Anal. Chem. 2000, 72, 4560-4567
Determination of Anionic and Nonionic Surfactants, Their Degradation Products, and Endocrine-Disrupting Compounds in Sewage Sludge by Liquid Chromatography/Mass Spectrometry Mira Petrovic´† and Damia` Barcelo´*
Department of Environmental Chemistry, IIQAB-CSIC, c/Jordi Girona 18-26, 08034 Barcelona, Spain
A comprehensive analytical method based on reversedphase liquid chromatography and mass spectrometry using both atmospheric pressure chemical ionization and electrospray ionization has been developed for the simultaneous determination of anionic and nonionic surfactants, their polar degradation products, and endocrinedisrupting compounds (EDCs) in sewage sludge. Extraction of target compounds, with recovery rates from 86% to nearly 100% for polyethoxylates and from 84 to 94% for polar degradation products, was achieved applying ultrasonic solvent extraction with a mixture of methanol/ dichloromethane (7:3, v/v). Cleanup of sample extracts was performed on octadecyl solid-phase extraction cartridges. Determination of less polar compounds: alcohol ethoxylates (AEOs), nonylphenol ethoxylates (NPEOs), coconut diethanol amides, poly(ethylene glycol)s, and phthalate esters was accomplished by reversed-phase LC-APCI-MS in positive ionization mode, while more polar compounds: nonylphenolcarboxylates, nonylphenol (NP), octylphenol, and bisphenol A were analyzed by ionpair LC-ESI-MS under negative ionization conditions. This protocol was successfully applied to the trace determination of anionic and nonionic surfactants, polar degradation products, and EDCs in sewage sludge collected from different sewage treatment plants. The analysis revealed the presence of NP at high concentration levels ranging from 25 to 600 mg/kg. Polyethoxylates (AEOs and NPEOs) were also found in all samples at parts-permillion levels (10-190 mg/kg AEOs and 2-135 mg/kg NPEOs, respectively). The use of sewage sludge as a fertilizer in agriculture is considered as one of the most acceptable disposal options. It varies, depending on country, from 15% in Japan up to 70% in Switzerland.1 However, the high concentrations of several persistent and toxic contaminants raised serious questions about its * Corresponding author: (tel) +34 93 400 6188; (fax) +34 93 204 59 04; (email)
[email protected]. † On leave from the Faculty of Chemical Engineering and Technology, University of Zagreb, Croatia. (1) Kloepffer, W. Chemosphere 1996, 33, 1067-1081.
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agricultural use. The spectrum of organic pollutants of anthropogenic origin occurring in sewage sludge is extremely wide and constantly changing, depending on locality and season and on the technology used in the treatment plant. Among the known organic contaminants, the highest concentrations are found for anionic surfactants (LAS), phthalates, polycyclic aromatic hydrocarbons (PAH), nonylphenol, polychlorinated biphenyls (PCB), and organotin compounds. Nonionic surfactants of alkylphenol (APEOs) and alcohol polyethoxylate (AEOs) type and coconut fatty acid diethanol amides (CDEAs) have been widely used in the last 40 years as detergents, emulsifiers, dispersants, antifoamers, and pesticide adjuvants. A large portion of them reach the sewage works and, even being degradable, reach sewage sludge in high concentrations due to the very high amounts present in the wastewaters and incomplete degradation during the sewage treatment process. For example, during wastewater treatment nonylphenol ethoxylates (NPEOs) are degraded to the mono- and diethoxylates by shortening of the hydrophilic ethoxylate chain. These compounds are further degraded in anaerobically stabilized sewage sludge to the fully deethoxylated 4-nonylphenol, more lipophilic and toxic compound resistant to further microbial degradation.2 This compound, and other APEOs degradation products, e.g., nonylphenolcarboxylates and octylphenol, are considered as endocrinedisrupting compounds (EDCs) that may potentially alter the normal hormone function and physiological status of animals.3 Sludge produced by municipal sewage treatment plants (STPs) can be considered as one of the most complex matrixes to be analyzed. However, the