Combining supercritical fluid extraction of soil herbicides with enzyme immunoassay analysis

Supercritical fluid extraction (SFE) of soil herbicides followed by enzyme immunoassay analysis (EIA) is explained in a step-by-step process. Extracted herbicides, include 2,4-D, simazine, atrazine, and alachlor. The herbicide, trifluralin was not successfully analyzed by EIA because of crossreacting metabolites. Problems with SFE, including uneven packing of cells, leaks, uneven flow and clogging, can largely be eliminated as the method parameters are optimized. It was necessary to add modifiers including methanol or acetone to the SF CO₂ to increase the solubility of the analytes. Detection limits of 2.5 ng/g soil for atrazine and alachlor and 15 ng/g soil for simazine and 2,4-D without concentration of the sample were achieved. Recoveries above 80% and relative standard deviations (RSDs) less than 15% for 2,4-D simazine, atrazine and alachlor were achieved. Atrazine and alachlor recoveries were above 90% with RSDs below 10%. Forty soil samples could be extracted and analyzed in an 8-h day.     

Interactions of atrazine and 2,4-D with human serum albumin studied by gel and capillary electrophoresis, and FTIR spectroscopy.

The herbicides 6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine (atrazine) and 2,4-dichlorophenoxyacetic acid (2,4-D) are widely used in agricultural practice to fight dicotyledon weeds mainly in maize, cereals, and lucerne. As a result, these compounds are found not only in the plants, soil, and water, but also in the cultivated ground in the following years as well as in agricultural products such as fruits, milk, butter, and sugar beet. The toxicological effects of herbicides occur in vivo, when transported to the target organ through the bloodstream. It has been suggested that human serum albumin (HSA) serves as a carrier protein to transport 2,4-D to molecular targets. This study was designed to examine the interaction of atrazine and 2,4-D with HSA in aqueous solution at physiological pH with herbicide concentrations of 0.0001-1 mM, and final protein concentration of 1% w/v. Gel and capillary electrophoresis, UV-visible and Fourier transform infrared spectroscopic methods were used to determine the drug binding mode, the drug binding constant, and the protein secondary structure in aqueous solution. Structural analysis showed that different types of herbicide-HSA complexes are formed with stoichiometric ratios (drug/protein) of 3:1 and 11:1 for atrazine and 4.5:1 and 10:1 for 2,4-D complexes. Atrazine showed a weak binding affinity (K=3.50 x 10(4) M(-1)), whereas two bindings (K(1)=2.50 x 10(4) M(-1) and K(2)=8.0 x 10(3) M(-1)) were observed for 2,4-D complexes. The herbicide binding results in major protein secondary structural changes from that of the alpha-helix 55% to 45--39% and beta-sheet 22% to 24--32%, beta-anti 12% to 10--22% and turn 11% to 12--15%, in the drug-HSA complexes. The observed spectral changes indicate a partial unfolding of the protein structure, in the presence of herbicides in aqueous solution.     

Degradation of atrazine and 2,4-dichlorophenoxyacetic acid by mycorrhizal fungi at three nitrogen concentrations in vitro.

Nine mycorrhizal fungi and free-living saprophytic microorganisms were tested for their ability to degrade two chlorinated aromatic herbicides at two herbicide concentrations and three nitrogen concentrations. Radiolabelled 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) were used as substrates at concentrations of 1 and 4 mM. After 8 weeks, none of the cultures tested grew at 4 mM 2,4-D. However, when the 2,4-D concentration was reduced to 1 mM, Phanerochaete chrysosporium 1767 had the highest level of 2,4-D mineralization and degradation under all nitrogen conditions. All cultures tested grew at both atrazine concentrations. In all cases, the ericoid mycorrhizal fungus Hymenoscyphus ericae 1318 had the highest level of atrazine carbon incorporated into its tissue. In general, as the nitrogen concentration increased, the total herbicide degradation increased. All of the cultures, except for Rhizopogon vinicolor 7534 and Sclerogaster pacificus 9011, showed increased degradation at 4 mM compared with 1 mM atrazine. The ability to degrade these two herbicides thus appeared to be dependent on the fungus and the herbicide, with no correlation to fungal ecotype (mycorrhizal versus free living).     

