Anal. Chem. 1997, 69, 1569-1576
Simultaneous Determination of Neutral and Acidic Pesticides in Natural Waters at the Low Nanogram per Liter Level Thomas D. Bucheli, Franca C. Gru 1 ebler, Stephan R. Mu 1 ller,* and Rene´ P. Schwarzenbach
Swiss Federal Institute for Environmental Science and Technology (EAWAG) and Swiss Federal Institute of Technology (ETH), CH-8600 Du¨ bendorf, Switzerland
A new method for the simultaneous identification and quantification of neutral and acidic pesticides at the low nanogram per liter concentration level in natural waters is presented. It has been validated for, and applied to, three important pesticide classes, namely, the triazines (e.g., atrazine and its major metabolites desethylatrazine and deisopropylatrazine), the acetamides (e.g., alachlor, metolachlor, and dimethenamid), and the phenoxy acids (e.g., 2,4-D, dichlorprop, and mecoprop). The method includes enrichment of the compounds by solid phase extraction on graphitized carbon black, followed by sequential elution of the neutral and acidic pesticides and derivatization of the latter fraction with diazomethane. Identification and quantification of the compounds is performed with GC/MS using atrazine-d5, [13C6]metolachlor, and [13C6]dichlorprop as internal standards. Absolute recoveries from Nanopure water spiked with 4-50 ng/L were 85 ( 10, 84 ( 15, and 100 ( 7% for the triazines, the acetamides, and the phenoxy acids, respectively. Recoveries from rainwater and lake water spiked with 2-100 ng/L were 95 ( 19, 95 ( 10, and 92 ( 14% for the triazines, the acetamides, and the phenoxy acids, respectively, and a groundwater contaminated with landfill leaches and pesticides from agricultural applications revealed similar numbers for most analytes. Average method precision determined with fortified rainwater (2-50 ng/L) was 6.0 ( 7.5% for the triazines, 8.6 ( 7.5% for the acetamides, and 7.3 ( 3.2% for the phenoxy acids, and method detection limits ranged from 0.1 to 4.4 ng/L. The method has been applied to analysis of various natural waters, including rainwater, roof runoff, surface water, and groundwater. It proved to be an excellent tool for routine multiresidue pesticide analysis even at low nanogram per liter concentrations of these analytes. Considering the tremendous global consumption of herbicides (∼2.5 million metric tons per year),1,2 it is not surprising that many of these compounds have been detected in natural waters, such * Corresponding author. Fax: +41-1-8235471. E-mail:
[email protected]. (1) WHO Public health impact of pesticides used in agriculture; World Health Organization/United Nations Environmental Program: Geneva, 1990. (2) Albert, R.; Baker, S.; Doull, J.; Butler, G.; Nelson, N.; Peakall, D.; Pimentel, D.; Tardiff, R. G. In Methods to assess adverse effects of pesticides on nontarget organisms; Scope 49, IPCS Joint Symposium 16, SGOMSEC 7, Tardiff, R. G., Ed.; John Wiley: Chichester, U.K., 1992; Chapter 1. S0003-2700(96)01059-1 CCC: $14.00
© 1997 American Chemical Society
as fog,3,4 precipitation,5-8 surface water,9,10 and groundwater (for a review, see Funari et al.11), and, therefore, have raised considerable concern both from a human health and from an environmental point of view. Of the ∼20 different classes of herbicides, the triazines, the acetamides, and the phenoxy acids are among the most widely used compounds. Prominent examples include atrazine, metolachlor, alachlor, and 2,4-D of which several tens of thousends of metric tons are used every year in the United States.12 In order to assess possible impacts of herbicides on aquatic ecosystems and drinking water supplies, analytical methods for the routine simultaneous determination of a large number of such compounds at trace concentrations in water samples are required. To date, the U.S. National Pesticide Survey has developed six different methods for the determination of over 100 pesticides in groundwater with limits of quantification ranging from 0.1 to ∼5 µg/L for most compounds.13 In addition, several other techniques for multiresidue pesticide analysis in natural waters have been applied to environmental samples within the last years, including solid phase extraction (SPE)-GC/MS,14 SPE-HPLC,15 LC/GC,16 or LC/MS,17,18 but only few methods allow the simultaneous determination of both neutral and acidic pesticides. One of the (3) Glotfelty, D. E.; Seiber, J. N.; Liljedahl, L. A. Nature 1987, 325, 602-605. (4) Schomburg, C. J.; Glotfelty, D. E.; Seiber, J. N. Environ. Sci. Technol. 