Journal of Environmental Quality 32:1258-1261 (2003)
© 2003 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
TECHNICAL REPORTS
Bioremediation and Biodegradation
Comparison of Triticonazole Dissipation after Seed or Soil Treatment
Elisabet Börjesson*,a,
John Stenströma,
Lennart Johnssonb and
Lennart Torstenssona
a Swedish University of Agricultural Sciences, Department of Microbiology, P.O. Box 7025, SE-750 07 Uppsala, Sweden
b Swedish University of Agricultural Sciences, Plant Pathology and Biocontrol Unit, P.O. Box 7035, SE-750 07 Uppsala, Sweden
* Corresponding author (Elisabet.Borjesson{at}mikrob.slu.se)
Received for publication September 26, 2002.
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ABSTRACT
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This study examined the long-term fate of the fungicide triticonazole (TTZ; 5-[(4-chlorophenyl)methylene]-2,2-dimethyl-1-(1H-1,2,4-triazol-1-ylmethyl)cyclopentanol) applied at a normal field dose (8.9 g ha-1) via seed treatment, which is the normal alternative in practice. The TTZ was applied to wheat (Triticum aestivum L.) grains as a disinfectant before sowing or spraying on bare soil for comparison and reference to the seed treatment. The seeds were germinated and grown in pots in a greenhouse at 22 ± 3°C. The dissipation of TTZ was studied by gas chromatography–mass spectrometry (GC–MS) analysis of the residues every fourth week until no TTZ could be detected. The recovery for analysis of TTZ in soil was between 98 and 131%, and the quantification level was 0.002 mg kg-1. After 56 d of incubation, 20 and 28% of the TTZ applied remained in the soil and seed treatments, respectively, with corresponding half-lives of 27 and 29 d. The microbial biomass initially decreased in the soil treatment but had recovered after 56 d. The active part of the biomass was not changed during the experimental time. Thus, with respect to dissipation of TTZ and its effect on the soil microbial biomass and activity, no long-lasting difference between soil and seed treatments could be found.
Abbreviations: TTZ, triticonazole
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INTRODUCTION
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TRITICONAZOLE IS A relatively new fungicide developed by Rhône–Poulenc (now Bayer CropScience, Monheim am Rhein, Germany) and patented in 1988. It is a broad-spectrum systemic triazole fungicide acting by inhibition of demethylation in the sterol biosynthesis pathway found in most fungi except Oomycetes. Triticonazole is used as a preventive seed disinfectant against seed-borne diseases and as a preventative treatment against a number of foliar pathogens such as rusts (Puccinia spp.), powdery mildew, leaf-spots, eye-spot, leaf and net blotch of cereals, seedling diseases, head smut of corn (Ustilago spp.), and bunt (Tilletia caries). Seed treatment combines disinfection of the seeds with long-term protection of the plant. The seeds are coated with a film of formulation containing the fungicide. Such targeted deposition of the fungicide allows for reduced rate doses and could minimize environmental risks.
The effect of commercial formulation adjuvants and application rates of TTZ (
0.2 mg kg-1) on the mineralization and dissipation of TTZ applied to soil has been studied under laboratory conditions (Beigel et al., 1999). It was found that the mineralization of TTZ is very slow and that its bioavailability for degradation is small due to strong and time-dependent sorption. It was also concluded in that study and in another study (Charnay et al., 2000a) that formulation additives at normal application rates had no significant effect on TTZ bioavailability.
A short-term study (7 d) has evaluated the release of TTZ from coated maize (Zea mays L.) seeds (Charnay et al., 2000b). It was concluded that the sorption of TTZ on soil is the key process governing its fate.
Thus, studies on the mineralization, degradation, and sorption of TTZ in soil have been published. However, there is a lack of information on the fate of TTZ when applied to seeds that have subsequently germinated. Therefore, this study was conducted to assess the long-term fate of TTZ applied to seeds at the recommended dose for disinfection. Soil spraying at the same dose level as in the seed application was used for comparison and as a reference to the seed treatment. A secondary aim was to compare the effect of TTZ on microbial amount and activity in the two treatments. In addition, the recommended dose for use in practical agriculture, 50 g TTZ per Mg of seeds (corresponding to initial concentrations of 0.01 to 0.02 mg kg-1 soil for the 7-cm upper layer in this study), requires analytical methods capable of quantifying very low concentrations. Analytical methods with limits of detection of 0.025 and 0.005 mg kg-1 soil have been described (Fischer et al., 1999), but due to the low application rate used in this study, it was necessary to develop and use a method with an even lower detection limit. Therefore, mass spectrometry was used for detection, which lowered the limit of quantification to 0.002 mg kg-1. This made it possible to study the degradation process over a reasonable range of concentration and time.
