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Mineralization of aged atrazine and mecoprop in soil and aquifer chalk
Chemosphere 45 (2001) 927±934
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Mineralization of aged atrazine and mecoprop in soil and aquifer chalk Gitte Bùggild Kristensen, Helle Johannesen, Jens Aamand
*
Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Thoravej 8, DK-2400 Copenhagen NV, Denmark Received 17 September 2000; received in revised form 5 January 2001; accepted 11 January 2001
Abstract The eect of ageing on the bioavailability and sorption of the herbicides atrazine and mecoprop was studied in soil and aquifer chalk sampled at an agricultural ®eld near Aalborg, Denmark. The herbicides were incubated in sterile soil or chalk up to 3 months prior to inoculation with 5 107 cells g 1 (dry weight) of a mecoprop degrading highly enriched culture (PM) or 1 109 cells g 1 (dry weight) of the atrazine degrading Pseudomonas sp. strain ADP. As a measure of the bioavailable residues accumulated 14 CO2 was measured for 2 months. In both soil and chalk ageing limited the rate of atrazine mineralization, and in chalk the extent of mineralization was reduced as well. The fraction of sorbed atrazine in the soil ranged between 50% and 62%, whereas a maximum of 12% was sorbed in chalk. No impact on the mineralization of aged mecoprop was seen as no sorption of this herbicide on either soil or chalk was measured. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Ageing; Herbicides; Pseudomonas sp.; Soil; Chalk
1. Introduction Pesticides introduced to soil are in some instances recalcitrant (Capriel and Haisch, 1983; Schroll et al., 1992; Perrin-Ganier et al., 1996) which results in long residence times and increases the risk of leaching to underlying aquifers. Sorption may reduce leaching of the compounds, and previous research has shown that sorption of organic chemicals to soils often entails an initial rapid process occurring over hours or a few days followed by a period of slow sorption on a time scale of months to years (Pignatello and Xing, 1996). Pignatello and Xing (1996) reviewed investigations dealing with the slow sorption of organic chemicals in general, and the slow sorbing fraction ranged between 14% and 90% of the total sorbed compound. Long residence times may
*
Corresponding author. Tel.: +45-381 42326; fax: +45381 42050. E-mail address: [email protected] (J. Aamand).
result in a larger sorbed fraction, and often lead to the formation of bound residues, chemical fractions that resist desorption (Capriel and Haisch, 1983; Reuter et al., 1999). Khan (1991) de®ned herbicide bound residues as chemical species originating from herbicide usage, which cannot be extracted by exhaustive extraction procedures used in residue analyses. The processes by which organic chemicals become increasingly resistant to desorption, sometimes termed chemical ageing are not fully understood. However, suggested mechanisms include slow diusion and entrapment within small pores in soil aggregates (Steinberg et al., 1987) or in solid organic matter (Brusseau et al., 1991), or formation of strong chemical bonds to soil constituents (Reuter et al., 1999). The mechanisms of ageing and methods for studying the process have recently been reviewed by Alexander (2000). Depending on the mechanism the result will range from permanent removal to slow desorption. Chemical ageing has been shown to decrease the bioavailability of, for instance, atrazine, phenanthrene, and
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 0 2 0 - 0
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G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934
naphthalene to earthworms (Kelsey and Alexander, 1997), simazine to plants (Scribner et al., 1992) and atrazine, phenanthrene, and naphthalene to microorganisms (Radosevich et al., 1997; Chung and Alexander, 1998; Guerin and Boyd, 1992). All of these studies have examined the bioavailability in soil from the plough layer. Throughout the last decade, pesticides including the triazine herbicide atrazine and the phenoxyalcanoic acid herbicide mecoprop have frequently been found in groundwater (Stockmarr, 1998), where the residence time can be long, due to the often observed slow degradation of pesticides in aquifers (Larsen et al., 2000). A long residence time emphasizes the importance of studies dealing with ageing. Only a few previous studies have dealt with the ageing of organic chemicals in aquifers. Hatzinger and Alexander (1995), however, investigated the availability of aged phenanthrene to Pseudomonas strain R in aquifer sand, and nutrient amended aquifer sand, and observed reduced availability in nutrient amended aquifer sand, whereas no eect of ageing was observed in unamended sand. To our knowledge no investigations regarding pesticide ageing in aquifers have been published. The purpose of this investigation is to study the eect of ageing on the bioavailability of the two herbicides atrazine and mecoprop in soil and aquifer chalk. Ageing may decrease the bioavailability in chalk signi®cantly, since slow diusion of the herbicides into the pores of the chalk matrix is likely, and with typical pore sizes in the range 0.3±0.6 lm (Price et al., 1993), these pores are considered to exclude most of the metabolically active microorganisms (Bakken, 1997). If desorption is slow, it may be limiting the overall degradation rate. The herbicides were incubated in sterile soil or chalk up to 3 months, prior to inoculation with a mecoprop degrading enriched culture (PM) or the atrazine degrading Pseudomonas sp. strain ADP. Rate and extent of mineralization was used as a measure of the bioavailable residues. Pure or enriched cultures were used to assure rapid degradation following inoculation of the soil or chalk. In soil and chalk ageing limited the rate of atrazine mineralization, and in chalk the extent was reduced as well. The fraction of sorbed atrazine in soil ranged between 50% and 62%, whereas a maximum of 12% was sorbed in chalk. No sorbed mecoprop was observed in either soil or chalk. Mineralization measurements may be hindered in chalk by the reduced eciency of 14 CO2 trapping caused by high pH and a high content of carbonates. In this study mineralization was measured by trapping the evolved 14 CO2 in an alkaline solution. The eciency of the used alkali trap was measured under dierent circumstances, and the capture of 14 CO2 was signi®cantly reduced at high mineralization rates both in soil and chalk samples. In buer-solutions the eciency was reduced at pH values exceeding 7.5.
