Environ. Sci. Technol. 1996, 30, 1822-1826
Supercritical Fluid Extraction of Atrazine and Polar Metabolites from Sediments Followed by Confirmation with LC-MS† S . P A P I L L O U D , * ,‡ W . H A E R D I , ‡ S. CHIRON,§ AND D. BARCELO Ä § De´partement de Chimie Analytique, University of Geneva, 30, quai E. Ansermet, 1211 Geneva 4, Switzerland, and Departamento de quimica ambiantal, Consejo Superior de Investigacio´nes Cientificas (C.S.I.C), 18-26 Jordi Girona, 08034 Barcelona, Spain
Atrazine is among the most widely used herbicides in intensive agriculture (corn fields). There is thus great incentive to monitor the concentration of this chemical as well as its degradation products, which have to be included in the balance of total toxicity. The metabolic pathway of atrazine in a freshwater sediment has been investigated in this paper. Atrazine was extracted by a supercritical fluid extraction method. The first step was to select the most efficient polar modifier: a polar mixture containing a strong nucleophilic agent (MeOH-H2O-Et3N) was too reactive, and atrazine was degraded into some of its metabolites during the extraction. An optimized nondegrading supercritical fluid extraction enabled the reproducible extraction of atrazine and its metabolites from the sediments, showing also that atrazine was metabolized in its hydroxylated analogue, involving chemical abiotic degradation. Our work reports for the first time the use of SFE followed by LCMS of the polar atrazine metabolites from an environmental matrix.
Introduction s-Triazines and in particular atrazine, which has been employed extensively worldwide for the last 25 years, are pre-emergent herbicides used mainly in the growth control of grasses in corn fields. Atrazine acts directly on target crops or is adsorbed on clay colloids and organic matter. Once in the environment, pesticides have a limited lifetime due to biological or chemical degradation processes; metabolites may also be toxic. It is thus of general concern for scientists to monitor concentration levels of both atrazine and its metabolites. Due to the high quantity of pesticides in use (average of 1 kg/ha), it is vital that † This work is dedicated to our late colleague and friend, Philippe Desmartin, who left us too early (November 10, 1995). * Corresponding author e-mail address:
[email protected]. ‡ University of Geneva. § Consejo Superior de Investigacio ´ nes Cientificas.
1822
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 30, NO. 6, 1996
phytotoxicity, bioaccumulation, bioavailability, formation of transformation products (TPs), and persistence in soils, sediments, and groundwaters be monitored (1, 2). Biodegradation is a pathway that generally gives rise to desalkylated compounds (desethylatrazine [DEA], deisopropylatrazine [DIA], and totally desalkylated atrazine [DAT]) and thus to partial or complete detoxification, as the TPs are generally thought to be less toxic. Bacteria [i.e. Arthobacter sp., Microbacterium sp., Pseudomonas sp. (3)] and fungi [i.e., A. fumigatus fres, Aspergillus niger, A. repeus, Cephalosporium acremonium, A. ustus, F. oxysporum (4, 5)] are generally involved in those processes. The distribution of the microfauna is determined by matrix characteristics and external conditions. Chemical process comprises the alternative pathway: hydroxylation is the most common reaction. UV photodecomposition, volatilization, and soil adsorption are other chemical processes described in literature, but they occur to a lesser extent than hydroxylation (2). In soils, the pathway strongly depends on intrinsic characteristics. For example, in corn fields containing a high quantity of organic matter and humic substances, hydroxyatrazine (HAT) was the only measurable metabolite of atrazine (6). In sediments, desalkylation pathways by microorganisms are reduced due to limited oxygenation, giving rise to HAT as the major measurable TP (7). Despite lower concentrations, metabolites DEA and DIA were found to be more mobile than atrazine due to their higher affinity for the aqueous fraction (8). SFE has been used for many years to extract various