Development and validation of a liquid chromatography-fluorescence-mass spectrometry method to measure glyphosate and aminomethylphosphonic acid in rat plasma

A simple and fast method has been developed and validated to measure glyphosate (GLYP) and aminomethylphosphonic acid (AMPA) in rat plasma based on reversed-phase high performance liquid chromatography (RP-HPLC) coupled to fluorescence (FLD) and electrospray ionization mass spectrometry (ESI-MS) detection. After protein precipitation with acetonitrile, GLYP and AMPA were derivatized with 9-fluorenylmethylchloroformate (FMOC-Cl) and then separated on a C(12) column (250mm×4.60mm i.d.) using a gradient of an ammonium formate (20mM, pH 8.5) and acetonitrile mobile phase. Selected ion monitoring (SIM) mode of the MS was used to obtain maximum sensitivity when quantifying GLYP and AMPA. The validation shows the method to be consistent and reliable, with an intra- and inter-day precision for GLYP and AMPA>9% for both detectors. For both compounds the accuracy ranged from 2.1% to 7.8% for the intra-day readings, and from 4.1% to 8.6% for the inter-day values. The efficacy of GLYP extraction ranged from 87% to 93% and it was between 76% and 88% for AMPA. Moreover, the limits of quantification (LOQ) for GLYP and AMPA were 5 and 10ng/mL, respectively with FLD, and 0.4 and 2ng/mL with ESI-MS. The method was successfully applied to simultaneously measure both compounds in rat plasma samples several days after oral administration of glyphosate.     

Degradation of glyphosate and other pesticides by ligninolytic enzymes

The ability of pure manganese peroxidase (MnP), laccase, lignin peroxidase (LiP) and horseradish peroxidase (HRP) to degrade the widely used herbicide glyphosate and other pesticides was studied in separate in vitro assays with addition of different mediators. Complete degradation of glyphosate was obtained with MnP, MnSO₄ and Tween 80, with or without H₂O₂. In the presence of MnSO₄, with or without H₂O₂, MnP also transformed the herbicide, but to a lower rate. Laccase degraded glyphosate in the presence of (a) 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), (b) MnSO₄ and Tween 80 and (c) ABTS, MnSO₄ and Tween 80. The metabolite AMPA was detected in all cases where degradation of glyphosate occurred and was not degraded. The LiP was tested alone or with MnSO₄, Tween 80, veratryl alcohol or H₂O₂ and in the HRP assay the enzyme was added alone or with H₂O₂ in the reaction mixture. However, these enzymes did not degrade glyphosate. Further experiments using MnP together with MnSO₄ and Tween 80 showed that the enzyme was also able to degrade glyphosate in its commercial formulation Roundup^® Bio. The same enzyme mixture was tested for degradation of 22 other pesticides and degradation products present in a mixture and all the compounds were transformed, with degradation percentages ranging between 20 and 100%. Our results highlight the potential of ligninolytic enzymes to degrade pesticides. Moreover, they suggest that the formation of AMPA, the main metabolite of glyphosate degradation found in soils, can be a result of the activity of lignin-degrading enzymes.     

Quantitative determination of glufosinate in biological samples by liquid chromatography with ultraviolet detection after p-nitrobenzoyl derivatization

We have established a new HPLC method for derivatizing and quantifying glufosinate (GLUF) in human serum and urine using p-nitrobenzoyl chloride (PNBC). The p-nitrobenzoyl derivative of GLUF (PNB-GLUF) was produced quantitatively over 10 min at room temperature. PNB-GLUF possesses the property of ultraviolet (UV) light absorption with a lambda(max) of 272.8 nm, and was isolated from biological specimens by reversed-phase chromatography using Inertsil Ph-3. In experiments at a UV wavelength of 273 nm, GLUF has a quantitative detection limit of 0.005 microg/ml, and when it was added to both serum and urine to yield concentrations of 0.1-1000 microg/ml, its recovery rate was quite satisfactory: at least 93.8% in all cases. Further, the measured amounts of GLUF in 23 serum samples from patients intoxicated by ingestion of GLUF compared favorably with those obtained by fluorescence derivatization-HPLC using 9-fluorenylmethyl chloroformate (R=0.998). This technique of analysis is, in addition, applicable for Glyphosat, which possesses a chemical structure resembling that of GLUF, and it will be of great use in the determination of these two compounds.     

