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Water Research Institute-CNR, via Reno 1, Roma, Italy
* Corresponding author (guzzella{at}irsa.rm.cnr.it)
Received for publication April 18, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: BTC, breakthrough curve • DDA, desethyl-deisopropylatrazine • DDAH, desethyldeisopropyl-atrazine-2-hydroxide • DEAH, desethyl-atrazine-2-hydroxide • DET, desethylterbuthylazine • DETH, deethylterbuthylazine-2-hydroxide • DIA, deisopropylatrazine • DIAH, deisopropylatrazine-2-hydroxide • TER, terbuthylazine • TH, terbuthylazine-2-hydroxide • TP, transformation product
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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In recent experiments (Dousset et al., 1997), degradation of [14C]terbuthylazine was investigated in laboratory studies with different topsoil to evaluate which soil characteristics would favor metabolite formation. After a 45-d incubation period, terbuthylazine concentration quickly decreased by 30%, whereas desethylterbuthylazine was simultaneously formed. The extrapolated terbuthylazine half-life varied between 88 and 116 d in different soils. The author proposed, according to other studies (Hance, 1970), that terbuthylazine is more readily adsorbed by soil than atrazine, and that this might protect the molecule from microbial attack. According to studies by Dousset et al. (1997), the prominent dealkylation product of terbuthylazine is DET. In comparison with DET, the deterbuthylated compound, deisopropylatrazine [DIA; 6-chloro-N-ethyl-(1,3,5)-triazine-2,4-diamine], is less concentrated. Thurman et al. (1994) hypothesized that the rate of deethylation is two to three times faster than the deisopropylation rate. Deethyldeisopropylatrazine [DDA; 6-chloro-(1,3,5)-triazine-2,4-diamine], produced by DIA deethylation or by DET deterbuthylation, was rarely detected (Winkelmann and Klaine, 1991a; Di Corcia et al., 1999). This compound has the highest capacity of binding to soil and the highest mineralization rate compared with the other metabolites (Winkelmann and Klaine, 1991a).
Dealkylated products can be considered hazardous contaminants for ground water pollution as they are generally more persistent and water soluble than the parent compound. On the contrary, hydroxylated compounds have lower solubility, preferentially accumulate in the first 10 cm of the soil layer, and therefore may be considered less potentially polluting for ground water. Hydroxylation is considered to be one of the major atrazine degradation pathways (Winkelmann and Klaine, 1991b; Jones et al., 1982).
In our study, terbuthylazine degradation pathways and its main metabolite formation were evaluated in different pluviometric situations, using suction cup lysimeters under open field conditions, to assess the influence of pluviometric condition in metabolite formation and mobilization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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A vacuum pump (Millipore, Bedford, MA) was used to establish a 15-kPa pressure in the lysimeter cups for collection of pore water samples. Manometer-type tensiometers were installed at the same depth as the lysimeter cups to control water saturation conditions of the different soil horizons. A 15-m-deep piezometer, bored between 3 and 15 m, was installed 2 m downstream from the plot. Ground water samples were collected with a submergible electric pump (7.6-cm-o.d. JS4/08; GEO Impianti, Bergamo, Italy). A manual pluviometer was also installed to measure precipitation events during the experiments. The rainfall events were recorded every day.
The bacterial concentration (cell number/g soil) was evaluated by collecting four soil samples (10 kg soil collected from the 5- to 15-cm soil layer) in March, June, and November 1999 and February 2000 near the experimental plot. Soil organic carbon content and temperature at 5 and 15 cm (mean values are shown in Table 2) were also recorded in the same samples. Organic carbon content was determined by back titration after oxidation with potassium dichromate in the presence of sulfuric acid (Walkley and Black, 1934).
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Experimental Conditions and Herbicide Application
Three experimental trials were performed in different hydraulic conditions to assess soil humidity and permeability influence on terbuthylazine degradation pathways. Herbicide and transformation product (TP) concentrations in water leachates and in soil were analyzed before every new experiment to avoid cross contamination between experiments.
The experimental field was decorticated and irrigated with 300 L of water (corresponding to a 40% topsoil humidity) before the herbicide application, to reduce the presence of clay crack in topsoil. Water preferential flow transport is typical of arid topsoil conditions and particularly critical for herbicide leaching and ground water contamination.