herbicides from environmental matrices because of its outstanding properties such as efficiency, selectivity, and rapidity (9-14). This method is becoming an interesting alternative to the traditional extractions such as the Soxhlet extraction or solid-liquid extractions, which are time consuming, nonselective, and may not be suitable for thermolabile compounds (Soxhlet). Extraction methods based on the use of supercritical fluids have already been applied to atrazine (10-14) and its desalkylated metabolites, but to our knowledge, no method based on supercritical fluid extraction (SFE) has been reported for the most polar metabolites (hydroxylated analogues). Atrazine metabolites have been extracted by solid-phase extraction and then similarly identified by HPLC-MS (15). In this paper, we will demonstrate that the SFE technique, which is generally used for nonpolar or moderately polar compounds, can also be applied to polar or very polar compounds. The aim of this work was to study the degradation of atrazine in the sediments taken from a small local lake, by supercritical fluid extraction. The sediment is artificially contaminated and left to age naturally. Thus, we will see the evolution of concentration and extractibility with time. The first step was to determine which mixture of polar modifiers would be the most efficient for the extraction of atrazine and whether the SFE technique is suitable on a variety of atrazine metabolites. Experiments showed that some conditions can lead to partial or total degradation during the extraction process. The second step was to monitor the degradation pathway unequivocally with the nondegrading method. Analyses were performed by HPLC equipped with a diode array
S0013-936X(95)00334-8 CCC: $12.00
1996 American Chemical Society
TABLE 1
Liquid Chromatographic Conditionsa component B (%) component C (%) time component A (%) CH3CN CH3OH (min) CH3COONH4 0.1 M (H2O) 0 9b 12
50 12.6 50
25 43.7 25
25 43.7 25
a A linear gradient is performed. b At t ) 9 min, the run is stopped. Initial conditions are re-established within 3 min.
FIGURE 1. Triazine herbicides: 1, atrazine; 2, desisopropylatrazine (DIA); 3, hydroxydesisopropylatrazine (HDIA); 4, desethylatrazine (DEA); 5, hydroxydesethylatrazine (HDEA); 6, hydroxyatrazine (HAT); 7, desethyl-desisopropyl-atrazine or desalkylated atrazine (DAT); 8, hydroxydesethyl-desisopropyl-atrazine (MET).
detector (DAD) and confirmed by HPLC combined with thermospray mass spectrometry (16).
Experimental Method Chemicals. Triazines (Figure 1) were purchased from Dr. Ehrensorfer (Augsburg, Germany). HPLC quality solvents were from J. T. Baker Company (Basel, Switzerland). Sample Preparation. The sediments come from a small eutrophic lake Lac de Breˆt (Lausanne Region, Switzerland). Sediments and the adjoining water were sampled from the bottom of the lake by means of a long weighted tube operated by a winch. Different sets of samples were prepared to monitor initial extractibility of atrazine and its polar metabolites (sed0) as well as extractibility and metabolization of atrazine in an aging spiked sediment (sed1 and sed2). Five different sets of sediments were sampled to study the extractibility and reproducibility of atrazine and all its metabolites, freshly spiked (sed0). The spiking method consists of adding a concentrated methanolic solution of atrazine and its seven metabolites and vigorously stirring the mixture for 2 h to homogenize the sample. The sample was spiked with 100 ppb, 1 ppm, and 10 ppm of each compound. The samples were dried and homogenized before extraction without any degradation or any significant loss of compound under investigation. Two other sets of sediments were prepared, spiked as usual, only with atrazine: (Sed1) the sediment and water were directly spiked, and (Sed2) the sediment was sterilized
