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Degradation of Herbicides Atrazine and Bentazone Applied Alone and in Combination in Soils1
Pedosphere 18(2): 265–272, 2008 ISSN 1002-0160/CN 32-1315/P c 2008 Soil Science Society of China ° Published by Elsevier Limited and Science Press
Degradation of Herbicides Atrazine and Bentazone Applied Alone and in Combination in Soils∗1 LI Ke-Bin1 , CHENG Jing-Tao2 , WANG Xiao-Fang1 , ZHOU Ying3 and LIU Wei-Ping4 1 Department
of Chemistry, Northwest University, Xi’an 710069 (China). E-mail: [email protected] of Chemistry, Shaanxi Institute of Education, Xi’an 710061 (China) 3 Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032 (China) 4 Research Center of Green Chirality, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China) 2 Department
(Received July 25, 2007; revised January 18, 2008)
ABSTRACT The application of a mixture of bentazone (3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one-2,2-dioxide) and atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) is a practical approach to enhance the herbicidal effect. Laboratory incubation experiments were performed to study the degradation of bentazone and atrazine applied in combination and individually in maize rhizosphere and non-rhizosphere soils. After a lag phase, the degradation of each individual herbicide in the non-autoclaved soil could be adequately described using a first-order kinetic equation. During a 30-d incubation, in the autoclaved rhizosphere soil, bentazone and atrazine did not noticeably degrade, but in the non-autoclaved soil, they rapidly degraded in both non-rhizosphere and rhizosphere soils with half-lives of 19.9 and 20.2 d for bentazone and 29.1 and 25.7 d for atrazine, respectively. The rhizosphere effect significantly enhanced the degradation of atrazine, but had no significant effect on bentazone. These results indicated that biological degradation accounted for the degradation of both herbicides in the soil. When compared with the degradation of the herbicide applied alone, the degradation rates of the herbicides applied in combination in the soils were lower and the lag phase increased. With the addition of a surfactant, Tween-20, a reduced lag phase of degradation was observed for both herbicides applied in combination. The degradation rate of bentazone accelerated, whereas that of atrazine remained nearly unchanged. Thus, when these two herbicides were used simultaneously, their persistence in the soil was generally prolonged, and the environmental contamination potential increased. Key Words:
atrazine, bentazone, degradation, herbicide combination
Citation: Li, K. B., Cheng, J. T., Wang, X. F., Zhou, Y. and Liu, W. P. 2008. Degradation of herbicides atrazine and bentazone applied alone and in combination in soils. Pedosphere. 18(2): 265–272.
INTRODUCTION Atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) is one of the most widely applied herbicides for pre- and post-emergence control of broadleaf and grassy weeds in maize and other crops. Bentazone (3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one-2,2-dioxide) is widely used for postemergence control of sedges and broadleaf weeds. The combination of both herbicides not only broadens their herbicidal spectrum but also reduces their respective application dosage. The combined application is recommended as a promising post-emergence herbicidal activity for the control of weeds in cornfields (Feng and Liu, 1990). Application of herbicide mixtures to enhance the herbicidal effect is a common practice in agriculture. However, when multi-contaminants exist in soils at the same time, the behavior of individual component may be affected. The common ingredients in pesticide formulations are surfactants, which would have complex effects on the behavior of other contaminants in the soil because they can enhance the water solubility of the pollutants and act as a catalyzer during their degradation ∗1 Project
supported by the Natural Science Basic Research Plan in Shaanxi Province of China (No. 2004K03-G3) and the Scientific Research Fund of the Department of Education of Shaanxi Province, China (No. 04JK234).
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processes owing to their special surface properties (Haigh, 1996). Although there have been several studies on the movement (Tindall and Vencill, 1995; Boesten and van der Pas, 2000), sorption (Lesan and Bhandari, 2003; Gaston et al., 1996; Li et al., 2003), and degradation of atrazine and bentazone in soils (Kruger et al., 1993; Gaston et al., 1996), few studies have been conducted regarding their individual degradation among complex systems, such as atrazine + bentazone and atrazine + bentazone + surfactant. Natural soils are rarely contaminated with only one xenobiotic compound; several organic contaminants may exist simultaneously. Recent observations show that the presence of structurally similar molecules causes competitive adsorption of herbicides and affects their degradation rates (Xing et al., 1996; Martins and Mermoud, 1998). Moreover, microorganisms do not rely on a single compound for growth, but rather on a wide range of natural or anthropic C sources. Therefore, investigation of the behaviors of a pesticide in multi-contaminant systems appears to be more realistic since it accounts for synergistic and antagonistic effects that may greatly affect the fate of a pollutant in the soil. The rhizosphere, the area in the immediate vicinity of plant roots, is a zone of intense microbial activity, which is caused by root exudates containing carbohydrates, amino acids, and organic acids (Curl and Truelove, 1986). It is likely that the rhizosphere has the potential to accelerate biodegradation of organic pollutants. Recently, there has been an increasing interest in the degradation of pollutants in rhizosphere soils. Several studies have found that organic contaminants generally disappear more quickly in planted soils than in unplanted soils, suggesting that vegetation has an important effect on the fate of pesticides in soils (Anderson et al., 1993). The objectives of the present study were to explore the effect of application of atrazine and bentazone in combination on the degradation of each individual herbicide, to evaluate the effect of a low-concentration surfactant on the degradation of the herbicides, and to investigate the rhizosphere effect on the degradation of the herbicides. MATERIALS AND METHODS Soil sampling and chemicals Rhizosphere and non-rhizosphere soils were collected from the agricultural field at Huajiachi Campus, Zhejiang University, Zhejiang Province, China. The soil in the field is a silty clay loam soil (241.8 g kg−1 clay, 5.3 g kg−1 sand, and 753 g kg−1 silt) with pH 6.1 and organic matter content of 36.4 g kg−1 . The sampling site was planted to corn for at least three consecutive years with no application of a combined formulation of atrazine and bentazone. High-performance liquid chromatography (HPLC) analysis