Published in J. Environ. Qual. 33:1771-1778 (2004).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA
TECHNICAL REPORTS
Organic Compounds in the Environment
Degradation of N,N'-Dibutylurea (DBU) in Soils Treated with only DBU and DBU-Fortified Benlate Fungicides
Linda S. Lee*,
Stephen A. Sassman,
Marianne Bischoff and
Ronald F. Turco
Department of Agronomy, Purdue University, West Lafayette, IN 47907-1150
* Corresponding author (lslee{at}purdue.edu).
Received for publication January 9, 2004.
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ABSTRACT
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N,N'-dibutylurea (DBU) is a breakdown product of benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate], the active ingredient in Benlate fungicides, and has been proposed to cause crop damage after the use of Benlate 50 DF fungicide (DuPont, Wilmington, DE). Our research focused on DBU persistence after application into soil. We assessed DBU persistence on direct application of DBU (carbonyl–14C) at two concentrations (0.08 and 0.8 µg DBU kg–1) to seven soils and two potting mixes in soil microcosms incubated at various combinations of soil water potential (–0.03 or –0.1 MPa) and temperature (23, 33, 44°C). For two soils at a subset of treatment variables we assessed DBU persistence in the presence of Benlate DF and SP fungicide formulations. Parent compounds, metabolites, and 14CO2 were tracked using chromatographic analysis with radioassay and UV detection, liquid scintillation counting, and post-extraction oxidation of the soil. DBU degradation was primarily microbial and for most soil–treatment combinations, half-lives were less than 2 wk. DBU degradation was retarded at the lower soil water potential and enhanced at 33°C. In the presence of the formulation, DBU degradation was slower for one soil type. The longest half-life observed in any case was less than 7 wk; therefore, long-term persistence of DBU applied to soils through a Benlate application is very unlikely.
Abbreviations: DBU, N,N'-dibutylurea • HPLC, high performance liquid chromatography • LSC, liquid scintillation counting
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INTRODUCTION
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BENOMYL IS THE ACTIVE INGREDIENT in Benlate fungicides that have been used by farmers and gardeners for many years. From 1987 through March 1991, DuPont sold benomyl as the Benlate 50 DF formulation (50 DF), which was the subject of crop damage claims by growers. One proposed cause of crop damage is N,N'-dibutylurea (DBU), a breakdown product of benomyl. In aqueous media, benomyl is converted to 2-benzimidazole-carbamate (MBC) and n-butylisocyanate (BIC). BIC reacts with water to form butylcarbamic acid, which quickly decarboxylates to give CO2 and n-butylamine (BA), which can react with any remaining BIC to form DBU (Calmon and Sayag, 1976; Moye et al., 1994; Tang and Song, 1996; Thorn et al., 1993; Tolson, 1996; Tolson et al., 1999). This sequence of reactions has been shown to occur in sealed ampules containing Benlate DF with no added moisture, since the inert ingredients contain enough water to hydrolyze BIC (Tang and Song, 1996). The USEPA has mandated that Benlate formulations contain no more than 1% DBU; however, improper storage and/or conditions of high humidity and temperature can result in DBU levels exceeding 5% in Benlate formulations (Tang and Song, 1996; Tolson et al., 1999). DBU also has been shown to form on plant leaves from deposition of BIC vapors from solution (Moye et al., 1994).