before fortification. The sample was placed for 1 h at 140°C. A small quantity of water was subsequently added to compensate for evaporation (2%). The sediment was spiked after the treatment. Sed1 and sed2 will be used over a period of 1 month. All sediments sets were left unattended, and aliquots were collected every 2-3 days. Sediments were mechanically stirred vigorously during 2 h before removal of the sample to ensure homogeneity. Extractions. Extractions were performed with a supercritical fluid extractor. This device has already been presented in detail in our previous works (12, 13). It consists of a 305 Gilson pump to deliver CO2 to the system and an auxiliary 302 Gilson pump used to add the polar modifier. A 805 Gilson manometric module was the pressure control unit of the system. Pressure was maintained at 300 bar. A metering valve served as the restrictor. The extraction cell was a cylindrical column made of stainless steel 14.5 cm in length with an internal diameter of 0.3 cm. The extraction vessel was heated to the desired temperature (65 °C) by an oven placed in the system. Extraction time was 45 min, and flow rate was set at 1 mL/min. Several modifiers were tested to investigate their suitability and efficiency. Sediment samples were spiked with atrazine only and immediately extracted, using the following modifier conditions: (1) 10% (volume) methanol containing 2% water; (2) 15% (volume) methanol containing 2% water; (3) 15% (volume) methanol containing 10% water; (4) 10% (volume) methanol:water:Et3N (78:2:20). HPLC Followed by Thermospray Mass Spectrometry Analysis. A Hewlett Packard Model 5988A TSP-LC-MS quadrupole mass spectrometer was used combined with a Hewlett Packard Model 59970C PC workstation for data acquisition and processing. The temperatures of the TSP system were 96, 220, and 270 for the stem, tip, and vapourand-source, respectively. The stem was temperature-time programmed: the final temperature of 96 °C was reached in 10 min (1 °C/min). Filament was on, and the ion mode was set on positive mode. The liquid chromatographic condition was programmed with a linear gradient as shown in Table 1. The flow rate was set at 1 mL/min, and the analytical column used was a C-18, 4 µm Licrosphere column, 125 mm × 4 mm i.d. (Merck, Darmstadt, Germany).
Results and Discussion Mass Spectrometry. Mass spectrometry, especially when coupled with thermospray technique, is widely used in environmental analysis to confirm contaminations (1618). This mild ionization technique gave rise to very low fragmentation, producing ions in the molecular region (19). The base peaks were protonated molecular ions [M + H]+. The other ions, in lower intensities, correspond to the formation of adducts. The most abundant adducts were
VOL. 30, NO. 6, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1823
TABLE 2
Ions Formed in Thermospray Mass Spectrometry compds
a
1).
b
mol mass
other important ions [relative intensities to base peak]a
base peak ion [M+H]+
atrazine HAT
215 197
216 198 [M+H]+
DEA DIA HDEA
187 173 155
188 [M+H]+ 174 [M+H]+ 156 [M+H]+
HDIA
169
170 [M+H]+
METb propazine
127 229
128 [M+H]+ 230 [M+H]+
257 [M + CH3CN + H]+ (15%) 239 [M + CH3CN + H]+ (10%) 256 [M + CH3CN + NH4]+ (8%) 229 [M + CH3CN + H]+ (72%) 215 [M + CH3CN + H]+ (100%) 197 [M + CH3CN + H]+ (83%) 214 [M + CH3CN + NH4]+ (22%) 211 [M + CH3CN + H]+ (56%) 228 [M + CH3CN + NH4]+ (41%) 247 [M+H+CH3CO2NH4]+ (19%) 169 [M + CH3CN + H]+ (40%) 271 [M + CH3CN + H+] (10%)
Italic ions are ions monitored in SIM mode. Eluent used was a mixture of water-ammonium acetate-acetonitrile and methanol (see Table Due to high background for m/z < 150, ion m/z 169 is selected for MET monitoring in SIM mode.
TABLE 3
Effect of Modifier in Supercritical Fluid Extraction of Sediment Spiked with Atrazine recoveriesa
distribution of herbicides found (%)
modifiers used
∑[herbicides]/[atrazine]initial (%)
atrazine
HAT
DIA
HDEA
HDIA
(1) 10% (v:v) methanol containing 2% water (2) 15% (v:v) methanol containing 2% water (3) 15% (v:v) methanol containing 10% water (4) 10% (v:v) methanol/water/Et3N (78:2:20)
28 ( 2 40 ( 3 78 ( 5 70 ( 5
100 20 ( 1 0 0
0 50 ( 2 100 50 ( 2
0 0 0 13 ( 1
0 0 0 3(1
0 30 ( 2 0 34 ( 1
a Recoveries are calculated taking into account the total amount of pesticides found compared to the initial amount of atrazine. HPLC analysis were done six times (n ) 6). Metabolites DAT, DEA, and MET were never found.
