Evaluating phytochemical and microbial contributions to atrazine degradation.

The inclusion of warm-season grasses, such as switchgrass (Panicum virgatum) and eastern gamagrass (EG) (Tripsacum dactyloides), in vegetated buffer strips has been shown to mitigate herbicide contamination in runoff and increase herbicide degradation in soil. The mode of action by which buffer strip rhizospheres enhance herbicide degradation remains unclear, but microorganisms and phytochemicals are believed to facilitate degradation processes. The objectives of this study were to: 1) screen root extracts from seven switchgrass cultivars for the ability to degrade the herbicide atrazine (ATZ) in solution; 2) determine sorption coefficients (Kd) of the ATZ-degrading phytochemical 2-ß-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DBG) to soil and Ca-montmorillonite, and investigate if DBG or ATZ sorption alters degradation processes; and 3) quantify ATZ degradation rates and soil microbial response to ATZ application in mesocosms containing soil and select warm-season grasses. Phytochemicals extracted from the roots of switchgrass cultivars degraded 44-85% of ATZ in 16-h laboratory assays, demonstrating that some switchgrass cultivars could rapidly degrade ATZ under laboratory conditions. However, attempts to isolate ATZ-degrading phytochemicals from plant roots were unsuccessful. Sorption studies revealed that DBG was strongly sorbed to soil (Kd = 87.2 L kg-1) and Ca-montmorillonite (Kd = 31.7 L kg-1), and DBG driven hydrolysis of ATZ was entirely inhibited when either ATZ or DBG were sorbed to Ca-montmorillonite. Atrazine degradation rates in mesocosm soils were rapid (t0.5 = 8.2-11.2 d), but not significantly different between soils collected from the two switchgrass cultivar mesocosms, the eastern gamagrass cultivar mesocosm, and the unvegetated mesocosm (control). Significant changes in three phospholipid fatty acid biomarkers were observed among the treatments. These changes indicated that different ATZ-degrading microbial consortia resulted in equivalent ATZ degradation rates between treatments. Results demonstrated that soil microbial response was the dominant mechanism controlling ATZ degradation in the soil studied, rather than root phytochemicals.

Chloro-triazine transport to streams-evaluating methods for partitioning deisopropylatrazine sources.

Streams in the Salt River Basin (SRB) of northeastern Missouri, USA, have been chronically contaminated by atrazine and metabolites, with peak annual transport occurring from spring to early summer. Since 2005, increased fall-applied simazine has introduced a second chloro-triazine herbicide that degrades to deisopropylatrazine (DIA), creating the need for a method to partition DIA between its two parent sources - i.e., DIA derived from atrazine (DIAATR) and that from simazine (DIASIM). Distinguishing DIA parent sources would extend current understanding of chloro-triazine transport, leading to more accurate risk assessments and improved watershed-scale load estimates. The objectives of this study were to evaluate proposed methods for DIA partitioning, and to apply the most effective method to estimate DIAATR and DIASIM concentrations and loads. Three DIA partition methods were developed and statistically evaluated: 1) edge-of-field (EOF) based on DIA and deethylatrazine (DEA) concentrations in runoff from atrazine treated fields; 2) DIA:DEA concentration ratios (D2R) in runoff from atrazine treated fields; and 3) concentration ratios of simazine:atrazine (SAR) in streams. Stream samples were collected year-round at 7 SRB stream sites from 2005 to 2010 and daily, quarterly, and annual concentrations and loads of atrazine, DEA, DIA, and simazine computed. The SAR method was superior to EOF and D2R in its ability to estimate concentrations and loads of DIASIM and DIAATR that were more accurate and highly correlated to observed transport of simazine, atrazine, and DIA. The SAR method results demonstrated the differences in DIASIM and DIAATR transport timing, with peak DIASIM transport occurring from mid-Nov to Apr and peak DIAATR transport from May to Jun. Dual season triazine applications within a watershed substantially increased the period of high chloro-triazine concentrations in streams from ~3 to ~8 months/yr, potentially increasing the risk of toxicity to aquatic ecosystems.