In all the experiments, herbicide application was performed by dissolving 112 g of PRIMAGRAM TZ in 600 L of water, making 28 mg/L terbuthylazine concentration. The commercial formulate PRIMAGRAM TZ (Ciba Specialty Chemicals, Basel, Switzerland) contains 15% terbuthylazine. The herbicide solution was applied with a water reservoir with a tube irrigation system to obtain a good uniformity in herbicide distribution on the plot. The different pluviometric simulations were performed one hour after the herbicide application, allowing the herbicide solution to penetrate in topsoil. Potassium chloride salt (1.5 g/L solution) was used in the first and second experiment as a tracer to study the mobility of nonreactive compounds transported downward by water flow.
The first experiment, with a constant 3-cm water head throughout the experiment, started on 9 July 1996 and was concluded at the end of August 1996. Topsoil water saturation was checked with the 30-cm tensiometer. The added water amount varied between 600 and 1200 L/d. The second experiment started on 16 June 1998 and was concluded at the end of July 1998 and simulated intermittent pluviometric conditions (30 mm/d of rain for 21 d) with a water drop raining system. Two 300-L rain events were simulated each day (with a flow of about 15 mm/h), one in the morning and the other in the afternoon.
The third experiment was performed from 10 May to 18 Nov. 1999, without an artificial water supply, except for the 600-L solution used for terbuthylazine application.
Considering the first 20 d since the herbicide application, the total amount of water added was 900 mm for the first experiment, 600 mm for the second, and 50 mm for the third. Natural precipitation events were recorded by the installed pluviometer.
Sample Collection, Extraction, and Analysis
Analytical reference standards of terbuthylazine (TER), DET, terbuthylazine-2-hydroxide [TH; 4-terbutylamino-6-ethylamino-(1,3,5)-triazine-2-OH], DIA, DDA, DETH, deethyl-atrazine-2-hydroxide [DEAH; 4-amino-6-isopropylamino-(1,3,5)-triazine-2-OH], deisopropylatrazine-2-hydroxide [DIAH; 4-amino-6-ethylamino-(1,3,5)-triazine-2-OH], and deethyldeisopropyl-atrazine-2-hydroxide [DDAH; 4,6-diamino-(1,3,5)-triazine-2-OH] were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) with a certified purity greater than 95%. Standard solutions (0.05, 0.1, 0.5, 1, and 5 mg/L) were prepared for high-performance liquid chromatography (HPLC) calibration in 25 mM KH2PO4. Methyl alcohol for pesticide residue analysis and HPLC, acetonitrile for HPLC, and ethyl acetate ACS for analysis were obtained from Carlo Erba (Milan, Italy). Chloride concentrations were determined by portable cuvette kits (Dr. Bruno Lange GmbH, Düsseldorf, Germany) with a 5 mg/L detection limit.
Leachate samples were collected by suction cup lysimeters in dark glass bottles (500 mL), while ground water samples were collected in 1-L dark glass bottles after 15 min of piezometer purging. All samples were kept at 4°C after their collection until laboratory analysis. Terbuthylazine and deethylterbuthylazine extraction from water was performed by eluting the water through Lichrolut EN 200-mg solid-phase extraction (SPE) cartridges (Merck, Darmstadt, Germany) (Pozzoni and Guzzella, 2000). The extract was concentrated to <1 mL by a gentle N2 flux and recovered to 1 mL or 0.25 mL in ethyl acetate, depending on the expected concentrations, for gas chromatography–nitrogen and phosphorous detector (GC–NPD) analysis. The extraction of the other TPs was performed on 0.5-g Carbograph SPE cartridges (Alltech Associates, Deerfield, IL) (Di Corcia and Marchetti, 1991; Bonfanti et al., 1998). The extract was concentrated to 10 mL by a N2 gentle flux and recovered to 1 mL with a 25 mM KH2PO4 solution.
