of rats. The sublethal exposure of CPF also causes decrease in the levels of reduced glutathione (GSH) and consequent increase in oxidized glutathione (GSSG) levels, resulting in a significant decrease in GSH/GSSG ratio in all the rat tissues tested. These results clearly indicate that CPF exposure causes oxidative stress in rat tissues. However, CPF exposure to rats fed with antioxidant vitamins (vitamin A, E, and C) for 1 month, prevented derangement of these antioxidant parameters. The accumulation of TBARS was also not seen in tissues of rats fed with antioxidant vitamins on CPF exposure. AChE activity, which is sensitive to OP pesticides, was also not significantly inhibited in these rats on CPF exposure. The present findings clearly show that oral intake of a mixture of vitamin A, E, and C, protects the rats from CPF induced oxidative stress and suggesting that this treatment alleviates the toxicity of this pesticide.

OP Pesticides, Organic Diets, and Children’s Health: Lu et al. Respond

OP Pesticides, Organic Diets, and Children’s Health: Lu et al. Respond

Effects of dimethoate on spiders from metal pollution gradient

In this study, an attempt has been made to assess whether a chronic exposure to metals in habitats under a strong industrial pressure might have equipped spiders with biochemical defensive mechanisms enabling them to survive an additional chemical stress. To check this, non-web-building wolf spiders Pardosa lugubris (Lycosidae) and funnel web Agelena labyrinthica (Agelenidae) were collected at five variously polluted meadows and, under laboratory conditions, intoxicated with either single or multiple dose of dimethoate (OP pesticide). Then the activities of detoxifying (carboxylesterase: CarE, glutathione S-transferase: GST), antioxidative (selene-dependent and selene-independent glutathione peroxidases: GPOX and GSTPx) enzymes as well as acetylcholinesterase as a biomarker of exposure to OP pesticides were measured. In web-building A. labyrinthica, even a single application of the pesticide caused the inhibition of CarE, GSTPx and GPOX in individuals from less polluted sites and AChE and GST in specimens pre-exposed to high metal concentrations. Multiple intoxication, irrespectively of the site, caused significant, in comparison to controls, decrease in CarE, AChE and GSTPx activities. Actively hunting P. lugubris seem more resistant to acute pesticide intoxication, since the spiders from each site had a constant level of GST and AChE. In individuals of this species from heavily polluted sites, the inhibition caused by multiple intoxication with dimethoate was stated only for glutathione peroxidases.

Analysis of Organophosphorus Pesticides in Whole Milk by Solid Phase Microextraction Gas Chromatography Method

Solid phase microextraction (SPME) was used for the extraction of residual coumaphos and dichlorvos in whole milk. The residues were analyzed by capillary gas chromatography equipped with nitrogen phosphorus detector (GC-NPD). A manual SPME holder with a 100-μm polyacrylate fiber was used. The optimized conditions for extraction by SPME method were: sample agitation, absorption temperature of 30°C, absorption time of 40 min, desorption time of 10 min, and sample volume was 16.0 mL in the vial. Under these conditions, the calibration graphs were linear in the range of 0.17 μgL−1 to 1.75 μgL−1 for coumaphos and 0.69 μgL−1 to 6.90 μgL−1 for dichlorvos. Precision was good with RSD values of 13% for coumaphos and 14% for dichlorvos. The detection limits (LOD) were 0.060 μgL−1 for dichlorvos and 0.052 for coumaphos. The quantification limits (LOQ) were 0.086 μgL−1 for dichlorvos and 0.066 μgL−1 for coumaphos. The results obtained in this study suggest that SPME is a suitable technique for residual pesticide analysis of milk. The data demonstrate that particular OP pesticides used in dairy farming in the region of Minas Gerais were found to contaminate cow whole milk, and the residues are not removed by treating the milk by boiling.

Concentration of contaminants in breeding bird eggs from the Colorado River Delta, Mexico

Organic contaminants (organochlorine [OC], organophosphorus [OP] pesticides and polychlorinated biphenyls [PCBs]), and metals (As, Cd, Hg, Pb, and Se) are a concern to avian health in the Colorado River delta, Mexico. We determined concentrations of contaminants in eggs of three breeding species of birds from the delta (mourning doves [Zenaida macroura], burrowing owls [Athene cunicularia], and marsh wrens [Cistothorus palustris]). We collected 27 eggs of mourning doves, eight eggs of burrowing owls, and 18 eggs of marsh wrens for analyses. Polychlorinated biphenyls, OC, and OP pesticides were analyzed by gas chromatography equipped with an electron capture detector, and metals were analyzed by inductively coupled plasma mass spectrometry. The non-ortho PCB congeners (PCB 77 and 126) were found in mourning dove and burrowing owl eggs at concentrations in which hatchability can be affected. Mean selenium concentration found in marsh wren eggs (5.6 microg/g dry wt) exceeded the level of concern. Arsenic and Cd were found at higher than normal concentrations, Hg concentrations did not exceed the level of concern in any of the species, and Pb concentrations were higher in eggs of species subject to hunting. With the exception of lead, marsh wren eggs contained the highest metal concentrations.