1991, 25, 155-160. (5) Richards, R. P.; Kramer, J. W.; Baker, D. B.; Krieger, K. A. Nature 1987, 327, 129-131. (6) Nations, B. K.; Hallberg, G. R. J. Environ. Qual. 1992, 21, 486-492. (7) Trevisan, M.; Montepiani, C.; Ragozza, L.; Bartoletti, C.; Ioannilli, E.; Del Re, A. A. M. Environ. Pollut. 1993, 80, 31-39. (8) Siebers, J.; Gottschild, D.; Nolting, H. G. Chemosphere 1994, 28, 15591570. (9) Buser, H.-R. Environ. Sci. Technol. 1990, 24, 1049-1058. (10) Bester, K.; Hu ¨ hnerfuss, H. Mar. Pollut. Bull. 1993, 26, 423-427. (11) Funari, E.; Donati, L.; Sandroni, D.; Vighi, M. In Pesticide risk in groundwater; Vighi, M., Funari, E., Eds.; CRC, Lewis: Boca Raton, FL, 1995; Chapter 1. (12) Gianessi, L. P.; Anderson, J. E. Pesticide use in U.S. crop production; National Center for Food and Agricultural Policy, Government Printing Office: Washington, DC, 1995. (13) Munch, D. J.; Graves, R. L.; Maxey, R. A.; Engel, T. M. Environ. Sci. Technol. 1990, 24, 1446-1451. (14) Benfenati, E.; Tremolada, P.; Chiappetta, L.; Frassanito, R.; Bassi, G.; Di Toro, N.; Fanelli, R.; Stella, G. Chemosphere 1990, 21, 1411-1421. (15) Schlett, C. Fresenius J. Anal. Chem. 1991, 339, 344-347. (16) Noij, T.; Vanderkooi, M. HRC, J. High Resolut. Chromatogr. 1995, 18, 535539. (17) Bagheri, H.; Brouwer, E. R.; Ghijsen, R. T.; Brinkman, U. A. T. J. Chromatogr. 1993, 647, 121-129. (18) Chiron, S.; Dupas, S.; Scribe, P.; Barcelo, D. J. Chromatogr., A 1994, 665, 295-305.
Analytical Chemistry, Vol. 69, No. 8, April 15, 1997 1569
Figure 1. Structure of the s-triazines (a), the acetamides (b), and the phenoxy acids (c). The substitutes R1, R2, R3, and R4 for different compounds analyzed are specified in Table 1.
first methods published by Cessna et al.19 used liquid/liquid extraction, derivatization of the acidic pesticides with diazomethane, and GC/ECD and GC/FID for separation and detection. More recently, SPE-HPLC20-22 and LC/MS23 were used to achieve that goal. However, the former method lacks a reliable, sufficiently sensitive, and highly specific detector, and the latter one is too sophisticated for routine analysis at trace concentrations. In this paper, we present a new method that attempts to overcome these drawbacks. The method allows the routine simultaneous determination of triazines (Figure 1a), acetamides (Figure 1b), and phenoxy acids (Figure 1c) in natural waters at the low nanogram per liter concentration level. Major emphasis was placed on the simplification of the usually cumbersome and tedious sample preparation, particularly for the analysis of acidic substances with GC/MS. EXPERIMENTAL SECTION Materials. The pesticides investigated are listed in Table 1. Dimethenamid (99.8%) was kindly provided by Sandoz Agro Ltd. (Basle, Switzerland). All other pesticides (purity >97%) were purchased from Riedl-de Hae¨n (Seelze, Germany). The internal standards pentadeuterioatrazine (ethyl-d5, 99%), ring-13C6-labeled metolachlor (99%), and ring-13C6-labeled dichlorprop (99%) were obtained from Cambridge Isotope Laboratories (Andover, MA). Methanol (MeOH), dichloromethane (MeCl2), toluene, and ethyl acetate (EA) (all with purity for pesticide residue analysis) were obtained from Burdick & Jackson (Muskegon, MI). Ascorbic acid (>99.5%), and trifluoroacetic acid (TFA, >98%) were from Fluka AG (Buchs, Switzerland). HCl (37%) was purchased from Merck (Darmstadt, Germany). Nitrogen (99.995%) was from (19) Cessna, A. J.; Grover, R.; Kerr, L. A.; Aldred, M. L. J. Agric. Food Chem. 1985, 33, 504-507. (20) Di Corcia, A.; Marchetti, M. Environ. Sci. Technol. 1992, 26, 66-74. (21) Liska, I.; Brouwer, E. R.; Ostheimer, G. L.; Lingeman, H.; Brinkman, U. A. T. Int. J. Environ. Anal. Chem. 1992, 47, 267-291. (22) Nouri, B.; Fouillet, B.; Toussaint, G.; Chambon, P. Analyst 1995, 120, 11331136. (23) Cappiello, A.; Famiglini, G.; Bruner, F. Anal. Chem. 1994, 66, 1416-1423.