of the soil extracts prior to the start of the study showed no detectable residue of bentazone and atrazine. At harvest, rhizospheres containing as much as possible associated roots were excavated by digging to a 20 cm depth around maize plants. Non-rhizosphere soil (NRS) samples were taken from the same site containing few roots. The rhizosphere, non-rhizosphere soil, and plant samples were placed in plastic bags and immediately transported to the laboratory and prepared within a few hours. Soil loosely adhering to the roots was removed by mild shaking. The remaining adherent soil, called rhizosphere soil (RS), was manually separated and collected in a bucket. Soil samples were sieved to 2 mm and then stored at 4 ◦ C for no more than 14 d prior to initiation of the study. A part of the soil samples was air-dried for 2 d at room temperature for property analysis using the procedures adopted by Liu et al. (2001). Bentazone (≥ 97%) was obtained from BASF (Ludwigshafen, Germany). Atrazine (≥ 99%) was purchased from Chemical Service (West Chester, USA). A nonionic surfactant, Tween-20 (chemically pure), was purchased from the Mingqing Chemical Reagent Plant (Wenzhou, China). Incubation experiments For investigating the degradation of the herbicides applied individually or in combination in the non-autoclaved RS and NRS, methanol solutions containing atrazine or bentazone or both of them were
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spiked with a portion of 25.0 g (dry weight) microbial-inactive soil which was sieved to 0.1 mm and dried at 110 ◦ C overnight. After evaporation of methanol and thorough mixing, the spiked portion was incorporated into an aliquot of 1 500 g RS (174 g kg−1 moisture content) or an aliquot of 1 500 g NRS (141 g kg−1 moisture content) at 5.03 g spiked portion per kg soil (dry weight). The final concentration of each herbicide was controlled at 10 mg kg−1 soil (dry weight), which was equivalent to 10 times the recommended application rate of the combined herbicide. To investigate the effect of surfactant, 0.4 g Tween-20 was also added together with the spiked portion, with an initial concentration of 1.1 g kg−1 in the aqueous phase. The soil moisture of all treatments was adjusted to 290 g kg−1 with the soil sieved several times to mix thoroughly. The homogenized samples were placed in six 2-L beakers and immediately sampled as day 0. The beakers were then covered with plastic film, which was pierced with a needle to allow air exchange, and incubated in the dark at 25 ◦ C. During the incubation period, the beakers were removed from the incubator every other day and brought to the original moisture content (290 g kg−1 ) by adding the required amount of sterile distilled water. At intervals of 2, 4, 6, 13, 21, 26, 36, and 47 days, duplicates of about 30 g soil from each treatment were sampled, weighed in a 50-mL high-density polyethylene centrifuge tube with screw caps, and then immediately stored at −20 ◦ C until analysis. The degradation of atrazine and bentazone alone in autoclaved RS was also investigated. Fifty grams (dry weight) RS was weighed in 100-mL Erlenmeyer flasks. After being plugged with cotton pads, the flasks were autoclaved at 120 ◦ C for 30 min each day in three consecutive days to remove microbial activity. Atrazine or bentazone 0.5 mg each was added to the autoclaved soil in 1 mL of methanol. Eleven flasks for each herbicide were incubated under the same conditions as described above, except that the spiking was performed in a laminar hood. At day 0.7, 3, 4, 6, 9, 12, 15, 17, 20, 26, and 30, one flask of each herbicide treated soil was taken out and stored at −20 ◦ C. Soil extraction and analysis At the end of the incubation, the frozen samples were thawed at room temperature. After transferring about 30 g of moist soil to 100 mL Erlenmeyer flasks, soil samples were extracted three times by shaking for 2 h with a 40 mL methanol and water mixture (9:1, v/v) each time. The combined extracts were concentrated in a rotary evaporator below 45 ◦ C under reduced pressure to remove the methanol fraction. The residual aqueous extracts were repartitioned three times with 20 mL dichloromethane (20 mL × 3). When bentazone was extracted with dichloromethane, the aqueous extract was adjusted to pH < 3.0. The dichloromethane fractions were pooled, passed through anhydrous sodium sulfate, and further concentrated to near dryness in a rotary evaporator. Then, N2 was used to dry the extract, and the residue was reconstituted in a 10 mL mobile phase composed of 66:44 methanol:water (pH adjusted to 2.8) for analysis on a Shimadzu HPLC equipped with a photodiode array detector (Kyoto, Japan). A YWG C18 reversed-phase column (10 µm; 4.6 mm × 250 mm) from Dalian Elite Analytical Instruments Co., Ltd., China, was used for separation. The detection wavelength was 218 nm and the flow rate was 0.8 mL min−1 . The injection volume was 10 µL. The HPLC detection limit for bentazone was 5.9 µg L−1 , and for atrazine, it was 3.8 µg L−1 . The revised first-order model used by Martins and Mermoud (1998) in their study was employed to analyze the herbicide degradation kinetics in this study: ln(ct /c0 ) = −k(t − tlag )
(1)
t1/2 = 0.693/k
(2)
where ct is the concentration of the herbicide at time t, c0 is the initial concentration of the herbicide, k is the first order degradation rate constant, tlag is the lag phase during which there is no apparent herbicide degradation, and t1/2 is the half-life. The parameter estimates were obtained for each degradation condition using linear regression. Regression analyses were performed with Origin 6.0. All
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experimental data used in the regression analysis were the mean value of duplicates except for those from the degradation of atrazine and bentazone in the autoclaved soil, which were single points without replicates. A t test on k was conducted to detect the differences in the effect of the incubation conditions on the pesticide degradation rates. RESULTS AND DISCUSSION Degradation of atrazine applied alone After application, the detected concentration of atrazine in the autoclaved soil during the 30-d incubation varied between about 83% and 118% of the initial concentration, which was comparable with the recovery of atrazine in freshly spiked soil of about 85% to 90.1% (data not shown). Conversely, the concentration of atrazine in the non-autoclaved RS decreased with incubation time after a short lag phase (Fig. 1a). After 47 d of incubation, approximately 30% of the added herbicide remained in the soil. The atrazine degradation kinetics in the non-rhizosphere soil was analogous to that of the rhizosphere soil (Fig. 1a), but it showed a decrease in degradation rate.
Fig. 1 Degradation kinetics of atrazine (a) and bentazone (b) in rhizosphere soil (RS) and non-rhizosphere soil (NRS). C0 and Ct are the initial herbicide concentration and the herbicide concentration at time t, respectively.