Relative importance of the effect of 2,4-D,glyphosate, and environmental variables on the soil microbial biomass

Two post-emergence herbicides (glyphosate and 2,4-D) were applied at field application levels to tilled field plots in a mixed cropping area in south-central Alberta. The effects of these chemicals on certain variables associated with microbial biomass and activity were monitored in these plots (as well as corresponding control plots) for 45 days. Glyphosate did not influence any of the microbial variables tested. Addition of 2,4-D significantly influenced all microbial variables investigated but these effects were transient, being detectable only within the first 1–5 days of herbicide addition. The effects of 2,4-D addition on the microbial variables tested, even when significant, were typically small and probably of little ecological consequence especially when spatial and temporal variation in these variables is taken into account.     

Effect of herbicide residues in a sandy loam on the growth,nodulation and nitrogenase activity (C2H2/C2H4) of Trifolium subterraneum

The herbicides 2,4-D, amitrole, atrazine, diclofop-methyl, diquat, paraquat and trifiluralin were applied at rates of 0, 2, 5 and 10 μg ai. g⁻¹ to a sandy loam soil and allowed to degrade for 120 days. After this period, subterranean clover seedlings were transplanted into treated soil and the effect of herbicide residues on plant growth, number of nodules formed and nitrogenase activity was investigated. At all rates of atrazine and chlorsulfuron, and at all rates of amitrole in excess of 2 mg ai g⁻¹ of soil, sufficient herbicide remained to be lethal to the seedlings. When amitrole was applied at the rate of 2 mg ai g⁻¹ of soil, plant growth, nodulation and nitrogenase activity of plants were reduced. Residues of diquat reduced all plant parameters studied while, residues of 2,4-D reduced plant growth and nodule formation, but plant nitrogenase activity was unaffected. Residues of trifluralin had no effect on plant growth parameters but the number of nodules formed per plant was reduced. Residues of paraquat and diclofop-methyl had no effect on any of the plant parameters studied.     

Effect of foliar application of herbicides,Benthiocarb, 2,4-D and Fluchloralin on phyllosphere microflora of potato

Three herbicides, Benthiocarb, 2,4-D and Fluchloralin were evaluated for their effect on leaf surface microflora of potato. In general, the application of herbicides resulted into a drop in the microbial population. Throughout the study herbicide treated leaves harboured less population compared to the untreated ones.     

Co-induction of a glutathione-S-transferase, a glutathione transporter and an ABC transporter in maize by xenobiotics

Glutathione conjugation reactions are one of the principal mechanisms that plants utilize to detoxify xenobiotics. The induction by four herbicides (2,4-D, atrazine, metolachlor and primisulfuron) and a herbicide safener (dichlormid) on the expression of three genes, ZmGST27, ZmGT1 and ZmMRP1, encoding respectively a glutathione-S-transferase, a glutathione transporter and an ATP-binding cassette (ABC) transporter was studied in maize. The results demonstrate that the inducing effect on gene expression varies with both chemicals and genes. The expression of ZmGST27 and ZmMRP1 was up-regulated by all five compounds, whereas that of ZmGT1 was increased by atrazine, metolachlor, primisulfuron and dichlormid, but not by 2,4-D. For all chemicals, the inducing effect was first detected on ZmGST27. The finding that ZmGT1 is activated alongside ZmGST27 and ZmMRP1 suggests that glutathione transporters are an important component in the xenobiotic detoxification system of plants.     