DBU has been shown to cause adverse effects to sensitive plants following various forms and rates of application. For Boston ferns, Moye et al. (1994) noted discoloring of the leaves within 24 h after foliar exposure to BIC vapors with 0.6 to 10.6 µg DBU g–1 found in the leaves. In the same study, spotted browning of the cotyledons in cucumber plants was noted by 24 h with 1.8 to 17.5 µg DBU g–1 found in the leaves. On a similar cucumber species, application of DBU as a soil drench resulted in a reduction in root and shoot growth rates at DBU rates greater than 0.04 mg kg–1 (estimated given the reported application rate of 935 L ha–1 using a solution of 94 mg L–1) (Gaffney et al., 1998). When applied directly to the roots, reduction in cucumber root and shoot growth and complete inhibition of hydrilla photosynthesis was observed after 1 h of exposure to 2.8 kg DBU ha–1 (1.25 mg kg–1) (Shilling et al., 1994). However, in the same study, no effect was observed on corn at even the highest DBU rate applied of 22.4 kg DBU ha–1 (9.6 mg kg–1), and no effect was observed on respiration of hydrilla, seed germination, or seed emergence for cucumbers until this maximum rate was applied (Shilling et al., 1994). Tang and Song (1996) noted inhibition of lettuce seedlings at 1920 mg L–1 DBU (0.16% of the applied Benlate DF of which the lowest treatment reported was 1.2 mg mL–1). Tolson et al. (1996) reported injury to ornamental peppers at DBU concentrations of <1% by weight of the Benlate drench rate, which translates to approximately 0.5 µg DBU g–1 soil. In studies assessing the effect of DBU on electron transport in isolated chloroplasts, a DBU concentration of 167 µM was estimated to cause a 50% inhibition of photo-induced ferricyanide reduction (Querns et al., 1998).
Most of the research related to DBU has focused on formation in solutions or in benomyl formulations, formation on plant leaves from BIC vapors from solution, and phytoxicity. A recent study by Sassman et al. (2004) investigated DBU formation after application of BIC and Benlate 50 DF and 50 SP fungicides to several soils at different water potentials and temperatures. DBU formation was observed in a few cases with the maximum DBU concentration observed of 0.41 µg g–1 (0.65 wt. % of applied benomyl at 62.8 µg g–1) after application of Benlate 50 DF at higher than the recommended drench rate. The DBU formed after Benlate application rapidly dissipated. This maximum observed concentration is well below those currently reported to cause adverse effects to plants. No work has been done to assess the persistence of DBU that may be concomitantly applied to soils in the application of Benlate fungicides. In the current study, our research focused on evaluating the persistence of DBU in soils and potting mixes of DBU incubated under various combinations of water potential (–0.03 or –0.1 MPa) and temperature (23, 33, and 44°C) when applied alone and with formulated material (50 DF and 50 SP).
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MATERIALS AND METHODS
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Materials
Di-n-butylurea (carbonyl–14C) (specific activity 55 mCi mmol–1) (DBU) dissolved in ethanol was purchased from NEN Life Science Products (Boston, MA). Benlate 50 SP (soluble powder) and Benlate 50 DF (dry flowable) fungicide formulations were provided by DuPont and stored at –80°C. ScintiSafe 30% liquid scintillation counting cocktail and sodium sulfate (>99%) were purchased from Fisher Scientific (Fair Lawn, NJ). Calcium chloride (ACS grade) was purchased from EM Science (Gibbstown, NJ). Acetonitrile (high performance liquid chromatography [HPLC] grade) and acetic acid (ACS grade) and potassium hydroxide (>99%) were purchased from Mallinckrodt (Paris, KY). Powdered cellulose was ACS grade and purchased from Sigma-Aldrich (St. Louis, MO). Combustaid, Carbosorb E, and Permafluor E+ scintillation cocktail were purchased from PerkinElmer (Wellesley, MA). Water was purified by reverse osmosis followed by filtration through a Barnstead (Dubuque, IA) Easy Pure LF ultrapure water system.
Soils
Six soils collected from agricultural fields in Costa Rica, where the soils are somewhat acidic (pH range 5.5 to 6.3), and one alkaline soil (pH = 7.9) collected from southern Florida were used. Soils were collected from areas where litigation issues with regard to Benlate use were pending. Although benomyl had been used extensively in these areas, the soils collected for this study were taken from agriculturally relevant sites that had not been treated with benomyl for at least the three years immediately before sampling. Each soil sample represented a mix of multiple subsamples taken from the surface (top 8 to 10 cm) at least 1 m apart from a 230-m2 sampling area until approximately 25 kg soil was obtained. Moist soils were passed through a 2-mm sieve, mixed well, and stored at 4°C before use. Soils were characterized by MDS Harris Laboratories (Lincoln, NE) using the following standard techniques. The pH was measured from a 1:1 water to soil ratio. The modified ammonium acetate method was used for cation exchange capacity (CEC), and organic matter was determined by loss on ignition. Selected soil characteristics including pH, organic carbon content, CEC, and nutrient analysis for N and P are summarized in Table 1. In addition to the soils, two potting soils (Fafard Professional Formula 2MIX and 3BMIX; BWI, Apopka, FL) were used in a subset of experiments where DBU was applied in the absence of formulation. The 2MIX media was composed of 55% Canadian sphagnum peat with the remaining 45% being a combination of perlite, and vermiculite. The 3BMIX media was composed of 45% Canadian sphagnum peat with the remaining 55% being a combination of processed bark, perlite and vermiculite. Soils used for sterile control experiments were irradiated with 3.2 MRad using a 60Co irradiator (Gammacell 220; MDS Nordian, Ottawa, ON, Canada).
