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Measurement of insecticide uptake and effective fraction in a beneficial insect using solid-phase microextraction.

The determination of insecticide uptake in beneficial insects is important for quantifying the doses that are responsible for the toxicological effects and to compare them with the doses that insects may absorb in treated fields. Because of the small size of some beneficial species, the amount of insecticide absorbed may be very low. Herein, we present a method that relies on the sensitivity and specificity of SPME (solid-phase microextraction) as a sampling technique that can be used to measure very small amounts of an organophosphorus insecticide in small insects. In our study, the method was applied to quantify the internal dose and free dissolved fraction of chlorfenvinphos in beneficial parasitoids exposed through a topical application. Up to 0.5 ng of the insecticide could be quantified in these fractions, that is, 10 times less than when using solvent extraction techniques. The penetration and elimination rates of the insecticide in the insect were also determined. The method proved to be suitable to quantify internal doses in parasitoids collected in a treated field.

Animals↗

Organophosphorus pesticide residues in Mexican commercial pasteurized milk.

A study was conducted to measure residues of 13 organophosphorus (OP) pesticides, widely used as dairy cattle ectoparasiticides or in crops used for animal feed, in homogenized and pasteurized Mexican milk samples. Four different milk brands with high distribution were collected biweekly during a 12 month period (n = 96) in supermarkets. OP pesticide residues were measured by gas chromatography with a flame photometric detector. Approximately 39.6% of the samples contained detectable levels of OP pesticide residues. Eight samples contained residues exceeding established maximum residue limits (MRL), and the OP pesticides present in these samples were dichlorvos (five samples), phorate, chlorpyrifos, and chlorfenvinphos (one sample, respectively). Average residues of 13 OP pesticides measured were below established MRLs ranging between 0.0051 and 0.0203 ppm.

Animals↗

Effect of commercial formulation of four organophosphorus insecticides on the ovaries of a freshwater teleost, Mystus vittatus (bloch)--a histological and histochemical study.

The chronic effect of an exposure to a sublethal concentration of commercial formulation of 4 organophosphorus insecticides such as birlane (chlorfenvinphos), gardona (tetrachlorvinphos), phosdrin (mevinphos) and malathion on midvitellogenic ovaries of Mystus vittatus was investigated using several histological and histochemical techniques. The loss of stage II and III oocytes accompanied by a significant decline in gonadosomatic index was recorded. The vitellogenesis in treated fishes was ceased as the oocytes did not advance to further stages as evidenced by the absence of lipid yolks and the lack of a PAS positive follicular layer. The RNA content of remaining stage I oocytes of exposed fishes resembled that of the stage I oocytes of the control fishes. The absence of delta 5-3 beta-hydroxysteroid dehydrogenase suggested the lack of steroidogenesis. Reduced glucose-6-phosphate dehydrogenase activity was also observed. The significance of these observations has been discussed in light of available literature.

3-Hydroxysteroid Dehydrogenases↗

Insecticide resistance in field strains of Lucilia cuprina.

A survey was conducted in 1977-78 to determine the level of insecticide resistance in field strains of Lucilia cuprina from a wide area of Queensland. Discriminating dose data showed that all 19 strains tested were resistant to diazinon, dichlofenthion, fenthion-ethyl and chlorfenvinphos, but resistance factors in all strains were low, the highest being 18.9 for diazinon. Six strains showed no resistance to butacarb. A strain collected in 1967 and selected with diazinon for 117 generations, attained resistance factors of organophosphorus compounds far higher than those of the present field strains.

Animals↗

The effect of arsenical dips on Parafilaria bovicola in artificially infected cattle in South Africa.

The possible adverse effect of arsenical tick control dips on Parafilaria bovicola infections was investigated in 48 artificially infected cattle. A treatment group of 24 cattle was dipped in a plunge dip containing 1600 ppm arsenic trioxide. A control group of the same size was dipped in an organophosphate dip containing a mixture of chlorfenvinphos and dioxathion. Regular weekly to 3-weekly dipping had no effect initially on the prevalence of ovipositional blood spots of P. bovicola in either group. However, from 4 months after bleeding commenced there was a significant reduction in blood spots in the arsenic-dipped cattle and, on slaughter at 12-14 months after infection, the arsenic group had significantly fewer live worms and fewer carcass lesions. Arsenic residues in muscle samples of treated cattle were 11.6 times higher than in the controls. It is proposed that arsenic residues in the sub-cutaneous muscle layers increase with repeated dipping until a level toxic to P. bovicola is finally reached. Older cattle would therefore be refractory to infection and their carcasses at slaughter would not be affected.

Animals↗

Neuromuscular effects of chronic administration of two organophosphorus insecticides to rats.

