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.     

Metabolism of twelve herbicides by Streptomyces

Experiments were conducted to assess the ability of Streptomyces (strain PS1/5) to metabolize twelve herbicides representing several different classes including: acetanilides, triazines, ureas, uracils, and imidazoles. Incubations in aqueous culture with dextrin as carbon source and either ammonium or Casamino acids as nitrogen source resulted in transformations (>50%) of eight of the herbicides tested: alachlor, metolachlor, atrazine, prometryne, ametryne, linuron, tebuthiuron, and bromacil; the remaining four herbicides (cyanazine, diuron, metribuzin, and imazapyr) were also transformed, but to a lesser extent. In most instances, biotransformations occurred concurrently with growth and results were consistent regardless of the nitrogen source (ammonium vs. Casamino acids). However, in some instances there were differences in rates of biotransformation as a consequence of the nitrogen source (e.g. alachlor, metribuzin), suggesting the selective induction of certain metabolic enzymes; in other instances biotransformations were not associated with growth, suggesting secondary metabolism. An experiment was also conducted to assess the ability of Streptomyces (strain PS1/5) to metabolize atrazine contaminated soil. Inoculation of soil amended with 20 μg/g of atrazine and 5% chitin as carbon source resulted in ca. 78% removal of atrazine within 28 days. These data suggest that Streptomyces species may be potential candidates for soil inoculation to bioremediate herbicide contaminated soils.     

An evaluation of the genotoxic properties of herbicides following plant and animal activation

Commercial and technical grades of 11 herbicides and 13 combinations of commercial grade herbicides were evaluated for their genotoxic properties with Salmonella typhimurium, Saccharomyces cerevisiae directly and following plant and animal activation, or with Zea mays. The herbicides were related by their use in commercial corn (maize) production. Commercial grade formulations of each herbicide and combination of herbicides were also evaluated in situ with the pollen waxy locus assay of Z. mays. Eradicane and bifenox were negative in all assays. Alachlor, propachlor, procyazine and SD50093 (a formulation of cyanazine plus atrazine) were positive in one assay. Cyanazine, dicamba and metolachlor were positive in 2 assays. Atrazine, simazine and butylate were tested only in situ. Atrazine and simazine were positive and butylate was negative. Of the combinations of herbicides evaluated with the 3 genetic assays, alachlor plus bifenox and procyazine plus metolachlor were positive in 1 assay and metolachlor plus atrazine was positive in 2 assays. Of the combinations of herbicides evaluated only in situ, butylate plus atrazine, eradicane plus atrazine, eradicane plus cyanazine and metolachlor plus cyanazine were positive while butylate plus cyanazine was negative.     

An Immunochromatographic Assay of 2,4-Dichlorophenoxyacetic Acid and Simazine Using Monoclonal Antibodies Labeled with Colloidal Gold

A method of the competitive immunochromatographic assay of the pesticides 2,4-D (2,4-dichlorophenoxyacetic acid) and simazine (2-chloro-4,6-bis(N-ethylamino)-1,3,5-triazine) in aqueous samples was developed. Monoclonal antibodies to these pesticides labeled with colloidal gold were used to visualize the results. The sensitivity of the 2,4-D and simazine assay is 12 ng/ml, and the time of analysis is 3–7 min. The method does not differ in sensitivity from the competitive EIA using conjugates of monoclonal antibodies to the pesticides with horseradish peroxidase; however, the time of the EIA is 1.5 h. The immunochromatographic method of the pesticide detection is available and simple and may be recommended for the development of assays of any other low-molecular compounds.     

Metabolism of twelve herbicides by Streptomyces

Experiments were conducted to assess the ability of Streptomyces (strain PS1/5) to metabolize twelve herbicides representing several different classes including: acetanilides, triazines, ureas, uracils, and imidazoles. Incubations in aqueous culture with dextrin as carbon source and either ammonium or Casamino acids as nitrogen source resulted in transformations (>50%) of eight of the herbicides tested: alachlor, metolachlor, atrazine, prometryne, ametryne, linuron, tebuthiuron, and bromacil; the remaining four herbicides (cyanazine, diuron, metribuzin, and imazapyr) were also transformed, but to a lesser extent. In most instances, biotransformations occurred concurrently with growth and results were consistent regardless of the nitrogen source (ammonium vs. Casamino acids). However, in some instances there were differences in rates of biotransformation as a consequence of the nitrogen source (e.g. alachlor, metribuzin), suggesting the selective induction of certain metabolic enzymes; in other instances biotransformations were not associated with growth, suggesting secondary metabolism. An experiment was also conducted to assess the ability of Streptomyces (strain PS1/5) to metabolize atrazine contaminated soil. Inoculation of soil amended with 20 g/g of atrazine and 5% chitin as carbon source resulted in ca. 78% removal of atrazine within 28 days. These data suggest that Streptomyces species may be potential candidates for soil inoculation to bioremediate herbicide contaminated soils.The U.S. Government's right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

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.     