2. Materials and methods 2.1. The ®eld site Samples were collected in the agricultural area of Drastrup, Denmark. The ®eld site is located on a gentle slope of a chalk hill, mounted by glaciers and meltwater streams during glacial periods. The sur®cial layer of sandy drift is approximately a meter thick. The chalk, coccolith mud with low Mg-content, was deposited in the shallow shelf sea, which took up the region during the Maastrictian period, and was uplifted to exposure during Neogene. The upper part of the chalk has a putty structure due to ancient glacial wear and recent weathering. 2.2. Soil and chalk Samples of soil from 0.2 to 0.3 m below surface (1.3% organic carbon, pH 6.7) and aquifer chalk from 2.8 to 3.2 m below surface (0.02% organic carbon, pH 7.4) were collected. Soil was obtained by digging, while aquifer chalk was collected as cores. At the time of sampling, February 1999, the water table was measured at 2.1 m below surface. The moisture content was determined gravimetrically by oven drying at 105°C for 24 h. Soil and chalk were sterilized by exposure to 22 kGy from a 10 MeV electron accelerator (Risù, Denmark). Irradiation was used for sterilization as it is reported to cause the smallest change in soil characteristics, e.g., speci®c surface area or cation exchange capacity (Stroetmann et al., 1994). Prior to irradiation the soil was passed through a 4 mm sieve at ®eld moisture content. The samples were stored at 4°C until used. 2.3. Bacterial strains and culture conditions One pure isolate was used in this study. Pseudomonas sp. strain ADP was isolated and characterized by Mandelbaum et al. (1995). The bacterium mineralizes atrazine (6-chloro-N-ethyl-N0 -isopropyl-1,3,5-triazine2,4-diamine), and is capable of using it as the sole source of nitrogen. ADP was grown in the original media used in enrichment (Mandelbaum et al., 1993), excluding sucrose, the salt stock solution, and the vitamin stock solution. 2 g l 1 citrate was used as the carbon source and 50 mg l 1 atrazine (>99% pure, Novartis) as the nitrogen source. The culture strain PM was enriched from a sandy aquifer near Karup, Denmark on mecoprop (()-2-(2-chloro-4-methylphenoxy) propionic acid) as the sole carbon source. PM was grown in a mineral medium containing: K2 HPO4 3H2 O 4:25 g l 1 ; NaH2 PO4 H2 O 1:0 g l 1 ; NH4 Cl 2:0 g l 1 , nitrilotriacetic acid 0:123 g l 1 ; MgSO4 7H2 O 0:20 g l 1 ; FeSO4 7H2 O 0:012 g l 1 ; ZnSO4 7H2 O 0:003 g l 1 ; MnSO4 H2 O 0:003 g l 1 , and 2 g glucose l 1 .
G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934
2.4. Chemicals Uniformly ring-labelled [14 C]atrazine (0:70 GBq mmol 1 , >95% radiochemical purity) was purchased from Sigma Chemical Company and uniformly ring-labelled [14 C] mecoprop 0:85 GBq mmol 1 ; > 95% from Institute of Isotopes, Budapest. Unlabelled herbicides ( >99% chemical purity) were obtained from Riedel-de H aen. Physico-chemical characteristics of the two herbicides are given in Table 1. 2.5. Mineralization of aged atrazine and mecoprop 10 g sterile soil or chalk (dry weight) was added to 100 ml airtight glass ¯asks and amended with an aqueous solution of atrazine or mecoprop to obtain an initial concentration of 2:5 mg kg 1 and an initial activity of 1:2 103 Bq. Moisture content in soil and chalk, herbicide amendment and additional water amendment resulted in a total liquid content of 7 ml. To prevent dissolution of chalk by the MilliQÓ water, the water added to these samples was saturated with chalk. A test tube containing 2 ml 0.5 M NaOH was placed in each ¯ask to detect if any mineralization occurred during ageing by trapping the 14 CO2 . All samples were incubated at 10°C in the dark. After a predetermined number of days, the same procedure was followed for the preparation of additional samples. The result was a group of samples aged for 1, 7, 30 or 90 days. Triplicate samples were prepared for each treatment. To determine bacterial mineralization the atrazine samples were inoculated with 1 ml of ADP cells in growth media (without atrazine) to obtain a density of 1:2 109 2:7 108 cells g 1 (dry weight) and the mecoprop samples with 1 ml of PM cells in growth media (excluding glucose) to a density of 4:7 107 1:2 107 PM cells g 1 (dry weight) determined by ac-
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ridin orange direct count. Flasks not inoculated with cells were included as controls. Prior to inoculation and at the time of sampling, NaOH in the alkali trap was replaced in a laminar ¯ow bench and ¯asks were left open for 5 min to replenish oxygen. The alkali was mixed with 10 ml of OptiPhase HiSafe scintillation ¯uid (Wallac, UK), left at least 10 h in darkness to eliminate chemo- and photoluminiscence, and counted for 10 min on a Wallac 1409 liquid scintillation counter. Calculations of accumulative mineralization were corrected for quenching and background radioactivity. 2.6. Mass balance At the termination of the experiment the distribution of 14 C into the following fractions was determined: (1) mineralized 14 C determined as accumulated 14 CO2 , (2) 14 C in aqueous solution, and (3) remaining 14 C. The samples were transferred to centrifugal ®lters with a 0.2 lm polyvinylidenedi¯ouride membrane (PVDF, Lida Manufacturing Corp.) and the liquid was separated from soil or chalk by centrifugation in 15 min at 1660 g. 14 C in solution was determined by liquid scintillation counting. Since an examination of sorption to ®lters showed only 96% recovery of atrazine after centrifugation, atrazine values were corrected for sorption to ®lters. To quantify the amount of mineralized atrazine, which was not caught in the alkali trap, sub-samples of the liquid and sub-samples of the soil or chalk were acidi®ed after centrifugation. A straightforward method was used on both the soil and liquid samples. 10 ml of 0.5 M HCl was added through a syringe needle to serum bottles containing 1 g soil or 3 ml liquid and an alkali trap. The samples were left overnight, and the radioactivity of the alkali was determined. Controls amended with NaH14 CO3 with an activity of 720 Bq were included, and more than 99% were recovered in the alkali.
Table 1 Physico-chemical characteristics of mecoprop and atrazine
a
Name
Molecular weight (g mol 1 )
Water solubility (g l 1 )
Kow a
Mecoprop
214.6
0.86
1.29
Atrazine
215.7
0.03
219
Octanol ± water partition coecient.
