Transcriptomic analysis of molecular mechanisms underlying the biodegradation of organophosphorus pesticide chlorpyrifos by Lactobacillus delbrueckii ssp.bulgaricus in skimmed milk

Bioremediation of organophosphorus pesticides in contaminated foodstuffs using probiotics has been increasingly under the spotlight in recent years,though the biodegradation mechanism and derived intermediate products remain unclear.This study aimed to help fill this knowledge gap and examined the degradation mechanism of organophosphorus pesticide,chlorpyrifos,in milk by Lactobacillus delbrueckii ssp.bulgaricus using gas chromatography-tandem mass spectrometry(GC-MS/MS)combined with transcriptome analysis.After the strain was cultured for 20 h in the presence of chlorpyrifos,differential expressions of 383 genes were detected,including genes probably implicated during chlorpyrifos degradation such as those related to hydrolase,phosphoesterase,diphosphatase,oxidoreductase,dehydratase,as well as membrane transporters.GC-MS/MS analysis revealed the changes of secondary metabolites in L.bulgaricus during milk fermentation due to chlorpyrifos stress.6-Methylhexahydro-2H-azepin-2-one,2,6-dihydroxypyridine and methyl 2-aminooxy-4-methylpentanoate as intermediates,along with the proposed pathways,might be involved in chlorpyrifos biodegradation by L.bulgaricus.

Study on the Toxicity Effect of Abamectin and Chlorpyrifos on Grass Carp(Ctenopharyngodon idllus)

[ Objective] The study aimed to discuss the toxicity effect of abamectin and ehlorpyrifos on grass carp （ Ctenopharyngdon idllus ） and their interrela- tions. [ Method] Taking healthy and active grass carp as the object, the acute toxicity test of single pesticide and the combined toxicity test of two pesticides on grass carp were carried out. [ Result] The LC50 of abamectin and chlorpyrifes and the mixture of two pesticides against grass carp at 24, 48, 72 and 96 h were as follows： abamectin： 0.54, 0.49, 0. 17 and 0.10 rag/L; chlorpyrifes ： 0.29, 0.21, 0.12 and 0.05 rag/L; the mixture of two pesticides ： 1.22, 1.08, 0.99 and 0. 86 mg/L. The safe concentration （SC） of ahamectin, chlorpyrifos and the mixture of two pesticides were 0.010, 0.005 and 0.086 rag/L, respectively. The tox- icity of the pesticides in sequence was ablorpyrifos 〉 ahamectin 〉 the mixture of two pesticides. [ Conclusion ] Different concentrations of abamectin, chlorpyrifos and the mixture of two pesticides had remarkable effect on the growth of grass carp, the higher the concentration was, the greater the toxicity effect was. Ahamectin at low toxicity intensity had certain relief function on the toxicity of ehlorpyrifos.

Synthesis and Characterization of Microcapsules with Chlorpyrifos Cores and Polyurea Walls

Microcapsules with chlorpyrifos cores and polyurea walls were synthesized with 2,4-tolylene diisocyanate as an oil-soluble monomer and ethylenediamine as a water-soluble monomer via an interracial polycondensation reaction. The products were characterized by means of Fourier transform infrared spectrometry, ^13C NMR spectrometry and ^31p NMR spectrometry. The morphology, the particle size and the particle size distribution, and the thermal properties were also evaluated. The prepared microcapsules exhibit clear and smooth surfaces and have a mean diameter of 28. 13 μm. These microcapsules also have a good thermal stability for long-term use, and have potential applications in minimizing the toxicity of chlorpyrifos through controlled release.

Degradation of chlorpyrifos alone and in combination with chlorothalonil and their effects on soil microbial populations

In practice, pesticides are usually applied simultaneously or one after another for crop protection, and this type of pesticide application often leads to a combined contamination of pesticide residues in the soil environment. A laboratory study was conducted to investigate the influence of chlorothalonil on chlorpyrifos degradation and its effects on soil bacterial, fungal, and actinomycete populations. Under the experimental conditions here, the half-lives of chlorpyrifos alone, and in combination with chlorothalonil, at the recommended and double dosages, were measured to be 3.24, 2.77, and 2.63 d, respectively. Chlorpyrifos degradation was not significantly altered by its combination with chlorothalonil. However, the inhibitory effect of chlorpyrifos on soil microorganisms was increased by its combination with chlorothalonil, and the increase was related to the levels of chlorothalonil added. Compared to those in the controls, the populations of bacteria, fungi, and actinomycetes were significantly reduced by 44.1%, 61.1%, and 72.8%, respectively, on the first day after treatment （DAT） by chlorpyrifos alone. With the addition of chlorothalonil, the inhibition was increased to 55.2%, 79.3%, and 85,8% at the recommended dosage, and 86.0%, 94.1%, and 90.8% at the double dosage, at one DAT, respectively. The results suggested that combined effects should be taken into account to assess the actual impacts of pesticide applications.

