Fungicide Dissipation and Impact on Metolachlor Aerobic Soil Degradation and Soil Microbial Dynamics☆

Science of the Total Environment 408 (2010) 1393–1402

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Science of the Total Environment

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Fungicide dissipation and impact on metolachlor aerobic soil degradation and soil microbial dynamics☆

Paul M. White a,b,⁎, Thomas L. Potter a, Albert K. Culbreath c a USDA-ARS Southeast Watershed Research Unit, 2381 Rainwater Road, Tifton, GA 31793, United States b Current address: USDA-ARS Sugarcane Research Unit, 5883 USDA Road, Houma, LA 70360, United States c Department of Plant Pathology, University of Georgia, Coastal Plain Experiment Station, Tifton, Georgia 31793, United States article info abstract

Article history: Pesticides are typically applied as mixtures and or sequentially to soil and plants during crop production. A common Received 21 September 2009 scenario is herbicide application at planting followed by sequential fungicide applications post-emergence. Received in revised form 25 October 2009 Fungicides depending on their spectrum of activity may alter and impact soil microbial communities. Thus there is a Accepted 8 November 2009 potential to impact soil processes responsible for herbicide degradation. This may change herbicide efficacy and Available online 16 December 2009 environmental fate characteristics. Our study objective was to determine the effects of 4 peanut fungicides, chlorothalonil (2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile), tebuconazole (α-[2-(4-chlorophenyl)ethyl]-α- Keywords: fl α fl α fl Metolachlor (1,1-dimethylethyl)-1H-1,2,4-triazole-1-ethanol), utriafol ( -(2- uorophenyl)- -(4- uorophenyl)-1H-1,2,4- Tebuconazole triazole-1-ethanol), and cyproconazole (α-(4-chlorophenyl)-α-(1-cyclopropylethyl)-1H-1,2,4-triazole-1-ethanol) Cyproconazole on the dissipation kinetics of the herbicide, metolachlor (2-chloro-N-(6-ethyl-o-tolyl)-N-[(1RS)-2-methoxy-1- Flutriafol methylethyl]acetamide), and on the soil microbial community. This was done through laboratory incubation of field Chlorothalonil treated soil. Chlorothalonil significantly reduced metolachlor soil dissipation as compared to the non-treated Soil microorganisms control or soil treated with the other fungicides. Metolachlor DT50 was 99 days for chlorothalonil-treated soil and PLFA 56, 45, 53, and 46 days for control, tebuconazole, flutriafol, and cyproconazole-treated soils, respectively. Significant reductions in predominant metolachlor metabolites, metolachlor ethane sulfonic acid (MESA) and metolachlor oxanilic acid (MOA), produced by oxidation of glutathione-metolachlor conjugates were also observed in chlorothalonil-treated soil. This suggested that the fungicide impacted soil glutathione-S-transferase (GST) activity.

Fungicide DT50 was 27–80 days but impacts on the soil microbial community as indicated by lipid biomarker analysis were minimal. Overall study results indicated that chlorothalonil has the potential to substantially increase soil persistence (2-fold) of metolachlor and alter fate and transport processes. GST mediated metabolism is common pesticide detoxiﬁcation process in soil; thus there are implications for the fate of many active ingredients. Published by Elsevier B.V.

1. Introduction et al., 2005; Jordan et al., 2009). Conclusions from these studies are that there is potential for interaction between herbicides and fungicides on Modern agriculture depends heavily on pesticides to control weed control and those interactions are compound and weed speciﬁc. weeds, plant diseases, and insect pests. Products are typically applied Increased soil persistence has also been observed (e.g., Kaufman as mixtures and or sequentially during the growing season. A common et al., 1970; Ferris and Lichtenstein, 1980; Fogg and Boxall, 2003). For scenario is application of one or more herbicides at planting followed example soil DT50 of the herbicide isoproturon increased 4-fold when it by sequential foliar fungicide applications post-emergence. This ap- was applied with the fungicide chlorothalonil (Fogg and Boxall, 2003). It proach is used almost universally by peanut (Arachis hypogaea L.) was suggested that the chlorothalonil impact was due to suppression of farmers in the Southeastern USA. non-target soil organisms by the parent compound and its primary soil Investigations in peanut and in other crops, and with other chemical degradate 4-hydroxy-chlorothalonil; however this was not investigated. combinations, e.g. herbicides and insecticides, have shown that interac- Results of studies examining the impact of chlorothalonil and degradates tions in terms of weed control between chemicals are possible (Lancaster on soil organisms have in some cases demonstrated a suppressive effect while in others there was no impact (Chen et al., 2001; Bending et al., 2007; Zhang et al., 2007). This is also the case with many other pesticides ☆ Mention of trade names or commercial products is solely for the purpose of including numerous fungicides, insecticides and herbicides. providing speciﬁc information and does not imply recommendation or endorsement by Regardless of the mechanism, herbicide soil persistence if the U.S. Department of Agriculture. increased has positive and negative implications. Negative outcomes ⁎ Corresponding author. Current address: USDA-ARS Sugarcane Research Unit, 5883 USDA Road, Houma, LA 70360, United States. Tel.: +1 985 853 3168; fax: +1 985 868 8369. include herbicide injury to agronomic crops and positive outcomes E-mail address: [email protected] (P.M. White). enhance weed control (Nash, 1967). Greater ecologic risk due to off-