Atrazine Transport through a Soil-Epikarst System.

Row crop and livestock production contaminate soils and groundwater of the karst aquifers within south-central Kentucky's Pennyroyal Plateau. Transport of atrazine from field application to the epikarstic drainage system beneath a field with active row-crop farming was investigated. The Crumps Cave study site is a shallow autogenic drainage system with a recharge area of ∼1 ha that contains two epikarst drains (WF-1 and WF-2) which were monitored for atrazine, deethylatrazine (DEA), and deisopropylatrazine (DIA) concentrations from January 2011 to May 2012. Atrazine concentrations in both drains did not increase above winter background levels for nearly 2 mo after application when levels suddenly spiked and reached peak concentrations for the study during an event in May 2011. Atrazine, DEA, and DIA were detected in 100% of samples, and metabolites accounted for 54 to 94% of the monthly total loads, except in May 2011. Median dealkylated metabolite/atrazine ratios (DMAR) were ∼5:1 at both sites, and seasonal DMAR patterns corresponded with changes in soil temperature. These data support the hypothesis that a combination of sorption and degradation in the soil column above the epikarst controlled the transport of atrazine and its metabolites. This resulted in delayed atrazine transport after application and prolonged transport of atrazine and its weakly sorbed metabolites to the epikarst aquifer. Management practices that reduce herbicide inputs, such as diverse crop rotations, cover crops, and use of low-rate and strong-sorbing herbicides, would improve groundwater quality in areas of the Corn Belt with intensive row cropping on karst topography.

Benzoxazinone-Mediated Triazine Degradation: A Proposed Reaction Mechanism.

The role of benzoxazinones (Bx, 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one) in triazine resistance in plants has been studied for over half a century. In this research, fundamental parameters of the reaction between DIBOA-Glc (2-ß-d-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one) and atrazine (ATR, 6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine) were examined. Through a series of experiments employing a variety of chromatographic and spectroscopic techniques, the DIBOA-Glc/ATR reaction was characterized in terms of reactant and product kinetics, stoichiometry, identification of a reaction intermediate, and reaction products formed. Results of these experiments demonstrated that the reaction mechanism proceeds via nucleophilic attack of the hydroxamic acid moiety of DIBOA-Glc at the C-2 position of the triazine ring to form hydroxyatrazine (HA, 2-hydroxy-4-ethylamino-6-isopropylamino-s-triazine), with associated degradation of DIBOA-Glc. Degradation of reactants followed first-order kinetics with a noncatalytic role of DIBOA-Glc. A reaction intermediate was identified as a DIBOA-Glc-HA conjugate, indicating a 1:1 DIBOA-Glc:ATR stoichiometry. Reaction products included HA and Cl(-), but definitive identification of DIBOA-Glc reaction product(s) was not attained. With these reaction parameters elucidated, DIBOA-Glc can be evaluated in terms of its potential for a myriad of applications, including its use to address the problem of widespread ATR contamination of soil and water resources.

Long-term agroecosystem research in the central Mississippi river basin: dissolved nitrogen and phosphorus transport in a high-runoff-potential watershed.

Long-term monitoring data from agricultural watersheds are needed to determine if efforts to reduce nutrient transport from crop and pasture land have been effective. Goodwater Creek Experimental Watershed (GCEW), located in northeastern Missouri, is a high-runoff-potential watershed dominated by claypan soils. The objectives of this study were to: (i) summarize dissolved NH-N, NO-N, and PO-P flow-weighted concentrations (FWC), daily loads, and yields (unit area loads) in GCEW from 1992 to 2010; (ii) assess time trends and relationships between precipitation, land use, and fertilizer inputs and nutrient transport; and (iii) provide context to the GCEW data by comparisons with other Corn Belt watersheds. Significant declines in annual and quarterly FWCs and yields occurred for all three nutrient species during the study, and the decreases were most evident for NO-N. Substantial decreases in first- and fourth-quarter NO-N FWCs and daily loads and modest decreases in first-quarter PO-P daily loads were observed. Declines in NO-N and PO-P transport were attributed to decreased winter wheat ( L.) and increased corn ( L.) production that shifted fertilizer application from fall to spring as well as to improved management, such as increased use of incorporation. Regression models and correlation analyses indicated that precipitation, land use, and fertilizer inputs were critical factors controlling transport. Within the Mississippi River Basin, NO-N yields in GCEW were much lower than in tile-drained areas, but PO-P yields were among the highest in the basin. Overall, results demonstrated that reductions in fall-applied fertilizer and improved fertilizer management reduced N and P transport in GCEW.

