Evaluation of O,O-Dialkyl O-Cyanophenyl Phosphates and Phosphorothioates as Insecticides

Various O,O-dialkyl O-cyanophenyl phosphates and phosphorothioates were prepared and their biological activities were examined. Among them, O,O-dimethyl O- (4-chloro-2-cyanophenyl) phosphorothioate was found to have selective and high toxicity to houseflies. O,O-Dimethyl O- (4-cyanophenyl) phosphorothioate, O,O-diethyl O- (4-cyanophenyl) phosphorothioate and O,O-diethyl O- (2-chloro-4-cyanophenyl) phosphorothioate showed high insecticidal activty to American cockroaches, though the former two were not so effective to houseflies. The dimethyl esters of these series exhibited markedly lowered mammalian toxicity. Among the O-ethyl O-cyanophenyl phenylphosphonothioates, O-ethyl O- (2-chloro-4-cyanophenyl) phenylphosphonothioate was highly effective to mites, while less effective to insects.     

The Isomerization of Alkyl Phosphorothionates Induced by Carboxylic Acid Amides

Alkyl phosphorothionates are isomerized to phosphorothiolates by the catalytic action of dimethylformamide. Methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothionate) and sumithion (O,O-dimethyl O-3-methyl-4-nitrophenyl phosphorothionate) are more reactive than ethyl parathion (O,O-diethyl O-p-nitrophenyl phosphorothionate). Saligenin cyclic methyl phosphorothionate (salithion) decomposed to give a complicated pattern of products on thin layer chromatography. Besides S-methyl isomer, desmethyl sumithion (O-methyl O-3-methyl-4-nitrophenyl hydrogen phosphorothioate), 3-methyl-4-nitrophenol, methyl formate and dimethylamine were detected as reaction products from sumithion. Some other carboxylic amides including dimethylacetamide, acetamide and urea are also active. A reaction mechanism is proposed.     

Metabolism of fensulfothion by a soil bacterium, Pseudomonas alcaligenes C1.

Fensulfothion (O,O-diethyl O-[4-(methylsulfinyl)phenyl]phosphorothioate), an organophosphorus pesticide used to control the golden nematode Heterodera rostochiensis, is used as a source of carbon by microorganisms isolated from soils treated with the pesticide. Two of the microbial isolates, Pseudomonas alcaligenes C1 and Alcaligenes sp. strain NC3, used more than 80% of the pesticide in 120 h in culture when supplemented as a source of carbon. P. alcaligenes C1, which showed maximal growth on fensulfothion, degraded the compound to p-methylsulfinyl phenol and diethyl phosphorothioic acid. The phenolic metabolite could be identified by conventional spectral analysis, whereas the spectral patterns of the phosphorus-containing metabolite suggested that the compound was complexed with some cellular molecules. However, utilization of the phosphoric acid ester and ethanol by P. alcaligenes C1 suggested that the microbe attacks fensulfothion by an initial hydrolysis of the compound and subsequent utilization of the phosphoric acid ester. The pathway of degradation of fensulfothion by P. alcaligenes is of great value in the detoxification of the pesticide residues and also in the environmentally stable phosphoric acid esters.     

Metabolism of fensulfothion by a soil bacterium, Pseudomonas alcaligenes C1

Fensulfothion (O,O-diethyl O-[4-(methylsulfinyl)phenyl]phosphorothioate), an organophosphorus pesticide used to control the golden nematode Heterodera rostochiensis, is used as a source of carbon by microorganisms isolated from soils treated with the pesticide. Two of the microbial isolates, Pseudomonas alcaligenes C1 and Alcaligenes sp. strain NC3, used more than 80% of the pesticide in 120 h in culture when supplemented as a source of carbon. P. alcaligenes C1, which showed maximal growth on fensulfothion, degraded the compound to p-methylsulfinyl phenol and diethyl phosphorothioic acid. The phenolic metabolite could be identified by conventional spectral analysis, whereas the spectral patterns of the phosphorus-containing metabolite suggested that the compound was complexed with some cellular molecules. However, utilization of the phosphoric acid ester and ethanol by P. alcaligenes C1 suggested that the microbe attacks fensulfothion by an initial hydrolysis of the compound and subsequent utilization of the phosphoric acid ester. The pathway of degradation of fensulfothion by P. alcaligenes is of great value in the detoxification of the pesticide residues and also in the environmentally stable phosphoric acid esters.     

