Environ. Sci. Technol. 2001, 35, 1610-1615
Biofiltration of Residual Fertilizer Nitrate and Atrazine by Rhizobium meliloti in Saturated and Unsaturated Sterile Soil Columns R E Z A M E H M A N N A V A Z , †,‡ SHIV O. PRASHER,† NARO MARKARIAN,† AND D A R A K H S H A N A H M A D * ,‡ Department of Agricultural and Biosystems Engineering, Macdonald Campus, McGill University, 21111 Lakeshore Road, Ste. Anne-de-Bellvue, Quebec, H9X 3V9 Canada, and Centre de Microbiologie et Biotechnologie, INRS-Institut Armand-Frappier, 245 Boulevard Hymus, Pointe-Claire, Quebec, H9R 1G6 Canada
This study was undertaken to investigate whether microbial bioaugmentation of subsurface soil with subsurface irrigation could be used as a biofiltration/biocontrol technology for agricultural pollutants. Nine Plexiglas columns, 458 mm long × 139 mm in diameter, were packed with a sterilized sandy loam soil. Subsurface irrigation, through a controlled water table management system, was used to deliver bacteria, Rhizobium meliloti A-025, to the soil and to maintain aerobic (unsaturated) or anaerobic (saturated) conditions in the columns. Nitrate and atrazine, a fertilizer and a corn herbicide, were applied to the soil surface, and leaching was affected by simulated rainfall events. The soil and drainage waters were analyzed for nitrate and atrazine residues after each rainfall simulation throughout the experimental period during which the soil was kept saturated for a total of 80 days and unsaturated for a total of 70 days. The monitoring of transport and survival of the implanted bacterial strain (A-025) showed that subsurface irrigation was successful in introducing and transporting the bacteria throughout the soil columns. During the saturated period, significantly more (95% probability) nitrate-N leached into the drainage waters from the control columns than from the bioaugmented columns; the increase being 450% or more for the abiotic control columns. The amount of atrazine that leached into the drainage waters during the unsaturated period was also significantly more from control columns as opposed to bioaugmented columns, with the increase being 262%.
Introduction Agricultural chemicals play a significant role in the production and protection of food and feed. It is estimated that corn yields in the second half of the 20th century quadrupled due to the use of fertilizers and pesticides (1). However, they have also gained notoriety for being one of the major nonpoint sources of groundwater pollution as they are * Corresponding author telephone: (514)630-8819; fax: (514)6308850; e-mail:
[email protected]. † McGill University. ‡ INRS-Institut Armand-Frappier. 1610
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subjected to drainage, surface runoff, and leaching under irrigation and rainfall. Nitrogen fertilizers are used very extensively in agriculture. It is estimated that 20-60% of the nitrogen fertilizer applied by farmers is lost through runoff, leaching, and denitrification (2) and that 30-60% of the nitrogen fertilizer applied in Quebec leaches into waterways and groundwater (3). The effects of water table management (WTM) on agricultural chemicals are also well documented (4-7). It has been suggested that controlling drainage increases the exposure time of chemicals to the degrading organisms and prolongs chemical leaching periods, subsequently decreasing pollution. For example, it was found that on farms with WTM systems, where water is pumped into the field for subirrigation and subsurface drainage methods are used, nitrate-N losses were decreased substantially from 34 to less than 20 kg ha-1 yr-1 (8). The other agricultural chemical investigated in this study, atrazine (2-chloro-4-(ethylamino)-6-isopropylamino-s-triazine), is one of the most extensively used herbicides for crops such as corn, sugarcane, pineapple, and fruit trees. Its presence in groundwater and its ecotoxicological impacts are well documented (9, 10). Various processes, such as hydrolysis, adsorption, volatilization, and photodegradation, govern its fate in the environment. However, the primary dissipation of atrazine is known to be through biological degradation at neutral pH and by chemical processes in acidic soils (11-13). Clay, organic matter, temperature, and pH are also important factors in the adsorption of atrazine. Adsorption increases as the clay content or organic matter content of the soil increases, whereas increasing temperature, soil water content, and pH reverses atrazine adsorption (14). Burkhard and Guth (15) reported that the rate of atrazine degradation by hydrolysis increases as the adsorption rate increases. Wenk et al. (16) showed that the rate of atrazine removal is proportional to soil water content. In the past decade, the microbial inoculation of soils for pest