The absorption, translocation and distribution of the herbicide glyphosate in maize expressing the CP-4 transgene

Maize (Zea mays L. var. Bonnie) transformed with a gene encoding a 5-enolpyruvylshikimate 3-phosphate synthase with altered sensitivity showed over 100-fold greater resistance to the herbicide glyphosate (N-[phosphonomethyl]glycine) in comparison with its non-transformed progenitor (parental control) at the third-leaf stage. Studies with [¹⁴C]-glyphosate at a dosage lethal to the parental control, but sublethal to the transgenic, revealed that a maximum of 45-65% of the applied dose was absorbed, with greater absorption occurring in transgenic plants. Translocation of glyphosate was closely related to its absorption (r value 0.956) with approximately 15% more of the applied dose being mobilized in transgenic plants than the parental controls. Analysis of electronic autoradiograms along the treated leaf lamina found discrete internal regions of glyphosate accumulation closely associated with the site of application. These regions contained lower amounts of glyphosate present in the treated leaf lamina was almost completely translocated in transgenic plants, while in the parental controls more remained and the leaf became necrotic. In both types of maize there was a small accumulation of herbicide in the tip region of the leaf which was not mobilized. Younger shoot tissues and roots were major sinks for translocated glyphosate accumulating approximately 25-40% of the applied dose depending upon treatment. In the parental control, equal amounts of glyphosate were found distributed between young shoot tissues and roots; while in transgenic plants, the young shoot tissue accumulated around three times more glyphosate than the roots. In both plant types, glyphosate was localized in the meristems and young, actively growing leaves. Specific glyphosate activity (the amount of glyphosate per unit dry weight of tissue) in the major sinks of the transgenic declined towards the end of the treatment period but remained relatively constant in the parental control. In conclusion, enhancing glyphosate resistance by genetic transformation influenced the absorption, translocation and distribution of this herbicide in whole plants.Keywords: Zea mays, glyphosate (N-[phosphonomethyl]-glycine), transgenic, absorption, translocation, source-sink.      

Expression of the hygromycin B phosphotransferase gene confers tolerance to the herbicide glyphosate

Escherichia coli cells and tobacco (cv. Xanthi) plants transformed with the hygromycin B phosphotransferase gene were able to grow in culture medium containing glyphosate at 2.0 mM. The growth of tobacco calli in media containing increasing glyphosate concentrations was measured. The ID₅₀ for glyphosate was 1.70±0.03 mM for hygromycin-B resistant plants, and 0.45±0.02 mM for control plants. Regenerated plants and progeny selected for resistance to hygromycin B were tested for glyphosate tolerance by spraying them with Faena herbicide (formulated glyphosate with surfactant) at a dose equal to 0.24 kg/ha. This was two times the dose required to kill 100 percent of the control plants. Phosphotransferase activity was measured in the extracts of the transformed leaves by the incorporation of ³²P from [^–32P]ATP and it was observed that hygromycin B phosphotransferase was able to recognize the molecule of glyphosate as substrate.Abbreviations (Hyg) Hygromycin - (Km) Kanamycin - (Glp) Glyphosate - (Sarc) Sarcosine - (AMPA) Aminomethylphosphonic acid     

Determination of Glyphosate and AMPA in Three Representative Agricultural Austrian Soils with a HPLC-MS/MS Method