Chemical structure
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As a further check on the method dierences between acidi®ed and non-acidi®ed liquid sub-samples were compared to the activity recovered in the alkali traps. This method of quanti®cation was not relevant in chalk samples, as large amounts of CO2 would evolve under acidi®cation. For this reason the serum bottle containing 1 g of chalk was connected by tubes and needles to a set-up comprising four serially interconnected serum bottles each containing 10 ml of NaOH. 10 ml of 2 M HCl was injected to the chalk and the produced CO2 bubbled through and got caught in the alkali of the four bottles. Liquid scintillation counting on 2 ml of alkali from each of the four serum bottles was undertaken. Duplicate chalk controls amended with NaH14 CO3 with an activity of 720 Bq were included, and 94% and 98% of the initially added activity were recovered in the alkali, respectively, less than 2% in the last serum bottle. To prevent backward ¯ow caused by low pressure a suciently high ¯ow of nitrogen was established in the ®rst serum bottle. The remaining 14 C was determined by combustion of sub-samples of 0.1±0.3 g in a Packard Oxidizer (model 507) with excess O2 and capture of 14 CO2 . The material was dried by means of air¯ow at room temperature before combustion of sub-samples, and values were corrected for the amount of 14 C originating from liquid remaining in the soil or chalk after centrifugation. 2.7. Sorption of aged atrazine and mecoprop A parallel experiment was performed to determine the amount of atrazine or mecoprop in solution and sorbed after ageing. The ageing was performed in the same way as in the mineralization experiment, however, after ageing the alkali trap was removed, and the experiment terminated by a determination of 14 C in solution and 14 C associated with soil or chalk by use of centrifugal ®lters, as described above. Controls were included to check the sterility and stability of pH during ageing. 2.8. Eciency of the alkali trap Since the eciency of 14 CO2 -capture may be reduced due to the relative high pH (7.4) and high content of carbonates in chalk the eciency of the alkali to trap 14 CO2 was examined under two dierent circumstances. The eciency was studied in buer-solutions within a range of pH-values, and in soil and chalk under conditions of fast mecoprop mineralization. 0.1 M potassium hydrogen phthalate or 0:1 M KH2 PO4 and 0.1 M HCl or 0.1 M NaOH were used to prepare buer-solutions with a range of pH-values between 3 and 10.5 (Weast et al., 1984). 10 ml of each buer-solution, an alkali trap (a test tube containing 2 ml 0.5 M NaOH), and NaH14 CO3 with an activity of
720 Bq were added to 100 ml airtight ¯asks. The samples were incubated at 10°C, and at regular intervals the NaOH was replaced and the radioactivity determined by liquid scintillation counting. The eciency of the trap was measured as the fraction of initially added activity recovered in the alkali. To elucidate the eect of soil or chalk (at natural pH) on the eciency of the alkali trap additional ¯asks with 10 g (dry weight) soil or chalk, MilliQâ water to a total of 7 ml, and NaH14 CO3 with an activity of 720 Bq were included. The eciency of the alkali trap under conditions of fast mineralization was studied by setting up ¯asks with soil or chalk resembling those aged for one day in the experiment with mecoprop. The ¯asks were inoculated with PM, and after 6, 24 and 51 h two soil and two chalk samples were sacri®cially terminated and the previously described methods of acidi®cation were used. 3. Results 3.1. Mineralization and sorption of aged atrazine Fig. 1(a) shows the accumulation of evolved 14 CO2 from the mineralization of atrazine with dierent residence times in soil prior to inoculation with Pseudomonas ADP. The initial mineralization rate, determined as 14 CO2 accumulated during the ®rst 24 h, declined signi®cantly as the time of atrazine residence in soil increased (ANOVA, P < 0:05). This rate was signi®cantly reduced, when the residence time exceeded 1 day, whereas no dierences were observed between mineralization rates of samples aged for 7, 30, and 90 days (Tukey test, P 0:05). This result is in agreement with the result of the sorption experiment (Table 2). Signi®cantly more atrazine was available in the aqueous-phase of the samples aged for 1 day, than any of the samples with longer residence times (Tukey test, P 0:05). Due to a large variation no signi®cant dierences were observed in sorbed 14 C between dierently aged samples (ANOVA, P > 0:05). At the termination of the mineralization experiment 63 days after inoculation the 14 C recovered as 14 CO2 in the dierently aged samples was in the range 82±87%, and the only signi®cant dierence was between samples aged for 1 day and 30 days (Tukey test, P 0:05). Fig. 1(b) shows the mineralization of atrazine aged in chalk, and the initial mineralization rate decreased as the residence time increased (ANOVA, P < 0:05). The rate was signi®cantly reduced, when the residence time exceeded 7 days, whereas no dierences were observed between samples aged for 30 and 90 days (Tukey test, P 0:05). Even after 63 days of incubation the recovered 14 CO2 was reduced with increasing residence time (ANOVA, P < 0:05). The extent of mineralization ranged between 59% and 71%. The sorption experiment
G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934
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added 14 C was recovered in the aqueous-phase, whereas 3±6% was recovered in the aqueous-phase of the chalk samples. 3.2. Mineralization and sorption of aged mecoprop No sorption of mecoprop were observed in either soil or chalk samples. In soil no signi®cant dierences in the initial mineralization rate was observed (ANOVA, P > 0:05). In chalk the initial mineralization rate of the samples aged for 1 and 7 days was lower than the rates of samples aged for 30 and 90 days (Tukey test, P 0:05). At the termination of the experiment 61 days after inoculation no signi®cant dierences in the 14 C recovered as 14 CO2 were observed between dierently aged chalk samples (ANOVA, P > 0:05). The total recovery of 14 C in the sorption and mineralization studies with mecoprop was in the range 88±100%. 3.3. Eciency of the alkali trap
Fig. 1. Accumulated mineralization by Pseudomonas sp. strain ADP of [ring-U-14 C]atrazine aged for 1 (), 7 (), 30 (N), and 90 days () in (a) soil and (b) chalk. Abiotic controls (). Points represent means of three replicates. Error bars indicate standard deviations.
(Table 2) showed signi®cantly increasing amounts of sorbed 14 C, when the residence time exceeded 7 days and decreasing amounts of 14 C in the aqueous-phase, when the residence time exceeded 1 day (Tukey test, P 0:05). However the maximum sorbed amount in chalk was 12% compared to 62% in soil. No mineralization occurred in the abiotic samples of soil or chalk. Fig. 2 shows the mass balance of the atrazine experiment, and the total recovery of 14 C was in the range 82±96%. In soil samples less than 1% of initially
The eciency of the alkali trap was investigated in buer-solutions with a range of pH-values between 3 and 10.5 (Fig. 3). Within 3 days 100% of the initially added 14 C was recovered in the alkali trap in buer-solutions with pH-values below 7.5. The eciency of the alkali trap at a given pH was reduced, when soil or in particular chalk was present in the system. In soil samples 84% of the initially added 14 C was captured in the trap after 3 days and 98% was captured after 12 days. In chalk only 13% of the initially added radioactivity was captured even after a period of 51 days. However, at the termination of the atrazine experiment results from the acidi®ed samples showed that the eciency of the alkali trap in soil was 100%, whilst it is being slightly reduced with an eciency between 92% and 99% in chalk. However, eciency of the alkali trap was markedly reduced in soil as well as in chalk under conditions of fast mineralization (Table 3). In the worst case, only 31% of the mineralized 14 C initially added as mecoprop was captured in the alkali trap 24 h after inoculation in chalk.
Table 2 Percentage of initially added [ring-U-14 C]atrazine sorbed and in solution after ageinga Ageing (days)
Soil Sorbed (% of added
1 7 30 90 a
50 7a 62 4a 60 14a 55 14a
Chalk 14
C)
Aqueous-phase (% of added 14 C)
Sorbed (% of added
51 6a 42 1b 41 1b 41 1b
22a 41a 111b 123b
14
C)
Values within columns followed by dierent letters are signi®cantly dierent (Tukey test, P 0:05).
Aqueous-phase (% of added 14 C) 1001a 902b 911b 931b
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G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934
Fig. 2. Mass balance for [ring-U-14 C]atrazine with dierent residence times prior to inoculation with Psedomonas sp. strain ADP in (a) soil and (b) chalk. Black: recovered as 14 CO2 , striped: 14 C in aqueous-phase, white: recovered after incineration. Error bars show the standard deviations of the total recovery of triplicates.
Fig. 3. The eciency of the alkali to trap 14 C 18 h after addition of NaH14 CO3 to buer-solutions (), soil (N), and chalk (). Points represent means of triplicates.