chemical composition, and the concentration of pollutants, especially highly lipophilic ones of low biodegradability and transformation products of partly degradable pollutants, should be determined in order to plan sludge disposal in landfills or its reuse in agriculture. Gas chromatography/mass spectrometry (GC/MS) is the most widely used technique for the quantification of trace organic compounds in sewage sludge. Prior to GC/MS quantification, a specific multistep cleanup procedure tailored to the compounds (2) Ahel, M.; Giger, W.; Koch, M. Water Res. 1994, 28, 1131-1142. (3) Sonnenschein, C.; Soto, A. M. J. Steroid Biochem. Mol. Biol. 1998, 65, 143150. 10.1021/ac000306o CCC: $19.00
© 2000 American Chemical Society Published on Web 08/25/2000
of interest followed by derivatization is usually needed. However, this technique gives only partial information and cannot be applied for the direct determination of polyethoxylates because of their low volatility. Liquid chromatography/mass spectrometry (LC/ MS) has turned out to be an effective analytical technique for the characterization and quantification of nonionic surfactants in complex mixtures, such as industrial wastewaters.4,5 STP influents and effluents,6,7 and river and marine sediments.8-10 Although, a substantial amount of data have been produced over the years, there is a need to develop analytical methods for the simultaneous isolation and low-ppm level determination of EDCs and anionic and nonionic surfactants and their polar degradation products in sewage slduges. In previous work from our group, we developed a solid-phase extraction (SPE) procedure followed by LC/MS11,12 that permits the determination of surfactants and polar degradation products in wastewater samples. In this work, we have clearly expanded the number of analytes to be determined, including several EDCs, and we have developed and applied the new method for the trace determination of the target analytes to sewage sludges. The procedure is designed to isolate and quantify a broad range of organic compounds, providing sensitivity and selectivity of detection. The method presented here is simple and allows an easy cleanup and analysis, avoiding the tedious and long cleanup and derivatization steps that currently take place when sewage sludges are analyzed by GC/MS. We focused our analytical efforts on the following target compounds: the nonionic surfactants, alcohol ethoxylates, nonylphenol ethoxylates, and coconut diethanol amides; polar degradation products, poly(ethylene glycol)s (PEGs); and the endocrinedisrupting compounds, nonylphenol, octylphenol, nonylphenolcarboxylates, phthalate esters, and bisphenol A (structures are shown in Figure 1). In addition, the method developed here allows one to determine the anionic surfactants LAS, which are always present in sewage sludge samples. EXPERIMENTAL SECTION Standards and Chemicals. All standards and chemicals used were of the highest purity commercially available. The individual polyethoxylated surfactants, corresponding to a mixture with an average number of ethoxy groups, were from Kao Corp. (Barcelona, Spain). The standards of alcohol ethoxylates were individual pure C10-C16 ethoxylates with an even number of carbon atoms and an average of four ethoxy units. The standard of nonylphenol polyethoxylate contains chain isomers and oligomers with an average of six ethoxy units. (4) Castillo, M.; Alpendurada, M. F.; Barcelo, D. J. Mass Spectrom. 1997, 32, 1100-1110. (5) de Voogt, P.; de Beer, K.; Wielen, F. Trends Anal. Chem. 1997, 16, 584595. (6) Cresenzi, C.; Di Corcia, A.; Samperi, R. Anal. Chem. 1995, 67, 1797-1804. (7) Schro ¨der, H. Fr.; Fytianos, K. Chromatographia 1999, 50, 583-595. (8) Shang, D. Y.; MacDonald, R. W.; Ikonomou, M. G. Environ. Sci. Technol. 1999, 33, 1366-1372. (9) Shang, D. Y.; Ikonomou, M. G.; Macdonald, R. W. J. Chromatogr., A 1999, 849, 467-482. (10) Kreisselmeier, A.; Duerbeck, H. W. J. Chromatogr., A 1997, 775, 187196. (11) Castillo, M.; Ventura, F.; Barcelo´, D. Waste Manage. 1999, 19, 101-110. (12) Castillo, M.; Alonso, M. C.; Riu, J.; Barcelo´, D. Environ. Sci. Technol. 1999, 33, 1300-1306.
Figure 1. Chemical structures of the target compounds.