Solutions and Sample Preparation
For the DBU-only degradation studies, 14C-DBU dissolved in ethanol was diluted with water for use in treating the soils. For DBU degradation when applied with formulations, soils were treated with aqueous solutions prepared by mixing 50 DF or 50 SP formulated material with 14C-DBU for 30 min. Analytical standards were prepared by serial dilution of the 14C-DBU source material with 30:70 (v/v) acetonitrile and water. Precise DBU concentrations were determined by scintillation counting immediately before reverse-phase liquid chromatography (RPLC) analysis. Analytical standards of carbendazim for determining benomyl and carbendazim residuals were prepared in 70:30 (v/v) water and acetonitrile. Potassium hydroxide solution (1 M) was prepared fresh with water that was boiled to remove dissolved gases and cooled to room temperature in a sealed container.
DBU Degradation Experiments
Laboratory studies were used to determine DBU persistence using standard soil microbial microcosm techniques (Horwath and Paul, 1994). Soils (approximately 10 g oven-dry equivalent weight) were pre-incubated at water potentials of –0.03 MPa (optimal) or –0.1 MPa (dry) and at 23 or 44°C in 100-mL glass jars with Teflon-lined screw caps for 3 d before treating with DBU. Soils were then fortified with carbonyl–14C labeled DBU at either 0.08 µg g–1 or 8.0 µg g–1, which is later referred to as low and high DBU applications, respectively. These two DBU levels were selected as representative of the DBU that may be applied if benomyl in the Benlate formulation used at the foliar (L) or drench (H) rates completely converted to DBU prior to application. A 15-mL vial containing 13 mL 1 M KOH solution was added to each jar (except for Day 0 and control samples) to trap 14CO2 resulting from the complete mineralization of DBU. The jars were quickly sealed with Teflon-lined closures and incubated at the desired temperatures. Carbon dioxide saturation of traps was avoided by frequent trap replacement. Traps were analyzed using liquid scintillation counting (LSC) methods. At specified time intervals, a set of three soil microcosms was extracted with ethyl acetate.
DBU (Carbonyl–14C) Enriched Benlate Fungicide Experiments
Soils (approximately 4.5 g dry equivalent weight) were pre-incubated at a specific temperature and moisture potential in 100-mL wide-mouth jars with Teflon-lined lids for 3 d before fortification with benomyl formulations (60–73 µg benomyl g–1 soil containing 0.25 to 0.29 µg DBU g–1 soil). This application rate is equivalent to 220 to 270% of the recommended drench rate (109 lb Benlate acre–1) for Benlate fungicides to nursery ornamentals, and was in between the high and low DBU application rate in the DBU-only soil treatments. Recoveries were performed in triplicate from soil and nonsoil control samples treated with 0.5 mL of fortified solution.
For Day 0 and nonsoil control samples, methanol (20 mL) was added before spiking and the soils were extracted as described below. For all other samples, a 15-mL vial containing 13 mL 1 M KOH solution was added to the jars for collection of 14CO2 resulting from mineralization of the radiolabel. The jars were quickly sealed with Teflon-lined closures and incubated at 23 or 33°C. Carbon dioxide traps were replaced at regular intervals and assayed using LSC. Soils were extracted at designated times followed by oxidation to determine non-extractable radiolabel.
