Environ. Sci. Technol. 1997, 31, 3682-3685
Automated Procedure for Extraction of Metolachlor from Soil† THERESA H. LEMME,* ALAN OLNESS, AND W. B. VOORHEES USDA, Agricultural Research Service, North Central Soil Conservation Research Laboratory, 803 Iowa Avenue, Morris, Minnesota 56267
Methods for extraction of metolachlor [2-chloro-N-(2-ethyl6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] from soil are tedious and time-consuming. Existing robotic stations were adapted to provide an automated multistep method of metolachlor extraction from soil. The method was examined using a fine-loamy, mixed Udic Haploboroll. Duration of equilibration (0-4 h), initial soil pH (5.07.1), temperature of evaporation (24, 35, and 50 °C), and gas used for perfusion (air or N2) were examined. Extraction efficiency was unaffected by duration of equilibration or initial soil pH. A temperature by perfusion gas interaction affected percent recovery. At 24 °C, N2 provided a small but measurable advantage in recovery, 71.1-74.2%. At 35 °C, a clear advantage in recovery, 50.5-69.8%, was obtained using compressed air. At 50 °C, recovery, which averaged only about 38%, was unaffected by perfusion gas. Relative recovery was unaffected by incubation time in 90% methanol over a 4-h range. Relative to the current extraction method, the serialized robotic method increased sample output by 267%. Relative costs of extraction were also compared. Hazardous waste generation was decreased by about 67%, and reagent cost was decreased by > 75% using the robotic method.
Introduction Metolachlor, a selective acetanilide herbicide, is used to control annual grasses and some broad leaved weeds in corn (Zea mays L.), soybeans (Glycine max L.), and other crops and vegetables. Evaluation of herbicide movement through soil requires analyses of a large number of samples. Current methods for metolachlor extraction from soil are long and tedious, involving multiple extractions, several solvents, long separation and evaporation steps, and analysis. Bouchard et al. (1) determined herbicide residue in soil using a 30-day pearl millet bioassay of metolachlor treated soil. A Soxhlet apparatus has been used for metolachlor extraction followed by centrifugation and radioassay (2) or cleanup on a Florisil column and analysis using capillary gas chromatography (GC) with an electron capture detector (ECD) (3). Weber and Swain (4) used radiochemical techniques to study metolachlor sorption by four soils. They found that sorption was correlated with organic carbon and humic matter and with clay content of the soils. Using a reversed-phase anion exchange solid-phase extraction method, Watts et al. (5) were able to manually extract six pesticides simultaneously. † Contribution from the USDA in cooperation with the Minnesota Agricultural Experiment Station, University of Minnesota, St. Paul, Science Journal Series No. 971250025. * Corresponding author tel: (320) 589-3411; fax: (320) 589-3787; e-mail:
[email protected].
3682 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 12, 1997
After benzene elution and evaporation under nitrogen (N2), they were able to manually process 20 samples per day for analysis by HPLC or GC. The manufacturer’s recommended method (6) for metolachlor extraction requires a 2-h methanol-water extraction in a Soxhlet apparatus, triplicate hexane partitioning of the methanol-water extract, removal of water by elution of the hexane extract through a column of Na2SO4, removal of hexane under vacuum in a rotary evaporator, and resuspension in hexane for analysis by GC. Automated robotic systems have been used for preparation of soil samples for atrazine [6-chloro-N-ethyl-N′-(1-methylethyl)-1,3,5-triazine-2,4-diamine] and alachlor [2-chloro-N(2,6-diethylphenyl)-N-(methoxymethyl)acetamide] analysis (7) and for the extraction of pesticide residues in other materials (8-10). A robotic method for metolachlor extraction has been used for soil extraction with methanol (11). The objective of this study was to develop an automated robotic procedure for the extraction of metolachlor from soil using an innovative solvent drying station. A station is a position in the robot domain where the robot has access to the instrumentation needed to complete a robotic task. Examples of some of these stations include the vortex station, the centrifuge station, a liquid-solid station (a sipping cannula to remove a precise quantity of extracting liquid above a centrifuged sample), and a master laboratory station that allows addition of known amounts of solvents. Robotic extractions involve several distinct steps or stations (Figure 1) within which or at which transfer of metolachlor may be less than complete. Because of this, recovery of metolachlor was evaluated under a range of conditions at several of the robotic stations at which loss might reasonably be expected to occur. In addition, three factors (evaporation temperature, gas used for perfusion at the evaporative drying station, and pH of the sample) were examined for their possible impact on recovery of metolachlor.