The mixture of coconut fatty acid diethanol amides was kindly supplied by H. Fr. Schro¨der. The proportional composition of the different homologues is as follows: C7, 7%; C9, 7.5%; C11, 60.9%; C13, 18%; C15, 6.6%. High-purity standard (98% pure) of 4-tert-octylphenol (OP) were obtained from Aldrich (Milwaukee, WI). Standards of 4-nonylphenol (NP) and nonylphenolcarboxylate (NPEC) were kindly supplied by AGBAR (Aigu¨es de Barcelona, Spain). Commercial linear alkylbenzenesulfonates with a low dialkyltetralinsulfonate (DATS) content (0.990). The recoveries (percent of standard added to sample recovered during extraction and cleanup) and reproducibility (relative standard deviation for triplicate analysis) of the method were determined by a spiking experiment. The sewage sludge spiked with 50 mg/kg standard mixture of nonionic surfactants, degradation products, phthalate esters, and bisphenol A was analyzed by applying the method described above, together with a blank sample (no spiked sample). The instrumental detection limits (LODinst) were calculated by a signal-to-noise ratio of 3 (the ratio between intensity of signal of each compound in standard solution obtained with SIM conditions and intensity of noise). Limits of detection of target compounds in sludge samples were calculated from LODinst, taking into account the amount of sample extracted, the volume of the extract analyzed, and the recovery rate obtained from a parallel assay of a spiked sample. The recoveries and limits of detection are reported in Table 2.
RESULTS AND DISCUSSION MS Detection. To quantify all target compounds, ranging in polarity from nonpolar (AEOs, NPEOs) to polar compounds (NPEC, BPA, LAS), the samples were analyzed using both APCI and ESI interface. All nonionic surfactants were detectable in positive ion mode using an APCI interface. The mixtures of polyethoxylated surfactants yield characteristic patterns showing [M + H]* ion for each compound and equidistant signals with mass differences of m/z +44 relative to the various ethoxylated oligomers. Together with these peaks, various clusters (Na+, NH4+, K+) also appear. Information on the oligomeric distribution of NPEO and AEO homologues can be readily obtained by extracting chromatograms for selected m/z values relative to oligomer components from the TIC chromatogram. However, the response of the MS detector for species having a different number of ethoxy units rapidly decreases as the number of ethoxy groups decrease from four to one. So, it can be expected that the present method underestimates the concentration of NPEOs, when the oligomer distribution of NPEO in the sample is substantially different from that of the calibrated standard solution, because the APCI-MS detector does not give an appreciable ion signal for compounds containing one and two ethoxy units. Different CDEA homologues were identified by their characteristic peak of m/z 106, and by molecular ions of from m/z 232 to 372 corresponding to the homologues with odd number of carbon atoms from C7 to C17. More polar compounds, such as APEO degradation products (NPEC, NP, OP) and bisphenol A were quantified using an ESI interface and negative ion ionization. The use of ESI provides a high level of specificity and sensitivity by employing single ion monitoring of the following molecular ions: m/z 277 (NPEC), 219 (NP), 205 (OP) and 227 (BPA). For this group of compounds, as determined by preliminary FIA, the sensitivity using an ESI interface was approximately 30-50 times higher than obtained with an APCI interface. Under the chromatographic conditions used, LAS were also determined. They eluted under ion-pairing conditions having retention time windows as follows: 11-12.3 (C10LAS), 12.5-14 (C11LAS), 13.3-15.8 (C12LAS), 15-17.8 (C13LAS), and 17.8-19.5 min (C14LAS). They were identified and quantified using ESI-NI mode, extracting ions m/z 297, 311, 325, 339, and 353 corresponding to molecular ions of homologues from C10 to C14, respectively. Recoveries and Limits of Detection. The analysis of surfactants in complex matrixes such as sewage sludge, STP influents, or effluents and industrial wastewaters is a challenging task. The nonionic surfactants and APEO degradation products (NPEC, NP, OP) are not easily extracted and eluted in a single step. Several protocols, employing sequential SPE13 on different sorbents, selective elution with solvents of different polarity,14 or their combination,11,12 were developed in order to extract and identify surfactants and their degradation products in complex aqueous matrixes. By the protocol employed in this work (twostep elution with solvent mixtures of different polarities), the target compounds are eluted in two fractions. The less polar compounds (AEOs, NPEOs, CDEAs, phthalate esters, NP, OP) are recovered (13) Fiehn, O.; Jekel, M. Anal. Chem. 1996, 68, 3083-3089. (14) Schro ¨der, H. Fr. Vom Wasser 1994, 82, 185-200.