Sample Extraction and Sample Preparation
Soil samples were preserved by freezing at –20°C until extraction. All samples were extracted and analyzed within 1 wk of the end of the incubation period. From each microcosm of the DBU-only experiments, the soil was divided into three subsamples of approximately 3.5 g dry weight each. Each subsample transferred to individual Teflon-lined 35-mL glass centrifuge tubes (Kimble-Kontes, Vineland, NJ) and extracted with ethyl acetate (20 mL). Centrifuge tubes were then placed on a rotary end-over-end mixer for 16 h. After mixing, the samples were centrifuged using a Jouan (Winchester, VA) CR 312 at 195 x g for 20 min. Samples for HPLC analysis were prepared by taking 10-mL aliquots of the ethyl acetate extracts and evaporating to dryness with a gentle stream of nitrogen under a weak vacuum (–0.07 MPa). Samples were then reconstituted in 3 mL of 45:55 (v/v) acetonitrile and water, subjected to sonication for 1 min to ensure complete dissolution, and filtered using a 5-mL glass syringe with a Teflon plunger fitted with a 0.45-µm Teflon filter (Alltech Associates, Deerfield, IL). To determine 14C loss during HPLC sample preparation, 1-mL aliquots of the ethyl acetate extracts before and after the evaporation–reconstitution–filtration step were transferred into 20-mL HDPE scintillation vials for LSC. Scintillation fluid (15 mL) was added to each scintillation vial and the vials were stored in the dark for 24 h before counting to suppress luminescence. The remaining filtered solution was transferred into a 2-mL Teflon-lined glass HPLC vial for HPLC analysis. Extraction recovery was 97.2 ± 2.9% for DBU control samples. The method detection limit for DBU was 1.2 µg DBU kg–1 soil.
For the Benlate fungicide formulation experiments, soil was extracted with methanol on application with 14C-DBU enriched Benlate applications and after subsequent incubations to recover the combined benomyl and carbendazim residues and any remaining DBU. Methanol extraction completely converts benomyl to carbendazim. The resulting carbendazim concentration, which represents the carbendazim originally present plus benomyl converted to carbendazim, is quantified in the extract using a chromatographic analysis. Methanol (20 mL) was added to the soil samples, which were extracted for 20 h on a rotary end-over-end mixer and centrifuged (600 x g for 15 min). The supernatant was decanted into a 100-mL wide mouth Teflon-lined amber jar. The extraction procedure was repeated and supernatants combined. Extracts were stored at 4°C in the dark until sample preparation and analysis could be performed (less than 1 wk). Extraction efficiency for benomyl and carbendazim ranged from 77 to 96%. The method detection limit for DBU was 5.3 µg kg–1. Values for the fraction of benomyl + carbendazim remaining were normalized to those at time 0 (time 0 results were assigned a value of unity).
Reverse-Phase Liquid Chromatography Analysis
Sample extracts (200 µL injection volume) were analyzed on a Shimadzu (Kyoto, Japan) HPLC system comprised of a SCL 10AVP system controller, a LC-10ATVP pump, a DGU-14A degasser unit, a FCV-11AL solvent selection valve, a SIL-10A autosampler, and a Raytest (Straubenhardt, Germany) Ramona 2000 radiodetector. For DBU-only studies, a Supelcosil LC-ABZ Plus (4.6 x 150 mm, 5-µm particle diameter; Supelco, Bellefonte, PA) analytical column was used for the analysis. The radiodetector was equipped with a Raytest glass scintillator flow cell (0.37-mL cell volume, 5.5-mm i.d.). The mobile phase consisted of 45:55 (v/v) acetonitrile and water at a flow rate of 1.0 mL min–1. Sample size was 200 µL. DBU retention time under these conditions was 4.5 min.
For DBU-enriched Benlate formulation studies, separations were performed on a reverse-phase C18 column (Luna 150-mm length x 4.6-mm i.d., 5-µm particle size; Phenomenex, Torrance, CA) with a pre-column guard (C18–ODS, 4.0-mm length x 3.0-mm i.d.; Phenomenex), and using UV and radioassay detectors in series. Solvent A was 15:85 (v/v) 50 mM sodium phosphate (pH 7.0) and water. Solvent B was 15:25:60 (v/v/v) 50 mM sodium phosphate (pH 7.0), water, and acetonitrile. Initial mobile-phase composition was 10% B followed by a linear gradient to 100% B over 18 min, back to 10% B by 19 min, and re-equilibration at initial conditions for 10 min. The flow rate was 1.2 mL min–1. Column temperature was maintained at 40°C using a Waters (Milford, MA) temperature controller. Carbendazim and DBU retention times under these conditions were 9 and 13.5 min, respectively. Carbendazim concentrations were measured with a UV detector at 280 nm.