4. Discussion In this study the rate of mineralization of atrazine was reduced in soil as an eect of ageing, however, the extent of mineralization reached after 63 days of incu-
bation did not dier much between the dierently aged samples. A reduction in the mineralization rate (of a test bacterium designated M91-3) due to ageing of atrazine has been observed before by Radosevich et al. (1997) in a no-till soil as well as in a conventional-till soil. However, their observations of decreased mineralization rates combined with a reduction in the extent of mineralization were not observed in this study. Perhaps dierent mechanisms have caused the resistance to desorption in the two experiments. In this study the eect of ageing is likely to be due to slow diusion or reversible bindings, since the extent of mineralization in differently aged samples does not dier, whereas irreversible bound atrazine cannot be excluded in the study of Radosevich et al. (1997). Chung and Alexander (1998) investigated the eect of atrazine ageing for 200 days on the extent of mineralization of M91-3 in 16 dierent soils and observed an eect in 15 of the soils. A high content of organic carbon in soils increased the eect of ageing. From the mass balance in Fig. 2, it can be observed that more than 80% of the atrazine was mineralized in soil at the termination of the experiment, whereas the sorption data in Table 2 show that more than 50% was sorbed at the time of inoculation. Consequently, some of the initially sorbed atrazine must have been mineralized. There are two possible explanations: either (1) the sorbed atrazine is available for the bacteria or (2) desorption is rapid. Since signi®cant dierences in rates between dierently aged atrazine samples have been observed at least part of the sorbed atrazine must have been initially unavailable. In the aquifer chalk the mineralization rate and extent was reduced with increasing residence time of atrazine. Ageing to our knowledge had never been investigated before for herbicides in aquifer material. However, the result is in accordance with the observations of Hatzinger and Alexander (1995) which showed a decrease in the bioavailability of phenanthrene after ageing in nutrient amended aquifer sand. Since only 12% of the atrazine was sorbed after 90 days of ageing (Table 2), the eect of ageing is surprising. However, if the results of the mass balance (Fig. 2) are examined, it seems as though a larger fraction is sorbed after inoculation of the samples. The most obvious explanation is the incorporation of 14 C into biomass, however, this is not the case, since no cellular synthesis can be supported from the fully oxidized carbon atoms of the triazine-ring (Radosevich et al., 1995). In any case atrazine must somehow be associated to bacteria, dead bacteria or exudates. Due to this sorbed fraction ranging from 16% to 23% the extent of mineralization in chalk did not reach the same level as in soil. An eect of mecoprop ageing could be expected, since sorption has been observed in both soil and chalk
G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934 Table 3 The eciency of the alkali to trap Incubation (h)
6 24 51 a b
14
933
CO2 produced from mineralization of mecoprop
Soil
Chalk 14
Caught in alkali (%)a
H CO3 (%)
0 19 48
0 11 5
a
b
Eciency (%)
Caught in alkalia (%)
H14 CO3 (%)
± 63 91
1 12 22
1 27 24
a
Eciencyb (%) ± 31 48
Percentage of initially added 14 C. Percentage of total mineralized caught in the alkali trap.
(Atalay and Hwang, 1999; Madsen et al., 2000). Atalay and Hwang (1999) measured the linear sorption distribution coecient (Kd ) of mecoprop in three dierent soils, and it ranged from 0.13 to 0.24. In chalk a Kd of 0.17 was measured by Madsen et al. (2000). Smith and Muir (1980) examined the extractability of aged 2,4-D, another phenoxyalcanoic acid herbicide, in three dierent soils and observed a rapid decrease in the amounts of extractable 2,4-D. However, in our experiment, no ageing of mecoprop was observed because no sorption was noted either in soil or chalk. The examination of eciency of the alkali trap elucidated that the capture of 14 CO2 was signi®cantly delayed at high mineralization rates particularly in chalk. This means that mineralization rates would be underestimated and caution should be taken not to compare soil and chalk samples. From a comparison of the soil and chalk samples with buer-solutions of the same pH, we can conclude, that the eciency of the alkali trap depends both on pH, trapping time, and soil type. To prevent degradation of the herbicides in the ageing period, the soil and chalk was sterilized before use. Depending on the sterilization method chosen dierent alterations of the material will result. Stroetmann et al. (1994) evaluated the eciency of dierent methods and examined changes in the speci®c surface area and cation exchange capacity. Irradiation seemed to be the least destructive method, since no changes in the two parameters were observed. The studies of bioavailability of each of the aged herbicides have been performed with a single bacterial strain or a highly enriched culture. This is an important simpli®cation of a natural system. Dierent organisms have dierent survival strategies, and in an enrichment procedure fast growing organisms with a low anity for the substrate may be favoured. Guerin and Boyd (1992) examined the bioavailability of naphthalene by using two dierent bacterial strains, and obtained dramatically dierent results which make generalizations regarding bioavailability of sorbed substrates inappropriate. Scribner et al. (1992) overcame the limitations of using pure cultures by performing their experiment with indigenous soil microbes, enabling more far-reaching
conclusions. They compared the degradation of ®eld aged and freshly added simazine, another triazine herbicide, and found freshly added simazine readily degradable, whereas aged simazine was biologically unavailable. Only few studies have dealt with the eects of ageing in aquifers. Our results show that ageing of the pesticides atrazine and mecoprop in samples from a chalk aquifer only caused a minor reduction in mineralization rates. Therefore, at least for these pesticides, ageing does not need to be taken into account when modelling their fate.
Acknowledgements This research was in part supported by the EC Environment and Climate Research Programme (Contract: ENV4-CT97-0441, Climate and Natural Hazards). Rene K. Juhler is thanked for discussions of the alkali trap. Hans-Jùrgen Albrechtsen is thanked for providing the radio-labelled NaH14 CO3 .
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Radosevich, M., Traina, S.J., Hao, Y.L., Tuovinen, O.H., 1995. Degradation and mineralization of atrazine by a soil bacterial isolate. Appl. Environ. Microbiol. 61, 297±302. Radosevich, M., Traina, S.J., Tuovinen, O.H., 1997. Atrazine mineralization in laboratory-aged soil microcosms inoculated with s-triazine-degrading bacteria. J. Environ. Qual. 26, 206±214. Reuter, S., Ilim, M., Munch, J.C., Andreux, F., Scheunert, I., 1999. A model for the formation of bound residues of the herbicide 14 C-isoproturon in soil. Chemosphere 39 (4), 627±639. Schroll, R., Langenbach, T., Cao, G., Doer¯er, U., Schneider, P., Scheunert, I., 1992. Fate of carbon-14 terbutylazine in soil±plant systems. Sci. Total Environ. 123±124, 377±389. Scribner, S.L., Benzing, T.R., Sun, S., Boyd, S.A., 1992. Desorption and bioavailability of aged simazine residues in soil from a continuous corn ®eld. J. Environ. Qual. 21, 115±120. Smith, A.E., Muir, D.C.G., 1980. Determination of extractable and non-extractable radioactivity from prairie soils treated with carboxylic- and ring-labelled [14 C]2,4-D. Weed Res. 20, 123±129. Steinberg, S.M., Pignatello, J.J., Sawhney, B.L., 1987. Persistence of 1,2-dibromethane in soils: entrapment in intraparticle micropores. Environ. Sci. Technol. 21 (12), 1201±1208. Stockmarr, J., 1998. Pesticider og nedbrydningsprodukter. In: Stockmarr J. (Ed.), Danish Monitoring Program 1998, Ministry of Environment and Energy. The Geological Survey of Denmark and Greenland, GEUS, pp. 41±60. Stroetmann, I., Kaempfer, P., Dott, W., 1994. Untersuchungen zur Ezienz von Sterilisationsverfahren an unterschiedlichen B oden. Eciency of dierent methods for sterilization of dierent soil types. Zentralbl. Hyg. Umweltmedizin 195, 111±120. Weast, R.C., Astle, M.J., Beyer, W.H., 1984. CRC Handbook of Chemistry and Physics, 64th ed. CRC Press, Boca Raton, Florida.