Methods and Materials Robot Modifications. A Zymark Zymate II core robotic system (Zymark Corp., Hopkinton, MA) was modified and programmed to perform metolachlor extractions from soil. The liquid-solid station of the Zymate II robot, originally configured to perform a pesticide cleanup by eluting the aqueous sample through a solid-phase extraction C-18 resin column, was modified as a solvent drying station with a custom-made cap assembly (12). A schematic diagram of the modified robot is given in Figure 1. Computer Programming Modifications. The robot is controlled by ‘easylab’ programs supplied by the manufacturer. Once the solvent drying station is installed in the liquid-solid station position within the robotic apparatus, the new station parameters, such as the station name and position, are programmed in the robot’s dictionary through the “Teaching screen”. Programming allows the robot to access the new position and interact with all other stations needed for the extraction. The Na2SO4 drying station required use of 50-mL centrifuge tubes to collect the large amount of filtrate generated in this procedure. The 13-mm tube holder in the output position on the liquid/solid station shuttle is replaced with a 50-mL centrifuge tube holder. Information on the change in tube size is entered into the program parameters associated with this station. Four centrifuge tube racks are required for this procedure; therefore, the robot is programmed to recognize each existing 40-position rack as two 20-position racks. Each 20-position rack requires that an identification code, clear positions, rack S0013-936X(97)00416-1 This article not subject to U.S. copyright. Published 1997 by the American Chemical Society.
FIGURE 1. Schematic diagram of the bench layout of Zymark robot stations for the extraction of metolachlor from soil: (1a) controller and computer; (1b) robotic core system; (2) capping and decapping station; (3) centrifuge; (4) dilute and dissolve station; (5) disposal station; (6) liquid/liquid extraction station; (7) evaporative drying station; (8) racks, 50-mL centrifuge tube; (9) hand, 20-30-mm tubes; (10) pipetting hand and 1-mL tip rack; (11) hand, 9-16-mm tubes; (12) solvent drying station; (13) rack, 16 by 100 mm tubes; (14) crimp capping station; (15) rack, 11-mm GC vials; (16) master laboratory stations.
FIGURE 2. Interaction of evaporation station temperature and perfusion gas on recovery of metolachlor (means of three metolachlor concentrations, equilibrated for 2 h with 90% methanol in water at 70 °C). Vertical lines represent standard deviations of means. indices, cap status, and volume for each rack be coded within programs to allow the robot to access each sample. Rack 1 tubes are capped and contain 10-g wet-frozen soil samples (corrections for soil moisture were made in the final calculations) with 10 mL of 90% methanol in water. Rack 2 tubes are uncapped and contain 1 mL of 6.6 M NaCl (water saturated with NaCl to aid formation of the methanol/hexane meniscus). Tubes in racks 3 and 4 are empty, uncapped, and receive extracts from initial tubes. The original extraction program automates robotic pesticide extraction by withdrawing liquid with a sipping cannula from above a centrifuged sediment or pad of soil (10-g soil sample in 10 mL of 90% methanol in water) at the liquidliquid station. Later in this procedure, it is necessary to also withdraw hexane from the organic hexane layer overlaying the water-methanol extracting solution. The sipping cannula depth is adjusted within programs for removing the methanol extract above the centrifuged soil sediment and for removing the hexane extract above the methanol layer. The centrifuge is programmed to use all swinging buckets. Two separate sets of balance tubes are needed: one set for the soil/methanol-water centrifugations and one set for the methanol-water/hexane centrifugations.