The revised first-order model used by Martins and Mermoud (1998) was found to well fit the degradation of atrazine in both RS and NRS (Table I). The short lag phases (tlag ) in RS and NRS may TABLE I Degradation parameters of atrazine and bentazone in soils with different treatments from the model ln(ct /c0 ) = −k(t−tlag ) and t1/2 = 0.693/k, where ct is the herbicide concentration at time t, c0 is the initial herbicide concentration, k is the first order degradation rate constant, tlag is the lag phase during which there is no apparent herbicide degradation, and t1/2 is the half-life Herbicide
Treatmenta)
k
Atrazine
RS NRS RSab RSabs RS NRS RSab RSabs
d−1 0.02694 0.02386 0.02249 0.02255 0.03432 0.03475 0.02744 0.03233
Bentazone
a) RS:
t1/2
tlag
r 2b)
2.0 0.0 6.7 3.2 0.0 6.8 6.6 4.2
0.99 0.97 0.99 0.95 0.98 0.95 0.97 0.96
d (0.00047)c) ad) (0.00213) b (0.00171) c (0.00339) c (0.00272) a (0.00568) a (0.00368) b (0.00430) a
25.7 29.1 30.8 30.7 20.2 19.9 25.2 21.4
rhizosphere soil; NRS: non-rhizosphere soil; ab: a mixture of atrazine and bentazone; abs: a mixture of atrazine, bentazone, and surfactant. b) Coefficient of determination. c) Values in parentheses are standard deviation. d) Means for a given herbicide followed by the same letter are not significantly different (P = 0.05) using t test.
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suggest that degrading microorganisms were initially present in the soil, and they were able to metabolize atrazine. This assumption was strongly supported by the fact that the degradation rate (k) of atrazine was significantly higher in RS than in NRS (Table I). The degradation in this study was also found to be more rapid than some measurements from other laboratories. For example, the half-lives obtained from this study were 25.7 and 29.1 d, respectively, whereas those reported by Martin-Neto et al. (1994) were from 1.5 months to 5 years. The difference may be caused by the different soil properties. Also, Chiarini et al. (1998) found that the plant development, cultivar, and soil type had an influence on the microbial colonization of plant roots. The faster degradation of atrazine in the non-autoclaved soil than in the autoclaved soil (Fig. 1a) indicated that biodegradation was involved. Atrazine degradation can occur via biotic and abiotic processes (Mandelbaum et al., 1993). N-Dealkylation, dechlorination, and ring cleavage are the major degradative processes for atrazine (Kruger et al., 1993; Houot et al., 1998). The dechlorination reaction is believed to be from a soil-catalyzed chemical process (Armstrong et al., 1967), but hydrolytic dechlorination by microbial consortia is also demonstrated (Mandelbaum et al., 1993). Forming of a bound residue is often observed when atrazine is incubated with soils for a long time (Kruger et al., 1993). The photodegradation and hydrolysis of atrazine could be prohibited owing to our experimental conditions. Although hydrolysis of atrazine is generally favored by extreme pH conditions and the autoclaved treatment can reduce the soil pH (Shaw et al., 1999), the pH used in this study was still not favorable for hydrolysis. It is generally accepted that sorption and a bound residue restrict the bioavailability of persistent organic pollutants in soils (Reid et al., 2000). Sorption of atrazine in soil is relatively weak and aqueous methanol solvent extraction can accurately determine the bioavailability of aging atrazine (Barriuso et al., 2004). Over a 30-d period of incubation, nearly all of the atrazine applied was extracted with aqueous methanol solvent from the autoclaved soil, thus suggesting that forming a bound residue contributed little or nothing to atrazine degradation in soils. All these implied that long-term incubation did not limit the bioavailability of atrazine, and that the biodegradation, at least in the initial phase of its degradation, was mainly responsible for atrazine degradation. Degradation of bentazone applied alone The decline of the total bentazone concentration in non-autoclaved RS, autoclaved RS, and NRS was similar to that of atrazine in the corresponding soils (Fig. 1b). Over a 30-d period of incubation, the concentration of bentazone in autoclaved RS remained at 80%–99% of the initial concentration, which was also comparable to the recovery of bentazone in the freshly spiked soil of about 95% to 105% (data not shown). It could also be concluded that biodegradation mainly caused the bentazone degradation. Eq. 1 was used to fit bentazone degradation in RS and NRS over time, and a good fit was obtained with r2 ranging from 0.95 to 0.98 (Table I). In NRS, a 6.8-d lag phase was observed in the degradation of bentazone, whereas no large lag phase was observed with RS. The degradation rate (k) of bentazone in RS was not significantly different from that in NRS (Table I). Therefore, no significant rhizosphere effect was observed in bentazone degradation, whereas a significant rhizosphere enhancement effect was found in atrazine degradation (Table I). The half-lives of bentazone in RS and NRS were 20.2 and 19.9 d, respectively. The calculated half-lives of this study, in most cases, were smaller than those reported in Gaston et al. (1996), which range from 38 to 88 d depending on the soils. The presence of a biostimulant, such as corn (Zea mays L.) residue, which can accelerate the degradation and mineralization of bentazone (Burauel and F¨ uhr, 1988), may have caused the relatively rapid degradation of bentazone in this study. Additionally, enzyme catalysis may also have contributed to the degradation of bentazone. For example, del Pilar Castillo et al. (2000) found that in a solid substrate fermentation system, bentazone was a substrate of pure lignin peroxidase and oxidized significantly faster as compared to the manganese peroxidase + Tween-80 system; however, phenols etc. may inhibit the activity of lignin peroxidase. Kim et al. (1997) observed that bentazone underwent apparent transformation by laccase or peroxidase in
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the presence of co-substrates such as humic monomer. Degradation of atrazine and bentazone applied in combination When atrazine and bentazone were applied together, a longer lag phase for the degradation was observed than with atrazine alone (Fig. 2a). Similar phenomenon was observed from bentazone degradation (Fig. 2b). When these two herbicides were applied in combination, the lag phase, using Eq. 1, for atrazine degradation increased from 2 to 6.7 d, and for bentazone degradation, it increased from 0 to 6.6 d (Table I). When compared to the mono-contaminant system, the degradation of each herbicide was slowed down and longer half-lives were observed in the binary-contaminant system.
Fig. 2 Degradation kinetics of atrazine (a) and bentazone (b) in the rhizosphere soil treated with single and combined herbicides. C0 and Ct are the initial herbicide concentration and the herbicide concentration at time t, respectively.