Monoclonal antibody-based enzyme-linked immunosorbent assays for the detection of the organophosphorus insecticide isofenphos

In our previous study, polyclonal antibodies against the organophosphorus insecticide isofenphos were generated and enzyme-linked immunosorbent assays (ELISAs) for this pesticide were developed. In this study, isofenphos ELISAs with higher performance were developed by using a monoclonal antibody and a larger number of immunoreagents. Using the antibody and a coating antigen, an indirect competitive ELISA for isofenphos was developed, which showed an IC 50 value of 35 ng/mL with a detection limit of 5.8 ng/mL. A direct competitive ELISA using an enzyme tracer was also developed, which showed an IC 50 value of 26 ng/mL with a detection limit of 4.8 ng/mL. The antibodies in both assays showed negligible cross-reactivity with other OP pesticides. Recoveries of isofenphos from fortified rice and lettuce samples ranged 30–328 and 52–197%, respectively. The bias in the recovery values was rationalized by using the standard curves obtained in the matrix extract.

Highly Sensitive and Selective Amperometric Microbial Biosensor for Direct Determination of p-Nitrophenyl-Substituted Organophosphate Nerve Agents

We report herein a whole cell-based amperometric biosensor for highly selective, highly sensitive, direct, single-step, rapid, and cost-effective determination of organophosphate pesticides with a p-nitrophenyl substituent. The biosensor was comprised of a p-nitrophenol degrader, Pseudomonas putida JS444, genetically engineered to express organophosphorus hydrolase (OPH) on the cell surface immobilized on the carbon paste electrode. Surface-expressed OPH catalyzed hydrolysis of the p-nitrophenyl substituent organophosphorus pesticides such as paraoxon, parathion, and methyl parathion to release p-nitrophenol, which was subsequently degraded by the enzymatic machinery of P. putida JS444. The electrooxidization current of the intermediates was measured and correlated to the concentration of organophosphates. The best sensitivity and response time were obtained using a sensor constructed with 0.086 mg dry weight of cells operating at 600 mV applied potential (vs Ag/AgCl reference) in 50 mM citrate--phosphate pH 7.5 buffer with 50 microM CoCl2 at room temperature. Under optimum operating conditions the biosensor measured as low as 0.28 ppb of paraoxon, 0.26 ppb of methyl parathion, and 0.29 ppb parathion. These detection limits are comparable to cholinesterase inhibition-based biosensors. Unlike the inhibition-based format, this biosensor manifests a selective response to organophosphate pesticides with a p-nitrophenyl substituent only, has a simplified single-step protocol with short response time, and can be used for repetitive/multiple and on-line analysis. The service life of the microbial amperometric biosensor was 5 days when stored in the operating buffer at 4 degrees C. The new biosensor offers great promise for rapid environmental monitoring of OP pesticides with nitrophenyl substituent.

Children's Exposure to OP Pesticides: Response to Fenske et al.

Children's Exposure to OP Pesticides: Response to Fenske et al.

Haemato-biochemical and immuno-pathophysiological effects of chronic toxicity with synthetic pyrethroid, organophosphate and chlorinated pesticides in broiler chicks