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MATERIALS AND METHODS
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A total of 96 Mitscherlich pots with a volume of 6.3 L each were prepared, 48 for the soil treatment and 48 for the seed treatment. The pots were placed in a greenhouse at 22 ± 3°C (light–dark 12 h–12 h; HPJ-T, 400 W MGR 102; Philips, Eindhoven, the Netherlands), watered every second to third day, and sampled every fourth week for chemical analysis.
Soil Preparation
Topsoil (0–20 cm) was sampled from an arable field under fallow [previous crop = barley (Hordeum vulgare L.)] in the county of Dalarna, central Sweden, on 26 June 2001 and transported to Uppsala, Sweden. The soil was placed under a tarpaulin until 18 August when it was sieved (4 mm) and the water content was determined (105°C) to 18.5% (±0.33, n = 3). The water content was then adjusted to 40% of maximum water-holding capacity. Physical and chemical properties of the soil were analyzed at the Department of Soil Science at the Swedish University of Agricultural Sciences (Table 1)
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Triticonazole Application to Seed
Winter wheat seed (cv. Stava, purchased from Svalöf Weibull AB, Svalöv, Sweden), with a germination capacity of 95% and a 1000-kernel weight of 41 g, was used. One kilogram of the seed was treated with 4.0 mL of a 1:1 mixture (v/v) of Premis 25 FS (Aventis CropScience, now Bayer CropScience) and distilled water corresponding to 50 mg kg-1 seed. The seeds were treated for 2 min in conventional seed treatment equipment (Ziro; Gamac Zweden, Hallsberg, Sweden).
Pot Preparation
The experiment was started 4 d after the seed treatment. A total of 96 Mitscherlich pots were used for the experiment, each containing 4800 g of moist soil. To each of 48 pots (A pots), 112 mL of a diluted Premis 25 FS suspension was sprayed on the soil surface, corresponding to 0.028 mg of TTZ. The Premis 25 FS suspension was made by mixing 1.0 mL of Premis 25 FS and 99 mL of distilled water. From this suspension 6.0 mL was taken and mixed with 5994 mL of distilled water. After the TTZ application, the soil surface in each pot was covered with 800 g of soil. Fourteen Premis 25 FS–treated wheat kernels, corresponding to 0.028 mg of TTZ, were placed in each of the other 48 pots (B pots) and similarly covered with 800 g of soil. Thereafter all pots were watered, as they also were immediately before the first sampling.
Plant Care
The pots were randomly placed in the greenhouse, where the temperature was recorded daily. The light was switched on for 12 h d-1. The watering of the pots was performed manually by a watering can from above and conducted at least every third day. The water content was controlled by weight and the plant pots were fertilized (0.04 g N per pot) once during the experiment.
Soil Sampling
Three pots with plants and three pots without plants were sampled at the start of the experiment and thereafter every fourth week. The soil samples were analyzed for TTZ on the day of sampling. Before sampling, the plants were cut off above the soil surface and after that the following schedule was conducted.
For the soil treatment pots (the A pots), the topsoil layer containing 2050 g of soil was taken from three randomly chosen pots. The sampling depth was determined to be 6 cm, the depth to which the first dose of water had drained. The next layer (1 cm), containing 300 g of soil, was then collected, and the remaining soil in one of the pots was finally sampled, for a total of seven samples.
For the seed treatment pots (the B pots), the topsoil layer (5 cm), containing 1750 g of soil, was taken from three randomly chosen pots. The remaining soil in one of the pots was then sampled, for a total of four samples.
Chemical and Biological Analyses
The amount of TTZ on the disinfected kernels was consequently analyzed by extracting 14 kernels with acetonitrile and quantified by high pressure liquid chromatography (HPLC). The equipment used was an HPLC 1100 instrument from Agilent Technologies Sweden AB (Kista, Sweden), comprising an autoinjector, a G1322A quaternary pump, and a G1314A variable wavelength UV detector.