We developed a novel method to quantify adsorbed glyphosate and AMPA in soils based on an extraction utilizing Na-tetraborate, an SPE clean-up step, and subsequent LC-MS detection. Reversed phase-based separation of glyphosate and AMPA was realized after FMOC-derivatization. The quantification involved external calibration and 1,2–¹³C, ¹⁵N- labeled glyphosate as well as ¹³C, ¹⁵N labeled AMPA as internal standards. The optimum recovery for extraction was obtained with 40 mM Na-tetraborate. The method was applied in three representative soils (Kirchberg, Phyra, and Pixendorf, Austria) where glyphosate was applied by standard agricultural practices. The recovery for glyphosate extracted with 40 mM Na-tetraborate buffer was 93.5% (RSD <2%) for glyphosate at Kirchberg-cambisol; 95.7% (RSD < 2%) at Pixendorf- chernozem and 79.1% (RSD <7%) at Phyra-stagnosol. The corresponding values for AMPA were 92.4% (RSD <2%) at Kirchberg, 98.1% (RSD <2%) at Pixendorf and 69.9% (RSD <4%) at Phyra. The limits of detection for glyphosate were 6.8 μg kg^?1(RSD <10%) at Kirchberg, 4.3 μg kg^?1 (RSD <10%) at Pixendorf, and 46.5 μg kg^?1 (RSD <7%) at Phyra. The limits of detection for AMPA were 26.7 μg kg^?1 (RSD <10%) at Kirchberg, 25.2 μg kg^?1 (RSD <10%) at Pixendorf, and 120.3 μg kg^?1 (RSD <9%) at Phyra. Accordingly, the limits of quantification were 22.7 μg kg^?1(RSD <5%) for glyphosate, and 88.9 μg kg^?1 (RSD <2%) for AMPA at Kirchberg and respectively 14.4 μg kg^?1 (RSD <6%) and 84 μg kg^?1 (RSD <5%) at Pixendorf and 13.8 μg kg^?1 (RSD <6%) and 87.2 μg kg^?1 (RSD <8%) at Phyra. Both substances in the soils were lower than the LOQ before applying the herbicide Roundup. The influence of higher contents of iron oxides, clay, and acidic pH, resulting in a more pronounced adsorption of glyphosate and AMPA in the soils of Phyra and Kirchberg, is demonstrated.     

Phytotoxicity of glyphosate soil residues re-mobilised by phosphate fertilisation

It has been repeatedly demonstrated that phosphate (P) and the herbicide glyphosate compete for adsorption sites in soils. Surprisingly, the potential consequences of these interactions for plants e.g. re-solubilisation of phytotoxic glyphosate residues in soils by application of P fertilisers or by root-induced mechanisms for P mobilization have not been investigated so far. In model experiments under greenhouse conditions, the potential for glyphosate re-mobilisation by P-fertiliser application was evaluated by bio-indication with soybean (Glycine max L.) cultivated on five contrasting soils with or without glyphosate application at 10?C35 days before sowing. Different levels of P-fertilisation (0, 20, 40, 80, 240 mg P kg^?1 soil) were supplied at the date of sowing. Visual symptoms of glyphosate toxicity, plant biomass, intracellular shikimate accumulation as physiological indicator for glyphosate toxicity and the plant nutritional status were determined. On glyphosate-treated soils, P application induced significant plant damage. Expression of damage symptoms declined in the order Arenosol > Acrisol ?? Ferralsol > Luvisol subsoil > Regosol. On the Arenosol, Ferralsol and Luvisol subsoil plant damage was associated with increased shikimate accumulation in the root tissue. On the Acrisol decline of germination and plant damage in absence of shikimate accumulation indicate toxicity of AMPA (aminomethylphosphonic acid) as the main metabolite of glyphosate in soils. On the Regosol, a growth-stimulating effect of glyphosate soil application (hormesis) was detected. The results suggest that re-mobilisation of glyphosate may represent an additional transfer pathway for glyphosate to non-target plants which is strongly influenced by soil characteristics such as P fixation potential, content of plant-available iron, pH, cation exchange capacity, sand content and soil organic matter.     

Pathway and rate-limiting step of glyphosate degradation by Aspergillus oryzae A-F02

Aspergillus oryzae A-F02, a glyphosate-degrading fungus, was isolated from an aeration tank in a pesticide factory. The pathway and rate-limiting step of glyphosate (GP) degradation were investigated through metabolite analysis. GP, aminomethylphosphonic acid (AMPA), and methylamine were detected in the fermentation liquid of A. oryzae A-F02, whereas sarcosine and glycine were not. The pathway of GP degradation in A. oryzae A-F02 was revealed: GP was first degraded into AMPA, which was then degraded into methylamine. Finally, methylamine was further degraded into other products. Investigating the effects of the exogenous addition of substrates and metabolites showed that the degradation of GP to AMPA is the rate-limiting step of GP degradation by A. oryzae A-F02. In addition, the accumulation of AMPA and methylamine did not cause feedback inhibition in GP degradation. Results showed that degrading GP to AMPA was a crucial step in the degradation of GP, which determines the degradation rate of GP by A. oryzae A-F02.     