0048-9697/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.scitotenv.2009.11.012 1394 P.M. White et al. / Science of the Total Environment 408 (2010) 1393–1402 site transport may be observed since increased soil residence time has recovered by vacuum filtration (Whatman 55-mm GF/F). The pro- the potential to increase runoff and leaching risks. The significance of cedure was repeated 2 times, extracts combined, and concentrated to pesticide interactions on soil persistence, although high, has not been 10 mL under an N2 gas stream. One gram subsamples were fortified with systematically investigated. The few published studies are limited to a 5μg of 2-chlorolepidine (internal standard), and analyzed by HPLC-MS narrow range of active ingredients and soil conditions. with a Thermoquest-Finnegan LCQ DECA ion trap system (Thermo-Fisher The current study focused on collecting soil persistence data which Scientific, San Jose, CA). Metolachlor, chlorothalonil, tebuconazole, can be used to guide weed and disease control management decisions flutriafol, and cyproconazole were analyzed by APCI using a Gemini® and evaluate environmental risk for peanut production on sandy soils C18 HPLC column, 150×4.6 mm, 0.5 μm, 110 Å (Phenomenex, Torrance, in the Southeastern USA. Specifically we examined the impact of four CA) with methanol (A) and 0.1% formic acid (B) gradient elution at fungicides, three triazoles and the chloronitrile, chlorothalonil, when 1mLmin−1. Initial conditions 10% A/90% B were changed to 90% A/10% B used individually on soil microbial dynamics of a Tifton loamy sand over 6 min, held for 5 min, and returned to initial conditions in 1 min. soil and the dissipation kinetics of a widely used herbicide, Positive ions (M+H)+ for metolachlor, cyproconazole, flutriafol, and metolachlor. These products are used on many other crops, thus tebuconazole were detected in the full scan mode (100–450 m/z). For findings are broadly applicable. chlorothalonil the negative ion corresponding to (M−Cl+O)− was monitored. Chlorothalonil is converted to its 4-hydroxy analog under 2. Materials and methods APCI conditions (Chaves et al., 2007). Full scan, positive ion scans were also used to test for metolachlor degradates hydroxymetolachlor, 2.1. Soil collection and incubation deschloroacetyl metolachlor propanol, metolachlor morpholinone, and phenyl alkyl-substituted metolachlor (keto-metolachlor) based on The investigation was conducted in conjunction with field-based previous reports (Sakkas et al., 2004; Hladik et al., 2005), and the peanut fungicide efficacy test conducted at a University of Georgia farm tebuconazole lactone degradate (Potter et al., 2005). Bayer CropScience located in south central Georgia (31° 30′N, 83° 32′W). Metolachlor was donated a reference lactone standard. Retentions times and ionization applied to the field on 21 May 2008 at 1.6 kg ha−1 as Dual Magnum® conditions for the metolachlor degradates were determined on the (Syngenta, Wilmington, DE). Peanut (cultivar Florida-07) was planted mixtures of degradates produced by hydrolysis in base (1 M KOH) or acid on 27 May 2008. After emergence the field was divided into replicate 2 (6 N HCl). Signal response was assumed equivalent to metolachlor. All by 8 m plots and four plots each were randomly selected for treatment analytes and ions monitored are summarized in Table 1. groups. Plots were treated with fungicides on 9 July 2008, 43 days after Two acidic metolachlor degradates, metolachlor ethane sulfonic planting. Timing reflected common practice among the region's peanut (2-[(2-ethyl-6-methylphenyl)(2-methoxy-1-methylethyl)amino]-2- growers. Soil (Tifton loamy sand; fine-loamy, kaolinitic, thermic, oxoethanesulfonic acid) acid, termed MESA, and metolachlor oxanilic Plinthic Kaniudult) was collected from 0 to 2 cm depth increment acid (2-[(2-ethyl-6-methylphenyl)(2-methoxy-1-methylethyl) from 4 untreated control plots and from plots treated immediately after amino]-2-oxoacetic acid) termed MOA, were separated on a Zorbax application of Folicur® 3.6 F (0.23 kg tebuconazole ha−1), Alto® 100 SL C8 HPLC Column, 50×2.1 mm, 3 μm, 110 Å (Agilent, Santa Clara, CA). (0.084 kg cyproconazole ha−1), Bravo® WeatherStik 720 F (1.26 kg Gradient elution at 0.6 mL