Degradation of chlorpyrifos in laboratory soil and its impact on soil microbial functional diversity

Degradation of chlorpyrifos at different concentrations in soil and its impact on soil microbial functional diversity were investigated under laboratory condition. The degradation half-live of chlorpyrifos at levels of 4, 8, and 12 mg/kg in soil were calculated to be 14.3, 16.7, and 18.0 d, respectively. The Biolog study showed that the average well color development （AWCD） in soils was significantly （P 〈 0.05） inhibited by chlorpyrifos within the first two weeks and thereafter recovered to a similar level as the control. A similar variation in the diversity indices （Simpson index lID and McIntosh index U） was observed, but no significant difference among the values of the Shannon-Wiener index H＇ was found in chlorpyrifos-treated soils. With an increasing chlorpyrifos concentration, the half-life of chlorpyrifos was significantly （P ≤ 0.05） extended and its inhibitory effect on soil microorganisms was aggravated. It is concluded that chlorpyrifos residues in soil had a temporary or short-term inhibitory effect on soil microbial functional diversity.

Dissipation of chlorpyrifos on pakchoi inside and outside greenhouse

The dissipation of chlorpyrifos on pakchoi inside and outside greenhouse was studied. The decline curve of chlorpyrifos on pakchoi could be described as first-order kinetic. The experimental data showed that both the hermetic environment of greenhouse and season affected dissipation rates of chlorpyrifos on pakchoi. Chlorpyrifos declined faster outside greenhouse than inside greenhouse. Chlorpyrifos residues at pre-harvest time were below the maximum residue limits(MRLs) fixed in China, whereas the values inside greenhouse were higher than those outside greenhouse by almost 50%. The recommended pre-harvest time established under conditions of open field might not always fit to greenhouse production.

Dissipation of chlorpyrifos in pakchoi-vegetated soil in a greenhouse

The dissipation of chlorpyrifos in pakchoi-vegetated soil was investigated in the summer and autumn in a greenhouse and field, respectively. The dissipation of chlorpyrifos in pakchoi-grown soil was comparatively described by fitting the residue data to seven models （lst-order, 1.5th-order, 2nd-order, RF lst-order, RF 1.5th-order, RF 2nd-order, and bi-exponential or two-compartment models）. Statistical analysis was performed using the SPSS 11.5 statistical package. The bi-exponential model was selected as the optimal model according to the coefficient of determination r^2. The dissipation half-lives （DT50） of chlorpyrifos in pakchoi-vegetated soil at the recommended dose in the summer and autumn, calculated by the bi-exponential model, were 0.6 and 1.2 d in a greenhouse, 0.4 and 1.0 d in a field, respectively; the corresponding values at double dose were 1.2 and 2.1 d in a greenhouse, 0.5 and 1.3 d in a field, respectively. The kinetic data indicate the dissipation of chlorpyrifos in pakchoi-grown soil in a greenhouse is slower than that in a field, and dissipates slower in the autumn than in the summer.

Toxicity assessment for chlorpyrifos-contaminated soil with three different earthworm test methods

Earthworm toxicity tests are useful tools for terrestrial risk assessment but require a hierarchy of test designs that differ in effect levels （behavior, sublethal, lethal）. In this study, the toxicity of chlorpyrifos contaminated soil on earthworms was assessed. In addition to the acute and chronic tests, an avoidance response test was applied. Earthworms were exposed to sublethal and lethal concentration of chlorpyrifos, and evaluated for acute toxicity, growth, fecundity and avoidance response after a certain exposure period. The test methods covered all important ecological relevant endpoints （acute, chronic, behavioral）. Concentration of 78.91 mg/kg, chlorpyrifos caused significant toxic effects in all test methods, but at lower test concentrations, only significant chronic toxic effects could be observed. In present study, chlorpyrifos had adverse effect on growth and fecundity in earthworm exposed to 5 mg/kg chlorpyrifos after eight weeks. The avoidance response test, however, showed significant repellent effects at concentration of 40 mg/kg chlorpyrifos. For chlorpyrifos, concentration affecting avoidance response was far greater than growth and fecundity, it seemed likely that earthworms were not able to escape from pesticide-contaminated soil into the clean soil in field and hence were exposed continuously to elevated concentrations of pesticides.