control and as fertilizer has attracted significant attention (17, 18). Rhizobia are used as inocula in many different countries for agricultural purposes because these symbionts, in the form of bacteroids, fix N2 in the roots of leguminous plants such as beans, clover, or alfalfa (19). Although the hallmark of rhizobia is N2 fixation, their ability to carry out denitrification via nitrate respiration during anaerobic growth has long been known (20) but never exploited. In fact, GarciaPlazaola et al. (21) have suggested that free-living rhizobia have the potential to remove fixed nitrogen from soil through denitrification under anaerobic conditions. They showed that oxygen, nitrate, temperature, moisture, and labile organic matter availability are the main factors that control denitrification by rhizobia. Denitrification is enhanced by temperatures from 15 to 25 °C and by saturation of pore spaces, which helps to reduce oxygen. Such anaerobic conditions are easily achieved in flooded soils after rainfall or irrigation. Three laboratories, including ours, have recently reported on the ability of rhizobia to transform atrazine (22-24). On the basis of these rationales, we attempted to evaluate one of our isolates of Rhizobium meliloti, strain A-025 (25), as a biofilter/biocontrol agent for two agricultural pollutants, nitrate-N and atrazine, using subirrigation for the bioaugmentation procedure.
Materials and Methods Soil Column Design and Setup. Nine Plexiglas columns, 458 mm long × 139 mm i.d., were packed with a sandy loam soil, S-VI (78% sand, 3% silt, 19% clay, and 3.7% organic matter, pH 6.17), excavated from the Macdonald Campus Farm of 10.1021/es0015693 CCC: $20.00
2001 American Chemical Society Published on Web 03/13/2001
FIGURE 1. Schematic diagram of a soil column. McGill University. The columns had a sampling port on the side (298 mm from the top) and were equipped with a delivery port at the bottom to supply water and bacterial inoculum. A schematic diagram of the setup is shown in Figure 1. The soil was well mixed and autoclaved for 1 h at 121 °C, three times each day, for two consecutive days before it was packed into the columns. A 20-mm sterilized gravel filter (size between 9.5 and 2.36 mm) was first packed at the bottom of the columns. The soil was then packed on top of the filter with 1.97 kg of soil every 100 mm, for a total of 8.44 kg of soil and a bulk density of 1300 kg m-3. Layers of sterilized cheesecloth were placed on top of the soil to minimize surface erosion during rain simulations. All columns, pipes, and tubes were sterilized by first washing them in 6.0% sodium hypochlorite (household bleach) and then rinsing with tap water that was also used in the experiment for irrigation. Preparation of Bacterial Inoculum. R. meliloti strain A-025 was grown in 5 mL of TYc (25) as a starter culture and incubated at 29 °C in a controlled environment incubator shaker (Psycrotherm, New Brunswick Scientific). Four-dayold cultures were inoculated into 300 mL of fresh TYc, then subcultured into six 2-L flasks after 24 h (each with 1 L of fresh TYc), and grown for another 24 h. The bacterial cells from the 1-L cultures (approximately 3.3 × 108 cell mL-1) were pooled and harvested by centrifugation (Dupont model RC5C, Sorvall Instruments) and plated on TYc and TYct (26) agar plates for microbial population counts and verification of purity. The collected cells were washed with 0.9% saline and resuspended in 200 mL of sterilized, deionized water and mixed well by vortexing before being introduced into the soil columns. Experimental Design. The three treatments, each in triplicate, were as follows: (i) NA: nitrate, atrazine, and bacterial inoculum; (ii) N: nitrate and bacterial inoculum, and (iii) abiotic control: nitrate, atrazine, and no bacterial inoculum. All soil columns first received 300 mL of tap water from the bottom delivery port (subirrigation), followed by 200 mL of cell suspension for those columns assigned for bacterial inoculum (i.e., treatment 1, NA; treatment 2, N). The columns were then saturated from the bottom by adding 2400 and 2600 mL of water to the treatment and the abiotic control columns, respectively. Eight days after the bacterial augmentation of the soil columns, 300 mg of calcium nitrate 4-hydrate, representing approximately 23 kg ha-1 of residual fertilizer nitrate, and 300 µL of atrazine (1000 ppm stock solution, 90% active) were uniformly applied to the soil surface. During the first 44 days after the atrazine and nitrate application, 20 mm (450 mL) of water was applied as rainfall simulation on the 9th day and on every 7th day thereafter so that all columns received a total of 120 mm of simulated rainfall. This is equivalent to the depth of rainfall that is expected to occur once every 25 yr in the month of May in Montreal, Canada. To observe the effect of a heavy rainfall, on the 80th day after chemical application, the equivalent