Physiological aspects of glyphosate degradation in Alcaligenes spec. strain GL

Alcaligenes spec. strain GL (IMET 11314) is able to grow on glyphosate (N-[phosphonomethyl]glycine) and other phosphonates as sole source of phosphorus. Degradation of glyphosate to inorganic phosphate and sarcosine by this strain is subject to several regulatory principles. While uptake and dephosphonation of glyphosate are regulated by P_i starvation, the intensity of glyphosate degradation is also controlled by the cellular ability to utilize the C-skeleton derived from glyphosate. Depending on the external concentration of glyphosate, the liberated sarcosine is differentially metabolised. Utilization of the sarcosine moiety and complete incorporation of 3-[¹⁴C]-label of glyphosate into cellular material occur only in cultures adapted to higher concentrations (5 mM) of the herbicide. At low concentrations of glyphosate (1 mM) only the P_i required by the growing cultures is utilized but not the sarcosine. Initially high rates of glyphosate uptake obtained after P_i-starvation decrease in the presence of low glyphosate concentrations. It is suggested that uptake and metabolism of glyphosate are connected with the expression of the sarcosine metabolizing capacity of the Alcaligenes cells.Abbreviation AMPA aminomethylphosphonic acid     

Circadian response of annual weeds in a natural setting to high and low application rates of four herbicides with different modes of actions

Four herbicides [glyphosate (GLYT), an amino acid synthesis inhibitor; glufosinate (GLUF), a glutamine synthetase inhibitor; fomesafen (FOME), a protoporphyrinogen oxidase inhibitor; and chlorimuron ethyl (CLIM), an acetolactate synthase inhibitor] were used to examine the influence of time of day of application on the control of a variety of annual broadleaf weeds in field studies conducted in Minnesota (five studies on GLYT and GLUF, three studies on FOME and CLIM). All herbicides were applied with an adjuvant at recommended high and low (half or quarter strength) rates every 3h between 06:00 and 24:00h local time. Visual ratings of percent weed control evaluated at 14d were analyzed by herbicide and application rate for each study and across studies for time-of-day effect by analysis of variance (ANOVA) and single cosinor. A circadian response to each herbicide was found, with greatest weed control observed between 09:00 and 18:00h. Increasing the herbicide application rate did not overcome the time-of-day effect (ANOVA: p≤0.008 for time-of-day effect for each herbicide and application rate). The least-squares fit of a 24h cosine was significant (p≤0.001) for each herbicide and application rate, with double amplitudes of 18-82% (units=% visual control) and estimated peaks (acrophases) near midday between 12:40 and 13:35h. Analysis of residuals obtained from multiple regression that included weed height, herbicide rate, temperature, and relative humidity as independent factors also found a significant time-effect by both ANOVA and cosinor for each herbicide and rate, with acrophases advancing significantly by 3 to 7h for GLYT and GLUF, but not for FOME or CLIM. These results suggest that the four herbicides, while belonging to different families with different modes of action, may reveal different peak times of efficacy when adjusting for environmental factors. Nonetheless, each displays similar circadian patterns when influenced by these factors under natural seasonal field conditions. The within-day rhythmic differences found in weed control are large enough to warrant consideration of the practical financial and environmental importance of the time-of-day that these and other herbicides are applied.     

Effect of glyphosate on the microbial activity of two Romanian soils

Glyphosate applied to soils potentially affect microbial activity. A series of field and laboratory experiments assessed the effect of this herbicide on soil microorganisms. The aim of experiments was to evaluate the effect of glyphosate application on the soil microbial community structure, function and their activity. We studied "in vitro", changes in the microbial activity of typical Chernozem and Gleysol soils, with and without applied glyphosate. The herbicide was applied at a rate of 2, respectively 4 mg kg(-1) of soil and microbial activity were measured by fluorescein diacetate (FDA) hydrolysis. We found an increase of 9 to 13% in FDA hydrolyses in the presence of glyphosate in rate of 2 mg kg (-1) compared with the same type of soil which had never received herbicide. The double quantity of glyphosate decrease soil microbial activity; the amount of hydrolyzed fluorescein is lower than the addition of 2 ppm. The greater decrease was observed in the Gleysol type where the fluorescein hydrolyzed is with 4, 85% lower than version control without glyphosate. Chemical characters of soil, influence soil biological activity when herbicide is added. In Chemozem case, rich in humus, whose predominant micro flora is represented by actinomycetes through glyphosate treatment these organisms growths of as major producers of antibiotics actinomycetes determine an inhibitory effect on eubacteria and micromycetes growth, which is highlighted by estimating a relatively small number of them. After 10 days, once with decreasing of glyphosate content in soil, decreases the number of active actinomycetes, therefore we are witnessing to a numerical growth of bacterial population. In Gleysol type the indigenous micro flora is represented by eubacteria, so when the glyphosate is added it was registered a high growth of these organisms fraction.     