Soil was sampled by pushing a 120-cm iron tube (5-cm i.d.) into the ground. Six soil sample cores were collected at different times after the herbicide application and divided into seven subsamples; only the inner part of the sample was kept to avoid contamination. All soil samples were frozen in sealed dark glass bottles and stored at -20°C. For the pesticide determination, the soil sample was defrosted and dehydrated under air conditions and ambient temperature (20–22°C) for 96 h. The water content of the air-dried soil sample was determined by drying 10 g of soil in a stove at 105°C for 24 h. The dry sample was sieved with a 2-mm sieve. The extraction procedure was performed according to the method developed by Guzzella et al. (1996), briefly described here: 10 to 50 g of air-dried sifted soil sample was extracted with pesticide free–grade methanol in a Soxhlet extraction apparatus for 8 h, and the extract was cleaned by passing it through a diatom power-filled column.
Terbuthylazine and desethylterbuthylazine determination was performed on a HRGC 5160 Mega Series (Carlo Erba) with an NPD 80-FL phosphorus–nitrogen detector, TS-2 ionic source, and Sil13-Chrompack capillary column (50-m length x 0.25-mm i.d., 0.2- to 0.25-µm film thickness; Varian, Palo Alto, CA) (Guzzella et al., 1996). Quantification employed an external standard method. Limit of detection was 0.005 µg/L for each compound when concentrating 1 L of the water sample to 0.25 mL.
The hydroxylated derivatives, DIA and DDA were identified with a DAD 1050 high-performance liquid chromatograph (HP, Palo Alto, CA) with an autosampler (HP), a Hicarbosphere 5 ODS-H column (250-mm length x 4.6-mm i.d.) (Mallinckrodt Baker, Phillipsburg, NJ), and a Lichrosorb RP8 (Merck) pre-column. Desethyl-deisopropylatrazine, DEAH, DIAH, DETH, and DDAH were quantified by the UV detector at 205 nm, DIA at 210 nm, and TH at 240 nm. Limits of detection of 1 L of the water sample concentrated to 1 mL were: 0.01 µg/L for DIAH, DDA, DIA, and TH; 0.03 µg/L for DEAH and DETH; and 0.05 µg/L for DDAH. All the analyses were conducted in duplicate and a standard deviation lower than 10% was accepted.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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In all the three experiments (Fig. 1), after the first few days, herbicide concentrations were lower at the 30- than the 90-cm depth. This can be explained on the basis of a rich clay content in the soil horizon between depths of 70 and 110 cm, which could have acted as a trap, capturing herbicide residuals leaching from the topsoil to the ground water.
In the first experiment, at both the 30- and 90-cm depths, BTCs showed only one main concentration peak with some relatively minor peaks. The very sharp peaks, which increased and decreased within a few hours, showed that preferential transport prevailed over matrix flow, that is, the herbicide moved mainly via macropore transport with low interaction with the topsoil horizon. On the contrary, in the other two experiments TER transport seemed to be driven mainly by matrix flow interactions and precipitation events. In the second experiment, BTCs showed many relatively high concentration peaks, indicating that herbicide transport had an impulsive behavior tightly bound to water transport in the unsaturated zone. At the 30-cm depth, the breakthrough curve had a similar trend to that in the first experiment and the terbuthylazine peak also was delimited in a short period of time. In the third experiment, BTCs showed many secondary peaks with similar concentrations at both 30- and 90-cm depths. The observed trend can be related to the natural precipitation events, that is, rainfall that happened mostly after the 80th day (as shown in Fig. 2) .
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With regard to ground water trend, in the first experiment one main terbuthylazine peak was determined just hours after herbicide application, reaching a concentration four times greater than the background value, while DET concentration seemed to increase during the first study, reaching 0.1 µg/L at the end of the experiment. In the other two experiments, no variation compared with the background value (0.05 µg/L) was observed.
Leaching of Transformation Products
In all the experiments, the presence of terbuthylazine TPs in water samples collected from lysimeters was monitored.