Nucleophilic N1-->N3 rearrangement of 5'-O-trityl-O2,3'-cycloanhydrothymidine

5'-O-Trityl-O2,3'-cycloanhydrothymidine (1) heated at 150 degrees C in the presence of O,O-diethyl phosphate or O,O-diethyl phosphorothioate anions undergoes rearrangement into N3-isomer (2); its structure was established by both advanced NMR methods and X-ray crystallographic studies. The most probable mechanism of 1-->2 rearrangement relies upon reversibility of glycosidic bond cleavage process.     

The Relationship Between Tobacco Yield and Time of Infection with Meloidogyne javanica

Yield of tobacco was related to the amount of infection by Meloidogyne javanica during the first month after transplanting. Six nematicidal treatments significantly reduced infection during this period and subsequently increased yield. However, during the second month after transplanting, infection in plots treated with O-ethyl S,S-dipropyl phosphorodithioate (V-C 9-104) and a mixture of 80% chlorinated C₃ hydrocarbons + 20% methyl isothioeyanate (DD + MENCS) was not significantly different from infection in untreated plots. After 3 months, root-knot indices in plots treated with V-C 9-104, DD + MENCS, O,O-diethyl O-[p-(methylsulfinyl) phenyl] phosphorothioate (B-25141), and 1,3-dichloropropene, 1,2-dichloropropane (DD) were not significantly different from those in untreated plots; reduced infection was present only in plots treated with ethylene dibromide (EDB) and 2-methyl-2 (methylthio) propionaldehyde O-(methylcarbamoyl) oxime (aldicarb). At the end of harvest (4 months after transplanting), root-knot indices in all plots were essentially equal.     

Hapten synthesis for enzyme-linked immunoassay of the insecticide triazophos

Two haptens of the insecticide triazophos (O,O-diethyl O-[1-phenyl-1H-1,2,4-triazol-3-yl] phosphorothioate) were synthesized by introducing appropriate spacers in the O-ethyl site of the analyte molecular structure. First, thiophosphoryl chloride (PSCl(3)) reacts with methanol at low temperature to give O-ethyl dichlorothiophosphate. After reacting with 1-phenyl-3H-1,2,4-triazol, the O-ethyl dichlorothiophosphate was transformed into the intermediate O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) chlorothiophosphate. Then the intermediate reacts with 4-aminobutyric acid and 6-aminobutyric acid to produce hapten I and hapten II, respectively. The molecule structures of the two haptens were identified by (1)H nuclear magnetic resonance spectrum and mass spectrum. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was also developed to evaluate the two haptens. Results showed that the monoclonal antibodies with high titers were obtained after immunizing with protein conjugates of these haptens and that the immunoassay has high affinity and specificity to triazophos. These results suggested that the haptens were synthesized successfully and could be used for immunoassay for the rapid screening and sensitive determination of this insecticide.     

Nucleophilic N1 → N3 Rearrangement of 5′-O-Trityl-O2,3′-Cycloanhydrothymidine

Abstract

5′-O-Trityl-O²,3′-cycloanhydrothymidine (1) heated at 150°C in the presence of O,O-diethyl phosphate or O,O-diethyl phosphorothioate anions undergoes rearrangement into N³-isomer (2); its structure was established by both advanced NMR methods and X-ray crystallographic studies. The most probable mechanism of 1→2 rearrangement relies upon reversibility of glycosidic bond cleavage process.     

Transition-state structures for enzymatic and alkaline phosphotriester hydrolysis.