Impact of Polyacrylamide Treatment on Sorptive Dynamics and Degradation of 2,4-D and Atrazine in Agricultural Soil

High-molecular-weight, anionic polyacrylamide (PAM) is added to irrigation water to reduce soil erosion during furrow irrigation of crops. The chemical nature of PAM, together with the observation that the polymer can be biotransformed by soil bacteria, led us to question the impact of PAM treatment on the fate of coapplied agrochemicals. The herbicides, atrazine (nonionic) and 2,4-D (anionic), were tested for pesticide sorption, desorption, and degradation in PAM-treated and untreated soils. Sorption of atrazine and 2,4-D in soil was unaffected by PAMtreatment, as was atrazine desorption. However, 2,4-D desorbedmore readily from the PAM-treated soil than from untreated soil. With respect to pesticide degradation, mineralization of the 2,4-D aromatic ring was not impacted by PAM treatment, but decarboxylation of the 2,4-D carboxylic acid side chain was significantly reduced in the PAM-treated soil. Limited mineralization (7 to 10%) of atrazine was observed in both soils. However, in PAM-treated soils atrazine conversion to ¹⁴CO₂ and bound residue components was significantly reduced, and there was an increase in the level of methanol extractable metabolites. These results may indicate that PAM application can alter the environmental fate of some pesticides in soils, especially under the high dose treatment conditions examined in this study.     

Influence of four herbicides on the algal flora of a prairie soil

Summary Four herbicides (2,4-D, trifluralin, MCPA and TCA) were applied at two concentration levels to isolated cores of a grassland loam soil. After herbicide contact times of 1, 5, and 20 days, samples were taken and the algal population estimated both quantitatively and qualitatively using two selective mineral salts media. Thirty one genera of algae were identified as occurring in the soil. Of these, Chlamydomonas, Chlorococcum, Hormidium, Palmella, and Ulothrix proved to be so sensitive to the four herbicides that they were rarely isolated from the cores after treatment. Other algal genera were found to be less sensitive, and the theoretical percentile sensitivity of fifteen genera was calculated. Chlorella, Lyngbya, Nostoc, and Hantzschia were found to be the most resistant algae, having percentile sensitivity to all four herbicides of less than 50%. Some algal genera varied in their sensitivity to each of the herbicides. Scytonema was sensitive to all of the herbicides except 2,4-D, while Tolypothrix showed a greater tolerance to MCPA. In the top cm of the soil, the reduction in cell numbers experienced by many algal genera after herbicide treatment was offset by an increase in the population of Chlorella. Stichococcus, Oscillatoria, and Spongiochloris all exhibited the ability to recover rapidly after a reduction in cell numbers resulting from the application of one of the herbicides. An overall reduction in cell numbers was noted for the algae growing preferentially on a nitrogen-free medium (i.e. potential nitrogen-fixers). re]19760511     

Herbicide Persistence in Seawater Simulation Experiments

Herbicides are detected year-round in marine waters, including those of the World Heritage listed Great Barrier Reef (GBR). The few previous studies that have investigated herbicide persistence in seawater generally reported half-lives in the order of months, and several studies were too short to detect significant degradation. Here we investigated the persistence of eight herbicides commonly detected in the GBR or its catchments in standard OECD simulation flask experiments, but with the aim to mimic natural conditions similar to those found on the GBR (i.e., relatively low herbicide concentrations, typical temperatures, light and microbial communities). Very little degradation was recorded over the standard 60 d period (Experiment 1) so a second experiment was extended to 365 d. Half-lives of PSII herbicides ametryn, atrazine, diuron, hexazinone and tebuthiuron were consistently greater than a year, indicating high persistence. The detection of atrazine and diuron metabolites and longer persistence in mercuric chloride-treated seawater confirmed that biodegradation contributed to the breakdown of herbicides. The shortest half-life recorded was 88 d for growth-regulating herbicide 2,4-D at 31°C in the dark, while the fatty acid-inhibitor metolachlor exhibited a minimum half-life of 281 d. The presence of moderate light and elevated temperatures affected the persistence of most of the herbicides; however, the scale and direction of the differences were not predictable and were likely due to changes in microbial community composition. The persistence estimates here represent some of the first appropriate data for application in risk assessments for herbicide exposure in tropical marine systems. The long persistence of herbicides identified in the present study helps explain detection of herbicides in nearshore waters of the GBR year round. Little degradation of these herbicides would be expected during the wet season with runoff and associated flood plumes transporting a high proportion of the original herbicide from rivers into the GBR lagoon.     