www.elsevier.com/locate/chemosphere
Mineralization of aged atrazine and mecoprop in soil and aquifer chalk Gitte Bùggild Kristensen, Helle Johannesen, Jens Aamand
*
Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Thoravej 8, DK-2400 Copenhagen NV, Denmark Received 17 September 2000; received in revised form 5 January 2001; accepted 11 January 2001
Abstract The eect of ageing on the bioavailability and sorption of the herbicides atrazine and mecoprop was studied in soil and aquifer chalk sampled at an agricultural ®eld near Aalborg, Denmark. The herbicides were incubated in sterile soil or chalk up to 3 months prior to inoculation with 5 107 cells g 1 (dry weight) of a mecoprop degrading highly enriched culture (PM) or 1 109 cells g 1 (dry weight) of the atrazine degrading Pseudomonas sp. strain ADP. As a measure of the bioavailable residues accumulated 14 CO2 was measured for 2 months. In both soil and chalk ageing limited the rate of atrazine mineralization, and in chalk the extent of mineralization was reduced as well. The fraction of sorbed atrazine in the soil ranged between 50% and 62%, whereas a maximum of 12% was sorbed in chalk. No impact on the mineralization of aged mecoprop was seen as no sorption of this herbicide on either soil or chalk was measured. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Ageing; Herbicides; Pseudomonas sp.; Soil; Chalk
1. Introduction Pesticides introduced to soil are in some instances recalcitrant (Capriel and Haisch, 1983; Schroll et al., 1992; Perrin-Ganier et al., 1996) which results in long residence times and increases the risk of leaching to underlying aquifers. Sorption may reduce leaching of the compounds, and previous research has shown that sorption of organic chemicals to soils often entails an initial rapid process occurring over hours or a few days followed by a period of slow sorption on a time scale of months to years (Pignatello and Xing, 1996). Pignatello and Xing (1996) reviewed investigations dealing with the slow sorption of organic chemicals in general, and the slow sorbing fraction ranged between 14% and 90% of the total sorbed compound. Long residence times may
*
Corresponding author. Tel.: +45-381 42326; fax: +45381 42050. E-mail address: [email protected] (J. Aamand).
result in a larger sorbed fraction, and often lead to the formation of bound residues, chemical fractions that resist desorption (Capriel and Haisch, 1983; Reuter et al., 1999). Khan (1991) de®ned herbicide bound residues as chemical species originating from herbicide usage, which cannot be extracted by exhaustive extraction procedures used in residue analyses. The processes by which organic chemicals become increasingly resistant to desorption, sometimes termed chemical ageing are not fully understood. However, suggested mechanisms include slow diusion and entrapment within small pores in soil aggregates (Steinberg et al., 1987) or in solid organic matter (Brusseau et al., 1991), or formation of strong chemical bonds to soil constituents (Reuter et al., 1999). The mechanisms of ageing and methods for studying the process have recently been reviewed by Alexander (2000). Depending on the mechanism the result will range from permanent removal to slow desorption. Chemical ageing has been shown to decrease the bioavailability of, for instance, atrazine, phenanthrene, and
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 0 2 0 - 0
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G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934
naphthalene to earthworms (Kelsey and Alexander, 1997), simazine to plants (Scribner et al., 1992) and atrazine, phenanthrene, and naphthalene to microorganisms (Radosevich et al., 1997; Chung and Alexander, 1998; Guerin and Boyd, 1992). All of these studies have examined the bioavailability in soil from the plough layer. Throughout the last decade, pesticides including the triazine herbicide atrazine and the phenoxyalcanoic acid herbicide mecoprop have frequently been found in groundwater (Stockmarr, 1998), where the residence time can be long, due to the often observed slow degradation of pesticides in aquifers (Larsen et al., 2000). A long residence time emphasizes the importance of studies dealing with ageing. Only a few previous studies have dealt with the ageing of organic chemicals in aquifers. Hatzinger and Alexander (1995), however, investigated the availability of aged phenanthrene to Pseudomonas strain R in aquifer sand, and nutrient amended aquifer sand, and observed reduced availability in nutrient amended aquifer sand, whereas no eect of ageing was observed in unamended sand. To our knowledge no investigations regarding pesticide ageing in aquifers have been published. The purpose of this investigation is to study the eect of ageing on the bioavailability of the two herbicides atrazine and mecoprop in soil and aquifer chalk. Ageing may decrease the bioavailability in chalk signi®cantly, since slow diusion of the herbicides into the pores of the chalk matrix is likely, and with typical pore sizes in the range 0.3±0.6 lm (Price et al., 1993), these pores are considered to exclude most of the metabolically active microorganisms (Bakken, 1997). If desorption is slow, it may be limiting the overall degradation rate. The herbicides were incubated in sterile soil or chalk up to 3 months, prior to inoculation with a mecoprop degrading enriched culture (PM) or the atrazine degrading Pseudomonas sp. strain ADP. Rate and extent of mineralization was used as a measure of the bioavailable residues. Pure or enriched cultures were used to assure rapid degradation following inoculation of the soil or chalk. In soil and chalk ageing limited the rate of atrazine mineralization, and in chalk the extent was reduced as well. The fraction of sorbed atrazine in soil ranged between 50% and 62%, whereas a maximum of 12% was sorbed in chalk. No sorbed mecoprop was observed in either soil or chalk. Mineralization measurements may be hindered in chalk by the reduced eciency of 14 CO2 trapping caused by high pH and a high content of carbonates. In this study mineralization was measured by trapping the evolved 14 CO2 in an alkaline solution. The eciency of the used alkali trap was measured under dierent circumstances, and the capture of 14 CO2 was signi®cantly reduced at high mineralization rates both in soil and chalk samples. In buer-solutions the eciency was reduced at pH values exceeding 7.5.