The primary and secondary 18O isotope effects for the alkaline (KOH) and enzymatic (phosphotriesterase) hydrolysis of two phosphotriesters, O,O-diethyl p-nitrophenyl phosphate (I) and O,O-diethyl O-(4-carbamoylphenyl) phosphate (II), are consistent with an associative mechanism with significant changes in bond order to both the phosphoryl and phenolic leaving group oxygens in the transition state. The synthesis of [15N, phosphoryl-18O]-, [15N, phenolic-18O]-, and [15N]-O,O-diethyl p-nitrophenyl phosphate and O,O-diethyl O-(4-carbamoylphenyl)phosphate is described. The primary and secondary 18O isotope effects for the alkaline hydrolysis of compound I are 1.0060 and 1.0063 +/- 0.0001, whereas for compound II they are 1.027 +/- 0.002 and 1.025 +/- 0.002, respectively. These isotope effects are consistent with the rate-limiting addition of hydroxide and provide evidence for a SN2-like transition state with the absence of a stable phosphorane intermediate. For the enzymatic hydrolysis of compound I, the primary and secondary 18O isotope effects are very small, 1.0020 and 1.0021 +/- 0.0004, respectively, and indicate that the chemical step in the enzymatic mechanism is not rate-limiting. The 18O isotope effects for the enzymatic hydrolysis of compound II are 1.036 +/- 0.001 and 1.0181 +/- 0.0007, respectively, and are comparable in magnitude to the isotope effects for alkaline hydrolysis, suggesting that the chemical step is rate-limiting. The relative magnitude of the primary 18O isotope effects for the alkaline and enzymatic hydrolysis of compound II reflect a transition state that is more progressed for the enzymatic reaction.     

Accelerated Mineralization of Two Organophosphate Insecticides in the Rhizosphere

Numerous xenobiotic compounds, including the organophosphate insecticides O, O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate (diazinon) and O, O-diethyl-O-p-nitrophenyl phosphorothioate (parathion), appear to be degraded in the soil environment by an initial cometabolic attack. Comparing the mineralization rates of radiolabeled diazinon and parathion in root-free and in rhizosphere soil, we tested our hypothesis that, because of the presence of root exudates, the rhizosphere is an especially favorable environment for such co-metabolic transformations. The insecticides were added individually at 5 μg/g to sealed flasks containing either soil permeated by the root system of a bush bean plant or identical soil without roots. Periodically, the flask atmospheres were flushed through traps and the evolved ¹⁴CO₂ was quantitated. Bush bean plant roots without associated rhizosphere microorganisms failed to produce a significant amount of ¹⁴CO₂. During 1 month of incubation, rhizosphere flasks mineralized 12.9 and 17.9% of the added diazinon and parathion radiocarbon, respectively, compared to 5.0 and 7.8% by the soil without roots. The mineralization of parathion but not of diazinon was stimulated in a similar manner when soil without roots was repeatedly irrigated with a root exudate produced in aseptic solution culture. Viable counts of microorganisms on soil extract agar were not significantly altered by root permeation or by root exudate treatment of the soil, leaving population selection and/or enhanced cometabolic activity as the most plausible interpretations for the observed stimulatory effects. Rhizosphere interactions may substantially shorten the predicted half-lives of some xenobiotic compounds in soil.     

Induction of oxidative stress and histopathological changes by sub-chronic doses of triazophos

The effect of triazophos (O, O-diethyl O-1-phenyl-1 H-1, 2, 4-triazol-3-yl phosphorothioate), a widely used insecticide was studied on the induction of oxidative stress and histological alterations at sub-chronic doses in male albino rats. Oral administration of triazophos at concentrations of 1.64, 3.2 and 8.2 mg/kg body wt for 30 days produced dose as well as time-dependent increase in the lipid peroxidation (determined by malondialdehyde levels) and glutathione-S-transferase (GST) activity in serum with aconcomitant decrease in ferric reducing ability of plasma (FRAP) and blood glutathione (GSH) content. Histopathological examination of liver of triazophos-treated rats showed significant and progressive degenerative changes as compared to control, which could be due to induction of oxidative stress. However, no significant histopathological changes were observed in spleen, kidney and brain at either dose of triazophos with respect to control. These results indicated that oral administration of triazophos was associated with enhanced lipid peroxidation and compromised antioxidant defence in rats in dose and time-dependent manner. Thus the present study demonstrated for the first time the role of oxidative stress as the important mechanism involved in the stimulation of hepatic histoarchitectural alterations at sub-chronic doses of triazophos in rats.     

Laboratory and field tests in 1966-67 on chemical control of wireworms (Agriotes spp.)