Colonisation of seedling roots by 2,4-D degrading bacteria: A plant-microbial model

The development of model plant-microbial associations between Gram negative soil microbes capable of degrading phenoxyacetate herbicides, such as 2,4-D and 2,4-D methyl ester, and the crops canola and wheat was described. Both an Acinetobacter baumannii pJP4 transconjugant and Alcaligenes eutrophus JMP 134 colonised non-parasitically on the roots of sterilised seedlings in a hydroponic system. Laser scanning confocal microscopy has shown that colonisation occurred both on the root surface and deeper inside the mucilage layer or inside some surface root cells. When 2,4-D was added to the hydroponic medium supporting the growth of those seedlings colonised by 2,4-D degrading bacteria, the gas chromatographic analysis showed a rapid decrease in the concentration of this herbicide. These bacteria colonising the root system were shown to be responsible for the degradation of 2,4-D. Plants inoculated with the 2,4-D degrading microbes were subsequently found to be less susceptible to damage by the herbicide in such hydroponic systems.     

Degradation of Herbicides in the Tropical Marine Environment: Influence of Light and Sediment

Widespread contamination of nearshore marine systems, including the Great Barrier Reef (GBR) lagoon, with agricultural herbicides has long been recognised. The fate of these contaminants in the marine environment is poorly understood but the detection of photosystem II (PSII) herbicides in the GBR year-round suggests very slow degradation rates. Here, we evaluated the persistence of a range of commonly detected herbicides in marine water under field-relevant concentrations and conditions. Twelve-month degradation experiments were conducted in large open tanks, under different light scenarios and in the presence and absence of natural sediments. All PSII herbicides were persistent under control conditions (dark, no sediments) with half-lives of 300 d for atrazine, 499 d diuron, 1994 d hexazinone, 1766 d tebuthiuron, while the non-PSII herbicides were less persistent at 147 d for metolachlor and 59 d for 2,4-D. The degradation of herbicides was 2–10 fold more rapid in the presence of a diurnal light cycle and coastal sediments; apart from 2,4-D which degraded more slowly in the presence of light. Despite the more rapid degradation observed for most herbicides in the presence of light and sediments, the half-lives remained > 100 d for the PS II herbicides. The effects of light and sediments on herbicide persistence were likely due to their influence on microbial community composition and its ability to utilise the herbicides as a carbon source. These results help explain the year-round presence of PSII herbicides in marine systems, including the GBR, but more research on the transport, degradation and toxicity on a wider range of pesticides and their transformation products is needed to improve their regulation in sensitive environments.     

A comparison of effects of several herbicides on photoautotrophic,photomixotrophic and heterotrophic cultured tobacco cells and seedlings

The effects of herbicides with different primary modes of action were examined on the growth of photoautotrophic, photomixotrophic, and heterotrophic cultures of tobacco cells. These responses were compared with those of tobacco seedlings to the same herbicides. Herbicides, which primarily inhibit or disturb photosynthetic processes, suppressed the growth of photoautotrophic cells most strongly, as compared to photomixotrophic and heterotrophic cells (atrazine, diuron, paraquat). Herbicides having a primary mode of action other than the inhibition of photosynthetic processes, suppressed the growth of all types of cultured cells at similar concentrations (2,4-D, diphenamid, glyphosate, dinoseb, sodium chlorate, bialaphos, DTP), but the photoautotrophic cells were still the most sensitive to all kinds of herbicides except sodium chlorate. Furthermore, photoautotrophic cells responded to most of the herbicides as did the seedlings, with the exception of glyphosate and diphenamid. The possibility of photoautotrophically cultured cells as a model system to study the effects of herbicides are discussed.Abbreviation bialaphos (2-amino-4-methylphosphinobutyryl)alanylalanine sodium salt - diuron 3-(3,4-dichloro-phenyl)-1,1-dimethyl-urea - 2,4-D 2,4-dichlorophenoxy-acetic acid - DTP 1,3-dimethyl-4-(2,4-dichlorobenzoyl)-5-hydoxy-pyrazolate - dinoseb 2-secbutyl-4,6-dinitrophenol     