Alleviating effects of melatonin on oxidative changes in the testes and pituitary glands evoked by subacute chlorpyrifos administration in Wistar rats

Objective:To evaluate the alleviating effects of melatonin on oxidative changes in the testes and pituitary gland induced by subacute chlopyrifos（CPF） exposure in rats.Methods:Forty adult male Wistar rats divided into 4 groups of 10 animals were used for the study.Croup I received soya oil（2 mL/kg） while group II was administered with melatonin（0.5 mg/kg）.Group III was administered CPF only(8.5 mg/kg l/10th of the LD50) while group Ⅳ was pretreated with melatonin（0.5 mg/kg） and then exposed to CPF（8.5 mg/kg）,10 min later.The regimens were administered by gavage once daily for a period of 28 d.At the end of the exposure period, the rats were sacrificed and the testicular tissues and pituitary glands were evaluated for the malonaldehyde（MDA） concentration and activities of superoxide dismutase（SOD） and catalase（CAT）.Results:CPF increased MDA concentrations and reduced the activities of SOD and CAT in the testes and pituitary gland.Melatonin pretreatment reduced the testicular and pituitary MDA concentrations and improves the SOD and CAT activities.Conclusions:the study showed that subacute CPF-induced oxidative stress in the testes and pituitary glands were alleviated by melatonin due to its antioxidant property.

Effect of chlorpyrifos on soil microbial populations and enzyme activities

Chlorpyrifos is an organophosphate pesticide that acts as an insecticide-acaricide by ingestion, contact, and inhalation, and is now widely used in greenhouse for the control of pests. The dissipation of chlorpyrifos in crops such as orange fruit （Martinez-Vidal et al., 1998）, tomatoes（Aysal et al., 1999）, soil and its effects on soil microbial characteristics （Singh et al., 2002） has been reported frequently. However, little information is available to describe impacts of chlorpyrifos on soil microbial population and enzyme activity in greenhouse. In this study, the effect of chlorpyrifos on soil bacteria, fungi, actinomycetes, and soil enzyme activity was investigated.

Degradation of Chlorpyrifos and Fipronil in Rice from Farm to Fork and Risk Assessment

Degradation of pesticide residues （chlorpyrifos and fipronil） in rice from farm to fork and risk assessment for human health were studied to reveal the magnitude of risks faced by different populations of interest, so that appropriate measures can be taken to control the risks, and to refine and update the human health risk assessment data while helping to determine the maximum residue level （MRL） value and harvest interval. Different dosages and treatments were used in field trials for the harvest residue test. Residue levels of postharvest-applied chlorpyrifos and fipronil during storage, exposure to sunlight, washing and boiling processes （boiled rice） were investigated for brown rice. The dietary exposure evaluation model （DEEM） was employed to estimate acute and chronic risks faced by different populations of interest. Percent of reference dose （POR） and margin of exposure （MOE） were calculated. A positive correlation between pesticide residues and the dosage and application frequency of pesticide was found in the field trials. Risk quotients indicate that multiple applications and double dosages of chlorpyrifos increase the risks to the entire population and prolong exposures to toxic concentrations. The concentration of pesticide residues decreased as a function of time, after sunlight exposure, storage, washing, and boiling processes. 91.6 and 96.16% degradations were achieved at the end of the experimental period for fipronil and chlorpyrifos, respectively. The boiling process played an important role in the degradation of these pesticides. The result of risk assessment to human health showed that harvest residues of chlorpyrifos in rice and acute dietary risks of chlopyrifos were of concern. The acute dietary （food only） risk estimated for chlorpyrifos as percent of acute population adjusted dose （aPAD） was frequently over 100%. The risk faced by boys under the age of 14 was higher than that for girls of the same age. For the subpopulation above age 14, the risk reversed. The chronic dietary risk from food alone showed that dietary exposures with fipronil were below the level of concern for the entire population, including children. The risk faced by rural residents was more serious than that for urbanite residents with the most sensitive populations being children and male residents who faced higher acute dietary risk than the other subpopulation groups. The harvest interval was found to be the critical measure to mitigate risk for all populations for safe rice eating. All risk levels decreased to acceptable levels when the harvest interval was extended to 14 d. To address these risks, a number of measures including reduced application rates （should not be doubled at single application）, increased retreatment intervals （longer than 7 d） and extended interval of harvest （at least 14 d） will be needed. The MRL for fipronil in rice is recommended to be 0.01 mg kg 1 in accordance with Codex （ref）.