Long-term agroecosystem research in the central Mississippi river basin: goodwater creek experimental watershed and regional herbicide water quality data.

Goodwater Creek Experimental Watershed (GCEW) has been the focus area of a long-term effort to document the extent of and to understand the factors controlling herbicide transport. We document the datasets generated in the 20-yr-long research effort to study the transport of herbicides to surface and groundwater in the GCEW. This long-term effort was augmented with a spatially broad effort within the Central Mississippi River Basin encompassing 12 related claypan watersheds in the Salt River Basin, two cave streams on the fringe of the Central Claypan Areas in the Bonne Femme watershed, and 95 streams in northern Missouri and southern Iowa. Details of the analytical methods, periods of record, number of samples, study locations, and means of accessing these data are provided. In addition, a brief overview of significant findings is presented. A key finding was that near-surface restrictive soil layers, such as argillic horizons of smectitic mineralogy, result in greater herbicide transport than soils with high percolation and low clay content. Because of this, streams in the claypan soil watersheds of northeastern Missouri have exceptionally high herbicide concentrations and relative loads compared with other areas of the Corn Belt.

Long-term agroecosystem research in the central Mississippi river basin: goodwater creek experimental watershed and regional nutrient water quality data.

We document the 20-yr-long research effort to study the transport of N and P to surface and groundwater in Goodwater Creek Experimental Watershed. We also document related efforts in nearby claypan watersheds and watersheds with contrasting soil and hydrologic conditions across the northern Missouri-southern Iowa region. Details of the analytical methods, instrumentation, method detection limits, and quality assurance program used to generate the data are described along with a brief overview of significant findings. Nutrient concentrations in streams were in the range associated with nuisance algal growth and presumed loss of aquatic invertebrate diversity. Incorporation of fertilizers was shown to be the most effective practice for reducing nutrient transport in runoff. Despite the claypan soils, NO leaching was a major fate for fertilizer N, and significant contamination of the glacial till aquifer has occurred when long-term fertilizer and manure N inputs exceeded crop N requirements. A key finding of these studies was that field areas with the poorest crop growth were also the most vulnerable to nutrient as well as sediment and herbicide transport.

Bioremediation of atrazine-contaminated soil by forage grasses: transformation, uptake, and detoxification.

A sound multi-species vegetation buffer design should incorporate the species that facilitate rapid degradation and sequestration of deposited herbicides in the buffer. A field lysimeter study with six different ground covers (bare ground, orchardgrass, tall fescue, timothy, smooth bromegrass, and switchgrass) was established to assess the bioremediation capacity of five forage species to enhance atrazine (ATR) dissipation in the environment via plant uptake and degradation and detoxification in the rhizosphere. Results suggested that the majority of the applied ATR remained in the soil and only a relatively small fraction of herbicide leached to leachates (<15%) or was taken up by plants (<4%). Biological degradation or chemical hydroxylation of soil ATR was enhanced by 20 to 45% in forage treatment compared with the control. Of the ATR residues remaining in soil, switchgrass degraded more than 80% to less toxic metabolites, with 47% of these residues converted to the less mobile hydroxylated metabolites 25 d after application. The strong correlation between the degradation of N-dealkylated ATR metabolites and the increased microbial biomass carbon in forage treatments suggested that enhanced biological degradation in the rhizosphere was facilitated by the forages. Hydroxylated ATR degradation products were the predominant ATR metabolites in the tissues of switchgrass and tall fescue. In contrast, the N-dealkylated metabolites were the major degradation products found in the other cool-season species. The difference in metabolite patterns between the warm- and cool-season species demonstrated their contrasting detoxification mechanisms, which also related to their tolerance to ATR exposure. Based on this study, switchgrass is recommended for use in riparian buffers designed to reduce ATR toxicity and mobility due to its high tolerance and strong degradation capacity.