Liquid Scintillation Counting
The LSC system was a Packard 1600TR (PerkinElmer). Aliquots (1 mL) of soil extracts were mixed with 15 mL ScintiSafe 30% in 20-mL scintillation vials. Vials were kept undisturbed in the dark for 24 h before LSC.
Post Extraction Oxidation
Extracted soils were analyzed for 14C content by combustion using a Packard Model 307 sample oxidizer (PerkinElmer). Soil (0.2–0.5 g dry weight equivalent) was weighed into a paper cup, mixed with approximately 200 mg powdered cellulose, placed in the instrument, and combusted for 3 min. Combustaid (0.1 mL) was added to moist soil samples before combustion to facilitate complete oxidation. Carbosorb E (10 mL) reagent was used to trap 14CO2 and Permafluor E+ scintillation cocktail (10 mL) was added to the sample. Samples were left in the dark for 24 h before 14C determination by LSC. Quenching was compensated by using a quench curve constructed with Carbosorb E and Permafluor E+.
Barium Chloride Precipitation
A barium chloride precipitation assay was used to ensure that the KOH traps had not reached saturation and to determine the fraction of 14C-label present as CO2. Aliquots (3 mL) of the solutions from each of the CO2 traps were transferred into 15-mL polypropylene centrifuge tubes and treated with 0.6 mL of 1.5 M BaCl2. The tubes were placed on a rotary end over end mixer for 1 h. After mixing, the tubes were centrifuged at 600 x g for 15 min. Parallel samples were processed except that water was added instead of BaCl2 to determine the radioactivity present immediately before precipitation. Samples (1 mL) were assayed by LSC to determine the fraction of radiolabel precipitated. The difference in the radiolabel measured in the absence and presence of precipitation was assumed to be CO2.
Statistical Analysis
Mineralization and degradation rates (k) and corresponding standard errors (SE) were estimated by fitting to the first-order rate law: ln Ct/Co = –kt, where Co and Ct are the concentrations at time 0 and time t, and k is the first-order rate constant. Data included in the first-order fits were limited to times where the percent DBU remaining was greater than 5 to 10%, because over subsequent times degradation did not appear first-order. For DBU-only experiments, triplicate microcosms were sampled at each time with the soil from each microcosm divided into three subsamples. Therefore, there were a total of nine measurements for each time used in finding degradation k values. For the DBU degradation studies in the presence of formulation, triplicate microcosms were sampled at each time, but soil from each microcosm was extracted in a single sample, thus only three measurements for each time. For the DBU-only experiments, one-tail t tests at probability levels of p < 0.05 were used to determine if increased soil water potential (decreased water content), increased DBU application rate, and increased temperature resulted in a larger amount of DBU remaining at a specified time (t) of 20 ± 1 d. Each pair of experiments tested were different by only one parameter (e.g., soil water potential, application rate, concentration, or temperature). Two-tailed t tests at probability levels of p < 0.05 were used to assess if the presence of formulation affected the amount of DBU remaining at 20 ± 1 d. Experiments were paired as with the DBU-only experiments, except that DBU concentrations could not be matched. The concentration of DBU used in the formulation studies was between the two application rates used in the DBU-only studies; therefore, comparisons were made with both the low and high DBU-only application rates when both were available.
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RESULTS AND DISCUSSION
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DBU-Only Degradation Studies
Representative curves for loss of DBU, 14CO2 evolution, and overall mass balance over time along with first-order rate law fits are shown in Fig. 1. First-order rate coefficients estimated for degradation and mineralization are summarized for the incubations at water potentials of –0.03 and –0.1 MPa in Tables 2 and 3, respectively, along with the percent DBU remaining at 20 ± 1 d. DBU degradation was relatively rapid with half-lives ranging between 1.4 and 16.5 d across all soil moisture–temperature treatments except for incubations at the highest temperature (44°C), for the high-pH soil from Florida (Soil 9RD) incubated under low moisture conditions, and sterile controls (not shown). For the two 60Co-irradiated sterilized soils incubated at 23°C and a –0.03 MPa water potential, little to no DBU was degraded or mineralized over the one month of incubation, indicating that microbial degradation is probably the primary process responsible for DBU loss in soils.