Immunochromatographic analysis of 2,4-dichlorophenoxyacetic acid and simazine using monoclonal antibodies labelled with colloidal gold

A method of the competitive immunochromatographic assay of the pesticides 2,4-D (2,4-dichlorophenoxyacetic acid) and simazine (2-chloro-4,6-bis(N-ethylamino)-1,2,5-triazine) in aqueous samples was developed. Monoclonal antibodies to these pesticides labeled with colloidal gold were used to visualize the results. The sensitivity of the 2,4-D and simazine assay is 12 ng/ml, and the time of analysis is 3-7 min. The method does not differ in sensitivity from the competitive EIA using conjugates of monoclonal antibodies to the pesticides with horseradish peroxidase; however, the time of the EIA is 1.5 h. The immunochromatographic method of the pesticide detection is available and simple and may be recommended for the development of assays of any other low-molecular compounds. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Effect of simazine and atrazine on the mineralization of fertilizer and manure nitrogen

Summary Laboratory experiments were carried out with alluvial sandy loam soil to study the effect of simazine and atrazine herbicides at four levels (0.5, 1.0, 1.5 and 2.0 kg/ha) on the mineralization of nitrogen (ammoniacal and nitrate production) from fertilizer urea and sludge sources. The herbicides stimulated nitrate production. No specific trend in total mineralized nitrogen, ammoniacal and nitrate nitrogen was observed by varying the levels of herbicides. Mineralization of total nitrogen (ammoniacal and nitrate nitrogen) in presence of simazine and atrazine from the different sources in the descending order was:Urea > Sludge + Urea > Sludge > No Nitrogen.     

Enzyme immunoassay determination of atrazine degradation in soil: Moisture,sterilization, and storage effects

Enzyme immunoassay (EIA) microtiter plate analysis was used to quantify atrazine (2‐chloro‐4‐ethylamino‐6‐isopropylamino‐1,3,5 triazine), fortified at 0, 50, and 500 or 549 ng/g, to Baxter and Maury silt loam soil sampled in 1965 and 1991. In the first experiment, aged soils (sampled in 1965 and stored air‐dried) were fortified with atrazine and then incubated in the dark at 0, 75, 150, 225 and 300 g/kg moisture for 15, 80, 154, and 289 d. In a second experiment, fresh soils were fortified with atrazine and incubated in the dark at 0, 150, and 300 g/kg moisture for 9, 15, 35, 55, 83, and 145 d. One half of the treatments in the second experiment were sterilized with 497 ng/g HgCl₂. Twenty milliliters of acetonitrile: water (9: 1) was used to extract 4 or 5 g of soil by vortex mixing at each sampling date. The soil extract was diluted, 80 μl incubated with antibody‐coated wells, and color development read using a microtiter plate reader. Recovery of atrazine from soil was 98% 5 d after fortification. Pesticide recoveries and first‐order degradation rates were dependent on the freshness and moisture content of the soil sample. Pesticide degradation was slower and recoveries higher in soil that had been air dried and stored since 1965, prior to fortification. More atrazine was extracted from soil maintained at 0 g/kg moisture than from soil maintained at 300 g/kg moisture over time.     

The effect of the glyphosate, 2,4-D, atrazine e nicosulfuron herbicides upon the Edaphic collembola (Arthropoda: Ellipura) in a no tillage system

The use of herbicides is a common and intensive practice in no tillage systems. The herbicides can influence, directly or indirectly, the population of edaphic arthropods. Collembola is a group that functions as a bio-indicator of soil conditions. The degree of abundance and diversity of Collembola provides the level of soil disturbance provoked by agricultural practices. This experiment was designed to compare the influence of herbicides on the population fluctuation of Collembola in a no-till soil preparation system. The work was conducted in a non irrigated no-till area at the Núcleo Experimental de Ciências Agrárias of the Universidade Federal de Mato Grosso do Sul (UFMS), Campus de Dourados, in soil planted with corn as a surface covering, during the period of December, 2002 to December, 2003. The data were analyzed according to a completely randomized model, in a split plot design. The plots received four types of herbicides: glyphosate, atrazine, 2,4-D and nicosulfuron. A fifth plot did not receive any herbicide (control), for a total of five treatment types. The sub plots were represented by their collection times (10, 20, 30 and 40 days after the herbicide applications). Both the type of herbicide and the time of data sampling influenced the Collembola population fluctuaction. The treatments with atrazine and 2,4-D caused the most reduction of the population of Collembola, depending on the time of application.     

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).     

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.     

Molecular characterization of corn glutathione S-transferase isozymes involved in herbicide detoxication

Corn ( Zea mays L.) glutathione S-transferases (EC 2.5.1.18) have attracted interest, in part, due to their involvement in the metabolism of several herbicides, including atrazine and alachlor. Three corn, glutathione S-transferases have been purified, and cDNA clones have been isolated and sequenced for two of these, GST I and GST III. In addition to showing some amino acid sequence similarity to each other, the two sequenced corn glutathione S-transferases also show some similarity to rat and human enzymes. The corn glutathione S-transferases responsible for atrazine tolerance have not yet been purified or cloned, but purification attempts indicate that corn has two glutathione S-transferases with activity towards atrazine. While many glutathione S-transferases from various organisms have been detected by using 1-chloro-2,4-dinitrobenzene as a substrate, the atrazine-specific glutathione S-transferases have very little or no activity with 1-chloro-2,4-dinitrobenzene. This shows the importance of assaying with a variety of substrates when characterizing glutathione S-transferases.     