min− 1 was with 0.1% formic acid (C) and chlorothalonil ha−1), and Topguard® 1.04 DC (0.13 kg flutriafol ha−1). acetonitrile+0.1% formic acid (D). Initial conditions, 90% C/10% D, Chlorothalonil and tebuconazole are among the most commonly used were changed linearly to 10% C/90% D in 5 min. Conditions were held fungicides in peanut production in the U.S. Flutriafol and cyproconazole for 2 min followed by return to initial conditions in 1 min. MESA and are not labeled for use on peanut in the U.S. Flutriafol has a section 18 MOA were detected after ESI ionization by MS2 with collision induced exemption for use on soybean in Georgia, and cyproconazole is used on dissociation (CID) of (M–H)− base peaks in MS2 spectra, 121 (MESA) peanut in other peanut producing countries. and 206 (OA) were used for quantitation. Soil was combined across the four field replicates to obtain a single Chlorothalonil's 4-hydroxy (4-hydroxy-2,5,6-trichloroisophthalo- composite sample for each fungicide treatment and sieved (No. 6 stainless nitrile) and an unidentified dichloro degradate were also analyzed steel 4-mm diameter). Water content was estimated on a portion of each using the Zorbax C8 HPLC Column and with negative ion ESI. Gradient sample by oven-drying at 90 °C for 1 h. Fifty g dry weight equivalent of elution at 0.6 mL min− 1 was with 2.0% acetic acid (E) and acetonitrile each was placed into twenty-seven 250-mL square glass bottles and water (F). Initial conditions, 80% E/20% F, were changed linearly to 20% E/ −1 content adjusted to 0.12 g H2Og soil with deionized water. An air-dried 80% D in 5 min. Conditions were held for 2 min followed by return to portion of each sample was characterized at the University of Georgia Soil, initial conditions over 1 min. The 4-hydroxy chlorothalonil degradate Plant, and Water Laboratory (Athens, GA). The soil pH (1:1 soil:water ratio) mean±standard deviation was 6.14±0.17, and Mehlich III extractable nutrients: phosphorus (20.9±4.42), calcium (883±165), Table 1 potassium (64.6±23.4), magnesium (47.1±13.9), manganese (5.3± Metolachlor, fungicides, and degradates analyzed in soil samples. Analyte # refers to Fig. 1. Primary ions used for quantification except for analytes 2 and 3. Values are m/z. 2.8), and zinc (2.25±0.49). Potassium chloride (2 M) extractable NH4-N and NO3-N were 3.03±4.78 and 9.26±1.58, respectively. Analyte # Compound Primary ion (secondary ion) Fifty mL of methanol was added to one set (n=3) of bottles for each 1 Metolachlor 284 treatment. These bottles were capped with Teflon-lined screw caps and 2 Metolachlor oxanilic acid 278 (206) immediately placed in a −20 °C chest freezer. The remaining bottles 3 Metolachlor ethane sulfonic acid 328 (121) were placed in a dark laboratory incubator maintained at 25±3 °C. On 4 Metolachlor morpholinone 234 5 Hydroxymetolachlor 266 3, 7, 14, 21, 28, 42, 56, and 105 days after the start of the incubation, 50- 6 Deschlorometolachlor 208 mL of methanol was added to 3 bottles from each treatment. Bottles 7 Desmethylmetolachlor 270 were re-capped, shaken and stored in the −20 °C freezer. For soil 8 Deschloroacetyl metolachlor propanol 194 microbial analysis a separate volume of soil was incubated in a 1 L bottle. 9 Phenyl alkyl-substituted metolachlor 298 At each sample interval a subsample was removed and frozen at −20 °C. 10 Tebuconazole 308 11 Tebuconazole lactone 224 12 Flutriafol 302 2.2. Soil pesticide analysis 13 Cyproconazole 317 14 Chlorothalonil 264 All bottles containing soil and methanol were brought to room 15 4-OH chlorothalonil 246 16 4,6-Dihydroxy chlorothalonil 228 temperature, shaken for 1 h, allowed to settle, and the methanol P.M. White et al. / Science of the Total Environment 408 (2010) 1393–1402 1395 was quantified using its base peak in full scan MS, m/z=245.5. The was based on MS2 analysis and GC–MS EI spectra. Analytical response MS spectrum of the other degradate had a base peak of m/z=241 and in HPLC analyses was assumed equal to 4-hydroxy-chlorothalonil. m/z=243 at 65% relative abundance indicating two chlorine atoms. Chlorothalonil, metolachlor, MESA, MOA, and deschloroacetyl Tentative identification, 4,6-hydroxy-2,5-dichloroisophthalonitrile, metolachlor propanol analytical standards were purchased from