1570 Analytical Chemistry, Vol. 69, No. 8, April 15, 1997
Carbagas (Ru¨mlang, Switzerland). Deionized water was further purified with a Nanopure water purification device (NANOpure 4, Skan, Basle, Switzerland). Diazomethane (∼0.4 M in diethyl ether) was freshly produced on the day before use as described in de Boer and Backer24 and stored at -20 °C. Beware: Diazomethane is carcinogenic and, under certain conditions, explosive. All manipulations should be carried out in a hood and with great care! Standard Solutions. For all pesticides, stock solutions of 1000 mg/L were prepared by dissolving 25 mg of each compound in 25 mL of MeOH (triazines and acetamides, except dimethenamid) or EA (phenoxy acids and dimethenamid). All solutions were further diluted with the respective solvents to obtain a final concentration of 20 mg/L per compound. For the triazines and the acetamides, respectively, standard mixtures (0.4 mg/L, except for atrazine, alachlor, and metolachlor, 1 mg/L) were produced from the above single-compound solutions. The phenoxy acid standard mixture contained 1 mg/L of each compound. Standard mixtures were used for calibrations and for the preparation of fortified samples. Each internal standard was diluted in toluene to yield a concentration of 7.5 mg/L. All solutions were stored at 4 °C in the dark. Sampling and Sample Preparation. Roof runoff and rain samples were collected in Tu¨ffenwies, an industrial area in the northwestern part of Zurich. Lake water was from Murtensee (western part of Switzerland). Groundwater samples were taken in the vicinity of the municipal landfill of Winterthur (Riet, Canton Zurich, Switzerland), and the landfill of Ko¨lliken (Canton Aargau, Switzerland). All samples were kept at 4 °C in the dark. Prior to analysis, water samples were allowed to reach room temperature. Natural water samples were filtered (cellulose nitrate filter, diameter 5 cm, pore size 0.45 µm; Satorius, Goettingen, Germany), and the exact volume of 1 L was spiked with 10 µL (75 ng/L) of each internal standard. For recovery studies, and/or internal calibration, Nanopure water and natural waters were spiked with the standard mixtures of all pesticide classes and/or internal standards. The samples were shaken vigorously and set aside over night. Solid Phase Extraction. Commercially available 6 mL glass cartridges were filled in the laboratory with 250 mg of graphitized carbon black (GCB; Carbopack B) between two Teflon frits and mounted on a 12-fold vacuum extraction box (all products from Supelco, Bellafonte, CA). Conditioning of the cartridges and extraction of the samples was carried out as described in Berg et al.25 Briefly, the solid phase was treated with 8 mL of MeCl2/ MeOH (80:20, v/v), 4 mL of MeOH, 20 mL of ascorbic acid solution (10 g/L, acidified with HCl to pH 2), and 10 mL of Nanopure water. Samples (1 L) were then drawn through the cartridges at a flow rate of 15 mL/min. Thereafter, the solid phase was washed with 5 mL of Nanopure water and 0.5 mL of MeOH and air-dried for 5 min. Sequential elution from the same cartridge was performed by first eluting the neutral fraction with 1 mL of MeOH and 6 mL of MeCl2/MeOH (80:20, v/v), followed by a second elution of the acidic fraction with 6 mL of MeCl2/EA (80:20, v/v) acidified with TFA (0.2%, v/v; solution produced immediately before use). After each of the two elution steps, cartridges were air-dried for 5 min (24) de Boer, T. J.; Backer, H. J. Org. Synth. Collect. 1963, 2, 250-253. (25) Berg, M.; Mu ¨ ller, S. R.; Schwarzenbach, R. P. Anal. Chem. 1995, 67, 18601865.