Improved HPLC-MS/MS method for determination of isoxaflutole (balance) and its metabolites in soils and forage plants.

A robust multi-residue procedure is needed for the analysis of the pro-herbicide isoxaflutole and its degradates in soil and plant materials at environmentally relevant (<1 microg kg-1) levels. An analytical method using turbo-spray and heat-nebulizer high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for the analysis of isoxaflutole (IXF) and its two metabolites, diketonitrile (DKN) and the benzoic acid metabolite (BA), at sub-microgram per kilogram levels in soil and plant samples. The average recoveries of the three compounds in spiked soil and plant samples ranged from 84 to 110% and 94 to 105%, respectively. The limits of quantification were validated at 0.06 microg kg-1 for soil and 0.3 microg kg-1 for plant samples. The limits of detection (LOD) for soil analysis were 0.01, 0.002, and 0.01 microg kg-1 for IXF, DKN, and BA, respectively. Corresponding LOD for the plant analysis method were 0.05, 0.01, and 0.05 microg kg-1. The developed method was validated using forage grass and soil samples collected from a field lysimeter study in which IXF was applied to each of four forage treatments. Forage plants and soils were sampled for analyses 25 days after IXF application to the soil. In soils, IXF was not detected in any treatment, and DKN was the predominant metabolite found. In forage plants, the concentrations of DKN and BA were 10-100-fold higher than that in soil samples, but IXF was not detected in any forage plants. The much higher proportion of BA to DKN in plant tissues (23-53%), as compared to soils (0-5%), suggested that these forages were capable of detoxifying DKN. The developed methods provided LODs at sub-microgram per kilogram levels to determine the fate of IXF and its metabolites in soils and forage plants, and they also represent considerable improvements in extraction recovery rates and detection sensitivity as compared to previous analytical methods for these compounds.

Reaction pathways of the diketonitrile degradate of isoxaflutole with hypochlorite in water.

Isoxaflutole (IXF; Balance) belongs to a new class of isoxazole herbicides. Isoxaflutole has a very short half-life in soil and rapidly degrades to a stable and phytotoxic degradate, diketonitrile (DKN). DKN was previously discovered to rapidly react with hypochlorite (OCl-) in tap water, yielding the benzoic acid (BA) degradate as a major product, but the complete reaction pathway and mechanism have not been elucidated. Thus, the objectives of this work were to (1) determine the stoichiometry of the reaction between DKN and OCl-; (2) identify products in addition to BA; and (3) propose a complete pathway and reaction mechanism for oxidation of DKN by OCl-. Stoichiometry of the reaction showed a molar ratio of OCl-/DKN of 2. In addition, two previously uncharacterized chlorinated intermediates were identified under conditions in which OCl- was the limiting reactant. The proposed chemical structure of a chlorinated benzoyl intermediate was inferred from a series of HPLC/MS and HPLC/MS/MS experiments and the use of mass spectral simulation software. A chlorinated ketone intermediate was also identified using ion trap GC/MS. Two additional end products were also identified: cyclopropanecarboxylic acid (CPCA) and dichloroacetonitrile (DCAN). On the basis of the reaction stoichiometry, the structure of the chlorinated intermediates, and the identification of the products, two reaction pathways are proposed. Both pathways involve a two-step nucleophilic attack and oxidation of the diketone structure of DKN, leading to formation of BA, DCAN, and CPCA.

The effect of five forage species on transport and transformation of atrazine and isoxaflutole (balance) in lysimeter leachate.