Fig. 1. Structures of metolachlor, tebuconazole, ﬂutriafol, cyproconazole, chlorothalonil and associated degradates evaluated in the experiment. Arrows imply metabolic pathways. Common names are listed in Table 1. 1396 P.M. White et al. / Science of the Total Environment 408 (2010) 1393–1402

Chem-Service (West Chester, PA), tebuconazole, flutriafol, and ground, and 5 g extracted with 100 mL Bligh and Dyer solution (2:1:0.8 cyproconazole from Sigma Aldrich and 4-hydroxy chlorothalonil ratio of methanol, chloroform, and 0.2 M disodium citrate buffer from Dr. Ehrenstorfer GmbH (Germany). The cyproconazole standard acidified to pH 4 with HCl). Seventy-five mL of the extract was was a ∼1:1 mixture of disteroisomers. transferred to a separatory funnel containing 25 mL of chloroform and Analytical recoveries of metolachlor, MESA, MOA, chlorothalonil, citrate buffer. Following overnight phase separation, the chloroform- tebuconazole, flutriafol, and cyproconazole were assessed after spiking methanol was recovered, and taken to dryness under a stream of N2 gas. untreated control plot soil at 0.2 μgg−1 and extraction in triplicate. Dried lipid extracts were fractionated on silica gel (Thermo Scientific, Another set of samples (n=3) were spiked post-extraction. Percent 500 mg, 6 mL volume) by sequential elution with chloroform, acetone, recovery was calculated by dividing the peak areas of the pre-extraction and methanol. NLFA were recovered in the chloroform and PLFA in by post-extraction spikes (Niessen et al., 2006). Mean±standard methanol, were to taken to dryness under N2 gas. Samples were deviations were: metolachlor (95±6), MESA (79±23), MOA (26±8), saponified and methylated in 0.2 M methanolic KOH at 60 °C for 1 h. chlorothalonil (77±10), tebuconazole (93±4), flutriafol (94±3), Fatty acid methyl esters (FAME) were recovered by addition of cyproconazole-1 (89±5), and cyproconazole-2 (91±1). Recovery deionized water and microextraction with hexane, extracts were studies were not conducted for metolachlor degradates 4–7and9, dried at 40 °C under N2 gas, re-dissolved in hexane, fortified with the chlorothalonil degradates 13 or 14, or the tebuconazole degradate 11 internal standard methyl nonadecanoate (Matreya #1029) and ana- (Fig. 1). Results were assumed to be comparable to parent compounds. lyzed by GC-FID and GC–MS. A DB5-MS column (30 m×250 μm Concentrations of analytes in samples were not corrected for percent ID×0.25 μm film thickness — Agilent #122-5532) was used in both recovery. GC-FID (Agilent model 6890) and GC–MS (Thermo Finnigan model DSQII) analyses with helium carrier gas (1.0 mL min−1 constant flow) 2.3. Microbial lipid analysis and oven temperature programmed as follows: 40 °C, hold for 1 min; 40°–170 °C at 20 °C min−1; 170°–270 °C at 5 °C min−1; and 270 °C hold Soil microbial phospholipid fatty acids (PLFA) and neutral lipid fatty 1 min. Splitless injections were at 220 °C, respectively. Bacterial fatty acids (NLFA) were analyzed according to published methods (Allison acid methyl esters mix (BAME — Matreya #1114) was used to identify and Miller, 2005; White and Ringelberg, 1998)withmodifications peaks based on retention time and MS-spectra (70 eV; electron impact). outlined elsewhere (White et al., 2009). Briefly, soil was lyophilized, The internal standard was used for quantitation.

Fig. 2. Linear regression of metolachlor remaining in (a) control soil and soil treated with (b) tebuconazole, (c) ﬂutriafol, (d) chlorothalonil, and (e) cyproconazole. Calculated DT50 are inserted in ﬁgure. P.M. White et al. / Science of the Total Environment 408 (2010) 1393–1402 1397

Mass spectral interpretation and comparison to the NIST Mass then conducted for each time period and means were separated using Spectral Database version 2.0 (NIST, Gaithersburg, MD) were used to pairwise t-tests by the pdiff procedure. tentative structural assignments of FAME peaks not present in the BAME Microbial PLFA and NLFA were subjectedtoa2-wayANOVAusingProc mix. The twenty one FAME were identified in the PLFA fraction, and Mixed with time and treatment as the independent variables and PLFA were grouped into saturated, gram positive and gram negative bacteria, and NLFA quantity (nmol g−1 soil) or mole fraction as the dependent and fungi according to McKinley et al. (2005):(a)saturated— 12:0, variable. Means were separated using pairwise t-tests with the pdiff 14:0, 15:0, 16:0, 17:0, 18:0, and 20:0; (b) gram positive bacteria — i15:0, procedure. Select PLFA indicating microbial stress, the ratio of cyclopro- a15:0, i16:0, i17:0, a17:0, and 10me18:0; (c) gram negative bacteria — panoic to monoeonic PLFA, were evaluated using linear regression. 2-OH 14:0, 16:1ω7c, cy17:0, 18:1ω7c, and cy19:0; and (d) fungi — 16:1ω5c, 18:1ω9c, 18:2ω6,9c. Six FAME from the eukaryotic neutral 3. Results and discussion lipid fraction were chosen to represent fungal biomass, and included 16:0, 16:1ω5c, 18:0, 18:1ω9c, 18:2ω6,9c, and 20:0. 3.1. Metolachlor dissipation kinetics To confirm FAME assignments, fungal colonies grown on selective agar plates were scraped and lyophilized and extracted using the above Metolachlor was applied 49 days before fungicide application and procedure and the FAME in the PLFA fractions analyzed. Fungal samples soil collection for the incubation study. Substantial dissipation due to a included colonies of the peanut pathogens Sclerotium rolfsii, Rhizoctonia combination of microbial degradation, volatilization, and leaching had solani,andCylindrocladium parasiticum grown on potato dextrose agar. occurred by this time. Calculations based on the target application rate Fungal PLFA samples contained only 16:0, 18:2ω6,9c, 18:1ω9c, and 18:0. indicated that about 0.4% of metolachlor applied was recovered in t=0 soil extractions. This scenario was similar to many crops that receive 2.4. Data analysis pre-emergence herbicide-treatment followed by post-emergence in- secticide or fungicides treatment. At the time of fungicide application Pesticide dissipation kinetics were evaluated by linear regression the metolachlor average±standard deviation concentration (n=12) of the natural log of the percent remaining over time with slopes was 0.073±0.01 nmol g−1. In the subsequent laboratory incubation tested for differences using an ANCOVA and pairwise comparisons chlorothalonil-treated soil exhibited the lowest metolachlor dissipation made using Tukey's test following a one-way ANOVA (GraphPad v. 5, rate, with a DT50 of 99 days (Fig. 2). Metolachlor DT50 was greater for the LaJolla, CA). A first order nonlinear regression model was also used to control soil than for the tebuconazole, flutriafol, and cyproconazole- evaluate natural log-transformed dissipation data (Gustafson and treated soils with values of 56, 45, 53, and 46 days, respectively. The