Atrazine movement in soil: Comparison of field observations and PRZM simulations

The predictive capability of the pesticide root zone model (PRZM) was investigated for herbicide atrazine [2‐chloro‐4‐(ethylamino)‐6‐(isopropylamino)‐s‐triazine] in corn production under no‐till (NT) and conventional‐till (CT) management practices. Simulation values of atrazine residues obtained using our site‐specific soil and environmental data were compared with the actual values measured in soil samples taken from the root zones of the NT and CT plots during three growing seasons: 1986, 1987, and 1988. The mean concentration of atrazine in soil at each sampling time and depth after application, for each tillage treatment plot (NT or CT), was estimated based on the type of distribution (i.e., normal or lognormal). Overall, the PRZMs simulated concentrations for the top 10 cm of soil compared well with the atrazine residues measured in the CT plots, but overestimated measurements in NT plots. For example, in 1986 the mean atrazine concentration measured in soil samples taken 6 d after application from the top 10 cm of CT plots was 548 μg/kg (S.E. 198 μg/kg), and the PRZM predicted value was 690 μg/kg. In contrast, the mean atrazine concentration for the same soil depth increment in NT plots was 385 μg/kg (S.E. 154 μg/kg), with a PRZM predicted value of 674 μg/kg. Although the PRZM prediction was closer to the measured mean for atrazine concentrations in the top 10 cm of the CT system, the model did not transport atrazine to the lower soil depths, as the actual values have indicated in all 3 years. The results of this model comparison, especially for the lower soil depths (20 to 30 cm) in the NT practice, indicated that the PRZM model does not account for the preferential transport of, and, consequently, underestimates the atrazine residue levels in the lower soil profile under NT management systems.     

Herbicide effects on the growth and nodulation potential ofRhizobium trifolii withTrifolium subterraneum L.

A study was made of the effect of the herbicides 2,4-D, amitrole, atrazine, chlorsulfuron, diclofop-methyl, diquat, glyphosate, paraquat and trifluralin on the nodulation of sub-clover (Trifolium subterraneum L. ‘Clare’), the growth ofR. trifolii TA1 in liquid nutrient medium and the ability of herbicide-treated inoculum to successfully nodulate sub-clover plants. As concentrations of amitrole, diclofop-methyl and glyphosate in the rooting environment increased from 0 to 20 mg ai L⁻¹, nodulation decreased linearly. The other herbicides at these concentrations caused more severe decreases in nodulation. Growth ofR. trifolii TA1 in nutrient broth was significantly retarded by all concentrations of diquat, 2 mg ai L⁻¹ of paraquat, 10 mg ai L⁻¹ of glyphosate and 2 mg ai L⁻¹ of chlorsulfuron. Other herbicides did not suppress rhizobial growth. Inoculation with TA1 that had been grown in the presence of amitrole, atrazine or glyphosate and then washed free of the herbicide decreased nodulation of sub-clover, indicating that these herbicides may physiologically influence the nodulating potential of certain strains of Rhizobium. The remaining herbicides showed no indications of this effect.     