The Effect of High-Pressure Arc Discharge Plasma on the Degradation of Chlorpyrifos

A study is conducted to determine the effect of a kind of high-pressure arc discharge plasma on the degradation rate and kinetic equations of chlorpyrifos in different solvents with the treated times and concentrations as variables. The degradation rate was sorted in different solvents as water, methanol, acetone and then acetoacetate. The tendencies of the degradation rates with treated time in water and methanol were optimally fitted with first-order kinetics equations while those in acetone and acetoacetate were fitted with zeroth-order kinetics equations. The difference was attributed to the stronger polarity of water and methanol. The weak correlation of the degradation rates with time was mainly because the high-temperature of the arc discharge tube and the chemically-active species generated by the discharge. The degradation half-life was extended with increase of chlorpyrifos concentration. A degradation half-life less than 3 min was achieved for chlorpyrifos in water and methanol when the initial concentration was less than 300 μg/ml.

Review of Toxicology of Atrazine and Chlorpyrifos on Fish

Atrazine （ATR） and chlorpyrifos （CPF） are widely used in agriculture, but have resulted in a series of toxicological and environmental problems. They were heavily used which have potential threat to fish and rodents. Several recent laboratory studies have shown ATR and CPF could lead to oxidative damage, immunocyte reduced and inhibit acetylcholinesterase （ACHE）. In order to clarify the toxicity of ATR and CPF, this paper summarized the adverse effects of ATR and CPF on reproduction, nerve and immune systems in fish.

Evaluation of the Genotoxicity of Chlorpyrifos in Common Indus Valley Toad, <i>Bufo stomaticus</i>Using Alkaline Single-Cell Gel Electrophoresis (Comet) Assay

Chlorpyrifos is a commonly used pesticide of organophosphate group, which causes toxicological effects in non-target organisms especially fish and frogs. In the present study, the genotoxicity of sublethal concentrations of chlorpyrifos was observed in the erythrocytes of common Indus valley toad, Bufo stomaticus, using the Alkaline Single-Cell Gel Electrophoresis (Comet) assay. In the first step, acute toxicity of chlorpyrifos was evaluated by exposing the tadpoles to high concentrations of the pesticide. The acute LC50 value of chlorpyrifos, calculated by Trimmed Spearman-Karber (TSK) in static bioassay, was found to be 930.0 μg/L. On the basis of acute LC50 value, the tadpoles were exposed to three sublethal concentrations (155, 233 and 465 μg/L) of chlorpyrifos for 96 h. Blood cells were collected at every 24 h interval and were subjected to the Alkaline Single-Cell Gel Electrophoresis assay. The observed DNA damage was concentration and time-dependent, and those levels of DNA damage in between the tested concentrations and times were significantly different (p < 0.05). The tadpoles exposed to different concentrations of chlorpyrifos also showed different morphological abnormalities. It was concluded that chlorpyrifos is a genotoxic pesticide causing DNA damage in Bufo stomaticus.

Dissipation Kinetics of Chlorpyrifos in Soils of a Vegetable Cropping System under Different Cultivation Conditions

Dissipation kinetics of chlorpyrifos in the near-neutral paddy soils of a vegetable cropping system under greenhouse, screenhouse and field conditions was studied using a rapid analytical method. The recoveries of chlorpyrifos were between 86.5% and 105.5% with relative standard deviations for repeatability between 6.6% and 9.1% at fortification levels of 0.01, 0.1 and 1 mg/kg in the soil. The limit of detection of the method was 0.004 mg/kg and the limit of quantification was found to be 0.01 mg/kg. The dissipation rates of chlorpyrifos followed the first-order kinetics and the half-life was 6.96, 6.04 and 5.20 days in the soil under greenhouse, screenhouse and field conditions, respectively. The dissipation rates of chlorpyrifos in the soil varied with different cultivation conditions. Chlorpyrifos in the soil dissipated slower in a greenhouse and screenhouse than in the open field, which was likely attributed to the hermetic environment in the greenhouse and screenhouse.