Holden, 1990). Model parameters (α, β) were obtained using Proc laboratory-calculated DT50 of the control sample suggested that about NLIN in SAS version 9.0 (Cary, N.C.) with the GAUSS–NEWTON 50% of the field applied metolachlor would have remained at the time of iterative setting. An improved fit(r2) between observed and modeled sampling. As noted, field dissipation of metolachlor was more rapid. data was obtained for tebuconazole and chlorothalonil using this The principal metolachlor degradates detected were MESA and approach. Results are explained in the text. Metolachlor, MESA, and MOA (Fig. 3). MESA concentrations increased almost 3-fold during MOA data were analyzed by a 2-way ANOVA using Proc Mixed. For incubations with relative amounts varying depending on treatment. significant 2-way interactions (pb0.05), the SLICE procedure was Cyproconazole-treated soil had the highest MESA levels in the sample used to compare means within a time period. A 1-way ANOVA was period 3–56 days, as compared to the other fungicides or the control

Fig. 3. Levels of metolachlor and degradates in soil: (a) metolachlor parent, (b) MESA, (c) MOA, and (d) hydroxymetolachlor. Error bars represent ±1 standard deviation. 1398 P.M. White et al. / Science of the Total Environment 408 (2010) 1393–1402 soil (Fig. 3). Between 14 and 56 days the soil treated with either tebuconazole and flutriafol had higher MOA levels, as compared to flutriafol or tebuconazole had higher MESA levels than soil treated other treatments. MOA concentration in cyproconazole and chlor- with chlorothalonil. The control soil contained more MESA than the othalonil-treated soils was similar after 3 days. At the end of the chlorothalonil-treated soil between 42 and 56 days sample periods. incubation MOA levels in tebuconazole and flutriafol-treated soils Overall chlorothalonil appeared to limit MESA formation. This were greater than in the cyproconazole-treated soil, but not the coincided with the slower metolachlor dissipation rate. control or chlorothalonil-treated soil. Again results suggested that The lower MESA formation rate and slower dissipation of suppression of metolachlor metabolism by chlorothalonil was due to metolachlor may be explained by a common mode of soil metabolism competition and suppression of soil GST metabolism. of metolachlor and chlorothalonil. Both compounds are readily The other metolachlor degradate commonly detected was hydro- metabolized via glutathione S-transferase to glutathione adducts. xymetolachlor. It was present in 48, 33, and 31% of soil samples The adduct formed by GST promoted substitution of glutathione for treated with tebuconazole, flutriafol, and chlorothalonil, respectively. the Cl on metolachlor's N-acetyl functional group is a precursor of Levels were about 8–10-fold lower than MESA and MOA, respectively. MESA (Aga et al., 1996). In chlorothalonil GST catalyzes substitution of No hydroxymetolachlor was observed in the control or the cyproco- glutathione for the Cl positioned ortho and para to the aromatic nitrile nazole-treated soil (Fig. 3). Hydroxymetolachlor was only detected groups (Kim et al., 2004). This Cl atom is labile and readily undergoes during the first 28 days. For the first 21 days, sufficient (N50%) sample nucleophilic substitution (Potter et al., 2001). Notably a reported numbers contained hydroxymetolachlor to calculate medians for chlorothalonil mode of action on plant pathogenic fungi is cellular tebuconazole, flutriafol, and chlorothalonil-treated soil. Values were glutathione depletion, thus it is likely that reaction kinetics with GST 0.005 to 0.008 nmol g− 1. Across treatments, values ranged from 0.003 are relatively rapid (Clarke et al., 1998). to 0.013 nmol g− 1. The low temporal levels may indicate a role for Like MESA MOA is reported to be a secondary product of GST hydroxymetolachlor as an intermediate between the parent and interaction with chloroacetanilides; acetochlor was used to derive MESA, MOA, or both. No other metolachlor degradates were detected. both ethane sulfonic acid and oxanilic acid derivatives via the GST Over time, tebuconazole (0.200 nmol g− 1)andflutriafol pathway (Feng, 1991). MOA and MESA generations were reported to (0.187 nmol g− 1) exhibited a greater molar mass of metolachlor be in a 70:30 ratio (Bayless et al., 2008). In our study, initial MOA residues, as compared to the control soil (0.149 nmol g− 1), the concentration was similar across treatments (p=0.6060) with an chlorothalonil (0.126 nmol g− 1) or the cyproconazole-treated soil average±standard deviation, 0.008 ±0.004 nmol g− 1 (Fig. 3). A (0.143 nmol g− 1).Molarsumsofmeansfortebuconazoleand rapid increase was observed at 3 days for MOA in control soil samples flutriafol were not different. The control and the cyproconazole- and soil treated with tebuconazole or flutriafol. By 7 days MOA treated soil were statistically similar but higher than chlorothalonil- concentration in cyproconazole-treated soil was similar to the control, treated soil. Across treatments an increase from during 0–105 days of but both were significantly lower than tebuconazole or flutriafol- 0.127 to 0.174 nmol g−1 was indicated. This was about 2-fold greater treated soil. Over the next 5 weeks of incubation soil treated with than the metolachlor concentration in time=0 samples suggesting that a precursor of these compounds was present but not accounted for in the analysis. A likely candidate was metolachlor's glutathione conjugate(s) produced by GST mediated soil metabolism. Its detection using our HPLC-MS conditions was unlikely (Perez et al., 2007).