Haemato- biochemical and immuno-pathophysiological changes following feeding of broiler chicks with 20 ppm fenvalerate (synthetic pyrethroid, SP), 2 ppm monocrotophos (organophosphate, OP) and 2 ppm endosulfan (chlorinated hydrocarbon, CH) were studied. Four groups of broiler birds (30 each) were fed poultry mash without (control) or mixed with pesticides for 8 weeks. Blood glucose, serum globulin and acetyl cholinesterase (AChE) activity level were decreased ( P<0.01) in all treated groups compared to control, but not the serum albumin and BUN. The total ATPase activity was enhanced ( P<0.01) in fenvalerate and monocrotophos than birds in control group. Body weight, total erythrocyte count, packed cell volume, haemoglobin, eosinophil and monocyte count did not show any changes. Total leucocytes and T-lymphocyte count was lower ( P<0.01) in all treated groups as compared to control group. B-cell count ( P<0.01), mean 2-4-dinitrofluorobenzene (DNFB) dermal sensitivity score and splenic indices from graft vs. host reaction ( P<0.05) were decreased in fenvalarate and endosulfan but the values for monocrotophos were intermediate between control and other treated groups. Pesticide intoxication reduced nitroblue tetrazolium (NBT) positive cells (active splenic macrophages) ( P<0.05) and spleen weight ( P<0.01). Whereas bursal weight was reduced only with endosulfan, thymic weight was reduced on endosulfan and fenvalerate-treated feed. Microscopic examination of these organs further revealed atrophy/hypoplasia, decrease in the size of follicles with depletion of lymphocytes and haemorrhages in thymus. The study concludes that the chronic exposure of chicks to small amount of SP, OP and CH pesticide leads to deleterious effects on metabolism and immune system of birds.

Acute Toxicity of Drainage Ditch Water from a Washington State Cranberry-Growing Region to Daphnia pulex in Laboratory Bioassays

High concentrations of organophosphorous insecticides resulting from cranberry bog applications were detected in the Grayland Drainage Ditch (GDD) system in Grayland, Washington State, during the 1994–1996 Washington State Department of Ecology Pesticide Monitoring Program. This drainage ditch system drains cranberry bogs and enters the Pacific Ocean via the North Cove and Supon Inlet. Concerns about the impact of these pesticides on human and environmental health led to this investigation of the potential impact on an indicator species, Daphnia pulex. To determine the toxic effects of multiple pesticides entering the GDD, standardized laboratory toxicity tests with D. pulex were conducted concurrently with the Washington State Department of Ecology pesticide sampling. Concentrations of three insecticides, diazinon, chlorpyrifos, and azinphos- methyl, were the highest ever detected in state waters. The GDD water was found to cause acute toxicity in 33− of the laboratory bioassays conducted. Regression analysis, however, detected a poor correlation between total insecticide detected and percentage mortality of D. pulex at the two drainage ditch sites studied, Grays Harbor County site and the Pacific County site. However, the relationship between mortality of D. pulex and detected concentrations of diazinon and chlorpyrifos were significant. Sampling schedules for chemical analysis and bioassay testing appear to be the primary reason that statistical analysis failed to correlate mortality with detected OP pesticide concentrations. Grab samples used in toxicity testing may over- or underestimate actual concentrations of contaminants present in the system being studied.

Cytogenetic response without changes in peripheral cholinesterase enzymes following exposure to a sheep dip containing diazinon in vivo and in vitro

Occupational exposure to organophosphorus insecticides (OPs), such as diazinon, may be monitored by the measurement of the activity of peripheral cholinesterase enzymes, including erythrocyte acetylcholinesterase (EAChE) and plasma or serum cholinesterase (plasma or serum ChE). Exposures have also been measured by the analysis of dialkyl phosphate metabolites of OPs in urine. The potential health risks associated with exposure, especially those of a neurological nature, may then be estimated, and appropriate measures to reduce or eliminate exposures can be implemented. There is evidence that some OP pesticides may have in vivo genotoxic effects, suggesting a possible link with cancer with long term or repeated heavy exposures. This paper describes work performed in 17 subjects with a single or two exposures to a sheep dip containing diazinon. Urine samples revealed OP metabolites dimethylphosphate (DMP), dimethylthiophosphate (DMTP), diethylphosphate (DEP) and diethylthiophosphate (DETP) in 37% of subjects at low levels which were not elevated after exposure. EAChE and plasma ChE were also unchanged before and after exposure, and were similar to those measured in unexposed control groups. Sister chromatid exchanges (SCE), a marker of chromosome damage, was significantly elevated in peripheral blood lymphocytes after exposure compared with before. SCE were unchanged in a group of non-occupationally exposed workers. In vitro studies with both authentic diazinon (98%) and diazinon in a sheep dip formulation (45%) showed increased SCE and decreased replicative indices, suggesting toxic and genotoxic effects of diazinon.

Pesticide use and residues on Queensland wool.