Of sixteen compounds applied to soil in laboratory tests, azinphos-ethyl, P2188 (O,O-diethyl S-chloromethyl phosphorothiolothionate), ‘Dursban’ (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate), P1973 (S-(N-methoxycarbonyl-N-methylcarbamoylmethyl) dimethyl phosphorothiolothionate), B77488 (O,O-diethylphosphorothioate O-esterwith phenylglyoxylonitrile oxime) and R42211 (O,O-diethyl O-(2-diethylamino-6-methyl-pyrimidin-4-yl) phosphorothioate) killed wireworms when first tested, but in second tests with the same soils only ‘Dursban’, P2188 and B77488 did so. Treating seeds with ‘Dyfonate’ (O-ethyl S-phenyl ethyl phosphonodithioate) or with ethion/γ-BHC mixtures killed few wireworms. Three field trials compared the organophosphorus insecticides ‘Dursban’, ‘Dyfonate’ and phorate with organochlorine standards. In trials with barley and potatoes the standard was 3 lb a.i./acre (3·36 kg/ha) of aldrin. The organophosphorus compounds increased plant stands of barley almost as much as aldrin, although they killed fewer wireworms; and they protected fewer potato tubers from wireworm damage. The third trial compared the organophosphorus compounds with 0·5 lb a.i./acre (0·56 kg/ha) γ-BHC sprayed on a site drilled with sugar beet seed dressed with dieldrin. The γ-BHC increased plant stands almost as much as did 3 lb a.i./acre of the organophosphorus insecticides, and killed as many wireworms.     

Involvement of two plasmids in fenitrothion degradation by Burkholderia sp. strain NF100

A bacterium capable of utilizing fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil. This bacterium was characterized taxonomically as being a member of the genus Burkholderia and was designated strain NF100. NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone. The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1.

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

模拟氮沉降对马尾松土壤微生物群落结构及温室气体释放的影响

以2年生马尾松(Pinus massoniana)盆栽苗土壤为对象,通过施氮肥模拟氮沉降对土壤理化性质、微生物群落结构及温室气体释放的影响,探明氮沉降对森林土壤温室气体释放的驱动机制。结果表明,模拟氮沉降处理显著提高了土壤速效氮含量和苗木根系氮含量;土壤微生物碳(SMBC)含量比对照显著下降78%,而土壤微生物氮(SMBN)则提高2.6倍。模拟氮沉降处理显著降低土壤中微生物群落总含量。施氮肥对马尾松土壤N_2O和CO_2的释放速率均有显著影响,增施氮肥不仅显著提高了土壤N_2O的释放速率,而且CO_2释放速率短期内也显著提高,但伴随微生物群落的下降,施肥后期CO_2释放速率表现下降趋势。相关分析表明,土壤CO_2和N_2O释放与土壤pH值、土壤温度、土壤湿度、土壤速效氮含量及SMBC、SMBN相关;逐步回归分析表明,土壤硝态氮含量的变化是驱动土壤温室气体释放的主导因子。3株种植单位土壤体积内根系生物量较高,增加了土壤水分的消耗速率和氮的吸收固定,因而减少N_2O的释放速率。以上研究阐明了氮沉降或过量施肥对土壤氮含量、土壤pH值、根系生物量及氮含量、土壤微生物群落结构等因子的影响,这些因子直接或间接影响土壤温室气体释放速率。氮沉降及施用氮肥是加快土壤温室气体(CO_2和N_2O)排放进程的重要因素。     

Woodchip and biochar amendments differentially influence microbial responses,but do not enhance plant recovery in disturbed semiarid soils

Restoration presents a global challenge in drylands (arid and semiarid ecosystems) where uses can range from exclusive conservation to open‐pit mining and restoration practices are constrained by scarce, unpredictable precipitation, and high ambient temperatures. Adding woodchip amendments to soils is a common strategy for mitigating soil degradation as amendments may enhance soil carbon and increase plant cover. We assessed the effect of surface or incorporated woodchip addition and incorporated wood‐derived biochar on soil carbon dynamics and microbial activities as well as plant cover in semiarid soils that had been removed and replaced. We found that woodchips at the soil surface increased soil organic carbon (SOC), and both surface and incorporated woodchips increased the dissolved organic carbon (DOC) content. The incorporation of woodchips inhibited plant cover yet increased soil CO₂ efflux and dissolved organic matter stoichiometry. Surface woodchips also significantly enhanced microbial activities but not plant cover. A significant amount of the soil efflux in response to incorporating woodchips was explained by plant cover and exoenzyme activities, but this was not the case for other amendment treatments. Biochar, thought to be more resistant to decomposition, neither stimulated nor reduced microbial activities or plant cover and did not influence SOC or DOC. Our findings demonstrate that the influence of woodchip amendments on microbial processes and soil carbon dynamics depends on the location of application and that coarse fast‐pyrolysis biochar has limited influence on soil processes over a 22‐month study in a water‐limited ecosystem.     