Groups of rats were fed chlorfenvinphos (at two dose levels) or dicrotophos. Whole blood and plasma cholinesterase activity was markedly reduced. Compared with control animals there was no change in muscle action potential amplitude (M response) to a single stimulus for periods of up to 3 months. Exposed animals developed a prolonged negative potential at the end of the spike potential, lasting up to 15 ms. Superimposed on this, repetitive activity (RA) developed with latency of approximately 4.5 ms. These abnormalities became more marked with time, even on constant dosing. Double and repetitive stimulation reduced or abolished the prolonged negative potential and RA.

Action Potentials↗

Acetylcholinesterase activity in several rat liver cell fractions after repeated poisoning with some organophosphates.

The reported investigations were carried out on female Wistar rats aged about 6 weeks, weighing 150 g. During 28 days the fats were given daily organophosphates (DFP, DDVP, Chlorfenvinphos, IPO-62, IPO-63, Phospholine) in a dose of 1/10 LD50 subcutaneously. The observations were conducted on groups of 8 rats. The body weight of the animals was noted on the 10th, 20th and 28th day of the experiment. On the 28th day the rats were killed and the activity of acetylcholinesterase (AChE) was determined by the biochemical method of Hestrin in liver homogenate and in the microsomal, mitochondrial and soluble fractions of liver cells. After 28 days of the experiment the body weight of the rats poisoned with these substances amounted to from 80.8% (DFP) to 90.7% (IPO-63) of that in control animals. The AChE activity was also reduced in relation to the control group ranging in the liver homogenate from 49.7% (DFP) to 75.6% (IPO-63), in the microsomal fraction from 33.0% (DFP) to 63.8% (IPO-63), in the mitochondrial fraction from 45.5% (DFP) to 72.9% (IPO-63), and in the soluble fraction from 52.8% (DFP) to 80.5% (DDVP).

Acetylcholinesterase↗

Effects of fertilizer on insecticides adsorption and biodegradation in crop soils.

Recent organic fertilizer treatments (cow manure, pig slurry, composts, or green manure) simultaneously increase insecticide adsorption onto soil and the insecticide soil persistence, indicating a mechanism of slow release of insecticide into soil by the organic matter. This occurred in sugar beet crops with aldicarb, thiofanox and imidacloprid; also, in leek, cauliflower and brussels sprouts crops with chlorpyrifos and chlorfenvinphos. In contrast, organic fertilizer treatments applied once or repeatedly in the past, have no significant influence on adsorption or persistence of insecticides; the same is observed for the old soil organic matter, when its soil concentrations change in limited ranges

Adsorption↗

Reactivation by various oximes of human erythrocyte acetylcholinesterase inhibited by different organophosphorus compounds.

The new bispyridinium oximes HI 6 and HLö 7 are promising antidotes against poisoning by highly toxic organophosphorus compounds, i.e. nerve agents. Until now, their ability to reactivate pesticide inhibited human acetylcholinesterase (AChE) has not been elucidated. For this purpose human erythrocyte AChE (EC 3.1.1.7) was inhibited (30 min) by chlorfenvinphos, dichlorvos, dicrotophos, heptenophos, mevinphos, monocrotophos, paraoxon, phosphamidon, trichlorfon, malaoxon, omethoate, oxydemeton-methyl or methamidophos by 85-98% of control. After removal of excess inhibitor, obidoxime, pralidoxime (2-PAM), HI 6 or HLö 7 (10, 30 or 100 mumol/l) were added and the AChE activity was measured spectrophotometrically at various times thereafter (5-60 min). The oximes significantly, but not completely, reactivated organophosphate inhibited AChE. The velocity and extent of reactivation were dependent on the oxime and its concentration. In all cases obidoxime was superior to the three other oximes, followed by HLö 7, 2-PAM and HI 6. In most cases obidoxime and HLö 7 were most effective at 10 or 30 mumol/l while 2-PAM and HI 6 needed 100 mumol/l. These data suggest that 2-PAM HI 6 and HLö 7 are less patent than obidoxime in reactivating human AChE inhibited by organophosphate pesticides.

Acetylcholinesterase↗

Pesticide toxicity assessment using an electrochemical biosensor with Pseudomonas putida and a bioluminescence inhibition assay with Vibrio fischeri.