2. Materials and methods 2.1. The ®eld site Samples were collected in the agricultural area of Drastrup, Denmark. The ®eld site is located on a gentle slope of a chalk hill, mounted by glaciers and meltwater streams during glacial periods. The sur®cial layer of sandy drift is approximately a meter thick. The chalk, coccolith mud with low Mg-content, was deposited in the shallow shelf sea, which took up the region during the Maastrictian period, and was uplifted to exposure during Neogene. The upper part of the chalk has a putty structure due to ancient glacial wear and recent weathering. 2.2. Soil and chalk Samples of soil from 0.2 to 0.3 m below surface (1.3% organic carbon, pH 6.7) and aquifer chalk from 2.8 to 3.2 m below surface (0.02% organic carbon, pH 7.4) were collected. Soil was obtained by digging, while aquifer chalk was collected as cores. At the time of sampling, February 1999, the water table was measured at 2.1 m below surface. The moisture content was determined gravimetrically by oven drying at 105°C for 24 h. Soil and chalk were sterilized by exposure to 22 kGy from a 10 MeV electron accelerator (Risù, Denmark). Irradiation was used for sterilization as it is reported to cause the smallest change in soil characteristics, e.g., speci®c surface area or cation exchange capacity (Stroetmann et al., 1994). Prior to irradiation the soil was passed through a 4 mm sieve at ®eld moisture content. The samples were stored at 4°C until used. 2.3. Bacterial strains and culture conditions One pure isolate was used in this study. Pseudomonas sp. strain ADP was isolated and characterized by Mandelbaum et al. (1995). The bacterium mineralizes atrazine (6-chloro-N-ethyl-N0 -isopropyl-1,3,5-triazine2,4-diamine), and is capable of using it as the sole source of nitrogen. ADP was grown in the original media used in enrichment (Mandelbaum et al., 1993), excluding sucrose, the salt stock solution, and the vitamin stock solution. 2 g l 1 citrate was used as the carbon source and 50 mg l 1 atrazine (>99% pure, Novartis) as the nitrogen source. The culture strain PM was enriched from a sandy aquifer near Karup, Denmark on mecoprop (()-2-(2-chloro-4-methylphenoxy) propionic acid) as the sole carbon source. PM was grown in a mineral medium containing: K2 HPO4 3H2 O 4:25 g l 1 ; NaH2 PO4 H2 O 1:0 g l 1 ; NH4 Cl 2:0 g l 1 , nitrilotriacetic acid 0:123 g l 1 ; MgSO4 7H2 O 0:20 g l 1 ; FeSO4 7H2 O 0:012 g l 1 ; ZnSO4 7H2 O 0:003 g l 1 ; MnSO4 H2 O 0:003 g l 1 , and 2 g glucose l 1 .
G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934
2.4. Chemicals Uniformly ring-labelled [14 C]atrazine (0:70 GBq mmol 1 , >95% radiochemical purity) was purchased from Sigma Chemical Company and uniformly ring-labelled [14 C] mecoprop 0:85 GBq mmol 1 ; > 95% from Institute of Isotopes, Budapest. Unlabelled herbicides ( >99% chemical purity) were obtained from Riedel-de H aen. Physico-chemical characteristics of the two herbicides are given in Table 1. 2.5. Mineralization of aged atrazine and mecoprop 10 g sterile soil or chalk (dry weight) was added to 100 ml airtight glass ¯asks and amended with an aqueous solution of atrazine or mecoprop to obtain an initial concentration of 2:5 mg kg 1 and an initial activity of 1:2 103 Bq. Moisture content in soil and chalk, herbicide amendment and additional water amendment resulted in a total liquid content of 7 ml. To prevent dissolution of chalk by the MilliQÓ water, the water added to these samples was saturated with chalk. A test tube containing 2 ml 0.5 M NaOH was placed in each ¯ask to detect if any mineralization occurred during ageing by trapping the 14 CO2 . All samples were incubated at 10°C in the dark. After a predetermined number of days, the same procedure was followed for the preparation of additional samples. The result was a group of samples aged for 1, 7, 30 or 90 days. Triplicate samples were prepared for each treatment. To determine bacterial mineralization the atrazine samples were inoculated with 1 ml of ADP cells in growth media (without atrazine) to obtain a density of 1:2 109 2:7 108 cells g 1 (dry weight) and the mecoprop samples with 1 ml of PM cells in growth media (excluding glucose) to a density of 4:7 107 1:2 107 PM cells g 1 (dry weight) determined by ac-
929
ridin orange direct count. Flasks not inoculated with cells were included as controls. Prior to inoculation and at the time of sampling, NaOH in the alkali trap was replaced in a laminar ¯ow bench and ¯asks were left open for 5 min to replenish oxygen. The alkali was mixed with 10 ml of OptiPhase HiSafe scintillation ¯uid (Wallac, UK), left at least 10 h in darkness to eliminate chemo- and photoluminiscence, and counted for 10 min on a Wallac 1409 liquid scintillation counter. Calculations of accumulative mineralization were corrected for quenching and background radioactivity. 2.6. Mass balance At the termination of the experiment the distribution of 14 C into the following fractions was determined: (1) mineralized 14 C determined as accumulated 14 CO2 , (2) 14 C in aqueous solution, and (3) remaining 14 C. The samples were transferred to centrifugal ®lters with a 0.2 lm polyvinylidenedi¯ouride membrane (PVDF, Lida Manufacturing Corp.) and the liquid was separated from soil or chalk by centrifugation in 15 min at 1660 g. 14 C in solution was determined by liquid scintillation counting. Since an examination of sorption to ®lters showed only 96% recovery of atrazine after centrifugation, atrazine values were corrected for sorption to ®lters. To quantify the amount of mineralized atrazine, which was not caught in the alkali trap, sub-samples of the liquid and sub-samples of the soil or chalk were acidi®ed after centrifugation. A straightforward method was used on both the soil and liquid samples. 10 ml of 0.5 M HCl was added through a syringe needle to serum bottles containing 1 g soil or 3 ml liquid and an alkali trap. The samples were left overnight, and the radioactivity of the alkali was determined. Controls amended with NaH14 CO3 with an activity of 720 Bq were included, and more than 99% were recovered in the alkali.
Table 1 Physico-chemical characteristics of mecoprop and atrazine
a
Name
Molecular weight (g mol 1 )
Water solubility (g l 1 )
Kow a
Mecoprop
214.6
0.86
1.29
Atrazine
215.7
0.03
219
Octanol ± water partition coecient.