of 60 mm of water (1350 mL) was applied, simulating the rainstorm that occurred in Montreal on July 14, 1987. On the 80th day after nitrate and atrazine application, the columns were drained, and three more rain simulations of 40 mm were applied on the 104th, 112th, and 150th days. The stages of experimental setup and the amounts and times of water applications are given in Table 1. Sample Collection and Analysis. Water samples were collected at the bottom of the columns after every simulated rainfall event for analysis of leached nitrate, atrazine, and microbial counts. Soil samples (20 g) were collected through the sampling ports on the sides of the columns before each water application during the unsaturated period to analyze nitrate and atrazine residues. Nitrate was measured by the Soil Testing Laboratory of the Natural Resources Science Department of Macdonald Campus of McGill University, using a Quikchem automated ion analyzer. Atrazine analysis was performed as described by Liaghat and Prasher (2) and Masse et al. (27). Water samples were extracted by mixing 200 mL of the sample with 50 mL of methylene chloride in a separatory funnel. The mixture was hand-shaken for 5 min, and the organic layer was collected. This process was repeated three times, and the extracts were pooled and evaporated to dryness. The residues were then dissolved in 10 mL of hexane and analyzed by gas chromatography (GC). Soil samples were extracted by shaking 10 g of soil in 100 mL of methanol for 60 min and then filtering them under suction. The filtrate was then evaporated to dryness in a rotary evaporator at 35 °C. The residues were dissolved in 10 mL of hexane and analyzed using a GC. The extraction efficiency of samples was estimated to be 88% ( 5% (2). The GC was a Varian model 3400 equipped with a TSD detector, an autosampler, and an integrator. The GC column was a 0.53 mm i.d. fused silica Megabore DB-5. The detector and injector were kept at 290 and 190 °C, respectively. The column temperature was maintained at 150 °C for 10 min and then raised to 180 °C at a rate of 2.5 °C min-1. The helium carrier gas flow was 15 mL min-1. Bacterial Population Counts. Serial dilutions of the drain water, up to 10-4, were spread on TYct agar plates (selective for R. meliloti) and incubated at 29 °C for a period of 15 days, while the number of colonies was determined periodically. The plates from the control treatment showed no growth of bacterial colonies on this selective medium. Statistical Analysis. Statistical analysis for nitrate and atrazine loss were done using the General Linear Models (GLM) procedure, repeated measures analysis of variance, tests of hypotheses for between subjects effects, and multiple pairwise comparison between treatments using the SAS System release 6.12 for Windows (SAS Institute, 1989).
Results and Discussion Microbial Bioaugmentation. The delivery and implantation of strain A-025 were performed through subirrigation. The population of strain A-025 in drainage water was determined on TYct agar plates at different times during the experimental period (Figure 2). The results indicated that more cells were transported during the saturation period (on the 44th and 80th days) in treatment 1 (NA, nitrate and atrazine) than that of treatment 2 (N, nitrate). On the 104th day, the last day of the saturated (anaerobic) period, the number of cells leached from treatment 1 was lower than that observed in treatment 2. However, during the unsaturated (aerobic) period (on the 112th day), there was a large increase in the population leached from treatment 1. These results suggest that subsurface irrigation may have good potential for bacterial bioaugmentation of subsurface soil as the bacterial cells were transported from the bottom of the soil columns, throughout the soil profile, up to the soil surface. Biofiltration of Applied Nitrate. The biofiltration of applied nitrate was investigated in this study by first applying VOL. 35, NO. 8, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Experimental Stages and Schedule of Simulated Rainfall Events treatment stage
application
1
2
3
1 2 3 4 5
soil packing (kg/column) subirrigation (mL) subsurface bacterial inoculation (mL) subirrigation (mL) surface application: atrazine (mL of 1000 ppm) nitrate (mg)
8.443 300 200 2400
8.443 300 200 2400
8.443 300 0.0 2600
0.3 300
0.0 300
0.3 300
day
water applied (mL)
9 16 23 30 37 44 80 104 112 150
450 450 450 450 450 450 1350 900 900 900
Stage 6: Rain Simulations rainfall (mm) 20 20 20 20 20 20 60 40 40 40
FIGURE 2. Leached population of bacterial cells, strain A-025, in the drain water during the experiment (cfu, colony forming unit).