Conventional Extraction Method. The manual method of metolachlor extraction (6) involves a 2-h extraction with a Soxhlet apparatus at 70 °C using 35 g of wet-frozen soil in 200 mL of 90% methanol in water (v/v). The soil-methanol mixture is filtered through nested filters (a Reeve Angel 802 filter inside a Whatman 2V filter) and brought to 200 mL volume. Methanol-water eluent, 30 mL, is placed in a separatory funnel along with 10 mL of 6.6 M NaCl and 100 mL of water. The aqueous methanol phase is partitioned three times with 50-mL portions of hexane. The hexane extracts are pooled and poured through a 30-g pad (granular layer or column) of anhydrous Na2SO4. The flask is rinsed with 40 mL of hexane, which is poured through the pad. The pad is rinsed twice with 25 mL of hexane. The hexane is evaporated from the pooled hexane-metolachlor extracts under vacuum in a rotary evaporator (dry ice and alcohol condensation). The herbicide residue is dissolved in two 2.5mL rinses of the evaporated flask, and a 1-mL portion is placed in a gas chromatographic vial for capillary gas chromatographic analysis with a nitrogen phosphorus detector. Robotic Extraction Method. A 10-g sample of soil is placed into a 50-mL screw-capped glass centrifuge tube (tube 1). Ten milliliters of 90% methanol in water (v/v) is added, and the tube is capped tightly and weighed. The suspension is equilibrated for 2 h in a 70 °C water bath, and 90% methanol is added to adjust for any loss from the original weight before the sample is placed on the robot for extraction. Within the scheduled program, samples are individually vortexed and centrifuged. A 5-mL portion of the extract is removed to a 50-mL test tube (tube 2) containing 1 mL of 6.6 M NaCl. Water, 5 mL, is added to the remaining sample in tube 1, which is again vortexed and centrifuged before a second 5-mL portion is removed to tube 2 (11 mL total). Six milliliters of hexane is added to the methanol extract in tube 2. The tube is vortexed and centrifuged before a 4-mL portion of the hexane supernatant solution is withdrawn and placed in tube 3 (4 mL total). An additional 5 mL of hexane is added to tube 2, which is vortexed and centrifuged after which a second 4-mL portion of hexane solution is withdrawn and pooled with the hexane extract in tube 3 (8 mL total). The pooled hexane extract is eluted through the anhydrous Na2SO4 drying station; tube 3 is rinsed with 2 mL of hexane and vortexed; and the contents are eluted through the drying station and pooled in tube 4 (10 mL total). Air pressure is used to expel the last traces of hexane from the Na2SO4 and into tube 4. The Na2SO4 pad is rinsed twice with 2 mL of hexane, and the eluate is pooled in tube 4 to make a final volume of 14 mL. Tube 4, containing the dehydrated hexane extract, is placed in the evaporative drying station where the hexane is volatilized with the aid of perfusing either a compressed air stream or N2 onto the liquid through an overhead cannula. When the tube is dry, the metolachlor residue is dissolved in 2 mL of hexane; the tube is vortexed; a 1-mL portion of the herbicide-hexane solution is pipetted into a gas chromatographic vial; and the vial is capped. Soil and Analytical Procedures. A 1 mg mL-1 stock solution of metolachlor was prepared by diluting 100 mg of analytical grade metolachlor (CGA 24705 Novartis Crop Protection, Inc., 99.7% purity) in a volumetric flask to a volume of 100 mL with hexane. Metolachlor standards (2.5-100 µg mL-1) were prepared by placing stock solution in a volumetric flask and bringing the volume to 50 mL with the addition of the appropriate volume of hexane. Surface soil from a Barnes loam [fine-loamy, mixed Udic Haploboroll, pH 7.1 (1:2 0.01 M CaCl2 (13)], organic carbon 29.1 g kg-1 (14), total Kjeldahl nitrogen 2.4 g kg-1 (15), and cation exchange capacity 298 mmol[+] kg-1 (16)] was used to evaluate the automated extraction of metolachlor. Soil pH was adjusted by the addition of AlK(SO4)2 to 400 g of soil in an evaporator dish. Six preparations (0.25, 0.5, 1.0, 2.0,
VOL. 31, NO. 12, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3683
TABLE 1. Recoveries of Metolachlor As Influenced by Modifications of Procedure in Sample Preparation metolachlor treatment
metolachlor added
reps
1 2 3ac 3bc 4a 4b
before centrifugationb to methanol before partitioning to hexane before drying station, air drying to hexane before drying station, N2 drying to dehydrated hexane extract before air drying to dehydrated hexane extract before N2 drying
4 4 5 4 4 3
recovered (%)a 79.5 ( 3.59 86.2 ( 2.21 89.5 ( 3.60 101 ( 1.70 99.5 ( 10.3 113 ( 3.26
a Recoveries are the mean ( standard deviation of all metolachlor concentrations. b Soil samples equilibrated with 90% methanol in water at 60 °C for 2 h before centrifugation. c Metolachlor was added to the hexane before dehydration in the Na2SO4 drying station and subsequent volatilization of the hexane under an air or N2 stream at 24 °C.