The above results indicated that the combination of bentazone and atrazine could result in a slight increase in the persistence of the individual herbicides in soil. The slower degradation rates in the binary-contaminant system can be caused by three potential interactive factors, toxicity, non-competitive inhibition, and competitive inhibition (Bielefeldt and Stensel, 1999). Since the two herbicides of this study were degraded in soils through different mechanisms, competitive inhibition had the least effect on the degradation of each herbicide. Thus, toxicity and non-competitive inhibition were presumed to be responsible for the decrease in the degradation rates of the pesticide in the mixture. This interpretation was consistent with soil respiration varying with the treatment as reported in Li et al. (2004). Surfactant effect on degradation of atrazine and bentazone applied in combination Pesticides are commonly applied as a formulation where the active ingredient can be present at higher rates in both solute and crystalline phases (Beigel et al., 1999). Formulation adjuvants may also affect the degradation of pesticides by modifying the availability of the compound; for example, some surfactants enhance the apparent solubility of weak water-soluble compounds and reduce their sorption to soils (Haigh, 1996). Thus, Tween-20, a nonionic surfactant, was selected in this study to investigate its effect on the degradation of atrazine and bentazone. Except for a shortened lag time, the degradation of atrazine in RS with bentazone + atrazine + Tween-20 showed no significant difference from that with bentazone + atrazine (Fig. 3a; Table I). However, bentazone had a shorter lag phase of degradation and a significantly more rapid degradation rate in RS with bentazone + atrazine + Tween-20 than in RS with bentazone + atrazine (Fig. 3b; Table I). The reasons why Tween-20 affected the degradation of atrazine and bentazone differently were still not clear. One possible reason could be that the surfactant enhanced the activity of bentazone degraders more than atrazine degraders. One previous study showed that the total CO2 release from RS at the beginning of incubation varied for different treatments in the order of atrazine + bentazone + Tween-20 > atrazine > atrazine + bentazone ≥ bentazone (Li et al., 2004). Nevertheless, further investigation is
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required to explain the effect of Tween-20.
Fig. 3 Degradation kinetics of atrazine (a) and bentazone (b) in the rhizosphere soil with binary (bentazone + atrazine) and ternary (bentazone + atrazine + Tween-20) contaminants. C0 and Ct are the initial herbicide concentration and the herbicide concentration at time t, respectively.
CONCLUSIONS When bentazone and atrazine were simultaneously applied to a soil at 10 mg kg−1 (dry soil) of each herbicide, the lag phases of their degradation were prolonged by 6.6 and 4.7 d, respectively, as compared with each herbicide alone. Their coexistence also resulted in a decrease in their degradation rate. Therefore, it was very important to prevent environmental pollution when these herbicides were used simultaneously. In the presence of a surfactant, Tween-20, in the bentazone + atrazine treated soil, there was a decrease in the lag phase of degradation of both herbicides, and the degradation rate of bentazone increased, whereas the degradation rate of atrazine remained unchanged. In addition, the rhizosphere effect significantly enhanced the degradation of atrazine, but had no significant effect on bentazone. REFERENCES Anderson, T. A., Guthrie, E. A. and Walton, B. T. 1993. Bioremediation in the rhizosphere. Environ. Sci. Technol. 27(13): 2 630–2 636. Armstrong, D. F., Chesters, G. and Harris, R. F. 1967. Atrazine hydrolysis in soil. Soil Sic. Soc. Am. Proc. 31: 61–66. Barriuso, E., Koskinen, W. C. and Sadowsky, M. J. 2004. Solvent extraction characterization of bioavailability of atrazine residues in soils. J. Agric. Food Chem. 52(21): 6 552–6 556. Beigel, C., Charnay, M. P. and Barriuso, E. 1999. Degradation of formulated and unformulated triticonazole fungicide in soil: Effect of application rate. Soil Biol. Biochem. 31: 525–534. Bielefeldt, A. R. and Stensel, H. D. 1999. Modeling competitive inhibition effects during biodegradation of BTEX mixtures. Wat. Res. 33(3): 707–714. Boesten, J. J. T. I. and van der Pas, L. J. T. 2000. Movement of water, bromide and the pesticides ethoprophos and bentazone in a sandy soil: The Vredepeel data set. Agric. Water Mgmt. 44: 21–42. Burauel, B. and F¨ uhr, F. 1988. The enhanced mineralization of simazine and bentazon in soil after plant uptake. J. Plant Nutr. Soil Sci. 151: 311–314. del Pilar Castillo, M., Ander, P., Stenstr¨ on, J. and Torstensson, L. 2000. Degradation of the herbicide bentazon as related to enzyme production by Phanerochaete chrysosporium in two solid substrate fermentation systems. World J. Microbiol. Biotechnol. 16: 289–295. Chiarini, L., Bevivino, A., Dalmastri, C., Nacamulli, C. and Tabacchioni, S. 1998. Influence of plant development, cultivar and soil type on microbial colonization of maize roots. Appl. Soil Ecol. 8: 11–18. Curl, E. A. and Truelove, B. 1986. The Rhizosphere. Springer-Verlag, Berlin and Heidelberg. 288pp. Feng, J. and Liu, T. 1990. Investigation on controlling weeds in cornfield by bentazone. Pesticides (in Chinese). 29(5): 59–61. Gaston, L. A., Locke, M. A. and Zablotowicz, R. M. 1996. Sorption and degradation of bentazon in conventional- and no-till Dundee soil. J. Environ. Qual. 25: 120–126. Haigh, S. D. 1996. A review of the interaction of surfactants with organic contaminants in soils. Sci. Total Environ. 185: 161–170.