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Table 1. Target Compounds and Their Main m/z Ions
in fraction A, while more polar analytes (NPEC, PEGs, BPA, LAS) are quantitatively recovered in fraction B. Although small portions of NPEOs and AEOs are found in fraction B, more than 90% recovery is obtained in fraction A. Polyethoxylated surfactants were quantitatively extracted from sewage sludge, enriched on C-18 cartridge, and desorbed with recoveries higher than 86% (Table 2). The overall recoveries of degradation products were 84% for NPEC, 88% for OP, and 92% for NP. Under the experimental conditions used, the LODs for sludge samples (based on a concentration factor of 2) were low-ppb level: 4-15 µg/kg for CDEAs; 15-50 µg/kg for phthalate esters; 65-90 µg/kg for NPEOs and AEOs; 130, 140, and 150 µg/kg for BPA, OP, and NP, respectively; and 560 µg/kg for PEGs. Such 4564
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detection limits easily attain the purpose of monitoring these compounds in sewage sludge. Concentrations of Target Compounds. The method was applied to the analysis of sewage sludge from five STPs. All receive domestic wastewaters mixed with industrial effluents. STP Igualada receives 30-40% of the industrial effluent of the tannery industry, STPs Abrera and Porto receive a significant fraction of industrial wastewater (textile industry), and STP Montornes also receives wastewater (∼50%) from the tannery sector and nearby chemical plant (detergent production). Three of the STPs (Montornes, Abrera, Porto) consist of a primary settlement involving only physicochemical treatment, and the STPs Igualada and Dresden apply biological sewage treatment.
Table 2. Mean Percent Recoveries, Repeatability (RSD for N ) 3), and Limits of Detection (LODs) Obtained in Selected Ion Monitoring (SIM) Mode compound poly(ethylene glycol)s (PEGx) alcohol ethoxylates (C10EO-C18EO)
nonylphenol ethoxylates (NPEOx) coconut diethanol amides (CDEA, n ) 7-15)
phthalates diethylphthalate (DEP) dibutylphathalate (DBP) bis(2-ethylhexyl)phthalate (DEHP) nonylphenol ethoxycarboxylate (NPEC) nonylphenol octylphenol bisphenol a a
recovery (RSD) 67 (7) 101 (9) C10 91 (5) C12 86 (6) C14 96 (13) 90 (12) (total CDEA)
87 (14) DEP 91(10) DBP 78 (9) DEHP 84 (11) 92 (11) 88 (9) 94 (8)
LODinst, ng (injected)
LODa (sludge), ng/g
15 2.5 2.5 3.0 2.5 0.50 C7 0.40 C9 0.35 C11 0.20 C13 0.15 C15
560 0 70 90 65 15 12 10 5.5 4
0.5 1.0 1.5 2.5 5.5 5 5
15 25 50 75 150 140 130
m(sludge) ) 2 g; V(extract) ) 1 mL; V(injected) ) 20 µL.
Figure 4. Full-scan LC-PCI-MS chromatogram (bottom trace) and SIM chromatogram for m/z 271, obtained under PI conditions by injecting the fraction A of sludge from STP Igualada. Inset: APCI-PI spectrum of a peak eluted at 16.4 min, corresponding to NPEO.
Table 3 lists the concentrations of target compounds determined. Almost all target compounds were identified in concentration levels ranging from several micrograms per kilogram to several grams per kilogram. Alcohol polyethoxylates (C10EO-C18EO) were found in all samples in concentrations from 10 to 190 mg/kg. The concentration of NPEO ranged from 2 mg/kg in sewage sludge from STP Porto to 135 mg/kg in sludge from STP Igualada. Figure 4. shows a PI full-scan LC-APCI-MS chromatogram of sludge from STP Igualada (fraction A) revealing the presence of NPEOs (x ) 4-11). The intensive ions that could be observed in the spectra are equally spaced (∆ m/z 44) molecular [M + H]+ ions (m/z 397627) and water adduct ions (m/z 414-722). The use of NPEOs in household detergent formulations has been voluntarily discontinued in many countries, because of the toxicity of their biodegradation products.15 The primary degradation product of NPEOs, nonylphenol, is detected in all samples, in concentrations ranged from 25 mg/kg in sludge from Dresden to more than 600
Figure 5. LC-ESI-MS chromatograms of sludge from STP Montornes obtained under NI conditions. (A) m/z 277 corresponds to NPEC in fraction B; (B) m/z 219 corresponds to NP in fraction A.