Chemical structure
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G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934
As a further check on the method dierences between acidi®ed and non-acidi®ed liquid sub-samples were compared to the activity recovered in the alkali traps. This method of quanti®cation was not relevant in chalk samples, as large amounts of CO2 would evolve under acidi®cation. For this reason the serum bottle containing 1 g of chalk was connected by tubes and needles to a set-up comprising four serially interconnected serum bottles each containing 10 ml of NaOH. 10 ml of 2 M HCl was injected to the chalk and the produced CO2 bubbled through and got caught in the alkali of the four bottles. Liquid scintillation counting on 2 ml of alkali from each of the four serum bottles was undertaken. Duplicate chalk controls amended with NaH14 CO3 with an activity of 720 Bq were included, and 94% and 98% of the initially added activity were recovered in the alkali, respectively, less than 2% in the last serum bottle. To prevent backward ¯ow caused by low pressure a suciently high ¯ow of nitrogen was established in the ®rst serum bottle. The remaining 14 C was determined by combustion of sub-samples of 0.1±0.3 g in a Packard Oxidizer (model 507) with excess O2 and capture of 14 CO2 . The material was dried by means of air¯ow at room temperature before combustion of sub-samples, and values were corrected for the amount of 14 C originating from liquid remaining in the soil or chalk after centrifugation. 2.7. Sorption of aged atrazine and mecoprop A parallel experiment was performed to determine the amount of atrazine or mecoprop in solution and sorbed after ageing. The ageing was performed in the same way as in the mineralization experiment, however, after ageing the alkali trap was removed, and the experiment terminated by a determination of 14 C in solution and 14 C associated with soil or chalk by use of centrifugal ®lters, as described above. Controls were included to check the sterility and stability of pH during ageing. 2.8. Eciency of the alkali trap Since the eciency of 14 CO2 -capture may be reduced due to the relative high pH (7.4) and high content of carbonates in chalk the eciency of the alkali to trap 14 CO2 was examined under two dierent circumstances. The eciency was studied in buer-solutions within a range of pH-values, and in soil and chalk under conditions of fast mecoprop mineralization. 0.1 M potassium hydrogen phthalate or 0:1 M KH2 PO4 and 0.1 M HCl or 0.1 M NaOH were used to prepare buer-solutions with a range of pH-values between 3 and 10.5 (Weast et al., 1984). 10 ml of each buer-solution, an alkali trap (a test tube containing 2 ml 0.5 M NaOH), and NaH14 CO3 with an activity of
720 Bq were added to 100 ml airtight ¯asks. The samples were incubated at 10°C, and at regular intervals the NaOH was replaced and the radioactivity determined by liquid scintillation counting. The eciency of the trap was measured as the fraction of initially added activity recovered in the alkali. To elucidate the eect of soil or chalk (at natural pH) on the eciency of the alkali trap additional ¯asks with 10 g (dry weight) soil or chalk, MilliQâ water to a total of 7 ml, and NaH14 CO3 with an activity of 720 Bq were included. The eciency of the alkali trap under conditions of fast mineralization was studied by setting up ¯asks with soil or chalk resembling those aged for one day in the experiment with mecoprop. The ¯asks were inoculated with PM, and after 6, 24 and 51 h two soil and two chalk samples were sacri®cially terminated and the previously described methods of acidi®cation were used. 3. Results 3.1. Mineralization and sorption of aged atrazine Fig. 1(a) shows the accumulation of evolved 14 CO2 from the mineralization of atrazine with dierent residence times in soil prior to inoculation with Pseudomonas ADP. The initial mineralization rate, determined as 14 CO2 accumulated during the ®rst 24 h, declined signi®cantly as the time of atrazine residence in soil increased (ANOVA, P < 0:05). This rate was signi®cantly reduced, when the residence time exceeded 1 day, whereas no dierences were observed between mineralization rates of samples aged for 7, 30, and 90 days (Tukey test, P 0:05). This result is in agreement with the result of the sorption experiment (Table 2). Signi®cantly more atrazine was available in the aqueous-phase of the samples aged for 1 day, than any of the samples with longer residence times (Tukey test, P 0:05). Due to a large variation no signi®cant dierences were observed in sorbed 14 C between dierently aged samples (ANOVA, P > 0:05). At the termination of the mineralization experiment 63 days after inoculation the 14 C recovered as 14 CO2 in the dierently aged samples was in the range 82±87%, and the only signi®cant dierence was between samples aged for 1 day and 30 days (Tukey test, P 0:05). Fig. 1(b) shows the mineralization of atrazine aged in chalk, and the initial mineralization rate decreased as the residence time increased (ANOVA, P < 0:05). The rate was signi®cantly reduced, when the residence time exceeded 7 days, whereas no dierences were observed between samples aged for 30 and 90 days (Tukey test, P 0:05). Even after 63 days of incubation the recovered 14 CO2 was reduced with increasing residence time (ANOVA, P < 0:05). The extent of mineralization ranged between 59% and 71%. The sorption experiment
G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934
931
added 14 C was recovered in the aqueous-phase, whereas 3±6% was recovered in the aqueous-phase of the chalk samples. 3.2. Mineralization and sorption of aged mecoprop No sorption of mecoprop were observed in either soil or chalk samples. In soil no signi®cant dierences in the initial mineralization rate was observed (ANOVA, P > 0:05). In chalk the initial mineralization rate of the samples aged for 1 and 7 days was lower than the rates of samples aged for 30 and 90 days (Tukey test, P 0:05). At the termination of the experiment 61 days after inoculation no signi®cant dierences in the 14 C recovered as 14 CO2 were observed between dierently aged chalk samples (ANOVA, P > 0:05). The total recovery of 14 C in the sorption and mineralization studies with mecoprop was in the range 88±100%. 3.3. Eciency of the alkali trap
Fig. 1. Accumulated mineralization by Pseudomonas sp. strain ADP of [ring-U-14 C]atrazine aged for 1 (), 7 (), 30 (N), and 90 days () in (a) soil and (b) chalk. Abiotic controls (). Points represent means of three replicates. Error bars indicate standard deviations.
(Table 2) showed signi®cantly increasing amounts of sorbed 14 C, when the residence time exceeded 7 days and decreasing amounts of 14 C in the aqueous-phase, when the residence time exceeded 1 day (Tukey test, P 0:05). However the maximum sorbed amount in chalk was 12% compared to 62% in soil. No mineralization occurred in the abiotic samples of soil or chalk. Fig. 2 shows the mass balance of the atrazine experiment, and the total recovery of 14 C was in the range 82±96%. In soil samples less than 1% of initially
The eciency of the alkali trap was investigated in buer-solutions with a range of pH-values between 3 and 10.5 (Fig. 3). Within 3 days 100% of the initially added 14 C was recovered in the alkali trap in buer-solutions with pH-values below 7.5. The eciency of the alkali trap at a given pH was reduced, when soil or in particular chalk was present in the system. In soil samples 84% of the initially added 14 C was captured in the trap after 3 days and 98% was captured after 12 days. In chalk only 13% of the initially added radioactivity was captured even after a period of 51 days. However, at the termination of the atrazine experiment results from the acidi®ed samples showed that the eciency of the alkali trap in soil was 100%, whilst it is being slightly reduced with an eciency between 92% and 99% in chalk. However, eciency of the alkali trap was markedly reduced in soil as well as in chalk under conditions of fast mineralization (Table 3). In the worst case, only 31% of the mineralized 14 C initially added as mecoprop was captured in the alkali trap 24 h after inoculation in chalk.