Before the analysis of TTZ in soil, each soil sample was thoroughly mixed and samples for determination of the water content were taken before TTZ analysis. Then, 50 g wet wt. of each sample was extracted with 200 mL of acetone and 30 mL of water by shaking for 30 min. The sample was filtered and the residues were reextracted with 200 mL of acetone. The extracts were pooled and adjusted to 500 mL with acetone and a portion of 50 mL was evaporated under vacuum to approximately 1 mL. The sample was then cleaned up by solid-phase extraction (SPE), first with a 500-mg CN column, then a 500-mg NH2 column (Scantec Lab, Partille, Sweden). The sample was dissolved in toluene before analysis. In this experiment, a gas chromatography–mass spectrometry (GC–MS) instrument from Agilent Technologies Sweden AB (GC 6890 and MSD 5973) was used to detect TTZ. The column used was a HP 5 for GC–MS (30-m length, 0.25-mm i.d., and 0.25-µm film thickness) from Hewlett-Packard (Palo Alto, CA). The injection temperature was 250°C, injection volume 1 µL, and column flow 1.6 mL helium min-1. The oven temperatures were 100°C for 2 min; 100 to 250°C at 20°C min-1; 250 to 260°C at 5°C min-1; and 260 to 280°C at 15°C min-1. The detector was a mass spectrometer with electron impact ionization with transfer line and manifold temperatures at 260°C and 230°C, respectively. The filament current was 10 µA and the electron multiplier voltage 1300 V. The total area from the masses 235, 236, and 299, measured by external standard, was used for quantification. The retention time was 13.1 min. The method was validated with soil samples spiked at the quantification levels 0.002, 0.01, and 0.02 mg kg-1 dry wt.
The soil samples were analyzed on the day of sampling every fourth week. When the concentration of TTZ in all pots from one treatment was below the quantification level at a sampling, no more analyses were made.
The microbial biomass was measured as substrate-induced respiration with the distribution between active and dormant microorganisms according to Stenström et al. (2001) in thawed samples from Days 0, 56, and 140 at the end of the study. The Day 0 sample from the seed treatment can be considered as a control sample in terms of microbial biomass as no kernels were present in this sample.
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RESULTS AND DISCUSSION
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Chemical Analysis
Recovery of TTZ in soil was measured at the spiking levels 0.002, 0.01, and 0.02 mg kg-1 and ranged between 99 and 131%. All values were calculated on a dry-weight basis. With the MS detection used in this study, the quantification level was 0.002 mg kg-1. The detection limit was 0.001 mg kg-1 and the area at the quantification level was five times above the baseline noise.
The 14 TTZ-treated kernels contained 0.022 (±0.001, n = 3) mg of TTZ when the study started. The theoretical concentration of TTZ applied to soil in the seed treatment can thus be calculated to 0.022/1.75 = 0.0123 mg kg-1 (wet wt.) of soil for the upper part (1750 g) of the pot.
The concentrations of TTZ in the topsoil layer on every sampling occasion are shown in Fig. 1
. The soil just beneath the surface was analyzed on the four first sampling occasions in both treatments. The main part of the TTZ apparently remained in the upper 10 cm of the soil in both treatments, since no TTZ was detected below this level. After 56 d, only about 20% of the TTZ applied was detected in the upper layer in both treatments and after 84 d no TTZ was detectable in the soil treatment.
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Fig. 1. Degradation of triticonazole (TTZ) in the soil and seed treatments where n = 3, except when n = 1 for Day 112 or n = 2 for Day 84 in the seed treatment.
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However, in the seed treatment, a small amount just above the quantification limit was detected until 140 d (Fig. 1), while the sampling after 168 d showed contents below the quantification level. It was probably the slow breakdown process of the kernels that decreased the TTZ degradation speed in this treatment. Some of the kernel shells were rather intact even after 140 d, but on Day 168 they were no longer discernible. The TTZ applied to the kernel surface was probably not available for microbial degradation. The most probable reason for this slow kernel breakdown process was the relatively low moisture content suggested by Aventis CropScience to be used in this study (40% of maximum water-holding capacity). When the small residue was observed after 140 d, the moisture content was increased to 50% of maximum water-holding capacity and kept at that level in all pots until the end of the study. This higher moisture level is probably closer to field conditions during the Swedish autumn.