Potential use of Lemna minor for the phytoremediation of isoproturon and glyphosate

Pesticides are being detected in water bodies on an increasingly frequent basis. The present study focused on toxicity and phytoremediation potential of aquatic plants to remove phytosanitary products from contaminated water. We investigated the capacity of Lemna minor (L. minor) to eliminate two herbicides isoproturon and glyphosate from their medium. Since phytoremediation relies on healthy plants, pesticide toxicity was evaluated by exposing plants to 5 concentrations (0-20 microg L(-1) for isoproturon and 0-120 microg L(-1) for glyphosate) in culture media for 4 d using growth rate and chlorophyll a fluorescence as endpoints. At exposure concentrations of 10 microg x L(-1) for isoproturon and 80 microg x L(-1) for glyphosate, effects on growth rate and chlorophyll fluorescence were minor (< 25%), so that this initial concentration was selected to study herbicide removal After a 4-d incubation, removal yields were 25% and 8% for isoproturon and glyphosate, respectively.     

Pre-harvest retting of flax: effects of water stress on uptake and efficacy of glyphosate

The effects of water stress on the uptake, translocation and efficacy of glyphosate in flax were investigated in relation to pre-harvest retting. Glyphosate (applied at a rate equivalent to 1.44 kg a.e. ha^-1 at 0, 10, 20 or 30 days after the start of flowering) caused little desiccation of flax grown in pots under restricted watering. Glyphosate application to well-watered plants caused the moisture content to decline from an initial value of 70 – 80% to approximately 40% at 3 wk after spraying. Glyphosate was applied 2 wk after the mid-point of flowering to flax grown in soil with moisture contents of 35, 31, 26, 22, 16 or 12%. Soil moisture levels (16% and 12%) which restricted evapotranspiration also reduced the efficacy of glyphosate but did not affect uptake of ¹⁴C-glyphosate. Translocation of ¹⁴C-glyphosate out of treated leaves was reduced only in the most severely stressed plants (12% soil moisture). Experiments with young plants (4 wk old) confirmed that water stress slightly reduced downward translocation of glyphosate. When the herbicide was applied to young plants under conditions which minimised differences in translocation, 10.8 μg glyphosate was sufficient to desiccate unstressed plants but 108 μg had little effect on stressed plants. This indicates that, in addition to any reduction in translocation which occurs during drought, water stress may reduce the susceptibility of flax to glyphosate. Thus only relief of plant water stress by irrigation is likely to improve response of the flax crop to glyphosate.     

Synergistic effect of the herbicides glyphosate and MCPA on survival of entomopathogenic nematodes

The survival and infectivity of the infective juveniles of two species of entomopathogenic nematodes, Steinernema feltiae (Rhabditida: Steinernematidae) Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), were determined after exposure for 72 h to two concentrations of the herbicides glyphosate and MCPA, as well as to the combination of the two herbicides (glyphosate + MCPA). For all herbicide treatments, concentrations and exposure times, S. feltiae was more tolerant to the herbicides than H. bacteriophora. The exposure of entomopathogenic nematodes to glyphosate + MCPA caused significantly higher mortality (26.33–57.33%) than glyphosate (0.67–15%) or MCPA (2.33–19%) alone. These results confirm the synergistic effect of the glyphosate + MCPA combination on the mortality in these nematodes. Nematode infectivity of Galleria mellonella larvae in response to the herbicides presence was evaluated in Petri dish assays containing sterile sand. Nematode infectivity was not significantly reduced by exposure to herbicides in S. feltiae but H. bacteriophora was less tolerant. Synergistic effect was obtained in the nematode mortality test but no synergistic effect was observed in the nematode infectivity assay. Our results suggest that possible synergistic effects of agrochemicals on survival of nematodes should be tested before mixing with entomopathogenic nematodes.     