A field lysimeter study with bare ground and five different ground covers was established to evaluate the effect of forage grasses on the fate and transport of two herbicides in leachate. The herbicides were atrazine (ATR; 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) and isoxaflutole [IXF; 5-cyclopropyl-4-(2-methylsulfonyl-4-trifluormethyl-benzoyl)isoxazole], which has the commercial name Balance (Aventis Crop Science, Strasbourg, France). The ground covers included orchardgrass (Dactylis glomerata L.), smooth bromegrass (Bromus inermis Leyss.), tall fescue (Festuca arundinacea Schreb.), timothy (Phleum pratense L.), and switchgrass (Panicum virgatum L.). The results suggested that the total IXF (parent + metabolites) showed higher mobility than ATR and its metabolites. Differences in the timing of transport reflected the rapid degradation of IXF to the more soluble, stable, and biologically active diketonitrile (DKN) metabolite in the system. Although grass treatments did not promote the hydrolysis of DKN, they significantly reduced its transport in the leachate through enhanced evapotranspiration. Grass treatments significantly enhanced ATR degradation in the leachates and soils, especially through N dealkylation, but they did not reduce total ATR transported in the leachate. Leachate from the orchardgrass lysimeters contained the highest proportion of ATR metabolites (64.2%). Timothy and smooth bromegrass treatments also displayed a significant increase in ATR metabolites in leachate. Grass-treated lysimeters showed higher microbial biomass carbon than bare ground. For ATR treatments, the proportion of metabolites in the leachate strongly correlated with the elevated soil microbial biomass carbon in forage treatments. In contrast, DKN degradation was poorly correlated with soil microbial biomass carbon, suggesting that DKN degradation is an abiotic process.

Watershed vulnerability to herbicide transport in northern Missouri and southern Iowa streams.

Herbicide contamination of streams has been well documented, but little is currently known about the specific factors affecting watershed vulnerability to herbicide transport. The primary objectives of this study were (1) to document herbicide occurrence and transport from watersheds in the northern Missouri/southern Iowa region; (2) to quantify watershed vulnerability to herbicide transport and relate vulnerability to soil properties; and (3) to compute the contribution of this region to the herbicide load of the Missouri and Mississippi Rivers. Grab samples were collected under baseflow and runoff conditions at 21 hydrologic monitoring stations between April 15 and July 15 from 1996 to 1999. Samples were analyzed for commonly used soil-applied herbicides (atrazine, cyanazine, acetochlor, alachlor, metolachlor, and metribuzin) and four triazine metabolites (deisopropylatrazine, deethylatrazine, hydroxyatrazine, and cyanazine amide). Estimates of herbicide load and relative losses were computed for each watershed. Median parent herbicide losses, as a percentage of applied, ranged from 0.33 to 3.9%; loss rates that were considerably higher than other areas of the United States. Watershed vulnerability to herbicide transport, measured as herbicide load per treated area, showed that the runoff potential of soils was a critical factor affecting herbicide transport. Herbicide transport from these watersheds contributed a disproportionately high amount of the herbicide load to both the Missouri and Mississippi Rivers. Based on these results, this region of the Corn Belt is highly vulnerable to transport of herbicides from fields to streams, and it should be targeted for implementation of management practices designed to reduce herbicide losses in surface runoff.

Atrazine remediation in wetland microcosms.

Laboratory wetland microcosms were used to study treatment of atrazine in irrigation runoff by a field-scale-constructed wetland under controlled conditions. Three experiments, in which 1 ppm atrazine was added to the water column of three wetland, one soil control, and one water control microcosm, were conducted. Atrazine dissipation from the water column and degradate formation (deethylatrazine [DEA]; deisopropylatrazine [DIA]; and hydroxyatrazine [HA]) were monitored. Atrazine dissipation from the water column of wetland microcosms was biphasic. Less than 12% of the atrazine applied to wetland microcosms remained in the water column on day 56. Atrazine degradates were observed in water and sediment, with HA the predominant degradate. Analysis of day 56 sediment samples indicated that a significant portion of the initial application was detected as the parent compound and HA. Most probable number (MPN) assays demonstrated that atrazine degrader populations were small in wetland sediment. Wetland microcosms were able to reduce atrazine concentration in the water column via sorption and degradation. Based on results from this study, it is hypothesized that plant uptake contributed to atrazine dissipation from the water column.