The degradation rate was almost the same in the soil treatment as in the seed treatment during the first 56 d (Fig. 1). This relatively fast TTZ degradation rate was followed by a much slower degradation at low residual concentrations, probably as an effect of a small availability of the TTZ by slow desorption and diffusion limitations (Pignatello and Xing, 1996). Such decreasing TTZ availability with time was also reported by Beigel et al. (1999). The lipophilic character of TTZ (partition coefficient octanol–water log P = 2.5; Rhône–Poulenc information) probably contributes to a strong binding to clay and organic matter. The values from the three first sampling dates were used to calculate the first-order rate constant (Fig. 2)
, after which time some of the analyzed concentrations were still >0.002 mg kg-1. However, these values did not fit the first-order model used.
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Fig. 2. Triticonazole (TTZ) degradation according to first-order kinetics during the first 56 d for the soil and seed treatments.
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The half-lives of TTZ in the soil and seed treatments were 27 and 29 d, respectively, which is much smaller than the 197 d obtained by Beigel et al. (1999) at 22°C and an initial dose of 0.2 mg TTZ kg-1, that is, a 16.2-times-higher initial concentration than used in our study. This big difference in degradation times can be due to the fact that they used soil that had been stored air-dry before the start of the study, and, as shown in their study, that the persistence of TTZ increases with increasing application rate. If the moisture content in our study had been at the optimum for degradation (60% maximum water-holding capacity instead of 40–50%) the half-lives would probably have been even shorter.
Microbial Biomass and Activity
The substrate-induced respiration, and consequently the total glucose-responsive biomass, was constant in the seed and soil treatments during the study period, except for a significantly (p < 0.05) smaller biomass in the soil treatment at Day 0, probably reflecting the larger exposure and toxicity of the fungicide when applied directly to bare soil (Fig. 3)
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Fig. 3. The substrate-induced respiration (the total height of each bar) and the distribution between the active (white) and dormant (black) organisms (n = 3). The term A refers to soil treatments, while B refers to seed treatments.
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In soils with no recent substrate addition, the distribution between active and dormant microorganisms is generally 5 to 20% active and 80 to 95% dormant (Stenström et al., 2001). However, in this experiment the active microorganisms represented a much larger fraction (29–42%) of the total biomass throughout the study period (Fig. 3), indicating a high soil carbon availability in the test soil.
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CONCLUSIONS
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Seed and soil treatment with TTZ at a normal field dose gave almost the same calculated degradation rates (t1/2 = 27 and 29 d, respectively). The faster degradation rate in this study compared with other studies is probably due to a high carbon availability and high microbial activity in the soil used and the low concentration of TTZ applied. However, a small resistant fraction was formed, especially in the seed treatment. The degradation of this fraction probably occurred concurrently with kernel degradation. No TTZ transport down to lower soil layers was observed in this experiment. The TTZ application, both in the soil and the seed treatments, did not give any persistent effect on microbial amount and activity.
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ACKNOWLEDGMENTS
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The financial support of Aventis CropScience Nordic AB is gratefully acknowledged.
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REFERENCES
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- Beigel, C., M.-P. Charnay, and E. Barriuso. 1999. Degradation of formulated and unformulated triticonazole fungicide in soil: Effect of application rate. Soil Biol. Biochem. 31:525–534.
- Charnay, M.-P., L. Tarabelli, C. Beigel, and E. Barriuso. 2000a. Modifications of soil microbial activity and triticonazole biodegradation by pesticide formulation additives. J. Environ. Qual. 29:1618–1624.[Abstract/Free Full Text]
- Charnay, M.-P., C. Verge, and E. Barriuso. 2000b. Influence of soil type and water content on release of triticonazole from coated maize seed. Pest Manage. Sci. 56:249–256.
- Fischer, R., R. Hänel, and J. Siebers. 1999. Rückstandsanalytik neuer Pflanzenschutzmittelwirkstoffe. Nachrichtenbl. Dtsch. Pflanzenschutzdienstes (Braunschweig) 51:285–292.
- Pignatello, J.J., and B. Xing. 1996. Mechanisms of slow sorption of organic chemicals to natural particles. Environ. Sci. Technol. 30:1–11.
- Stenström, J., K. Svensson, and M. Johansson. 2001. Reversible transition between active and dormant microbial states in soil. FEMS Microbiol. Ecol. 36:93–104.[Medline]
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ABSTRACT
INTRODUCTION