TABLE 2. Results of General Linear Model Procedures for Nitrate and Atrazine Concentrations at Different Experimental Periods Pr > F nitrate-N period
saturated
unsaturated
day
drainage water
9 16 23 30 37 44 80 104 112 150
0.3864 0.9959 0.8622 0.5053 0.4229 0.3859 0.1524 0.6292 0.9541 0.3075
atrazine
soil
drainage water
soil
0.0204 0.7844 0.4843
0.3693 0.1160 0.5882 0.9141 0.2337 0.6481 0.3800 0.2580 0.0047 0.3245
0.9912 0.2190 0.4307
the GLM procedure to nitrate-N residues in drainage waters of both saturated and unsaturated periods and in soil, as presented in Table 2. During the saturated period of the experiment (the first 80 days), the nitrate-N residues in the drainage waters of all treatments were not significantly different from each other on the days on which the samples were taken (Figure 3). The same can also be said for the samples of the drainage waters or soil samples taken during the unsaturated period (Table 2). The only exception is the nitrate-N residues in soil on day 104. This more or less implies that, if we compare the nitrate-N residues in the drainage 1612
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soil condition saturated saturated saturated saturated saturated saturated saturated unsaturated unsaturated unsaturated
waters or the soil samples of the various treatments of the same day during the saturated or unsaturated periods, we may arrive at the conclusion that there is no treatment effect. However, we would have arrived at this conclusion by making treatment comparisons on specific days, and the impact of repeated measurements made over time would not have been considered. Thus, a repeated measures analysis of variance was carried out to investigate the impact of different treatments over time (Table 3). Again, there was no significant treatment effect, although the nitrate-N residues in drainage waters during the unsaturated period were significantly different with respect to time. The combined effects of treatment and time for both saturation periods and in soil were also not significant. To investigate this further, the nitrate-N concentrations in drainage waters and soil over the experimental period were summed for each saturation period, and a protected LSD test was performed to investigate variations among the different treatments by doing multiple pairwise comparisons of the overall means of each treatment (Table 4). During the saturated period, the summed nitrate-N concentrations in drainage waters were significantly different (R ) 0.05) between the treatment columns and control. The values in the control columns were significantly higher than those in the nitrate only and nitrate plus atrazine soil columns. The summed concentration of nitrate (seven measured values after the 1st to the 7th rainfall simulations) in drainage waters was 18 ppm for the control columns, as opposed to 4.0 ppm for the atrazine-nitrate and 2.0 ppm for the nitrate soil columns (Table 4). These results suggest that anaerobic conditions during the saturated period would have initiated denitrification in the bioaugmented columns, but not in the abiotic control columns. Since all columns were autoclaved in the beginning and the two types of treatment columns were bioaugmented with R. meliloti A-025, the differences in nitrate-N residues in drainage waters can be attributed only to bioaugmentation. It is possible that the bacterial bioaugmentation could be helping either in the denitrification process or in immoblizing the nitrate residues in soil. Since there is no significant increase in the nitrate-N residues in soil after the saturated period, this appears to be less likely. In any case, by looking at the results presented in Table 4, it can be stated with confidence that subirrigation was able to introduce bacteria into soil (Figure 2) and that this
FIGURE 3. Concentration of nitrate-N in the drainage waters during (a) the saturated period, (b) the unsaturated period, and (c) in the unsaturated soil after the saturated period. Results represent the mean and SD of three replicates. NA, treatment 1 (bacteria, nitrate, and atrazine), N, treatment 2 (bacteria and nitrate). introduction significantly reduced nitrate-N pollution over the saturated period of the experiement. Zablotowicz et al. (20) and Garcia-Plazaola et al. (21) have suggested the following sequence as the likely pathway for denitrification in anoxic environments by different species of rhizobia: nitrate ion (NO3) f nitrite ion (NO2) f nitric oxide (NO) f nitrous oxide (N2O) f nitrogen gas (N2). However, there is no report of these bacteria oxidizing nitrite ion (NO2) back into nitrate ion (NO3), or atrazine-NH3 into NO3-, in a