Fig. 4. Chlorothalonil dissipation in soil over time: (a) observed and modeled data Fig. 5. Tebuconazole dissipation in soil over time: (a) observed and modeled data including both linear and nonlinear models, and (b) parent compound and degradate including both linear and nonlinear models, and (b) parent compound and lactone concentrations during the incubation. Error bars represent ±1 standard deviation. derivative concentrations. Error bars represent ±1 standard deviation. P.M. White et al. / Science of the Total Environment 408 (2010) 1393–1402 1399

Tebuconazole dissipation was also nonlinear, with a modeled r2 value of 0.971 compared to r2 =0.8733 for the linear model (Fig. 5a). Tebuconazole exhibited a lag phase with negligible dissipation from 0 to 7 days (linear regression not different from 0; p=0.8914). Calculated

tebuconazole DT50 and DT90, based on the nonlinear model, were 27 and 70 days. When the lag phase was taken into account, DT50 and DT90 are 35 and 77 days, respectively. The tebuconazole lactone degradate was detected in the incubation samples between 21 and 56 days (Fig. 5b). The lactone was not detected at 105 days, presumably due to degradation. Other tebuconazole degradates reported in the literature were not observed in the present study (Potter et al., 2005). Flutriafol dissipation followed a linear ﬁrst order model but not well (r2 =0.713) (Fig. 6). Nonlinear model estimations did not

converge. Linear model DT50 and DT90 were 78 and 260 days. Flutriafol Fig. 6. Cyproconazole and ﬂutriafol dissipation in soil over time including observed and DT50 listed in the Footprint database is 1358 days (Footprint database, modeled data. Only linear model-derived values are expressed due to inability to adequately ﬁt nonlinear equations to observed data. 2009). Data for clay loam and a sandy loam soil were 1650 and 1444 days, respectively, when incubated at 18 °C (Bromilow et al.,

1999a). Flutriafol was persistent in field trials as well, with a DT50 N400 days in either a clay loam or sandy loam soil fallowed or cropped to spring barley (Hordeum vulgare L cv Cooper) (Bromilow et al., 3.2. Fungicide dissipation 1999b). No degradates of flutriafol were observed in the full scan HPLC-APCI-MS analysis of the soil sample extracts. Chlorothalonil dissipation was rapid and exhibited nonlinear Chromatographic separation permitted evaluation of the two characteristics. The better fit of the nonlinear model was indicated by cyproconazole stereoisomers separately (Fig. 6). Both exhibited linear 2 2 the difference in r , 0.946 vs. 0.900 respectively (Fig. 4). Rapid first order dissipation kinetics with r valuesN0.825. The DT50 and metabolism of chlorothalonil, DT50, b1to3.5days,inTiftonloamy DT90 for cypro-1 (77, 256 days) and cypro-2 (77, 257 days) were sand was reported previously (Potter et al., 2001). In that study and in similar in magnitude to flutriafol. The relative abundance of each the current investigation, the primary chlorothalonil degradation isomer was similar throughout the incubation with a mean ratio of pathway was conversion to 4-hydroxy chlorothalonil. On a molar cypro-1 to cypro-2 of 0.940 and standard deviation, 0.054 indicating basis this compound represented a 90–95% of the initial soil chlor- no preferential dissipation of stereoisomers. Published cyproconazole othalonil by 21 days (Fig. 4). The dissipation pattern was similar to dissipation rates vary widely (DT50 = 5 to 223 days) but were another study where up to 49% of soil-applied chlorothalonil was between 21 and 49 days in an agricultural soil (Gardner et al., 2000; transformed into 4-hydroxy chlorothalonil (Van der Pas et al., 1999). Buerge et al., 2006). Stereoisomers were found to dissipate at different The compound exhibited similar persistence as observed in a peanut rates elsewhere (1.4 fold difference), and at wider differences when field experiment with multiple applications (Potter et al., 2001). Levels incubated separately (1.8 fold difference) (Buerge et al., 2006). As was of the other chlorinated metabolite, tentatively identified as 4,6- the case with flutriafol, no degradates were detected. hydroxy-2,5-dichloroisophthalonitrile were low, b10% of total chlorothalonil residues at all time points. The more persistent 4-hydroxy 3.3. Microbial community dynamics chlorothalonil has been reported to act as a broad spectrum microbial inhibitor (Montonaga et al., 1998). Thus, 4-hydroxy chlorothalonil may Soil PLFA and NLFA (nmol g−1 soil) FAMEs were combined to have slowed metolachlor metabolism in chlorothalonil-treated soil. estimate recoverable microbial biomass during lab incubations (Fig. 7).