To determine practices for control of louse infestation and blowfly strike in Queensland sheep flocks that are associated with organophosphorous and synthetic pyrethroid residues on wool. Information on residues was obtained from a survey of Queensland wool clips. Information on pesticide use was obtained from a trace-back postal survey. The association between pesticide use and residues was assessed using generalised linear models, controlling for potential confounding by flock location. Between 1995 and 1997 Queensland wool clips were randomly sampled. Samples were tested for the presence and amount (mg per kg of greasy wool) of organophosphorous and synthetic pyrethroid pesticides. A questionnaire seeking information on flock characteristics and pesticide use was sent to the manager of each flock from which a wool sample was tested. The median amount of OP and SP residue was 0.8 and 0.25 mg/kg, respectively, and 91 and 95% of wool samples contained < 8 mg/kg of OP and SP residues, respectively. The frequency of OP pesticide use for louse control was significantly (P = 0.005) associated with mean OP residue amount, and the timing of SP use for louse control, in relation to shearing, was significantly (P < 0.001) associated with mean SP residue amount. Most Queensland wool clips have acceptable amounts of residues after the use of OP and SP pesticides, but wool growers can further reduce residues by effectively controlling louse infestation with pesticide applications early after shearing and the use of non-chemical methods of ectoparasite control.

Behavioral and physiological effects of acute sublethal exposure to dimethoate on wood mice, Apodemus sylvaticus [I—Laboratory studies

The effects on behavior and cholinesterase (ChE) of an OP pesticide, dimethoate, were examined in wood mice under laboratory conditions. Mice were administered 0, 5, 15, or 50 mg/kg intraperitoneal dimethoate and their behavior was recorded in an open field for one hour. In a second experiment, using only the 0 and the 50 mg/kg dose, mice were subjected to 10-min open field tests repeated at various time intervals during a 24-h period. Shortly after administration of dimethoate, there was a general, dose-dependent, behavioral depression that was characterized by increased inactivity and decreased grooming, rearing, and sniffing. The introduction of a novel object in the open field failed to elicit any reaction in mice treated with the two highest doses of dimethoate. The behavioral impairment completely disappeared 6 h after treatment. A stereotyped compulsive grooming was also observed in the first 30 min after administration of the two highest doses. Exposure to dimethoate caused a dose-dependent decrease in ChE activity in the brain and in serum. Behavioral impairment was associated with maximum levels of ChE inhibition of 65-75% (brain) and 75-85% (serum). Recovery of ChE activity lagged behind that of behavioral impairment and started 3-6 h after dimethoate administration. The possible implications for free-living wood mice which inhabit cereal fields and may be exposed to OPs are discussed.

Enhanced expression of a bacterial gene for pesticide degradation in a common soil fungus

The wide dispersion of toxic chemicals throughout the varied soil and water structures of our earth has stimulated the development of bioremediation technologies which may be applied to many different environmental situations. One of the new developing technologies involves the genetic engineering of natural microorganisms with new degradative capabilities for bioaugmentation of select, contaminated environments. In these studies, a hybrid genetic system formed by the combination of a bacterial structural gene in a fungal expression system is developed and productively introduced into the common soil fungus, Gliocladium virens. The hybrid gene was formed by combining the constitutive promoter and translational signals from a fungal gpd gene (encoding glyceraldehyde-3-phosphate-dehydrogenase) with the structural region of the bacterial opd gene (organophosphate degrading gene). The fungal expression region of gpd included 1071 bp of the 5′-flanking region (the promoter) and 735 bp of the 3′-flanking region (the terminator). A set of opd expression vectors was constructed containing different length of promoter and terminator regions and used to transform a native strain of G. virens, a hardy soil-inhabiting fungus. Organophosphate hydrolase (OPH, encoded by the opd gene) activity was detected in several of the genetic transformants, and the levels of expression were greatly enhanced (over twenty-fold) as compared to transformants with nonhomologous fungal genetic sequences. Furthermore, it was possible to increase the enzymatic activity another fifteen to thirty-five fold by adding 0.1 mM concentrations of Co +2 in the growth medium. Thus, these studies have demonstrated the ability to genetically augment the metabolic capabilities of a naturally-occurring fungus, rendering them capable of ex situ environmental remediation of a variety of widely used neurotoxic, OP pesticides.

Organophosphorus compounds in the river PO and in the northern Adriatic

Two classes of organophosphorus compounds, OP pesticides and phosphate esters used for industrial purposes were investigated in a monitoring program carried out at the mouth of the river Po and along the Adriatic coast. Contamination by OP pesticides resulted rather low and of land based origin. Non‐pesticides, on the contrary, can reach considerable contamination levels both in river and in sea water. Local sources of pollution seem to be present along the coast and to cause in some cases a more severe pollution in sea than in river water.