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Houot, S., Barriuso, E. and Bergheaud, V. 1998. Modifications to atrazine degradation pathways in a loamy soil after addition of organic amendments. Soil Biol. Biochem. 30(14): 2 147–2 157. Kim, J. E., Wang, C. J. and Bollag, J. M. 1997. Interaction of reactive and inert chemicals in the presence of oxidoreductases: Reaction of the herbicide bentazon and its metabolites with humic monomers. Biodegradation. 8(6): 387–392. Kruger, E., Somasundaram, L. and Kanwar, R. S. 1993. Persistence and degradation of [14 C]atrazine and [14 C]deisopropylatrazine as affected by soil depth and moisture conditions. Environ. Toxicol. Chem. 12: 1 959–1 967. Lesan, H. M. and Bhandari, A. 2003. Atrazine sorption on surface soils: time-dependent phase distribution and apparent desorption hysteresis. Water Res. 37(7): 1 644–1 654. Li, K. B., Cai, X. Y. and Liu, W. P. 2004. Influences of single and combined herbicides on soil microbial activity. Journal of Agro-Environment Science (in Chinese). 23(2): 392–396. Li, K. B., Liu, W. P., Xu, D. M. and Lee, S. 2003. Influence of organic matter and pH on bentazone sorption in soils. J. Agric. Food Chem. 51(18): 5 362–5 366. Liu, W. P., Wang, Q. Q., Yang, W. C. and Wang, X. R. 2001. Adsorption of acetanilide herbicides on soil and its components: IV. Sorption of acetanilide herbicides on soils and its correlation with soil properties. Pedosphere. 11(3): 217–226. Mandelbaum, R. T., Wackett, L. P. and Allan, D. L. 1993. Rapid hydrolysis of atrazine to hydroxyatrazine by soil bacteria. Environ. Sci. Technol. 27(9): 1 943–1 946. Martin-Neto, L., Vieira, E. M. and Sposito, G. 1994. Mechanism of atrazine sorption by humic acid: A spectroscopic study. Environ. Sci. Technol. 28(11): 1 867–1 873. Martins, J. M. and Mermoud, A. 1998. Sorption and degradation of four nitroaromatic herbicides in mono- and multi-solute saturated/unsaturated soil batch systems. J. Contam. Hydrol. 33: 187–210. Reid, B. J., Jones, K. C. and Semple, K. T. 2000. Bioavailability of persistent organic pollutants in soils and sediments—a perspective on mechanisms, consequences and assessment. Environ. Pollut. 108: 103–112. Shaw, L. J., Beaton, Y., Glover, L. A., Killham, K. and Meharg, A. A. 1999. Re-inoculation of autoclaved soil as a non-sterile treatment for xenobiotic sorption and biodegradation. Appl. Soil Ecol. 11: 217–226. Tindall, J. A. and Vencill, W. K. 1995. Transport of atrazine, 2, 4-D, and dicamba through preferential flowpaths in an unsaturated claypan soil near Centralia, Missouri. J. Hydrol. 166(1–2): 37–59. Xing, B., Pignatello, J. J. and Gigliotti, B. 1996. Competitive sorption between atrazine and other organic compounds in soils and model sorbents. Environ. Sci. Technol. 30(8): 2 432–2 440.
Degradation of Herbicides Atrazine and Bentazone Applied Alone and in Combination in Soils∗1 LI Ke-Bin1 , CHENG Jing-Tao2 , WANG Xiao-Fang1 , ZHOU Ying3 and LIU Wei-Ping4 1 Department
of Chemistry, Northwest University, Xi’an 710069 (China). E-mail: [email protected] of Chemistry, Shaanxi Institute of Education, Xi’an 710061 (China) 3 Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032 (China) 4 Research Center of Green Chirality, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China) 2 Department
(Received July 25, 2007; revised January 18, 2008)
ABSTRACT The application of a mixture of bentazone (3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one-2,2-dioxide) and atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) is a practical approach to enhance the herbicidal effect. Laboratory incubation experiments were performed to study the degradation of bentazone and atrazine applied in combination and individually in maize rhizosphere and non-rhizosphere soils. After a lag phase, the degradation of each individual herbicide in the non-autoclaved soil could be adequately described using a first-order kinetic equation. During a 30-d incubation, in the autoclaved rhizosphere soil, bentazone and atrazine did not noticeably degrade, but in the non-autoclaved soil, they rapidly degraded in both non-rhizosphere and rhizosphere soils with half-lives of 19.9 and 20.2 d for bentazone and 29.1 and 25.7 d for atrazine, respectively. The rhizosphere effect significantly enhanced the degradation of atrazine, but had no significant effect on bentazone. These results indicated that biological degradation accounted for the degradation of both herbicides in the soil. When compared with the degradation of the herbicide applied alone, the degradation rates of the herbicides applied in combination in the soils were lower and the lag phase increased. With the addition of a surfactant, Tween-20, a reduced lag phase of degradation was observed for both herbicides applied in combination. The degradation rate of bentazone accelerated, whereas that of atrazine remained nearly unchanged. Thus, when these two herbicides were used simultaneously, their persistence in the soil was generally prolonged, and the environmental contamination potential increased. Key Words:
atrazine, bentazone, degradation, herbicide combination
Citation: Li, K. B., Cheng, J. T., Wang, X. F., Zhou, Y. and Liu, W. P. 2008. Degradation of herbicides atrazine and bentazone applied alone and in combination in soils. Pedosphere. 18(2): 265–272.
INTRODUCTION Atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) is one of the most widely applied herbicides for pre- and post-emergence control of broadleaf and grassy weeds in maize and other crops. Bentazone (3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one-2,2-dioxide) is widely used for postemergence control of sedges and broadleaf weeds. The combination of both herbicides not only broadens their herbicidal spectrum but also reduces their respective application dosage. The combined application is recommended as a promising post-emergence herbicidal activity for the control of weeds in cornfields (Feng and Liu, 1990). Application of herbicide mixtures to enhance the herbicidal effect is a common practice in agriculture. However, when multi-contaminants exist in soils at the same time, the behavior of individual component may be affected. The common ingredients in pesticide formulations are surfactants, which would have complex effects on the behavior of other contaminants in the soil because they can enhance the water solubility of the pollutants and act as a catalyzer during their degradation ∗1 Project
supported by the Natural Science Basic Research Plan in Shaanxi Province of China (No. 2004K03-G3) and the Scientific Research Fund of the Department of Education of Shaanxi Province, China (No. 04JK234).