TABLE 2. Effect of Method on Time Required for Individual Metolachlor Analysis method
timea steps (h) (number)
bioassay 720 extraction with Soxhlet apparatus 24 14C exchange 10 water and methanol extraction 2 supercritical fluid extraction 3 robotic extraction 3.4
3 6 3 9 5 5
ref 1 3 4 5 18 this report
a Time required for analysis of a single sample. Sequential programming and multiple samples reduce the average time for analysis within a procedure. Four samples are simultaneously in various stages of the extraction within the robotic extraction.
3.0, and 4.0 g) of AlK(SO4)2 were dissolved in 120 mL of distilled-deionized water. The solutions were added to six soil preparations, mixed thoroughly, covered, and allowed to equilibrate at room temperature. Additional water (20 mL) was added on the fifth and tenth day of equilibration after which both of the soil preparations were again mixed. After 2 weeks, the soils were dried at 105 °C for 12 h and ground for metolachlor extraction. For each metolachlor assay, four replicates of 10 g of dry soil were placed in 50-mL centrifuge tubes, metolachlor standard in methanol was added to the soil (5 ng mg-1), and the mixture was then vortexed and allowed to equilibrate 16 h at room temperature before robotic extraction. The effect of temperature during the evaporation step (Figure 2) was studied by setting the temperature of the evaporative heating block at 24 (room temperature), 35, or 50 °C (five replications). The effect of the perfusion gas used at the evaporation station was examined with compressed air and commercial N2. To evaluate station recoveries (Table 1), metolachlor was added to treatment 1, the soil before initial centrifugation; treatment 2, the methanol extract before hexane partitioning; treatment 3, the hexane extract before removal of water at the drying station; or treatment 4, the dehydrated hexane extract just before the evaporative drying station. Metolachlor was added to the soil to effect a final concentration of 5, 10, and 20 ng mg-1 soil, the sample was thoroughly mixed, and the metolachlor was allowed to equilibrate for 16 h; then, 10 mL of 90% methanol were added and the soil sample in 90% methanol was equilibrated at 70 °C. After the methanol equilibration, the extraction procedure was initiated. The equilibration time for soil in 90% methanol was evaluated for periods of 0, 0.25, 0.5, 1, 2, and 4 h. Data from the various tests were subjected to statistical evaluation using SAS programs including PROC TTEST, PROC GLM, and PROC ANOVA (17). A probability level of 5% was chosen for significance.