Copper and zinc fractions affecting microorganisms in long-term sludge-amended soils

The influences of Zn and Cu on soil enzyme activities (acid phosphatase, alkaline phosphatase, arylsulfatase, cellulase, dehydrogenase, protease (z-FLase), urease, beta-D-glucosidase and beta-D-fructofuranosidase (invertase)) and microbial biomass carbon were investigated in agricultural soils amended with municipal sewage sludge or compost since 1978. The trace metals in the soils were fractionated using a sequential extraction method. Long-term application of the sewage sludge and composts caused accumulations of Cu and Zn in the soils, ranging from 140 to 144 and from 216 to 292 mg kg(-1), respectively. The percentage of Cu was highest in the NaOH- and HNO3-extractable fractions (44-51% and 38-46%, respectively), while the percentage of Zn was highest in the HNO3- and EDTA-extractable fractions (65-83% and 11-32%, respectively). Although the percentage of the bioavailable fractions (sum of KNO3 + H2O-, NaOH-, and EDTA-extractable amounts) of Cu (53-64%) was higher than that of Zn (15-37%), the percentage of the most labile fractions (KNO3 + H2O) of Zn (2.1-5.9%) was larger than that of Cu (1.1-2.4%). The size of the microbial biomass carbon increased with the application of sewage sludge or compost. For some enzymes, however, the ratio of the enzyme activity to microbial biomass was lower in the soils amended with sewage sludge or compost than that in the control soil. The soil enzyme activities were more adversely affected by Zn than by Cu. From a multiple regression analysis, it was found that dehydrogenase, urease, and beta-D-glucosidase activities were reduced by the KNO3 + H2O-extractable fraction of Zn in the soils. These microbial activities seem to be sensitive to Zn stress, indicating the possibility that they might be useful bioindicators for evaluation of the toxic effects of Zn on microorganisms in the soils.     

Controlled release of DNA/polyamine complex by photoirradiation of a solid phase presenting o-nitrobenzyl ether tethered spermine or polyethyleneimine

Various gene transfer and automated/monitorized analytical applications require the controlled release of nucleic acid. A solid phase with spermine or polyethyleneimines (PEI, 600 MW) tethered by o-nitrobenzyl linkages was synthesized with polyethylene oxide beads (ArgoGel-NH(2)). The photolysis of test compound O-2-nitrophenethyl O,O-diethyl phosphate or solid phase with o-nitrobenzyl group as synthetic linker was completely degradable with photoirradiation at 365 nm for 10-18 min at 3.5 mW/cm(2). DNA binding with polyamine of the solid phase and releasing of DNA/polyamine were monitored by UV measurement and gel electrophoresis. The potential exists to employ a DNA-loaded solid phase for spatially, temporally, or dose-controlled release of DNA, at extracellular or intracellular sites.     

The role of the O antigen in adjuvant activity of lipopolysaccharide

Adjuvant activities of isogenic Salmonella enterica, serovar Typhimurium, O-6,7 and O-4,5,12 lipopolysaccharide (LPS), lipid A and Bordetella pertussis LPS were compared by immunizing groups of mice subcutaneously with diphtheria and tetanus toxoid vaccine alone or mixed with one of the LPS derivatives. Five weeks later the mice were bled and the tetanus and diphtheria antibodies in the sera were measured. All the LPS derivatives efficiently increased the antibody responses when compared to the vaccine alone, but the mannose-rich O-6,7 LPS and lipid A were significantly more potent than O-4,5,12 LPS and B. pertussis LPS. We conclude that the quality of the O antigen influences the adjuvant activity of LPS.