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processes owing to their special surface properties (Haigh, 1996). Although there have been several studies on the movement (Tindall and Vencill, 1995; Boesten and van der Pas, 2000), sorption (Lesan and Bhandari, 2003; Gaston et al., 1996; Li et al., 2003), and degradation of atrazine and bentazone in soils (Kruger et al., 1993; Gaston et al., 1996), few studies have been conducted regarding their individual degradation among complex systems, such as atrazine + bentazone and atrazine + bentazone + surfactant. Natural soils are rarely contaminated with only one xenobiotic compound; several organic contaminants may exist simultaneously. Recent observations show that the presence of structurally similar molecules causes competitive adsorption of herbicides and affects their degradation rates (Xing et al., 1996; Martins and Mermoud, 1998). Moreover, microorganisms do not rely on a single compound for growth, but rather on a wide range of natural or anthropic C sources. Therefore, investigation of the behaviors of a pesticide in multi-contaminant systems appears to be more realistic since it accounts for synergistic and antagonistic effects that may greatly affect the fate of a pollutant in the soil. The rhizosphere, the area in the immediate vicinity of plant roots, is a zone of intense microbial activity, which is caused by root exudates containing carbohydrates, amino acids, and organic acids (Curl and Truelove, 1986). It is likely that the rhizosphere has the potential to accelerate biodegradation of organic pollutants. Recently, there has been an increasing interest in the degradation of pollutants in rhizosphere soils. Several studies have found that organic contaminants generally disappear more quickly in planted soils than in unplanted soils, suggesting that vegetation has an important effect on the fate of pesticides in soils (Anderson et al., 1993). The objectives of the present study were to explore the effect of application of atrazine and bentazone in combination on the degradation of each individual herbicide, to evaluate the effect of a low-concentration surfactant on the degradation of the herbicides, and to investigate the rhizosphere effect on the degradation of the herbicides. MATERIALS AND METHODS Soil sampling and chemicals Rhizosphere and non-rhizosphere soils were collected from the agricultural field at Huajiachi Campus, Zhejiang University, Zhejiang Province, China. The soil in the field is a silty clay loam soil (241.8 g kg−1 clay, 5.3 g kg−1 sand, and 753 g kg−1 silt) with pH 6.1 and organic matter content of 36.4 g kg−1 . The sampling site was planted to corn for at least three consecutive years with no application of a combined formulation of atrazine and bentazone. High-performance liquid chromatography (HPLC) analysis of the soil extracts prior to the start of the study showed no detectable residue of bentazone and atrazine. At harvest, rhizospheres containing as much as possible associated roots were excavated by digging to a 20 cm depth around maize plants. Non-rhizosphere soil (NRS) samples were taken from the same site containing few roots. The rhizosphere, non-rhizosphere soil, and plant samples were placed in plastic bags and immediately transported to the laboratory and prepared within a few hours. Soil loosely adhering to the roots was removed by mild shaking. The remaining adherent soil, called rhizosphere soil (RS), was manually separated and collected in a bucket. Soil samples were sieved to 2 mm and then stored at 4 ◦ C for no more than 14 d prior to initiation of the study. A part of the soil samples was air-dried for 2 d at room temperature for property analysis using the procedures adopted by Liu et al. (2001). Bentazone (≥ 97%) was obtained from BASF (Ludwigshafen, Germany). Atrazine (≥ 99%) was purchased from Chemical Service (West Chester, USA). A nonionic surfactant, Tween-20 (chemically pure), was purchased from the Mingqing Chemical Reagent Plant (Wenzhou, China). Incubation experiments For investigating the degradation of the herbicides applied individually or in combination in the non-autoclaved RS and NRS, methanol solutions containing atrazine or bentazone or both of them were
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spiked with a portion of 25.0 g (dry weight) microbial-inactive soil which was sieved to 0.1 mm and dried at 110 ◦ C overnight. After evaporation of methanol and thorough mixing, the spiked portion was incorporated into an aliquot of 1 500 g RS (174 g kg−1 moisture content) or an aliquot of 1 500 g NRS (141 g kg−1 moisture content) at 5.03 g spiked portion per kg soil (dry weight). The final concentration of each herbicide was controlled at 10 mg kg−1 soil (dry weight), which was equivalent to 10 times the recommended application rate of the combined herbicide. To investigate the effect of surfactant, 0.4 g Tween-20 was also added together with the spiked portion, with an initial concentration of 1.1 g kg−1 in the aqueous phase. The soil moisture of all treatments was adjusted to 290 g kg−1 with the soil sieved several times to mix thoroughly. The homogenized samples were placed in six 2-L beakers and immediately sampled as day 0. The beakers were then covered with plastic film, which was pierced with a needle to allow air exchange, and incubated in the dark at 25 ◦ C. During the incubation period, the beakers were removed from the incubator every other day and brought to the original moisture content (290 g kg−1 ) by adding the required amount of sterile distilled water. At intervals of 2, 4, 6, 13, 21, 26, 36, and 47 days, duplicates of about 30 g soil from each treatment were sampled, weighed in a 50-mL high-density polyethylene centrifuge tube with screw caps, and then immediately stored at −20 ◦ C until analysis. The degradation of atrazine and bentazone alone in autoclaved RS was also investigated. Fifty grams (dry weight) RS was weighed in 100-mL Erlenmeyer flasks. After being plugged with cotton pads, the flasks were autoclaved at 120 ◦ C for 30 min each day in three consecutive days to remove microbial activity. Atrazine or bentazone 0.5 mg each was added to the autoclaved soil in 1 mL of methanol. Eleven flasks for each herbicide were incubated under the same conditions as described above, except that the spiking was performed in a laminar hood. At day 0.7, 3, 4, 6, 9, 12, 15, 17, 20, 26, and 30, one flask of each herbicide treated soil was taken out and stored at −20 ◦ C. Soil extraction and analysis At the end of the incubation, the frozen samples were thawed at room temperature. After transferring about 30 g of moist soil to 100 mL Erlenmeyer flasks, soil samples were extracted three times by shaking for 2 h with a 40 mL methanol and water mixture (9:1, v/v) each time. The combined extracts were concentrated in a rotary evaporator below 45 ◦ C under reduced pressure to remove the methanol fraction. The residual aqueous extracts were repartitioned three times with 20 mL dichloromethane (20 mL × 3). When bentazone was extracted with dichloromethane, the aqueous extract was adjusted to pH < 3.0. The dichloromethane fractions were pooled, passed through anhydrous sodium sulfate, and further concentrated to near dryness in a rotary evaporator. Then, N2 was used to dry the extract, and the residue was reconstituted in a 10 mL mobile phase composed of 66:44 methanol:water (pH adjusted to 2.8) for analysis on a Shimadzu HPLC equipped with a photodiode array detector (Kyoto, Japan). A YWG C18 reversed-phase column (10 µm; 4.6 mm × 250 mm) from Dalian Elite Analytical Instruments Co., Ltd., China, was used for separation. The detection wavelength was 218 nm and the flow rate was 0.8 mL min−1 . The injection volume was 10 µL. The HPLC detection limit for bentazone was 5.9 µg L−1 , and for atrazine, it was 3.8 µg L−1 . The revised first-order model used by Martins and Mermoud (1998) in their study was employed to analyze the herbicide degradation kinetics in this study: ln(ct /c0 ) = −k(t − tlag )
(1)
t1/2 = 0.693/k
(2)
where ct is the concentration of the herbicide at time t, c0 is the initial concentration of the herbicide, k is the first order degradation rate constant, tlag is the lag phase during which there is no apparent herbicide degradation, and t1/2 is the half-life. The parameter estimates were obtained for each degradation condition using linear regression. Regression analyses were performed with Origin 6.0. All
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experimental data used in the regression analysis were the mean value of duplicates except for those from the degradation of atrazine and bentazone in the autoclaved soil, which were single points without replicates. A t test on k was conducted to detect the differences in the effect of the incubation conditions on the pesticide degradation rates. RESULTS AND DISCUSSION Degradation of atrazine applied alone After application, the detected concentration of atrazine in the autoclaved soil during the 30-d incubation varied between about 83% and 118% of the initial concentration, which was comparable with the recovery of atrazine in freshly spiked soil of about 85% to 90.1% (data not shown). Conversely, the concentration of atrazine in the non-autoclaved RS decreased with incubation time after a short lag phase (Fig. 1a). After 47 d of incubation, approximately 30% of the added herbicide remained in the soil. The atrazine degradation kinetics in the non-rhizosphere soil was analogous to that of the rhizosphere soil (Fig. 1a), but it showed a decrease in degradation rate.