nitrification process, under oxic conditions. It is therefore difficult to explain the higher concentration of summed nitrate-N in the drainage water of treatment 1 in the presence of atrazine. Under unsaturated (aerobic) conditions, the summed nitrate-N concentrations (3 measured values after three rainfall simulations on days 104, 112, and 150) in the drainage waters, contrary to those measured during the saturated period, were not significantly different, nor were those in the VOL. 35, NO. 8, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Repeated Measures Analysis of Variance of Nitrate-N and Nitrate-N and Atrazine Treatments (r ) 0.05) Pr > F nitrate-N
atrazine
drainage water
soil
drainage water
soil
source
saturated
unsaturated
unsaturated
saturated
unsaturated
unsaturated
treatment time time × treatment
0.3030 0.2603 0.5284
0.2719 0.0015 0.5568
0.2290 0.1457 0.8953
0.2616 0.0242 0.5991
0.0269 0.0007 0.0069
0.1471 0.1762 0.7171
TABLE 4. Summed Drainage Waters and Soil Concentrations of Nitrate-N and Atrazine during Different Periods of the Experimenta concn during different periods chemical
treatment
saturated (drainage water)
unsaturated (drainage water)
unsaturated (soil)
nitrate-N (ppm)
1 (NA) 2 (N) 3 (control) 1 (NA) 3 (control)
4 ( 3a 2 ( 1a 18 ( 14b 5.9 ( 0.9 7(1
109 ( 11 98 ( 25 86 ( 10 4.2 ( 0.8c 11 ( 3d
2.6 ( 0.6 2.3 ( 0.8 1.7 ( 0.4 19 ( 4 25 ( 4
atrazine (ppb)
a Different letters indicate significant difference between treatments (R ) 0.05) based on the protected LSD test in the multiple pairwise comparisons of overall means among treatments. a and b for nitrate-N, c and d for atrazine. NA stands for nitrate and atrazine treatment, and N stands for nitrate treatment.
soil (Figure 3b; Table 4). This is to be expected since denitrification is known to take place under saturated conditions, and there are significant differences in the summed nitrate-N concentrations between bioaugmented and control soil columns. Biofiltration of Applied Atrazine. Atrazine residues in drainage waters and soil were analyzed along the same lines as the nitrate-N. First the GLM procedure was used to compare atrazine concentrations in different treatments (Table 2) found on specific days during the saturated and unsaturated periods (Figure 4). Since the measurements were repeated several times during each saturation period, a repeated measures analysis of variance was also applied to the measured data. And last, summed atrazine levels from each treatment and each experimental period were analyzed by using the protected LSD test in multiple pairwise comparisons of the overall means of various treatments. The results of the GLM procedure are given in Table 2. Like nitrate-N, there is no statistical difference in atrazine concentrations in drainage waters or in soil on measurement days during both saturated and unsaturated periods, with the exception of day 112 during the unsaturated period. The repeated measures analysis of variance was applied next to study the effect of different treatments over time. For the saturated period, there is no treatment effect. In general, there is little microbial activity under saturated conditions, and thus these results are to be expected. The atrazine concentrations significantly vary over time in all treatments, and this is also to be expected since atrazine will be getting sorbed/desorbed onto soil particles or organic matter over time, thus variably leaching out of soil. However, during the unsaturated (abiotic) period, there is a significant treatment effect and a highly significant time and treatment × time effect (Table 3). Since all columns were autoclaved in the beginning and there is a significant treatment effect, it clearly means that bioaugmentation has worked and that the bacterial strain used in the study, R. meliloti A-025, is causing atrazine degradation in a significant way (Figure 4) thereby leading to significantly different atrazine concentrations in drainage waters during the unsaturated period. Like nitrate-N, atrazine residues were also summed over two saturation periods, and the analysis results are given in Table 4. The protected LSD test was applied to perform the multiple pairwise comparisons of the overall means among 1614