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Figure 6. Full-scan LC-APCI-MS chromatogram (bottom trace) of sludge from STP Abrera and SIM LC-APCI-MS chromatograms corresponding to different CDEA homologues. Insets: (A) APCI-PI mass spectra of C11DEA, (B) C13DEA, (C) C15DEA, and (D) C17DEA.
mg/kg in sludge from STP Montornes. (Table 3). Nonylphenolcarboxylate was also detected in some samples, at significantly lower concentration level. The extracted chromatograms of m/z 277, showing the presence of NPEC, and m/z 219, showing the presence of NP in sludge from STP Montornes, are depicted in Figure 5A and B, respectively. Ahel and co-workers2 experimentally determined the distribution coefficients of NP and NPEC in the secondary treatment phases of STP Uster (Switzerland). The distribution coefficients (log k), defined as the ratio between their concentrations in the sludge and in secondary effluent, were for NP 4.2 and for NPEC only 2.7. They also estimated that 92-96% of nonylphenol is discharged from the STPs via digested sludge and only 4-8% via secondary effluents. The maximum permissible concentration in sludge for substances of similar toxicity, such as cadmium, is set at ∼30 mg/kg.16 Schnaak and co-workers17 determined so-called “standard values” of various organic pollutants in sewage sludge when applied to soil. These ecotoxicologically based standard values present maximum tolerable residual concentrations in sewage sludge for agricultural use. The standard value for nonylphenol was set at 60 mg/kg. Concentrations found in sludge from STPs in the Catalonian region and in the sample from STP Porto are much higher, leading to the conclusion that disposal of these sludges should be planned carefully in order to minimize the environmental risk. Similar values of NP are reported by other researchers. Lee and Peart,18 using supercritical extraction and GC/MS, found high concentrations of NP in sludge samples collected inside the Toronto STPs (470 and 137 mg/kg). The (15) Bennie, D. T.; Sullivan, C. A.; Lee, H. B.; Peart, T. E.; Maguire, R. J. Sci. Total Environ. 1997, 193, 263-275. (16) Giger, W.; Schaffner, C. Science 1984, 225, 623-625. (17) Schnaak, W.; Kuechler, T.; Kujawa, K. P.; Henschel, D.; Susenbach, D. R. Chemosphere 1997, 35, 5-11. (18) Lee, H. B.; Peart, T. E. Anal. Chem. 1995, 67, 1976-1980.
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Table 3. Levels of Target Compounds Found in Sludges Collected in Different STP concentration (mg/kg dry weight) compound
Igualada
Montornes
Abrera
Porto
Dresden
PEGx C10EOx C12EOx C14EOx C16EOx C18EOx NPEOx C7DEA C9DEA C11DEA C13DEA C15DEA C17DEA DEP DBP DEHP NPEC NP OP BPA C10LAS C11LAS C12LAS C13LAS C14LAS
17.6 23 49 77 40 nf 135 nf 0,09 4.5 5.5 3.3 1.8 0.4 2 9.3 nf 172 nf nf 79 763 671 547 nf
3.9 32 1 7 1.8 nf 21 nf nf 0.6 2.8 1.1 nf nf 0.25 10.5 14 601 nf nf 210 2850 4130 4340 182
4.4 70 2.5 12.6 5.9 nf 100 nf 0.05 1.8 10.5 7 5.5 0.2 0.6 15.0 nf 325 nf nf 152 2540 3925 3320 88
1.7 5.7 1.3 4.5 1.3 nf 2.1 nf nf 0.3 0.2 0.2 nf nf 0.7 27 10 234 nf nf 129 1860 3020 2650 40
31 nfa 8.5 23 98 20 21 nf 0.2 6.2 4.2 nf nf 1.9 9.7 8 nd 25.5 nd nd 78 906 1055 940 nd
a
nf, not found; nd, not determined.
concentrations of OP in these samples were 9.2 and 12.1 mg/kg. Bennett and Metcalfe19 analyzed sewage sludge from the secondary treatment facility of the local STP (Ontario) and found 370 mg/kg NP and 20 mg/kg OP. In contrast to these findings, (19) Bennett, E. R.; Metcalfe, C. D. Environ. Toxicol. Chem. 1998, 17, 12301235.