Two different toxicity tests, an electrochemical biosensor Cellsense and a bioluminescence inhibition assay ToxAlert were performed in order to establish and compare the acute toxicity responses of different types of raw and spiked water for a selected group of pesticides. The selected compounds were endosulfan, chlorfenvinphos, dimethoate, fenamiphos, ametryn, deltamethrin and alpha-cypermethrin; all of them are used in large quantities for agricultural purposes. In the first step, the study of the toxicity responses for each individual pesticide with Milli-Q water was carried out. Next, the toxic responses of different mixtures of these pesticides in different water matrices, i.e., Milli-Q water, surface water, groundwater and wastewater were studied in order to evaluate (i) device advantages and limitations for the toxicity evaluation of real environmental samples, (ii) antagonistic or synergistic effects and (iii) the influence of the water matrices. The survey of pesticides in real samples was carried out using a combined method involving both chemical analysis and toxicity bioassays. Chemical analysis involved the use of solid-phase micro-extraction (SPME) followed by gas chromatography with electron capture detection (GC/ECD) or thermoionic specific detection (GC/TSD) with mass spectrometric confirmation (GC/MS).

Biosensing Techniques↗

Determination of pesticides by solid phase extraction followed by gas chromatography with nitrogen-phosphorous detection in natural water and comparison with solvent drop microextraction.

The European Union specifies that drinking water can contain pesticide residues at concentrations of up to 0.1 microg/L each and 0.5 microg/L in total, and that 1-3 microg/L of pesticides can be present in surface water, but the general idea is to keep discharges, emissions and losses of priority hazardous substances close to zero for synthetic substances. Therefore, in order to monitor pesticide levels in water, analytical methods with low quantification limits are required. The method proposed here is based on solid phase extraction (SPE) followed by gas chromatography with a nitrogen-phosphorous detector (GC-NPD). During method development, six organophosphate pesticides (azinphos-ethyl, chlorfenvinphos, chlorpyriphos, ethoprophos, fenamiphos and malathion) and two organonitrogen pesticides (alachlor and deltamethrin) were considered as target analytes. Elution conditions that could influence the efficiency of the SPE were studied. The optimized methodology exhibited good linearity, with determination coefficients of better than 0.996. The analytical recovery for the target analytes ranged from 70 to 100%, while the within-day precision was 4.0-11.5%. The data also showed that the nature of the aqueous matrice (ultrapure, surface or drinking water) had no significant effect on the recovery. The quantification limits for the analytes were found to be 0.01-0.13 microg/L (except for deltamethrin, which was 1.0 microg/L). The present methodology is easy, rapid and gives better sensitivity than solvent drop microextraction for the determination of organonitrogen and organophosphate pesticides in drinking water at levels associated with the legislation.

Chromatography, Gas↗

Sensitive detection of organophosphates in river water by means of a piezoelectric biosensor.

A highly sensitive piezoelectric biosensor has been developed for detection of cholinesterase inhibitors. The inhibitor benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on a monolayer of 11-mercaptomonoundecanoic acid (MUA) self-assembled on the gold surface of the sensor. The binding of high-molecular-weight cholinesterase to the immobilized cocaine derivative was monitored with a mass sensitive piezoelectric quartz crystal (quartz crystal nanobalance; QCN). In the presence of an inhibiting substance in the sample, the binding of cholinesterase to the immobilized inhibitor was reduced. The decrease of the rate of mass change was proportional to the concentration of free inhibitor in the sample. This way the affinity sensor followed anti-cholinesterase toxicity and the enzyme activity of ChE was not addressed. A assay for detection of organophosphates (OP) was optimized. Regeneration of the sensor surface was achieved with 1 mol L(-1) formic acid, which enabled 40 measurements with one sensor. All assays were carried out in a flow-through arrangement. The total measurement time (binding+regeneration) was 25 min and the detection limit for different OP (paraoxon, diisopropylfluorophosphate, chlorpyriphos, and chlorfenvinphos) was down to 10(-10) mol L(-1) (0.02 microg L(-1)). This sensor was used for determination of organophosphate (diisopropylfluorophosphate) levels in river water samples.

Biosensing Techniques↗

Matrix solid-phase dispersion extraction procedure for multiresidue pesticide analysis in oranges.

A multiresidue extraction method based on matrix solid-phase dispersion (MSPD) is optimized for the extraction and gas chromatographic screening of eighteen insecticides (aldrin, carbophenothion, captafol, chlorpyriphos, chlorfenvinphos, diazinon, dicofol, alpha-endosulfan, beta-endosulfan, ethion, fenitrothion, folpet, methidathion, malathion, methyl-azinphos, methyl-parathion, phosmet, and tetradifon) from oranges. After optimization of different parameters, such as type of solid phase used and the amount of solid phase or eluent, recoveries ranged from 67 to 102% with relative standard deviations ranging from 2 to 10%. The limits of detection, calculated as 3 times the baseline noise ranged from 2 to 171 micrograms/kg. These limits of detection were about 10 times lower than the maximum residue levels established by the European Community. Compared with classical methods, the described procedure is simple, less labour intensive and does not require preparation and maintenance of equipment. Troublesome emulsions, such as those frequently observed in liquid-liquid partitioning did not occur.