Denitrifying capacity of rhizobial strains of Argentine soils and herbicide sensitivity

Agrochemical application in soils is a matter of environmental concern, and among soil microorganisms, rhizobia and their action before different pesticides are interesting to study, due to their taxonomic and functional diversity. The objectives of the present work were to assess the capacity of rhizobial populations to use herbicides as source of nutrients, as well as their ability to reduce nitrates and / or denitrify. Eighty-one strains belonging to four populations of different genera of rhizobia (Rhizobium, Mesorhizobium, Ensifer and Bradyrhizobium) were assessed. The effect of glyphosate, 2,4-dichlorophenoxyacetic acid, and atrazine on growth of the strains, as well as the ability of the strains to act on herbicide transformation to reduce nitrate and denitrify, were evaluated. The genera studied showed different responses to pesticides. Bradyrhizobium had the greater capacity to utilize the herbicides and among the compounds evaluated, atrazine was the most used as a source of energy. To conclude, some Bradyrhizobium strains were able both to denitrify and to use the atrazine herbicide. The results obtained in this study increase expectations of the use of rhizobia as inoculants, causing changes at the agricultural and environmental level and allowing an appropriate management of agricultural soil fertilization, efficiency in nitrogen fixation and a faster biodegradation of pesticides in soil.     

Importance of the type of soil for the induction of micronuclei and the growth of primary roots of Vicia faba treated with the herbicides atrazine, glyphosate and maleic hydrazide.

Research was carried out on the genotoxic effects (induction of micronucleated cells in primary root tips) and toxic effects (reduction in primary root growth) in young plants of Vicia faba grown in soils with different organic matter contents and treated with the herbicides atrazine, glyphosate and maleic hydrazide. The data obtained show that the genotoxic effects are noticeably influenced by the interactions between the herbicide and the type of soil in which the Vicia faba have grown. While maleic hydrazide proved to be highly clastogenic for young plants grown in both soils, atrazine was genotoxic only in young plants grown in soil poor in organic matter. Glyphosate did not induce micronuclei under either soil condition, but induced a significant toxic effect.     

Bacterial, Fungal, and Actinomycete Populations in Soils Receiving Repeated Applications of 2,4-Dichlorophenoxyacetic Acid and Trifluralin

Soil samples were collected from an untreated plot and plots receiving repeated applications of 2,4-dichlorophenoxyacetic acid (2,4-D) and alpha,alpha,alpha-trifluoro-2, 6-dinitro-N,N-dipropyl-p-toluidine (trifluralin); they were then plated on media specific for bacteria, fungi, and actinomycetes. The actinomycete colony count in the trifluralin-treated plot was greater than the control, but the same as the control in the 2,4-D-treated plot. The bacterial count was lower in both treated plots. Fungal colonies in the trifluralin-treated plots were greater than the control, but not different from the control in the 2,4-D-treated plot.     

Influence of temperature on phytotoxicity of triazine herbicides to pine seedlings

The effects of temperature, over a range of 5 to 30 C, on phytotoxicity of simazine, atrazine, propazine, prometryne, prometone, and ipazine to young Pinus resinosa seedlings were investigated in growth chambers. Herbicides were applied to the soil surface and then mixed into the soil before pine seeds were planted. Development of recently germinated seedlings was then studied for 7 weeks. High temperatures greatly accelerated herbicide toxicity, but the effects of temperature varied greatly among herbicides. Atrazine and simazine were more toxic than other herbicides tested at all temperatures. Toxicity of simazine and atrazine was apparent early, whereas effects of propazine, prometryne, prometone, and ipazine were somewhat delayed. After 7 weeks maximum dry-weight production of shoots under each herbicide treatment and control occurred at 20 C, with some decreases noted at lower temperatures and marked decreases at progressively higher ones. At 20 C final seedling dry weights following treatment with simazine or atrazine were only one-third as high as in control plants. Growth was also reduced in lesser amounts by propazine, prometryne, prometone, or ipazine. Variations in phytotoxicity of different triazine herbicides appeared to be related more to their structural differences than their solubilities. Under the constant environmental conditions of the experiments, toxicity symptoms in plants treated with triazine herbicides appeared more rapidly and decisively than in previous field experiments under fluctuating environments. The influence of high temperatures in enhancing triazine toxicity appeared to involve complex interactions of physiological activity of plants and temperature effects on herbicide uptake.