Microalgae fiber optic biosensors for herbicide monitoring using sol–gel technology

Three microalgal species (Dictyosphaerium chlorelloides (D.c.), Scenedesmus intermedius (S.i.) and Scenedesmus sp. (S.s.)) were encapsulated in silicate sol–gel matrices and the increase in the amount of chlorophyll fluorescence signal was used to quantify simazine. Influence of several parameters on the preparation of the sensing layers has been evaluated: effect of pH on sol–gel gelation time; effect of algae density on sensor response; influence of glycerol (%) on the membrane stability. Long term stability was also tested and the fluorescence signal from biosensors remained stable for at least 3 weeks. D.c. biosensor presented the lowest detection limits for simazine (3.6 μg L⁻¹) and the broadest dynamic calibration range (19–860 μg L⁻¹) with IC₅₀ 125 ± 14 μg L⁻¹. Biosensor was validated by HPLC with UV/DAD detection. The biosensor showed response to those herbicides that inhibit the photosynthesis at photosystem II (triazines: simazine, atrazine, propazine, terbuthylazine; urea based herbicides: linuron). However, no significant increases of fluorescence response was obtained for similar concentrations of 2,4-D (hormonal herbicide) or Cu(II). The combined use of two biosensors that use two different genotypes, sensitive and resistant to simazine, jointly allowed improving microalgae biosensor specificity.     

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.     

Optical whole-cell biosensor using Chlorella vulgaris designed for monitoring herbicides

An optical biosensor was designed for determination of herbicides as aquatic contaminants. Detection was obtained with immobilised Chlorella vulgaris microalgae entrapped on a quartz microfibre filter and placed in a five-membrane-home-made-flow cell. The algal chlorophyll fluorescence modified by the presence of herbicides was collected at the tip of an optical fibre bundle and sent to a fluorimeter. A continuous culture was set up to produce algal cells in reproducible conditions for measurement optimisation. Effects of flow rate, algal density, temperature, and pH on the biosensor response to atrazine were studied. Reversibility and detection limits were determined for DNOC and atrazine, simazine, isoproturon, diuron. Detection of photosystem II (PSII) herbicides was achieved at sub-ppb concentration level.     

Characterization of Arthrobacter nicotinovorans HIM, an atrazine-degrading bacterium, from agricultural soil New Zealand

Arthrobacter nicotinovorans HIM was isolated directly from an agricultural sandy dune soil 6 months after a single application of atrazine. It grew in minimal medium with atrazine as sole nitrogen source but was unable to mineralize 14C-ring-labelled atrazine. Atrazine was degraded to cyanuric acid. In addition to atrazine the bacterium degraded simazine, terbuthylazine, propazine, cyanazine and prometryn but was unable to grow on terbumeton. When added to soil, A. nicotinovorans HIM did enhance mineralization of 14C-ring-labelled atrazine and simazine, in combination with naturally occurring cyanuric acid degrading microbes resident in the soil. Using PCR, the atrazine-degradation genes atzABC were identified in A. nicotinovorans HIM. Cloning of the atzABC genes revealed significant homology (>99%) with the atrazine degradation genes of Pseudomonas sp. strain ADP. The atrazine degradation genes were held on a 96 kbp plasmid.     

Monitoring natural vegetation for herbicide-induced chromosomal aberrations

The average frequency of spontaneous mitotic chromosome aberrations, determined in 12 weed species growing under natural conditions, was 0.4%. Herbicides induced significant increases in this frequency in five species, Ambrosia artemisiifolia L., Pastinaca sativa L., Solidago canadensis L., Solidago nemoralis Ait., and Vicia cracca L. The auxin herbicides — 2,4-D, picloram, and 2,4-D + 2,4,5-T — induced a larger proportion of lagging chromosomes and a smaller proportion of fragmented chromosomes than found among spontaneous aberrations. The non-selective herbicides, simazine and diuron, produced multipolar spindles that were not observed among untreated cells. Clastogenic effects of nonselective herbicides in Pastinaca sativa were short-lived, giving highest frequencies of aberrant cells in June and July and lower aberration rates in August and September. The opposite trend was found in untreated flower buds of this species, suggesting that the spontaneous aberration rate is higher in flower buds from older plants.     

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     

Metabolism of the herbicide atrazine by Rhodococcus strains.

Rhodococcus strains were screened for their ability to degrade the herbicide atrazine. Only rhodococci that degrade the herbicide EPTC (s-ethyl-dipropylthiocarbamate) metabolized atrazine. Rhodococcus strain TE1 metabolized atrazine under aerobic conditions to produce deethyl- and deisopropylatrazine, which were not degraded further and which accumulated in the incubation medium. The bacterium also metabolized the other s-triazine herbicides propazine, simazine, and cyanazine. The N dealkylation of triazine herbicides by Rhodococcus strain TE1 was associated with a 77-kb plasmid previously shown to be required for EPTC degradation.