In the first experiment DET was detected at both depths (Fig. 4A) , whereas the other TPs were measured only at the 90-cm depth. At the 30-cm depth, DET was determined since the 10th day (Fig. 4A) and its maximum concentration was reached on the 22nd day. The DET concentration curve at the 90-cm depth (Fig. 5A) had the same trend as shown at 30 cm but the highest concentration was twice as large, indicating that this compound has a faster leaching behavior, compared with its parental compound. The presence of DET at 90 cm was mainly dominated by its leaching from topsoil rather than an in situ biodegradation. In this experiment, TH was the second-most abundant metabolite, showing the maximum concentration on the 43rd day (Fig. 5B) and then decreasing progressively. Terbuthylazine-2-hydroxide is a lypophilic compound that is rarely detectable in water samples and is generally absorbed by organic-rich soil. Deethylterbuthylazine-2-hydroxide, formed by DET hydroxylation or TH deethylation, behaved like TH, suggesting that DETH is mainly derived from TH degradation. Deisopropylatrazine, the deterbuthylated metabolite, was scarcely found in this experiment. Its formation is generally limited by steric hindrance of the ter-buthyl substituent and, furthermore, it degrades rapidly to DIAH and DDA. Deisopropylatrazine-2-hydroxide was measured in concentrations greater than those of DIA, demonstrating that DIA was generally underestimated because of its low persistence. Desethyl-deisopropylatrazine, formed by degradation of both DIA and DETH, showed an accumulative behavior: its concentration was still increasing at the end of the experiment.
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In the second experiment, the breakthrough curves at 30 and 90 cm (Fig. 4B and 5C) showed an early appearance of DET just after a few days after herbicide application; concentrations 10 times greater compared with the first experiment were measured and the maximum concentration peaks occurred later at both depths. The other TPs (Fig. 4C, 4D, and 5B) also reached their maximum concentration peaks later than in the first experiment. Deethylterbuthylazine-2-hydroxide and DDA were the main metabolites observed after DET. At the 30-cm depth, the highest concentration was measured between the 72nd and 79th days both for DET and other TPs, while at the 90-cm depth metabolite maximum cumulative concentration was reached on the 101st day. The phenomenon might be related to natural precipitation events that occurred after the simulated rain. At the 90-cm depth, TP concentrations were 2.5 times higher than in the first experiment, due to the lower dilution of the metabolites compared with the first experiment.
The TP concentrations in water samples from the third experiment were higher than in the other two experiments, since the dilution effect was less relevant and degradation processes were favored (Fig. 4E, 4F, 4G, 5E, and 5F). The DET maximum concentration at the 30-cm depth occurred on the 37th day, corresponding to a natural precipitation event (30 mm of rain; Fig. 5E). At the 90-cm depth, DET showed a breakthrough curve with many peaks: the first one was in agreement with the one measured at 30 cm. In this experiment, DET concentrations were 70 and 10 times greater than in the first experiment at 30- and 90-cm depths, respectively. Terbuthylazine-2-hydroxide, at the 30-cm depth, was detected irregularly (as shown in Fig. 4G), only when rain events allowed its solubilization. In contrast, DETH was detected throughout the whole experiment with relatively high peaks, reaching the maximum concentration on the 190th day. The DETH peak measured on the 37th day could be related to DET degradation, while TH degradation could have played an important role in the DETH formation later on (Fig. 4G). Desethyl-deisopropylatrazine, together with DIA and DIAH, showed the maximum cumulative concentration on the 46th day and other two peaks on the 175th and 202nd, which could be related to DETH. At the 90-cm depth (Fig. 5E and 5F) TH was irregularly detected, while DETH showed a trend similar to the one showed at 30 cm, but with an accumulative behavior, reaching its maximum concentration at the end of the third experiment.
This research showed that TP concentrations in water samples increased from the first to the third experiment. This was not only due to a lower dilution of the chemicals, but also to the important role played by the degradation processes: the greater pesticide residence time increased the opportunity for pesticide degradation. The main degradation pathways showed in these experiments were: TER > DET > DETH > DDAH and TER > TH > DETH > DDAH.
Desethylterbuthylazine to Terbuthylazine Ratio
In this study the role played by the water flow regime conditions in influencing DET mobility was also evaluated. Because of increasing DET concentrations with time, due to TER degradation, and decreasing TER concentrations, due to both degradation and adsorption by organic matter in the topsoil, the DET to TER ratio increased with time at both depths (Fig. 6)
. The simulated hydraulic condition in the second experiment facilitated both degradation and leaching of DET, as showed by the DET to TER ratio in Fig. 6, which was greater than in the other two situations at both depths.
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With regard to the comparison between DET to TER ratios in the water leachates (Fig. 6) and in the soil samples collected at the 30-cm depth (Fig. 7) , the values measured in the middle of the experiments (60–80 d after the herbicide application) are reported in Table 3.