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Fig. 1. Representative trends in N,N'-dibutylurea (DBU) degradation experiments for two soils fortified with 0.08 µg DBU g–1 soil incubated at two moisture potentials and at 23°C with fits of the first-order rate law.
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Table 2. Summary of N,N'-dibutylurea (DBU) (carbonyl–14C) degradation and mineralization rates in soil incubated at a water potential of –0.03 MPa, where L and H refer to application of 0.08 or 0.8 µg DBU g–1 soil, respectively.
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Table 3. Summary of N,N'-dibutylurea (DBU) (carbonyl–14C) degradation and mineralization rates in soil incubated at a water potential of –0.1 MPa, where L and H refer to application of 0.08 or 0.8 µg DBU g–1 soil, respectively.
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The effect of soil water potential (–0.03 and –0.1 MPa), temperature (23, 33, and 44°C), applied DBU concentration (0.08 and 0.8 µg DBU g–1), and media (various soils and potting mixes) on DBU degradation was assessed by whether differences in the amount degraded at 20 ± 1 d were significant at the 95% confidence interval. Soil water potential had the greatest and most consistent impact on DBU degradation with significantly less being degraded under drier conditions (–0.1 MPa soil water potential) in all 16 paired treatments except one (Table 3). At the more optimal soil water potential (–0.03 MPa) and temperature (23 to 33°C) with regard to both microbial activity and plant growth, DBU half-lives ranged between 1.4 and 9.4 d.
The effect of temperature was investigated at 23, 33, and 44°C for two soils (2AC and 9RD) under both moisture conditions, and for one potting media (3MIX) at 23 and 33°C at –0.03 MPa. The percent DBU remaining on Day 20 when incubated at 33°C was significantly less than at 23°C incubations (Table 2 and 3). However, at 44°C DBU mineralization and degradation were substantially slower with significantly less degraded by Day 20 compared with either 23 or 33°C. Therefore, very high temperature and dry conditions, which are both generally unfavorable for microbial activity and plant health, retard DBU degradation, which further supports that DBU degradation is driven by microbial processes.
The effect of applied DBU concentration from 0.08 to 0.8 µg DBU kg–1 was assessed for five paired treatments for each soil water potential. For soils incubated at –0.03 MPa soil water potential, differences in DBU degradation with an increase in applied DBU concentration were small and in most cases not significant. Under drier conditions (–0.1 MPa soil water potential) in two of the five paired experiments (Soils 1AC and 8R), much less DBU (5 to 10 times) was degraded by Day 20 at the 95% confidence interval. For the other three experimental pairs, more DBU (within a factor of 2) was degraded by Day 20 at the higher DBU concentration. Overall, no consistent trend was observed between DBU degradation and application rates.
Of the soil chemical properties, only pH appeared to have a significant effect on DBU degradation. All but one soil in this study were from Costa Rica and weakly acidic (pH 5.5 to 6.3), which is typical of tropical soils. For the soil from Florida, which was also the only basic soil (pH = 7.9), degradation was much slower for all paired conditions. Soil pH, geographic location, and land use can all affect microbial DBU degradation; therefore, attributing the difference in Soil 9RD to only pH is highly speculative. Soil organic matter (OM) ranged from 3.5 to 9.1%; however, all soils had between 3.5 and 4.1% OM except Soil 7CB, which had the highest percent organic matter at 9.1. No apparent trend between DBU degradation and percent organic matter was noted. Clay content ranged from 6.8 to 52.8%; however, there was no significant trend in degradation as a function of clay content. For a given soil moisture content, increasing clay content will decrease soil water potential (decrease water availability) to microbes and plants (Hillel, 2004, p. 114–115). Clearly, soil water potential did impact DBU degradation with degradation being significantly slower at the lower soil water potential (Table 3). In the current study, soil moisture was adjusted such that the energy status of water was constant across soil type; therefore, the effect that clay may have on water availability and subsequent degradation was not observed. Note that in the field where soil water potential is not controlled, DBU degradation in soils with higher clay content will have a greater likelihood to be negatively impacted by reduced rainfall or irrigation events.