Acetates↗

Effect of commercial formulation of four organophosphorus insecticides on the LH-induced germinal vesicle breakdown in the oocytes of a freshwater teleost, Mystus vittatus (Bloch)--a preliminary in vitro study.

Effect of commercial formulation of four organophosphorus insecticides such as malathion, phosdrin (mevinphos), birlane (chlorfenvinphos), and gardona (tetrachlorvinphos) on LH-induced in vitro germinal vesicle breakdown (GVBD) in the oocytes of Mystus vittatus was investigated using three concentrations for each insecticide. All of these insecticides could significantly inhibit the LH-induced GVBD in all of their concentrations except two lower concentrations of birlane. A probable mechanism of inhibition of reproduction by these insecticides is discussed in the light of present findings.

Animals↗

Piperonyl butoxide as a tool in aquatic toxicological research with organophosphate insecticides.

Experiments were conducted to determine the effects of piperonyl butoxide, a synthetic methylenedioxyphenyl inhibitor of cytochrome(s) P450, on the toxicity of organophosphate insecticides to three cladoceran test species: Ceriodaphnia dubia. Daphnia magna, and Daphnia pulex. Coadministration of piperonyl butoxide effectively reduced the acute toxicity of four metabolically activated organophosphates (parathion, methyl parathion, diazinon, and malathion) and did not affect the toxicity of three organophosphates not requiring metabolic activation (dichlorvos, chlorfenvinphos, and mevinphos). These results indicate that piperonyl butoxide may be an effective tool in toxicological research focused upon identifying specific compounds responsible for toxicity in complex aqueous mixtures.

Animals↗

Cytogenetic effects on human lymphocytes of a mixture of fifteen pesticides commonly used in Italy.

Lymphocytes obtained from 5 healthy donors were incubated with a mixture of 15 pesticides commonly found in foods of central Italy (dithiocarbamates (20.7%), benomyl (19.6%), thiabendazole (14.9%), diphenylamine (14.4%), chlorthalonil (13.1%), procymidone (8.0%), methidathion (2.3%), chlorpyrifos-ethyl (2%), fenarimol (1.9%), parathion-methyl (1%), chlorpropham, parathion, vinchlozolin, chlorfenvinphos and pirimiphos-ethyl (< 1%)). The percent of each pesticide in the mixture was proportional to its average concentration in foods. Incubated with the lymphocytes at a concentration of 1-20 micrograms/ml the pesticide mixture did not induce significant variations in the number of hypodiploid, hyperdiploid and polyploid cells or in the number of chromosome and chromatid aberrations. On the contrary, we observed a dose-dependent increase in the number of nonsynchronous centromeric separations which reached the level of 37.9% at 20 micrograms/ml of pesticide mixture in the incubation medium. This effect was not observed when benomyl was excluded from the mixture. These data show that the removal of benomyl could decrease the toxicity of pesticide residues present in human food.

Adult↗

Differences in the mode of lethality produced through intravenous and oral administration of organophosphorus insecticides in rats.

This study was undertaken to investigate the possibility that mechanisms other than cholinesterase (ChE) inhibition account for the acute toxicity of organophosphorus insecticide. Both the P = O type insecticide (direct ChE inhibitors: chlorfenvinphos and dichlorvos) and the P = S type insecticide (indirect ChE inhibitors: diazinon and fenthion) were employed. Rats treated with lethal doses of intravenous and oral P = O type insecticides and oral P = S type insecticides exhibited typical signs of anti-ChE poisoning along with marked inhibition of brain and erythrocyte ChE activity. In contrast, rats given lethal doses of intravenous P = S type insecticides exhibited tonic convulsions and opisthotonos, with only slight inhibition of ChE activities. When P = O type insecticides were intravenously administered to anesthetized and conscious rats, animals exhibited typical anti-ChE poisoning signs in cardiorespiration: hypertension and apnea which were antagonized by atropine. After administration of lethal doses of P = O type insecticides, breathing disappeared before the cessation of heart beats. Rats receiving lethal doses of intravenous P = S type insecticides did not show hypertension, but exhibited transient cessation of breathing and heart beats. Breathing was observed after the disappearance of heart beats. The electroencephalogram (EEG) was characterized by spike and wave complexes. The EEG and cardiorespiratory changes were not antagonized by atropine. It was concluded that lethality following intravenous P = S type insecticides may be independent of ChE inhibition.

Administration, Oral↗