Table 2 Percentage of initially added [ring-U-14 C]atrazine sorbed and in solution after ageinga Ageing (days)
Soil Sorbed (% of added
1 7 30 90 a
50 7a 62 4a 60 14a 55 14a
Chalk 14
C)
Aqueous-phase (% of added 14 C)
Sorbed (% of added
51 6a 42 1b 41 1b 41 1b
22a 41a 111b 123b
14
C)
Values within columns followed by dierent letters are signi®cantly dierent (Tukey test, P 0:05).
Aqueous-phase (% of added 14 C) 1001a 902b 911b 931b
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G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934
Fig. 2. Mass balance for [ring-U-14 C]atrazine with dierent residence times prior to inoculation with Psedomonas sp. strain ADP in (a) soil and (b) chalk. Black: recovered as 14 CO2 , striped: 14 C in aqueous-phase, white: recovered after incineration. Error bars show the standard deviations of the total recovery of triplicates.
Fig. 3. The eciency of the alkali to trap 14 C 18 h after addition of NaH14 CO3 to buer-solutions (), soil (N), and chalk (). Points represent means of triplicates.
4. Discussion In this study the rate of mineralization of atrazine was reduced in soil as an eect of ageing, however, the extent of mineralization reached after 63 days of incu-
bation did not dier much between the dierently aged samples. A reduction in the mineralization rate (of a test bacterium designated M91-3) due to ageing of atrazine has been observed before by Radosevich et al. (1997) in a no-till soil as well as in a conventional-till soil. However, their observations of decreased mineralization rates combined with a reduction in the extent of mineralization were not observed in this study. Perhaps dierent mechanisms have caused the resistance to desorption in the two experiments. In this study the eect of ageing is likely to be due to slow diusion or reversible bindings, since the extent of mineralization in differently aged samples does not dier, whereas irreversible bound atrazine cannot be excluded in the study of Radosevich et al. (1997). Chung and Alexander (1998) investigated the eect of atrazine ageing for 200 days on the extent of mineralization of M91-3 in 16 dierent soils and observed an eect in 15 of the soils. A high content of organic carbon in soils increased the eect of ageing. From the mass balance in Fig. 2, it can be observed that more than 80% of the atrazine was mineralized in soil at the termination of the experiment, whereas the sorption data in Table 2 show that more than 50% was sorbed at the time of inoculation. Consequently, some of the initially sorbed atrazine must have been mineralized. There are two possible explanations: either (1) the sorbed atrazine is available for the bacteria or (2) desorption is rapid. Since signi®cant dierences in rates between dierently aged atrazine samples have been observed at least part of the sorbed atrazine must have been initially unavailable. In the aquifer chalk the mineralization rate and extent was reduced with increasing residence time of atrazine. Ageing to our knowledge had never been investigated before for herbicides in aquifer material. However, the result is in accordance with the observations of Hatzinger and Alexander (1995) which showed a decrease in the bioavailability of phenanthrene after ageing in nutrient amended aquifer sand. Since only 12% of the atrazine was sorbed after 90 days of ageing (Table 2), the eect of ageing is surprising. However, if the results of the mass balance (Fig. 2) are examined, it seems as though a larger fraction is sorbed after inoculation of the samples. The most obvious explanation is the incorporation of 14 C into biomass, however, this is not the case, since no cellular synthesis can be supported from the fully oxidized carbon atoms of the triazine-ring (Radosevich et al., 1995). In any case atrazine must somehow be associated to bacteria, dead bacteria or exudates. Due to this sorbed fraction ranging from 16% to 23% the extent of mineralization in chalk did not reach the same level as in soil. An eect of mecoprop ageing could be expected, since sorption has been observed in both soil and chalk
G.B. Kristensen et al. / Chemosphere 45 (2001) 927±934 Table 3 The eciency of the alkali to trap Incubation (h)
6 24 51 a b
14
933
CO2 produced from mineralization of mecoprop
Soil
Chalk 14
Caught in alkali (%)a
H CO3 (%)
0 19 48
0 11 5
a
b
Eciency (%)
Caught in alkalia (%)
H14 CO3 (%)
± 63 91
1 12 22
1 27 24
a
Eciencyb (%) ± 31 48
Percentage of initially added 14 C. Percentage of total mineralized caught in the alkali trap.
(Atalay and Hwang, 1999; Madsen et al., 2000). Atalay and Hwang (1999) measured the linear sorption distribution coecient (Kd ) of mecoprop in three dierent soils, and it ranged from 0.13 to 0.24. In chalk a Kd of 0.17 was measured by Madsen et al. (2000). Smith and Muir (1980) examined the extractability of aged 2,4-D, another phenoxyalcanoic acid herbicide, in three dierent soils and observed a rapid decrease in the amounts of extractable 2,4-D. However, in our experiment, no ageing of mecoprop was observed because no sorption was noted either in soil or chalk. The examination of eciency of the alkali trap elucidated that the capture of 14 CO2 was signi®cantly delayed at high mineralization rates particularly in chalk. This means that mineralization rates would be underestimated and caution should be taken not to compare soil and chalk samples. From a comparison of the soil and chalk samples with buer-solutions of the same pH, we can conclude, that the eciency of the alkali trap depends both on pH, trapping time, and soil type. To prevent degradation of the herbicides in the ageing period, the soil and chalk was sterilized before use. Depending on the sterilization method chosen dierent alterations of the material will result. Stroetmann et al. (1994) evaluated the eciency of dierent methods and examined changes in the speci®c surface area and cation exchange capacity. Irradiation seemed to be the least destructive method, since no changes in the two parameters were observed. The studies of bioavailability of each of the aged herbicides have been performed with a single bacterial strain or a highly enriched culture. This is an important simpli®cation of a natural system. Dierent organisms have dierent survival strategies, and in an enrichment procedure fast growing organisms with a low anity for the substrate may be favoured. Guerin and Boyd (1992) examined the bioavailability of naphthalene by using two dierent bacterial strains, and obtained dramatically dierent results which make generalizations regarding bioavailability of sorbed substrates inappropriate. Scribner et al. (1992) overcame the limitations of using pure cultures by performing their experiment with indigenous soil microbes, enabling more far-reaching
conclusions. They compared the degradation of ®eld aged and freshly added simazine, another triazine herbicide, and found freshly added simazine readily degradable, whereas aged simazine was biologically unavailable. Only few studies have dealt with the eects of ageing in aquifers. Our results show that ageing of the pesticides atrazine and mecoprop in samples from a chalk aquifer only caused a minor reduction in mineralization rates. Therefore, at least for these pesticides, ageing does not need to be taken into account when modelling their fate.
Acknowledgements This research was in part supported by the EC Environment and Climate Research Programme (Contract: ENV4-CT97-0441, Climate and Natural Hazards). Rene K. Juhler is thanked for discussions of the alkali trap. Hans-Jùrgen Albrechtsen is thanked for providing the radio-labelled NaH14 CO3 .
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