Results Both temperature of the evaporation station and the gas used for perfusion in the evaporation station affected relative
3684
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 12, 1997
recovery of metolachlor (Figure 2). When evaporation was at 24 °C, recovery of metolachlor added to the soil was slightly greater with compressed N2 (74.2 ( 6.0%) than with air (71.1 ( 6.2%; p < 0.05). Increasing the temperature of the evaporative drying station increased the rate of volatilization and shortened the time required to process each sample. The effects of the gas appear to be complex because at the intermediate temperature of 35 °C greater recovery was obtained with air (69.8 ( 7.8%) than with N2 (50.5 ( 4.7%; p < 0.05). The reason for the temperature by gas-composition interaction at the evaporation station is unclear. At a temperature of 50 °C, the recoveries were low, 38 ( 4.6%, and were indistinguishable between the gases. The reduced recovery of metolachlor added before centrifugation and to the methanol before hexane partitioning (treatments 1 and 2, Table 1) is expected because of adsorption due to partitioning with soil (79.5 ( 3.59% and 86.2 ( 2.21%, respectively). Little loss of metolachlor occurred when water was removed from hexane at the sodium sulfate station (treatment 3). Recoveries of metolachlor added to the dehydrated hexane at the solvent drying station ranged from 89.5 ( 3.6% when subsequently evaporated with compressed air to 101 ( 1.7% with N2 (24 °C). Recoveries of metolachlor added at the evaporative drying station (treatment 4) distinctly benefited from the use of N2 when evaporation occurs at 24 °C (p < 0.05). Recovery of metolachlor from soil, which ranged from 68 to 74% when the dried hexane was volatilized with compressed air and from 68 to 78% when volatilized with N2, was unaffected by duration of equilibration with the 90% methanol in water which ranged from 0.25 to 4 h before centrifugation (data not shown). Lengthy equilibration is usually effective in releasing more tightly bound residues that have been intimately associated with the soil surfaces for an extended period of time. Here, metolachlor was added only a few hours before extraction, and conversion of readily extractable residues to ‘bound’ residues was apparently minimized. Addition of the six rates of AlK(SO4)2 to the Barnes soil (original pH 7.1) resulted in a pH range of 5.0-7.0 (pH values: 5.0, 5.2, 6.1, 6.5, 6.8, and 7.0). Recovery of metolachlor added to soil in the extracting solution, which ranged between 64 and 72% when volatilized with compressed air and from 74 to 78% when volatilized with N2, was unaffected by soil pH (data not shown). Soil pH was modified to determine if it affected the relative recovery of metolachlor after extraction. This differs from soil that has been treated to establish different pH values and to which metolachlor has been added and allowed to equilibrate for the purpose of observing relative adsorption effects. Under most circumstances, the pH of the soil used in the extraction seems unlikely to effect any loss of extracted metolachlor during individual steps in the automated method. The recovery of about 70% of recently added metolachlor with the robotic procedure (Figure 2) appears consistent with that of other studies. For example, assuming usual bulk
TABLE 3. Time, Chemical Use, and Cost Comparison of Soxhlet Apparatus and Robotic Extraction Methods Soxhlet apparatusa
robotic chemical step
time (h)
chemical costb ($)
step
form
vol (mL)
initial extraction
0.8
methanol
90
0.60c
hexane partitioning
0.8
dehydration
0.45
hexane methanol hexane sodium sulfate
11 40 6
0.11 0.25 0.06 0.05
evaporation dissolution total
0.95 0.40 3.4d
2 149
0.02 1.09
hexane
time (h)
form
Soxhlet extraction filtration hexane partitioning
3.0 0.5 0.25
methanol (2 filters) hexane
dehydration
0.50
sodium sulfate
rotary evaporation dissolution
0.50 0.25 5.0
hexane
vol (mL)
costb ($)
200
1.25 0.50 2.35
240
0.50 5 445
0.05 4.65
a Current method developed by registrant. b Costs exclude hazardous waste disposal expense. c Costs are current estimates from a variety of commercial sources. Individual suppliers and volume discounts contribute to variance in actual costs. Total cost does not include robotic or Soxhlet apparatus or operating costs. d Individual determination. Sequential programming and volume preparations permit several samples to be processed simultaneously.