Absorption and efflux of glyphosate by cell suspensions

The absorption and efflux of [¹⁴C]-glyphosate (N-[phosphonomethyl]glycine) was studied in maize (Zea mays L. cv. Aussie) and soybean (Glycine max L. Merr. cv. Maple Arrow) cell suspensions. Glyphosate absorption was complex: at low external herbicide concentrations (3-250 M) there was evidence for a single active uptake system with an apparent Km of 31 M and Vmax of 11 nmol g^-1 fr. wt. 2 h^-1. The system was inhibited by carbonylcyanide m-chlorophenyl hydrazone (CCCP), orthovanadate, diethylstilbestrol (DES), phosphate, and phosphonoformic acid (PFA) suggesting the glyphosate carrier to be a phosphate transporter energized by the plant plasmalemma ATPase. At higher external glyphosate concentrations the operation of this carrier was masked as passive diffusion became the dominant absorption mechanism. Any non-specific binding of glyphosate to the cell surface during absorption was low (0.02-0.02 nmol g^-1 fr. wt). Efflux kinetics of [¹⁴C]-glyphosate suggests the herbicide to be located in the cells in three kinetically distinct compartments: after 24 h uptake of radiolabelled herbicide, 71% of absorbed glyphosate was found in the slow compartment (t1/2 162 h), 19% in the medium (t1/2 185 min) and 10% in the fast (t1/2 27 min). The implications of these results in relation to the delivery of glyphosate to its subcellular target site and subsequent phytotoxicity are discussed.Keywords: Zea mays, Glycine max, glyphosate (N-[phosphonomethyl]glycine), absorption, compartmentation.      

Circadian Response of Annual Weeds in a Natural Setting to High and Low Application Rates of Four Herbicides with Different Modes of Action

Four herbicides [glyphosate (GLYT), an amino acid synthesis inhibitor; glufosinate (GLUF), a glutamine synthetase inhibitor; fomesafen (FOME), a protoporphyrinogen oxidase inhibitor; and chlorimuron ethyl (CLIM), an acetolactate synthase inhibitor] were used to examine the influence of time of day of application on the control of a variety of annual broadleaf weeds in field studies conducted in Minnesota (five studies on GLYT and GLUF, three studies on FOME and CLIM). All herbicides were applied with an adjuvant at recommended high and low (half or quarter strength) rates every 3h between 06:00 and 24:00h local time. Visual ratings of percent weed control evaluated at 14d were analyzed by herbicide and application rate for each study and across studies for time-of-day effect by analysis of variance (ANOVA) and single cosinor. A circadian response to each herbicide was found, with greatest weed control observed between 09:00 and 18:00h. Increasing the herbicide application rate did not overcome the time-of-day effect (ANOVA: p≤0.008 for time-of-day effect for each herbicide and application rate). The least-squares fit of a 24h cosine was significant (p≤0.001) for each herbicide and application rate, with double amplitudes of 18–82% (units=% visual control) and estimated peaks (acrophases) near midday between 12:40 and 13:35h. Analysis of residuals obtained from multiple regression that included weed height, herbicide rate, temperature, and relative humidity as independent factors also found a significant time-effect by both ANOVA and cosinor for each herbicide and rate, with acrophases advancing significantly by 3 to 7h for GLYT and GLUF, but not for FOME or CLIM. These results suggest that the four herbicides, while belonging to different families with different modes of action, may reveal different peak times of efficacy when adjusting for environmental factors. Nonetheless, each displays similar circadian patterns when influenced by these factors under natural seasonal field conditions. The within-day rhythmic differences found in weed control are large enough to warrant consideration of the practical financial and environmental importance of the time-of-day that these and other herbicides are applied.     

Enzymological basis for herbicidal action of glyphosate

The effects of 1 millimolar glyphosate (N-[phosphonomethyl]glycine) upon the activities of enzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata [L.] Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. The activities of prephenate dehydrogenase and arogenate dehydrogenase were also not sensitive to inhibition. Two molecular species of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase were resolved, one stimulated several-fold by Mn²⁺ (DAHP synthase-Mn), and the other absolutely dependent upon the presence of Co²⁺ for activity (DAHP synthase-Co). Whereas DAHP synthase-Mn was invulnerable to glyphosate, greater than 95% inhibition of DAHP synthase-Co was found in the presence of glyphosate. Since Co²⁺ is a V_max activator with respect to both substrates, glyphosate cannot act simply by Co²⁺ chelation because inhibition is competitive with respect to erythrose-4-phosphate. The accumulation of shikimate found in glyphosate-treated seedlings is consistent with in vivo inhibition of both 5-enolpyruvylshikimic acid 3-phosphate synthase and one of the two DAHP synthase isozymes. Aromatic amino acids, singly or in combination, only showed a trend towards reversal of growth inhibition in 7-day seedlings of mung bean. The possibilities are raised that glyphosate may act at multiple enzyme targets in a given organism or that different plants may vary in the identity of the prime enzyme target.     