those of acetonitrile or cation amonium delivered in the HPLC mobile phase. Table 2 shows the different ions obtained in thermospray mass spectrometry. The mass spectrometer was set to record masses ranging from 150 to 400 amu to avoid interferences below 150 amu, due to high matrix background. In this respect, MET is determined by selecting the acetonitrile adduct (m/z ) 169) and not the base peak (m/z ) 128). HAT, whose base peak is ion m/z )198 [HAT +H]+, could also have a contribution from [HDEA+ CH3CN + H]+, m/z ) 197, but the retention times are different (HAT: retention time is 2.6 min, and HDEA retention time is 2.1 min). Calculation of concentration was made possible with a standard (propazine). The method is reproducible and not hindered by coextracted matter from the matrix. Choice of an Adequate Modifier for SFE. Although the use of polar modifiers with polar analytes by SFE has been attempted (20), very polar or very strong nucleophilic agents may lead to in-situ degradation. Our experiments have shown that using high quantities of methanol and water improve the extraction recoveries but also lead to partial hydrolysis of atrazine. Spectacular results are obtained: triethylamine in aqueous methanol solution and 15% of MeOH, used as modifiers, lead to complete degradation of atrazine. HAT is the principal product of the reaction. High pressure and temperature combined with strong nucleophilic power of the polar modifier (Et3N, H2O, and MeOH) seem to explain this phenomenon. Table 3 shows the insitu chemical degradation occurring during the extraction. As the aim of this work is to follow a degradation pathway and not to optimize the extractibility, it is essential to extract all our compounds without any chemical alteration. Indeed, the emphasis here is placed on quantifying the natural metabolites of atrazine using nondegrading extraction conditions. Although low recovery is obtained, the method
1824
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 30, NO. 6, 1996
TABLE 4
Extraction of Atrazine and Metabolites in Freshly Spiked Sediment (n ) 6)a recovery (%) compd
100 ppb
1 ppm
10 ppm
atrazine HAT DEA DIA HDEA HDIA DAT MET
24 ( 3 20 ( 2 15 ( 1 29 ( 1 19 ( 2 14 ( 4 17 ( 2 8(1
26 ( 3 20 ( 1 17 ( 1 26 ( 1 19 ( 2 22 ( 1 20 ( 4 9(2
28 ( 2 23 ( 3 25 ( 3 26 ( 4 29 ( 3 35 ( 3 27 ( 2 10 ( 3
a Sediment was spiked at three different levels and extracted immediately. The extraction conditions were as follow: supercritical CO2 modified with 10% of methanol containing 2% of water was used at 300 bar and 65 °C, during 45 min.
is statistically reproducible (standard deviation 70%, according to the American EPA recommendation). Such recoveries were not achieved without degradation in this case. Degradation of Atrazine in Sediments. All samples of Sed1 and Sed2 were extracted at 300 bar by supercritical CO2 modified with 10% of methanol containing 2% of water (condition 1, Table 3) and then analyzed by HPLC equipped with DAD and confirmed by HPLC/MS. No degradation is reported during the analyses by chromatography. All base peaks of atrazine metabolites have been searched for in SIM mode (see Table 2 and chromatograms in Figure 2). Only atrazine and HAT were detected. The presence of those two compounds was confirmed by the exact matching of the full mass spectra of the investigated peaks on the chromatogram and those of the pure substances. Low recoveries are due to the strong adsorption of atrazine to
the sediments. This can be explained by the high content of organic matter (8.3%) and clay (27.6%) in the sediment. These compounds have very strong adsorption capacity. Figure 3 shows the time evolution of atrazine and HAT concentrations in Sed1 samples. Results of Sed2 displays only minor variations (