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FIGURE 4. Concentration of atrazine in the drainage waters during (a) the saturated period, (b) the unsaturated period, and (c) in the unsaturated soil following the saturated period. Results represent the mean and the SD of three replicates. different treatments. As expected, while the summed atrazine concentrations (seven measured values after the 1st to the 7th rainfall simulations) did not vary significantly during the saturated (anaerobic) period of 80 days, the values in the NA
treatment (Table 4) were significantly lower than those in the control during the drainage events that took place in the unsaturated (aerobic) period of 70 days. The concentrations were 11 ppb for the control and 4.2 ppb for the bioaugmented soil columns (Table 4), with the largest difference occurring on the 112th day (Figure 4b). During this aerobic period, atrazine concentration in the bioaugmented soil samples was also lower (Figure 4c), and the bacterial population showed a large increase (Figure 2). The summed concentration of atrazine in the drainage waters from control soil columns was 262% higher than that from bioaugmented columns. These results reaffirm that R. meliloti, strain A-025, may be used to decontaminate soils that are contaminated with atrazine. The loss of atrazine in bioaugmented soil columns might have been different if nitrate had not been added or if aerobic conditions had been maintained over the entire experimental period. Wilber and Parkin (28) and Crawford et al. (12) have shown that the transformation of atrazine is decreased in the presence of nitrate, and Topp et al. (29) reported that atrazine was not degraded under anaerobic or denitrifying conditions. Many studies have investigated the transport and fate of agricultural chemicals through saturated and unsaturated soil profiles (30-32). Saturated conditions are the worst-case scenario for chemical movement in groundwater systems (33). Therefore, to maximize the leaching of nitrate and atrazine, the initial stage of this experiment was performed using saturated soil columns. In these worst-case scenarios, with rain simulations of 20 mm, low concentrations of atrazine were detected in drainage waters. Atrazine has been shown to have very low leachability through soil columns (2, 34). Smith et al. (35) suggested that long periods of water application are required in order to affect the atrazine concentration in a soil profile. Therefore, the higher concentrations of both nitrate and atrazine observed in the drainwater collected after the 80th day (onset of the unsaturated period) might have resulted from the increased depth of rain simulations applied after this date. In conclusion, subsurface irrigation was successful in introducing and translocating bacteria in the soil columns. Under saturated conditions, the strain A-025 significantly (95% probability) reduced nitrate-N leaching as compared to the control. The difference in nitrate-N leaching from the two bioaugmented treatments however was not significant. Furthermore, the atrazine loadings from bioaugmented soil columns were significantly (95% probability) less than that from the control columns under unsaturated conditions. Other transformation products of atrazine were not measured in this study, and the soil used had been sterilized and was therefore devoid of detectable soil microflora and plants. Hence, until further investigations are conducted, these data should not be extrapolated to predict the fate of nitrate-N or atrazine in agricultural systems. The overall results of this study indicate that bioaugmentation of agricultural soils with ecotoxicologically and ecopathologically safe and suitable bacterial strains, such as Rhizobium, using a WTM-based subsurface bioaugmentation system to biofilter/biocontrol/ biodegrade agrochemicals such as nitrate and atrazine before they reach aquatic systems, may be a feasible, effective, and sustainable solution for the reduction of leaching farm pollutants.
(2) (3) (4) (5) (6) (7) (8) (9)
(10) (11) (12) (13)
(14) (15) (16)
(17) (18) (19) (20) (21) (22) (23)
(24) (25) (26) (27) (28) (29) (30) (31) (32) (33)
Acknowledgments This work was partly supported by NSERC research grants to D.A. and S.O.P. and by NSERC and INRS graduate fellowships to R.M.
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Received for review August 9, 2000. Revised manuscript received January 30, 2001. Accepted February 2, 2001. ES0015693 VOL. 35, NO. 8, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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