octylphenol was not found in samples tested in this study (LOD 140 µg/kg). These data may indicate that octylphenol polyethoxylates are not used for industrial applications in areas covered by this study. Coconut fatty acid diethanol amide surfactants with an alkyl chain between 9 and 17 carbon atoms are present in all samples tested. Concentrations found were generally lower than 15 mg/ kg. The TIC chromatogram, extracted chromatograms for m/z values corresponding to each homologue of CDEA and their mass spectra are depicted in Figure 6, parts A-D. The shift in the alkyl chain length toward increased hydrophobicity is observed. In the commercial mixture, the most abundant homologue is C11, while in the sludge sample, the profile differs, and the most abundant homologue groups are C13 and C15. These compounds are mainly used in households in textile washing and hand dishwashing formulations, and their widespread use has led to their ubiquitous occurrence in wastewaters. In three STPs from Catalonia, the concentrations found in influents varied from 270 (Abrera) to 475 µg/L (Igualada), while in effluents, concentrations rarely exceeded 10 µg/L, indicating elimination rates higher than 97%.20 The presence of phthalates (diethyl, dibutyl, d bis(2-ethylhexyl)) has been detected in all samples using APCI interface and PI mode. In samples tested in this study, diethylphthalate (DEP) was found in concentrations varying from undetectable to 1.9 mg/kg, dibutylphthalate (DBP) from 0.25 to 9.7 mg/kg, and bis(2-ethylhexyl)phthalate (DEHP) from 8 to 27 mg/kg. These compounds are used as a plasticizer of PVC and as emulsifiers for paints and cosmetics, and they usually enter the wastewater path diffusely by many small sources of pollution. The concentrations of BPA were below detection limit (130 µg/kg). This compound is widely used in industry for a variety of applications such as in the production of polycarbonate polymers and epoxy resins, and it has been identified as an estrogenic compound. However, sludge is modest sink for BPA due to its moderate solubility (120 mg/L) and low Kow (log Kow 3.32).21 Beside that, BPA is easily degraded in biological wastewater treatment systems (over 99%) and it is not likely to be found in a sewage sludge. Anionic surfactants LAS can be also analyzed by this methodology using an ESI interface in the NI mode, as shown in Figure 7. They were found at extremely high concentrations in all samples: Sludge from STP Igualada contained (total LAS) 2.1 g/kg, STP Dresden 3 g/kg, STP Porto 7.7 g/kg, STP Abrera 10 g/kg, and STP Montornes 11.7 g/kg. The longer alkyl chain homologues are preferentially sorbed, because of the higher hydrophobicity of these compounds, and consequently, an increase in proportion of C12, C13, and C14 homologues is observed. Linear alkylsulfonates are the major surfactant class used in detergents throughout the world, because of their effectiveness and environmental safety. A significant proportion of the LAS is (20) Petrovic, M.; Barcelo´, D. Unpublished results. (21) Staples, C. A.; Dorn, P. B.; Klecka, G. M.; O’Block, S. T.; Harris, L. R. Chemosphere 1998, 36, 2149-2173.
Figure 7. TIC and SIM LC-ESI-MS chromatograms and UV trace (220 nm) of sludge from STP Igualada (fraction B) obtained under NI conditions, corresponding to linear alkylbenzene sulfonates.
removed by adsorption onto sewage solids during primary settlement and sewage sludge, as a final product, contains high concentrations of LAS, usually on the order of 2-10 g/kg. These levels are of concern since the new draft European Union Directive on sludge-amended soil requires a maximum level of 2.6 g/kg LAS. CONCLUSIONS The present methodology permits the simultaneous and unequivocal determination of anionic and nonionic surfactants, their polar degradation products, and endocrine-disrupting compounds in predominately organic matrixes. For optimum detection of all target compounds, ranging in polarity from nonpolar to polar compounds, both APCI under positive ionization conditions and ESI under negative ionization conditions have to be used. Using the specificity of a MS detector, low detection limits were achieved, permitting the monitoring of these compounds in sewage sludge. This protocol can be also applied to a variety of sewage sludge samples from different origins. ACKNOWLEDGMENT This work has been supported by the EU Environment and Climate program through the Waste Water Cluster project SANDRINE (ENV4-CT98-0801) and by CICYT (AMB1999-1705CE).. We thank Merck (Darmstadt) for supplying the SPE cartridges and LC column, respectively. M.P. acknowledges the grant from Spanish Ministry of Education and Culture (SB97B09092411). Received for review March 15, 2000. Accepted July 5, 2000. AC000306O
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