Fig. 1 Degradation kinetics of atrazine (a) and bentazone (b) in rhizosphere soil (RS) and non-rhizosphere soil (NRS). C0 and Ct are the initial herbicide concentration and the herbicide concentration at time t, respectively.
The revised first-order model used by Martins and Mermoud (1998) was found to well fit the degradation of atrazine in both RS and NRS (Table I). The short lag phases (tlag ) in RS and NRS may TABLE I Degradation parameters of atrazine and bentazone in soils with different treatments from the model ln(ct /c0 ) = −k(t−tlag ) and t1/2 = 0.693/k, where ct is the herbicide concentration at time t, c0 is the initial herbicide concentration, k is the first order degradation rate constant, tlag is the lag phase during which there is no apparent herbicide degradation, and t1/2 is the half-life Herbicide
Treatmenta)
k
Atrazine
RS NRS RSab RSabs RS NRS RSab RSabs
d−1 0.02694 0.02386 0.02249 0.02255 0.03432 0.03475 0.02744 0.03233
Bentazone
a) RS:
t1/2
tlag
r 2b)
2.0 0.0 6.7 3.2 0.0 6.8 6.6 4.2
0.99 0.97 0.99 0.95 0.98 0.95 0.97 0.96
d (0.00047)c) ad) (0.00213) b (0.00171) c (0.00339) c (0.00272) a (0.00568) a (0.00368) b (0.00430) a
25.7 29.1 30.8 30.7 20.2 19.9 25.2 21.4
rhizosphere soil; NRS: non-rhizosphere soil; ab: a mixture of atrazine and bentazone; abs: a mixture of atrazine, bentazone, and surfactant. b) Coefficient of determination. c) Values in parentheses are standard deviation. d) Means for a given herbicide followed by the same letter are not significantly different (P = 0.05) using t test.
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suggest that degrading microorganisms were initially present in the soil, and they were able to metabolize atrazine. This assumption was strongly supported by the fact that the degradation rate (k) of atrazine was significantly higher in RS than in NRS (Table I). The degradation in this study was also found to be more rapid than some measurements from other laboratories. For example, the half-lives obtained from this study were 25.7 and 29.1 d, respectively, whereas those reported by Martin-Neto et al. (1994) were from 1.5 months to 5 years. The difference may be caused by the different soil properties. Also, Chiarini et al. (1998) found that the plant development, cultivar, and soil type had an influence on the microbial colonization of plant roots. The faster degradation of atrazine in the non-autoclaved soil than in the autoclaved soil (Fig. 1a) indicated that biodegradation was involved. Atrazine degradation can occur via biotic and abiotic processes (Mandelbaum et al., 1993). N-Dealkylation, dechlorination, and ring cleavage are the major degradative processes for atrazine (Kruger et al., 1993; Houot et al., 1998). The dechlorination reaction is believed to be from a soil-catalyzed chemical process (Armstrong et al., 1967), but hydrolytic dechlorination by microbial consortia is also demonstrated (Mandelbaum et al., 1993). Forming of a bound residue is often observed when atrazine is incubated with soils for a long time (Kruger et al., 1993). The photodegradation and hydrolysis of atrazine could be prohibited owing to our experimental conditions. Although hydrolysis of atrazine is generally favored by extreme pH conditions and the autoclaved treatment can reduce the soil pH (Shaw et al., 1999), the pH used in this study was still not favorable for hydrolysis. It is generally accepted that sorption and a bound residue restrict the bioavailability of persistent organic pollutants in soils (Reid et al., 2000). Sorption of atrazine in soil is relatively weak and aqueous methanol solvent extraction can accurately determine the bioavailability of aging atrazine (Barriuso et al., 2004). Over a 30-d period of incubation, nearly all of the atrazine applied was extracted with aqueous methanol solvent from the autoclaved soil, thus suggesting that forming a bound residue contributed little or nothing to atrazine degradation in soils. All these implied that long-term incubation did not limit the bioavailability of atrazine, and that the biodegradation, at least in the initial phase of its degradation, was mainly responsible for atrazine degradation. Degradation of bentazone applied alone The decline of the total bentazone concentration in non-autoclaved RS, autoclaved RS, and NRS was similar to that of atrazine in the corresponding soils (Fig. 1b). Over a 30-d period of incubation, the concentration of bentazone in autoclaved RS remained at 80%–99% of the initial concentration, which was also comparable to the recovery of bentazone in the freshly spiked soil of about 95% to 105% (data not shown). It could also be concluded that biodegradation mainly caused the bentazone degradation. Eq. 1 was used to fit bentazone degradation in RS and NRS over time, and a good fit was obtained with r2 ranging from 0.95 to 0.98 (Table I). In NRS, a 6.8-d lag phase was observed in the degradation of bentazone, whereas no large lag phase was observed with RS. The degradation rate (k) of bentazone in RS was not significantly different from that in NRS (Table I). Therefore, no significant rhizosphere effect was observed in bentazone degradation, whereas a significant rhizosphere enhancement effect was found in atrazine degradation (Table I). The half-lives of bentazone in RS and NRS were 20.2 and 19.9 d, respectively. The calculated half-lives of this study, in most cases, were smaller than those reported in Gaston et al. (1996), which range from 38 to 88 d depending on the soils. The presence of a biostimulant, such as corn (Zea mays L.) residue, which can accelerate the degradation and mineralization of bentazone (Burauel and F¨ uhr, 1988), may have caused the relatively rapid degradation of bentazone in this study. Additionally, enzyme catalysis may also have contributed to the degradation of bentazone. For example, del Pilar Castillo et al. (2000) found that in a solid substrate fermentation system, bentazone was a substrate of pure lignin peroxidase and oxidized significantly faster as compared to the manganese peroxidase + Tween-80 system; however, phenols etc. may inhibit the activity of lignin peroxidase. Kim et al. (1997) observed that bentazone underwent apparent transformation by laccase or peroxidase in
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the presence of co-substrates such as humic monomer. Degradation of atrazine and bentazone applied in combination When atrazine and bentazone were applied together, a longer lag phase for the degradation was observed than with atrazine alone (Fig. 2a). Similar phenomenon was observed from bentazone degradation (Fig. 2b). When these two herbicides were applied in combination, the lag phase, using Eq. 1, for atrazine degradation increased from 2 to 6.7 d, and for bentazone degradation, it increased from 0 to 6.6 d (Table I). When compared to the mono-contaminant system, the degradation of each herbicide was slowed down and longer half-lives were observed in the binary-contaminant system.