Fig. 7. Total soil microbial FAME (the sum of phospholipid and neutral lipid derived fatty acids) during the experiment. Bars are means and lines are ±1 standard deviation. Statistical comparisons are discussed in the text. 1400 P.M. White et al. / Science of the Total Environment 408 (2010) 1393–1402

Total FAME were higher in the control soil or soil amended with conducted in this laboratory, 4 times the recommended maximum tebuconazole or chlorothalonil at 20 h with a reciprocal trend at 45 h, yearly field rate of tebuconazole (320 μgg−1) reduced the fungal PLFA suggesting that fungicide impact on microbial communities was small. A 18:2ω6,9c, but only briefly(b10 days). trend of reduced FAME was observed at b12 h which could be attributed PLFA data indicated that fungicides used in the experiment had to disturbance associated with sampling and sieving soil. Depletion of little effect on the fungal community. The fungi PLFA mole fraction soil substrates combined with no input (crop residue, root exudates, fluctuated at 0.25±0.06 during the entire experiment (Table 2). The and/or manure) may have lead to low available carbon sources and a time×treatment interaction (p=0.0079) indicated the control soil reduced microbial population observed at N29 days. In the case of (42 and 105 days) and chlorothalonil-treated soil (105 days) had flutriafol failure to observe a response was presumably due to the higher fungal PLFA. The control soil exhibited a mole fraction 50% relatively low flutriafol dose. The compound was found to impact soil higher than the treated soils at 42 days and the control and cellulose degradation at 170 μgg−1,or103 times the recommended chlorothalonil-treated soil exhibited 90 and 43% higher fungal mole field application rate (Munier-Lamy and Borde, 2000). In that study, fraction than the other treatments (Table 2). The persistence of 4-OH high rates of flutriafol resulted in higher bacteria to fungi ratio as chlorothalonil could partially explain the higher fungal PLFA mole evidenced by microscopic evaluation and increased cellulose mineral- fraction, as the compound has shown to exhibit antibacterial activity ization. The relative dose associated with the agronomically-based (Montonaga et al., 1998). application rate may also have played a role in our failure to detect a Gram positive bacteria accounted for the greatest PLFA mole tebuconazole impact on soil organisms. In a preliminary study fraction during the incubation, averaging 0.42±0.06. Treatment

Table 2 Soil microbial PLFA mole fractions for a) fungi, b) gram positive bacteria, and c) gram negative bacteria. Time is in hours and days, and all data is expressed as a mole fraction. Bolded time periods contain signiﬁcant differences between treatments. *Means within a time period followed by the same letter are not different (pb0.05).

Time Control Tebuconazole Flutriafol Chlorothalonil Cyproconazole

Mean Std. Dev Mean Std. Dev Mean Std. Dev Mean Std. Dev Mean Std. Dev

a) Fungi 0 0.21 0.01 0.25 0.01 0.22 0.02 0.20 0.03 0.23 0.03 6 h 0.24 0.04 0.25 0.01 0.30 0.05 0.21 0.02 0.19 0.01 12 h 0.22 0.01 0.25 0.00 0.26 0.02 0.22 0.02 0.27 0.08 20 h 0.32 0.09 0.26 0.05 0.23 0.02 0.25 0.07 0.21 0.01 45 h 0.28 0.05 0.25 0.02 0.26 0.04 0.24 0.02 0.26 0.04 3 days 0.24 0.02 0.30 0.07 0.24 0.01 0.21 0.01 0.23 0.01 7 days 0.22 0.02 0.22 0.02 0.25 0.03 0.22 0.04 0.20 0.00 14 days 0.23 0.02 0.22 0.01 0.30 0.02 0.25 0.07 0.26 0.02 21 days 0.31 0.09 0.33 0.08 0.48 n=1 0.33 0.08 0.33 0.14 29 days 0.33 0.13 0.34 0.06 0.30 0.02 0.29 0.08 0.41 n=1 42days 0.37 a* 0.07 0.27 b 0.02 0.24 b 0.03 0.23 b 0.00 0.24 b 0.03 105days 0.40 a 0.17 0.19 b 0.01 0.21 b 0.02 0.30 a 0.10 0.23 b 0.03 Mean 0.28 0.09 0.26 0.05 0.26 0.06 0.25 0.06 0.25 0.06

b) Gram positive bacteria 0 0.45 0.02 0.41 0.01 0.41 0.01 0.47 0.01 0.48 0.03 6 h 0.44 b 0.03 0.42 b 0.01 0.40 c 0.02 0.47 ab 0.02 0.50 a 0.02 12 h 0.48 0.00 0.44 0.00 0.40 0.01 0.46 0.03 0.45 0.06 20 h 0.41 0.06 0.41 0.02 0.44 0.02 0.42 0.07 0.48 0.00 45 h 0.39 0.04 0.39 0.03 0.40 0.03 0.39 0.02 0.41 0.01 3 days 0.44 0.02 0.40 0.04 0.41 0.01 0.44 0.01 0.46 0.02 7 days 0.45 0.02 0.44 0.02 0.43 0.02 0.45 0.02 0.48 0.01 14 days 0.46 0.01 0.43 0.02 0.42 0.06 0.40 0.06 0.45 0.02 21 days 0.35 0.06 0.32 0.04 0.24 n=1 0.34 0.05 0.34 0.07 29 days 0.35 0.09 0.32 0.05 0.37 0.02 0.37 0.04 0.29 n=1 42 days 0.32 0.06 0.39 0.02 0.40 0.04 0.39 0.02 0.41 0.08 105days 0.33 c 0.08 0.48 a 0.02 0.46 ab 0.05 0.36 bc 0.07 0.40 abc 0.09 Mean 0.41 0.07 0.41 0.05 0.41 0.05 0.41 0.06 0.44 0.06