Development of Ecologically Acceptable Chlorpyrifos Formulation for Effective and Safe Application

Application of the commercial formulation of chlorpyrifos has resulted in considerable environmental contamination. This study was designed to develop environmentally acceptable chlorpyrifos formulation for safe and effective application. This involved the modification of clay surfaces from hydrophilic to hydrophobic via ion-exchange reaction with organic cations. The resulting organo-clay complexes were tested for adsorption and release of chlorpyrifos. HPLC and FTIR measurements showed considerable adsorption and strong interaction between chlorpyrifos molecules and the organo-clay complexes. Basal spacing results emphasized the formation of suitable micro-pores for chlorpyrifos molecules. HPLC and bioassay techniques confirmed the slow release of chlorpyrifos. Leaching potential showed retention of chlorpyrifos in the top soil. Therefore application of organoclay formulation of chlorpyrifos may reduce their potential environmental contamination and produce safe applications.

Genistein and daidzein reduced chlorpyrifos induced damage in PC12 cell

The present study preliminarily evaluated neurotoxicity injuries induced by chlorpyrifos in PC12 cell,which were used as a model for nervous cell system.In cultured PC12 cell,application of soy isoflavones(genistein,daidzein,monomer and mixture)significantly reduced chlorpyrifos induced toxicity,a widely used pesticide,and resulted in a better cell survival rate.Treatments with isoflavones reduced malondialdehyde content,reactive oxygengeneration and acetylcholine level in medium,and maintained mitochondrial membrane potential integrity.Daidzein enhanced endogenous antioxidant system in PC12 cell with an increasing in superoxide dismutase perunit activity.Genistein reduced acetylcholine content in the medium.Daidzein and genistein showed different effects,and their combined effect were greater than individual.In conclusion,soy isoflavones as an antioxidant and neuroprotectant,enhanced choline metabolism,which effectively mitigated disadvantageous influence of PC12 cell caused by chlorpyrifos.

Effects of melatonin on changes in cognitive performances and brain malondialdehyde concentration induced by sub-chronic coadministration of chlorpyrifos and cypermethrin in male Wister rats

Objective:To evaluate the ameliorative effect of melatonin on sub-chronic chlorpyrifos(CPF)and cypermethrin(CYP)-evoked cognitive changes in male Wistar rats.Methods:Fifty adult male Wistar rats,divided into five groups of ten rats each,were used for the study.GroupsⅠandⅡwere given distilled water and soya oil(2 ml/kg)respectively.GroupⅢwas administered with melatonin at 0.5 mg/kg only.GroupⅣwas administered with CPF[7.96 mg/kg(1/10th LD_(50))]and CYP[29.6 mg/kg(1/10th LD_(50))].and Group V was administered with CPF[7.96 mg/kg(1/10th LD_(50)]and CYP[29.6 mg/kg(1/10th LD_(50)]30 min after melatonin(0.5 mg/kg).The regimens were administered by gavage once daily for 12 weeks.Thereafter,cognitive performances were determined and the brain was evaluated for malonaldehyde concentration.Results:CPF and CYP induced cognitive deficits and increased brain malonaldehyde concentration,which were all ameliorated by melatonin.Conclusion:Cognitive deficits elicited by CPF and CYP was mitigated by melatonin due to its antioxidant property.

Development of the Artificial Antigens for the Organophosphorus Insecticide Chlorpyrifos

This study reported that the hapten of the organophosphorus insecticide chlorpyrifos, O,O-diethyl-O-[3,5-dichloro-6-(2-carboxyethyl)thio-2-pyridyl]phosphorothioate(named AR) wassynthesized by using technical grade chlorpyrifos reacted with 3-marcapropanoic acid in hotalkaline solution. The hapten was conjugated to bovine serum albumin (BSA) with the modifiedactive ester method to form artificial immune antigen. The ratio of AR:BSA was 39:1. Theartificial coating antigen for chlorpyrifos was synthesized by conjugating AR to ovalbumin(OVA) with the mixed-anhydride method, and the ratio was 13:1. The anti-chlorpyrifos polyclonalantibodies were obtained by using the artificial immune antigen (AR-BSA) to immune in therabbits.

Study on Immunochemical Assays for the Organophosphorus Insecticide Chlorpyrifos

The anti-chlorpyrifos polyclonal antibodies were obtained by using the artificial immuneantigen to immune in New Zealands white rabbits. The enzyme-tagged antibodies wereprepared by coupling horseradish peroxidase (HRP) to the purified antibody with themodified sodium periodate method. The indirect competitive enzyme linked immuno-sorbentassays (ELISA) and the HRP-tagged antibody direct ELISA (E-Ab) were established, respectively.The limit of detection (LOD) for the indirect ELISA and E-Ab were 0.0033 and 0.0042 gmL-1, respectively. The linear detection ranged well from 0.005 to 2.0 g mL-1.