Glyphosate degradation by immobilized bacteria: laboratory studies showing feasibility for glyphosate removal from waste water.

To evaluate immobilized bacteria technology for the removal of low levels of glyphosate (N-phosphonomethylglycine) from aqueous industrial effluents, microorganisms with glyphosate-degrading activity obtained from a fill and draw enrichment reactor inoculated with activated sludge were first exposed to glyphosate production wastes containing 500-2000 mg glyphosate/L. The microorganisms were then immobilized by adsorption onto a diatomaceous earth biocarrier contained in upflow Plexiglas columns. The columns were aerated, maintained at pH 7.0-8.0, incubated at 25 degrees C, supplemented with NH4NO3 (50 mg/L), and exposed to glyphosate process wastes pumped upflow through the biocarrier. Glyphosate degradation to aminomethylphosphonic acid was initially > 96% for 21 days of operation at flows yielding hydraulic residence times (HRTs) as short as 42 min. Higher flow rate studies showed > 98% removal of 50 mg glyphosate/L from the waste stream could be achieved at a HRT of 23 min. Glyphosate removal of > 99% at a 37-min HRT was achieved under similar conditions with a column inoculated with a pure culture of Pseudomonas sp. strain LBr, a bacterium known to have high glyphosate-degrading activity. After acid shocking (pH 2.8 for 18 h) of a column of immobilized bacteria, glyphosate-degrading activity was regained within 4 days without reinoculation. Although microbial growth and glyphosate degradation were not maintained under low organic nutrient conditions in the laboratory, the low levels of degradable carbon (45-94 mg/L) in the industrial effluent were sufficient to support prolonged glyphosate-degrading activity. The results demonstrated that immobilized bacteria technology is effective in removing low levels of glyphosate in high-volume liquid waste streams.     

Metabolism of glyphosate in Pseudomonas sp. strain LBr

Metabolism of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. strain LBr, a bacterium isolated from a glyphosate process waste stream, was examined by a combination of solid-state 13C nuclear magnetic resonance experiments and analysis of the phosphonate composition of the growth medium. Pseudomonas sp. strain LBr was capable of eliminating 20 mM glyphosate from the growth medium, an amount approximately 20-fold greater than that reported for any other microorganism to date. The bacterium degraded high levels of glyphosate, primarily by converting it to aminomethylphosphonate, followed by release into the growth medium. Only a small amount of aminomethylphosphonate (about 0.5 to 0.7 mM), which is needed to supply phosphorus for growth, could be metabolized by the microorganism. Solid-state 13C nuclear magnetic resonance analysis of strain LBr grown on 1 mM [2-13C,15N]glyphosate showed that about 5% of the glyphosate was degraded by a separate pathway involving breakdown of glyphosate to glycine, a pathway first observed in Pseudomonas sp. strain PG2982. Thus, Pseudomonas sp. strain LBr appears to possess two distinct routes for glyphosate detoxification.     

草甘膦对空心莲子草叶甲Agasicles hygrophila成虫3种代谢酶活性的影响

采用不同浓度的草甘膦0.41 g/L、0.82 g/L、1.23 g/L、1.64g/L、2.05 g/L分别以胃毒和触杀法处理空心莲子草叶甲Agasicles hygrophila成虫,测定其乙酰胆碱酯酶(AChE)、羧酸酯酶(CarE)和谷胱甘肽S-转移酶(GSTs)比活力.试验结果表明:两种处理,草甘膦对AChE活力均有不同程度的抑制作用;对CarE活力影响较为显著,在2.05 g/L浓度下,胃毒处理CarE对α-乙酸萘酯(α-NA)和β-乙酸萘酯(β-NA)水解能力分别是对照组的50%和57%,触杀处理CarE对α-乙酸萘酯(α-NA)和β-乙酸荼酯(β-NA)水解能力分别是对照组的53%和59%;胃毒处理埘酶活力影响大于触杀处理,草甘膦对GSTs的活力影响不明显.