densities, it appears that 60-80% of freshly applied metolachlor was recovered in a field study on a loamy sand 24 h after application (11). Adsorption of metolachlor is positively correlated with soil organic matter content and soil clay content (2), and the Barnes loam is relatively rich in organic matter. An initial incubation time of 2 h was chosen, since time of equilibration made no difference in the recovery, for equilibration of soil samples in 90% methanol in water at 70 °C in the automated robotic method. The extracted metolachlor in hexane is dehydrated in the solvent drying station before the hexane is volatilized at 24 °C in the evaporative drying station since there was no real time advantage in the integrated program to heating the station to 35 °C. Nitrogen gas was used as the perfusive gas at the evaporative drying station. Manual handling (2 h day-1) is limited to weighing wetfrozen soils into centrifuge tubes, adding the initial 10 mL of 90% methanol in water, placing the tubes in the 70 °C waterbath and removing them after the 2-h equilibration, and placing the tubes on the robot. Further manual handling is associated with glassware cleanup and assurance that robotic reservoirs (hexane, methanol, and water) contain enough solvents for the extractions. The present robotic method greatly reduced the time required for sample analysis (Table 2). From the method of Watts et al. (5), the total extraction time was 2 h sample-1. By serializing the procedure, the author could run 20 samples day-1. Using the presented method, the total time to extract a sample is 3.4 h, but by serializing the procedure, the time required for robotic extraction is about 1.5 h. Reagent costs are reduced by 75%, and hazardous waste generation is reduced by 67% (Table 3).
Acknowledgments The metolachlor extraction program is available on disk from the USDA Agricultural Research Service, North Central Soil Conservation Research Laboratory. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap. Manufacturer’s and trade names are included in the text as a convenience to the reader and do not constitute any preferential endorsement of those products over other similar products.
Literature Cited (1) Bouchard, D. C.; Lavy, T. L.; Marx, D. B. Weed Sci. 1982, 30, 629-632. (2) Peter, C. J.; Weber, J. B. Weed Sci. 1985, 33, 874-881. (3) Sauer, T. J.; Fermanich, K. J.; Daniel, T. C. J. Environ. Qual. 1990, 19, 727-734. (4) Weber, J. B.; Swain, L. R. Soil Sci. 1993, 156, 171-177. (5) Watts, D. W.; Bogus, E. R.; Hall, J. K.; Mumma, R. O. J. Environ. Qual. 1994, 23, 383-386. (6) Wurz, R. E. M. Novartis Crop Protection, Inc., personal communication, 1994. (7) Koskinen, W. C.; Jarvis, L. J.; Dowdy, R. H.; Wyse, D. L.; Buhler, D. D. Soil Sci. Soc. Am J. 1991, 55, 561-562. (8) Becker, J. M.; Culligan, J. F., Jr. Proceedings of an International Symposium on Laboratory Automation and Robotics, Boston; Zymark Corp.: Boston, 1992; pp 508-517. (9) Brennecke, R. Proceedings of an International Symposium on Laboratory Automation and Robotics, Boston; Zymark Corp.: Boston, 1991; pp 454-472. (10) Goodwin, P. A. Proceedings of an International Symposium on Laboratory Automation and Robotics, Boston; Zymark Corp.: Boston, 1989; pp 185-194. (11) Burgard, D. J.; Koskinen, W. C.; Dowdy, R. H.; Cheng, H. H. Weed Sci. 1993, 41, 648-655. (12) Lemme, T. H.; Olness, A.; Voorhees, W. B. Proceedings of an International Symposium on Laboratory Automation and Robotics, Boston: Zymark Corp.: Boston, 1997. (13) Thomas, G. W. In Methods of Soil Analysis, Part 3; Sparks, D. L., Ed.; American Society of Agronomy: Madison, WI, 1996; pp 475490. (14) Yeomans, J. C.; Bremner, J. M. Commun. Soil Sci. Plant Anal. 1988, 19, 1467-1476. (15) Bremner, J. M. In Methods of Soil Analysis, Part 3; Sparks, D. L., Ed.; American Society of Agronomy: Madison, WI, 1996; pp 1085-1122. (16) Sumner, M. E.; W. P. Miller. In Methods of Soil Analysis, Part 3; Sparks, D. L., Ed.; American Society of Agronomy: Madison, WI, 1996; pp 1201-1229. (17) Ray, A. A. SAS User’s Guide: Statistics; Statistical Analysis Systems Institute: Cary, NC, 1982. (18) Topp, E.; Smith, W. N.; Reynolds, W. D.; Khan, S. U. J. Environ. Qual. 1994, 23, 693-700.
Received for review May 13, 1997. Revised manuscript received September 9, 1997. Accepted September 13, 1997.X ES970416L X
Abstract published in Advance ACS Abstracts, October 15, 1997.
VOL. 31, NO. 12, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3685