Table 1. Investigated Pesticides and Their Structures, Retention Times, and Ions Monitored substituent in positiona compound triazines atraton atrazine atrazine-d5b desethylatrazine deisopropylatrazine propazine simazine terbuthylazine acetamides acetochlor alachlor dimethenamid metalaxyl metazachlor metolachlor [13C6]metolachlorc propachlor phenoxy acids 2,4-D dichlorprop [13C6]dichlorpropc MCPA mecoprop 2,4,5-T 2,4,5-TP
R1
R2
R3
R4
OCH3 Cl Cl Cl Cl Cl Cl Cl
CH2CH3 CH2CH3 CD2CD3 H CH2CH3 CH(CH3)2 CH2CH3 CH2CH3
CH3 CH2CH3 CH3 CH3 CH2CH3 CH2CH3 H
Cl CH2OCH2CH3 Cl CH2OCH3 for molecule structure, see Figure 2 OCH3 CHCH3COOCH3 Cl CH2N2(CH)3 Cl CHCH3CH2OCH3 Cl CHCH3CH2OCH3 Cl CH(CH3)2
H H H H H Cl Cl
Cl Cl Cl CH3 CH3 Cl Cl
CH(CH3)2 CH(CH3)2 CH(CH3)2 CH(CH3)2 H CH(CH3)2 CH2CH3 C(CH3)3
CH2 CH(CH3) CH(CH3) CH2 CH(CH3) CH2 CH(CH3)
CH2CH3 CH2CH3 CH3 CH3 CH3 CH3 H
ret time (min)
massesd (m/z)
11.8 13.9 13.8 14.4 15.2 13.1 14.7 13.7
196, 211 200, 215 205, 220 172, 187 173, 158 214, 229 186, 201 214, 229
15.1 15.9 15.6 17.9 24.4 17.8 17.8 10.1
162, 146 160, 188 154, 230 206, 249 132, 209 162, 238 168, 244 120, 176
9.7 8.4 8.4 8.0 7.3 12.5 10.5
234, 199 248, 162 254, 168 214, 141 228, 171 233, 268 198, 282
a Substituents of the structures in Figure 1a-c. b Internal standard for GC/MS. c Internal standards for GC/MS, ring-marked isotopes. d First number, quantification mass; italic number, mass of the molecular ion (or the respective methyl ester for phenoxy acids).
to allow maximum solvent elution and a minimum of interferences between the two elution solvents. Both fractions were separately collected in conical glass vials (7.5 mL; Supelco, Bellafonte, CA). Both eluates were concentrated by evaporating the solvent to a final volume of 200 ( 50 µL at ambient temperature using a gentle nitrogen stream for ∼30 min. To diminish the remaining MeOH content, 200 µL of EA was then added to the neutral fraction and the volume was again reduced to 200 ( 50 µL. Diazomethane solution (500 µL to 3 mL, varying from sample to sample) was slowly added to the acidic fraction until the solution kept the yellow color of the derivatization reagent. After 15 min, the volume was carefully reduced to 200 ( 50 µL again, which also removed the excess derivatization agent, and the solution was passed through a 0.45 µm filter (Spartan 13, Schleicher & Schuell, Dassel, Germany). GC/MS Analysis. Separation of both the neutral and acidic analytes was carried out with a Fisons Instruments HRGC 8000 Series on a homemade fused-silica capillary column [32 m, 0.25 mm i.d., OV240OH (33% cyanopropyl, 66% methyl), 0.3 µm film thickness], using helium as carrier gas (150 kPa, 2.6 mL/min). The neutral, or the acidic sample (1 µL) was injected with split/ splitless mode. Injector temperature was 200 °C. The oven temperature was programmed as follows: 1 min at 120 °C, to 195 °C at 20 °C/min, to 225 °C (212 °C for acidic fraction) at 1.5 °C/ min, to 260 °C at 5 °C/min (20 °C/min for acidic fraction), and 2 min at 260 °C. The interface temperature was 250 °C. Detection was performed with a FI MD 800 mass spectrometer in the electron impact mode (EI+, 70 eV) and single-ion monitoring (SIM). Identification of a given analyte was assured by using two compound-specific ions (see Table 1) with identical retention times and a mass ratio similar to the one determined with internal calibration (