Fig. 2 Degradation kinetics of atrazine (a) and bentazone (b) in the rhizosphere soil treated with single and combined herbicides. C0 and Ct are the initial herbicide concentration and the herbicide concentration at time t, respectively.
The above results indicated that the combination of bentazone and atrazine could result in a slight increase in the persistence of the individual herbicides in soil. The slower degradation rates in the binary-contaminant system can be caused by three potential interactive factors, toxicity, non-competitive inhibition, and competitive inhibition (Bielefeldt and Stensel, 1999). Since the two herbicides of this study were degraded in soils through different mechanisms, competitive inhibition had the least effect on the degradation of each herbicide. Thus, toxicity and non-competitive inhibition were presumed to be responsible for the decrease in the degradation rates of the pesticide in the mixture. This interpretation was consistent with soil respiration varying with the treatment as reported in Li et al. (2004). Surfactant effect on degradation of atrazine and bentazone applied in combination Pesticides are commonly applied as a formulation where the active ingredient can be present at higher rates in both solute and crystalline phases (Beigel et al., 1999). Formulation adjuvants may also affect the degradation of pesticides by modifying the availability of the compound; for example, some surfactants enhance the apparent solubility of weak water-soluble compounds and reduce their sorption to soils (Haigh, 1996). Thus, Tween-20, a nonionic surfactant, was selected in this study to investigate its effect on the degradation of atrazine and bentazone. Except for a shortened lag time, the degradation of atrazine in RS with bentazone + atrazine + Tween-20 showed no significant difference from that with bentazone + atrazine (Fig. 3a; Table I). However, bentazone had a shorter lag phase of degradation and a significantly more rapid degradation rate in RS with bentazone + atrazine + Tween-20 than in RS with bentazone + atrazine (Fig. 3b; Table I). The reasons why Tween-20 affected the degradation of atrazine and bentazone differently were still not clear. One possible reason could be that the surfactant enhanced the activity of bentazone degraders more than atrazine degraders. One previous study showed that the total CO2 release from RS at the beginning of incubation varied for different treatments in the order of atrazine + bentazone + Tween-20 > atrazine > atrazine + bentazone ≥ bentazone (Li et al., 2004). Nevertheless, further investigation is
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required to explain the effect of Tween-20.
Fig. 3 Degradation kinetics of atrazine (a) and bentazone (b) in the rhizosphere soil with binary (bentazone + atrazine) and ternary (bentazone + atrazine + Tween-20) contaminants. C0 and Ct are the initial herbicide concentration and the herbicide concentration at time t, respectively.
CONCLUSIONS When bentazone and atrazine were simultaneously applied to a soil at 10 mg kg−1 (dry soil) of each herbicide, the lag phases of their degradation were prolonged by 6.6 and 4.7 d, respectively, as compared with each herbicide alone. Their coexistence also resulted in a decrease in their degradation rate. Therefore, it was very important to prevent environmental pollution when these herbicides were used simultaneously. In the presence of a surfactant, Tween-20, in the bentazone + atrazine treated soil, there was a decrease in the lag phase of degradation of both herbicides, and the degradation rate of bentazone increased, whereas the degradation rate of atrazine remained unchanged. In addition, the rhizosphere effect significantly enhanced the degradation of atrazine, but had no significant effect on bentazone. REFERENCES Anderson, T. A., Guthrie, E. A. and Walton, B. T. 1993. Bioremediation in the rhizosphere. Environ. Sci. Technol. 27(13): 2 630–2 636. Armstrong, D. F., Chesters, G. and Harris, R. F. 1967. Atrazine hydrolysis in soil. Soil Sic. Soc. Am. Proc. 31: 61–66. Barriuso, E., Koskinen, W. C. and Sadowsky, M. J. 2004. Solvent extraction characterization of bioavailability of atrazine residues in soils. J. Agric. Food Chem. 52(21): 6 552–6 556. Beigel, C., Charnay, M. P. and Barriuso, E. 1999. Degradation of formulated and unformulated triticonazole fungicide in soil: Effect of application rate. Soil Biol. Biochem. 31: 525–534. Bielefeldt, A. R. and Stensel, H. D. 1999. Modeling competitive inhibition effects during biodegradation of BTEX mixtures. Wat. Res. 33(3): 707–714. Boesten, J. J. T. I. and van der Pas, L. J. T. 2000. Movement of water, bromide and the pesticides ethoprophos and bentazone in a sandy soil: The Vredepeel data set. Agric. Water Mgmt. 44: 21–42. Burauel, B. and F¨ uhr, F. 1988. The enhanced mineralization of simazine and bentazon in soil after plant uptake. J. Plant Nutr. Soil Sci. 151: 311–314. del Pilar Castillo, M., Ander, P., Stenstr¨ on, J. and Torstensson, L. 2000. Degradation of the herbicide bentazon as related to enzyme production by Phanerochaete chrysosporium in two solid substrate fermentation systems. World J. Microbiol. Biotechnol. 16: 289–295. Chiarini, L., Bevivino, A., Dalmastri, C., Nacamulli, C. and Tabacchioni, S. 1998. Influence of plant development, cultivar and soil type on microbial colonization of maize roots. Appl. Soil Ecol. 8: 11–18. Curl, E. A. and Truelove, B. 1986. The Rhizosphere. Springer-Verlag, Berlin and Heidelberg. 288pp. Feng, J. and Liu, T. 1990. Investigation on controlling weeds in cornfield by bentazone. Pesticides (in Chinese). 29(5): 59–61. Gaston, L. A., Locke, M. A. and Zablotowicz, R. M. 1996. Sorption and degradation of bentazon in conventional- and no-till Dundee soil. J. Environ. Qual. 25: 120–126. Haigh, S. D. 1996. A review of the interaction of surfactants with organic contaminants in soils. Sci. Total Environ. 185: 161–170.
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