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Adams and Thurman (1991) suggested that the desethylatrazine to atrazine ratio, also called the DAR ratio, can be used, for atrazine, as a measure of the age of the herbicide residue and the amount of interaction between atrazine and the soil. Goolsby et al. (1997) stated that a DAR ratio less than 0.1 in the pore water indicates a residue that has not interacted significantly with the surrounding soil, whereas higher values (median about 0.4) are detected after considerable degradation of the herbicide in the soil.
Extrapolating these consideration to terbuthylazine showed a similar degradation pathway, and it is possible to conclude that the obtained DER ratios (DET to TER) suggest that TER degradation occurred in the second and the third experiments, while it was scarce in the first. This conclusion is also in agreement with the results regarding the other TPs. At the 90-cm depth (Fig. 5), the amount of the TPs was five times greater in the third experiment compared with the first, and two times higher in the second compared with the first. Treviglio topsoil was used to calculate the Koc value for DET, which was found to be higher compared with terbuthylazine (42 versus 99) (Bottoni et al., 1996). All these considerations lead to the conclusion that DET can be considered a high-risk pollutant for ground water contamination.
Herbicide Content in Soil Samples
Terbuthylazine and DET concentrations in soil samples collected at the 30-cm depth are reported in Fig. 7. Although soil samples were taken from different depths, this section will concentrate on the analysis at 30 cm, so that comparison can be made to the lysimeter samples. In the first experiment one core was sampled 60 d after herbicide application, whereas in the other two experiments, six cores were collected at different times. In the first experiment, TER and DET concentrations reached values of 70 and 8 µg/kg, respectively, 60 d after the application. The great limitation of the data on herbicide concentration in the first experiment makes impossible any remarks.
The second and third experiments showed similar starting TER concentrations but terbuthylazine and DET seemed to be retained in soil for a longer time in the second experiment than in the third one. An opposite trend was shown in water samples, where higher TER and DET concentrations occurred in the third experiment. In conclusion, in the third experiment, terbuthylazine demonstrated a lower persistence.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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In this study, BTCs showed significant differences in the three experimental conditions and, inside each one, at different depths (Fig. 1). The constant water saturation and the natural precipitation enhanced infiltration rates, while the herbicide concentrations in water reached the maximum values. Furthermore, the natural precipitation condition generated the highest risk condition for ground water contamination, because TER passed through the clay soil horizon at 70 to 90 cm and its highest concentrations were recorded at the 90-cm depth. In these conditions the herbicide might quickly reach the first aquifer.
With regard to the TP formation, results showed that DET is the prominent dealkylated terbuthylazine metabolite, whereas DIA was found only at trace concentrations, in agreement with the results of Dousset et al. (1997). The high levels of DET and the low levels of DIA observed in the experiments might be a consequence of the different rates between deethylation and deterbuthylation. Similar to atrazine (Mills and Thurman, 1994), DIA can be regarded as a minor-importance metabolite in TER degradation reactions. However, C14 studies have shown that DIA has shorter dissipation rate than DET, indicating that its limited concentrations might be due to a rapid turnover in the unsaturated zone rather than to its absence (Winkelmann and Klaine, 1991b). The presence of a detectable DIAH concentration in the present experiments (Fig. 4 and 5) agrees with this hypothesis.
In this research significant concentrations of hydroxylated derivatives were measured in the pore water samples. The analysis of the 90-cm samples (Fig. 5) showed a mobilization of TH in water flow saturation conditions and of DETH in unsaturated steady-state flow condition. There is, therefore, a real possibility that these chemicals can move through the topsoil horizon toward the ground water, as Schiavon (1988) also suggested.
In conclusion, the present experiments demonstrated that soil dryness (Fig. 3) facilitates the formation of dealkylated and hydroxylated derivatives of terbuthylazine, while rainfall events promote DET leaching, the most mobile among TPs. The DER ratio, as was demonstrated for DAR, can give useful information on the age of the herbicide residue and the amount of interaction between parental compound and topsoil.
The main degradation pathways evidenced were: TER > DET > DETH > DDAH and TER > TH > DETH > DDAH; among TPs, DET and DETH showed a high mobility from agricultural topsoil to ground water and can be considered potential leaching compounds.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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