c) Gram negative bacteria 0 0.34 0.01 0.34 0.01 0.37 0.03 0.32 0.02 0.30 0.05 6 h 0.32 0.02 0.33 0.00 0.30 0.03 0.32 0.00 0.31 0.01 12 h 0.30 0.01 0.31 0.01 0.34 0.02 0.33 0.01 0.28 0.03 20 h 0.27 0.03 0.33 0.05 0.33 0.01 0.33 0.02 0.31 0.01 45 h 0.33 0.03 0.36 0.02 0.34 0.03 0.37 0.01 0.32 0.04 3days 0.32 0.01 0.30 0.02 0.35 0.01 0.34 0.01 0.31 0.01 7days 0.33 0.01 0.33 0.00 0.31 0.05 0.33 0.03 0.31 0.01 14days 0.31 0.01 0.35 0.01 0.28 0.04 0.34 0.06 0.29 0.00 21days 0.35 0.03 0.35 0.05 0.28 n=1 0.33 0.04 0.33 0.07 29days 0.31 0.04 0.34 0.00 0.33 0.00 0.34 0.04 0.30 n=1 42days 0.31 0.02 0.34 0.03 0.36 0.02 0.38 0.02 0.35 0.05 105days 0.27 0.09 0.33 0.02 0.34 0.03 0.34 0.04 0.37 0.07 Mean 0.31 c 0.04 0.34 a 0.03 0.33 ab 0.03 0.34 a 0.03 0.32 bc 0.04

Fungi Gram positive bacteria Gram negative bacteria

Effect F ratio p-value Effect F ratio p-value Effect F ratio p-value

Time 6.72 b0.0001 Time 14.16 b0.0001 Time 1.77 0.0693 Trt 2.44 0.0513 Trt 2.75 0.0319 Trt 4.81 0.0013 Time×trt 1.8 0.0079 Time×trt 1.89 0.0045 Time×trt 1.26 0.1696 P.M. White et al. / Science of the Total Environment 408 (2010) 1393–1402 1401

Table 3 Gram negative stress indicator – the ratio of cy19:0 to 18:1w7c – in soil during the experiment. Main effects of time (pb0.0001) and treatment (pb0.0001) were significant; The 2- way time × treatment interaction was not significant (p=0.1837). LSD0.05 for trt is 0.07 and time is 0.12. Bolded values highlight significant main treatment effects.

Time Control Tebuconazole Flutriafol Chlorothalonil Cyproconazole Mean Std. Dev. Mean Std. Dev. Mean Std. Dev. Mean Std. Dev. Mean Std. Dev. Mean Std. Dev.

0 0.76 0.02 0.62 0.06 0.57 0.08 0.70 0.10 0.86 0.06 0.70 0.12 6 h 0.70 0.06 0.49 0.06 0.47 0.09 0.70 0.06 0.83 0.00 0.63 0.15 12 h 0.69 0.03 0.52 0.02 0.36 0.12 0.58 0.17 0.65 0.13 0.58 0.14 20 h 0.63 0.21 0.58 0.02 0.56 0.03 0.68 0.13 0.89 0.12 0.67 0.16 45 h 0.58 0.07 0.55 0.08 0.56 0.09 0.64 0.03 0.67 0.11 0.60 0.09 3 days 0.72 0.05 0.55 n=1 0.56 0.02 0.58 0.12 0.71 0.07 0.58 0.20 7 days 0.92 0.06 0.70 0.02 0.68 0.14 0.77 0.36 0.91 0.18 0.80 0.20 14 days 0.93 0.04 0.74 0.04 0.59 0.08 0.71 0.19 0.80 0.16 0.76 0.15 21 days 0.97 0.22 0.75 0.19 0.64 n=1 0.67 0.02 1.11 0.61 0.84 0.28 29 days 1.00 0.13 0.71 0.06 0.55 0.03 0.74 0.15 0.63 n=1 0.72 0.18 42 days 0.68 0.05 0.85 0.04 0.84 0.06 0.99 0.10 1.19 0.06 0.89 0.17 105 days 1.08 0.47 1.16 0.08 1.01 0.05 0.91 0.19 1.30 0.23 1.09 0.25 Trt avg 0.81 0.21 0.69 0.20 0.62 0.18 0.72 0.18 0.88 0.26 differences were only observed during the 6 h and 105 days sample laboratory and field assistance. Funding was provided by the USDA- times (Table 2). Gram negative PLFA constituted a smaller fraction Agricultural Research Service Southeast Watershed Research Unit in than gram positive bacterial PLFA (Table 2). Soil samples were taken Tifton, GA. The use of trade names does not imply endorsement by the between rows in the bulk soil, and gram negative bacteria are known USDA. to be more abundant in rhizosphere soil (Kennedy, 2005). An indicator of gram negative bacteria stress and subsequent slow growth is an References increase in the ratio of cyclopropanoic to monoeonic PLFA (Calderón et al., 2000; Villanueva et al., 2004). Both the control and cyprocona- Aga DS, Thurman EM, Yockel ME, Zimmerman LR, Williams TD. Identification of a new zole-treated soil exhibited a higher cy19:0 to 18:1ω7c ratio across time sulfonic acid metabolite of metolachlor in soil. Environ Sci Technol 1996;30(2):592–7. Allison VJ, Miller RM. 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