DBU Degradation in the Presence of the Formulations
Degradation of DBU in 9RD and 2AC soils under a subset of conditions used in the DBU-only studies was measured in the presence of Benlate DF and SP at an application rate of 60 to 73 µg g–1 benomyl and a DBU application rate of 0.25 to 0.29 µg g–1, which was in between the low and high application rate used in the DBU-only studies. Curves for loss of DBU and 14CO2 evolution with time are represented in Fig. 2. Overall mass balance of applied 14C-DBU ranged between 93 and 99% (not shown) in the formulation studies. Estimated first-order rate coefficients for DBU mineralization and degradation are summarized in Table 4 along with the percent DBU and combined benomyl and carbendazim remaining at Day 20 ± 1. For Soil 9RD, DBU degradation was slower in the presence of formulation with significantly less DBU being degraded by Day 20 (Table 4; Fig. 2B). No effect on DBU degradation was observed from the formulated material in Soil 2AC. Also in the two paired experiments comparing formulation type (DF versus SP), no significant differences in DBU degradation was observed. The percent DBU remaining at Day 20 was significantly less for soil incubated at 33°C compared with 23°C, and at –0.03 MPa soil water potential compared with –0.1 MPa, similar to what was observed for the DBU-only experiments (i.e., in the absence of formulation).
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Fig. 2. Comparative trends in N,N'-dibutylurea (DBU) degradation in the presence and absence of formulation (DF and SP) at –0.03 MPa soil water potential and 23°C for (A) Soil 2AC and (B) Soil 9RD. Application rates of DBU at the low and high concentrations were 0.08 and 0.8 µg g–1, respectively, and 0.25 to 0.29 µg DBU g–1 soil for the Benlate formulations.
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Table 4. Summary of degradation and mineralization rates of N,N'-dibutylurea (DBU) (carbonyl–14C) enriched Benlate DF and SP fungicides applied to soils at 0.25 to 0.29 µg g–1 DBU and 60 to 73 µg g–1 benomyl.
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For Soil 9RD, the percent remaining of the combined amounts of benomyl and carbendazim at Day 19 was similar to the percent DBU remaining (Table 4). For Soil 2AC, benomyl-carbendazim degradation was much slower; percent benomyl-carbendazim remaining at 19 d was much greater than observed for DBU. Benomyl-carbendazim degradation also increased with an increase in temperature from 23 to 33°C, but to a lesser extent than observed for DBU. For both soils across treatments, half-lives for benomyl-carbendazim combined were 2 to 3 mo, which is within the range of half-lives (1–6 mo) observed for various soil conditions (Baude et al., 1974; Liu and Hsiang, 1994; Yarden et al., 1985). Fungicide formulation type (50 DF or 50 SP) had little effect on either mineralization or benomyl degradation rates. At the end of the incubation period, between 34 to 69% of the applied benomyl still remained.
Environmental Significance
DBU degradation was primarily microbial and for most soil–treatment combinations, half-lives were less than 2 wk. DBU degradation was retarded at the lower soil water potential, enhanced at 33°C, and retarded at the highest temperature investigated of 44°C. In the presence of the formulation, DBU degradation was slower for one soil type. The longest half-life observed in any case was less than 7 wk; therefore, long-term persistence of DBU applied to soils through a Benlate application is very unlikely. In the presence of plants, root growth is likely to enhance microbial activity in the rhizosphere, which in turn may enhance DBU degradation. In an overall assessment of the potential effect of DBU on crop production, several factors must be considered including levels of DBU required to negatively impact plant growth, the actual amount of DBU applied with the formulation, which is likely to be orders of magnitude less than what was used in this study, and soil fertility. Results from this study suggest that for most soils under conditions that would be conducive to commercial plant production, less than 10% of DBU would be remaining after 6 wk. DBU may persist longer (6–7 wk) if soil conditions are adverse to normal microbial activity, which would also be adverse to plant growth.
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ACKNOWLEDGMENTS
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This work was funded in part by EI DuPont de Nemours and Company. Approved for publication as Purdue Agricultural Research Programs Journal Ser. 17310. A special thanks to Judy Santini for help in performing the statistical analysis.
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REFERENCES
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