Investigation of the Fate of Linear Alkyl Benzenesulfonates and

Major coproducts of commercial mixtures of linear alkyl benzenesulfonate (LAS) surfactants are dialkyl tetralin- sulfonates (DATS) and methyl-branched...
1 downloads 0 Views 92KB Size
Download PDF
Environ. Sci. Technol. 1999, 33, 4112-4118

Investigation of the Fate of Linear Alkyl Benzenesulfonates and Coproducts in a Laboratory Biodegradation Test by Using Liquid Chromatography/Mass Spectrometry A N T O N I O D I C O R C I A , * ,† FRANCESCA CASASSA,† CARLO CRESCENZI,† ANTONIO MARCOMINI,‡ AND ROBERTO SAMPERI† Dipartimento di Chimica, Universita` “La Sapienza”, Piazza Aldo Moro 5, 00185 Roma, Italy, and Dipartimento di Scienze Ambientali, Universita` di Venezia, Calle Larga S. Marta, I-30123 Venezia, Italy

Major coproducts of commercial mixtures of linear alkyl benzenesulfonate (LAS) surfactants are dialkyl tetralinsulfonates (DATS) and methyl-branched isomers of LAS (isoLAS). As a total, DATS and iso-LAS can account for up to 15% of LAS. Unlike LAS, little and contrasting information on the fate of DATS and iso-LAS is available. We have used liquid chromatography/mass spectrometry with an electrospray interface to follow biotransformation of LAS coproducts. Structure elucidation of their breakdown products was obtained by in-source collision-induced decomposition (CID) spectra. However, metabolites of LAS and isoLAS could not be distinguished from each other by their CID spectra. According to the OECD 301 B protocol, a laboratory biodegradation experiment of LAS and coproducts was conducted. DATS were more resistant than iso-LAS to primary biodegradation. Biotransformation of both LAStype compounds and DATS produced, besides expected sulfophenyl alkyl monocarboxylated (SPAC) LAS and sulfotetralin alkylcarboxylated (STAC) DATS metabolites, significant amounts of dicarboxylated (SPADC and STADC) species. SPADCs were less persistent than STADCs. After more than 5 months from the beginning of the experiment, 40% and 35% of the initial amounts of DATS and isoLAS, respectively, were not mineralized. About 64% of refractory SPACs contained 2-5 alkanoyl carbons in the alkyl chain, while the number of alkanoyl carbons in the free alkyl chains of refractory STACs and STADCs averaged respectively 2.4 and 1.1. On the basis of the results of this study and LAS consumption, we roughly estimated that 200 000 ton of refractory organics is each year dispersed in the environment as the result of use of the above surfactants.

Introduction After soaps, linear alkyl benzenesulfonates (LAS) are the most widely used surfactants in domestic and industrial detergents. * Corresponding author fax: +39-06-490631; e-mail: dicorcia@ axrma.uniroma1.it. † Universita ` “La Sapienza”. ‡ Universita ` di Venezia. 4112

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 22, 1999

FIGURE 1. Examples of structures and acronyms of linear alkyl benzenesulfonate surfactants, coproducts, and related catabolic products. In 1995, the global production was ca. 2.8 × 106 ton (1). Commercial LAS are complex mixtures of essentially C10C14 alkyl homologues and 26 major phenyl positional isomers. These isomers arise from the fact that the aromatic ring can attach to every point of the alkyl chain, with the exception of position one. Positional isomers with the aromatic ring linked to the more external carbon atoms of the alkyl chain are usually called external isomers, while internal isomers are those isomers with the benzene ring attached to the internal carbon atoms of the alkyl chain. Commercial LAS also contain coproducts called dialkyl tetralinsulfonates (DATS) and iso-LAS. Because of the possible formation of cis-trans isomers and of the relative position of the alicyclic moiety and the sulfonated group on the aromatic ring, 70 major isomers of DATS can be present in commercial LAS (2). The so-called iso-LAS are isomers of LAS having mainly a single methyl branching in one of the various attachment points of the alkyl chain (3). This class of isomers have been grouped into two main subclasses: iso-LAS type I are species with a methyl branch attached to nonbenzylic carbons of the alkyl chain, while iso-LAS type II have the methyl branch located at the benzylic carbon of the alkyl chain (3). Relative concentrations of DATS in commercial formulations are reported to account for 6-8% of LAS (4, 5), while relative concentrations of iso-LAS can range between 3 and 6% (6). Some examples of structures of LAS, DATS, and iso-LAS are visualized in Figure 1 together with their related biotransformation products. Under aerobic conditions, rate and pathway of LAS biodegradation have been objects of many studies, which have been reviewed by Swisher (7) and Scho¨berl (8). Although the biodegradation pathway of LAS has been not yet fully elucidated, there are many evidences showing that the first cycle of biotransformation (primary biodegradation) begins with oxidation of the external methyl groups (ω-oxidation) followed by stepwise shortening of the alkyl chain via oxidative cleavage of C2 units (β-oxidation). This process leads 10.1021/es9905952 CCC: $18.00

 1999 American Chemical Society Published on Web 09/30/1999

to the formation of sulfophenylcarboxylic acids (SPACs, where A stands for the residual intact alkyl chain and a deponent number eventually following the letter A indicates the number of alkanoyl carbon atoms in the carboxylated alkyl chain). The second cycle (ultimate biodegradation or mineralization) involves opening of the aromatic ring and/or desulfonation of SPACs leading ultimately to CO2, H2O, inorganic salts, and biomass. It is generally accepted that also DATS and iso-LAS form carboxylated intermediates upon biodegradation. Carboxylated DATS have been identified by mass spectrometric detection in a sewage-contaminated groundwater (9) and in aqueous samples of sewage treatment plants (5). Because of the enormous amounts of LAS consumed each year, significant amounts of LAS coproducts can reach the environment. Although commercial LAS mixtures were introduced in the mid-1960s, only recently the fate of DATS and iso-LAS has received some attention. Results of studies aimed at assessing mineralization yields of these two classes of chemicals are contrasting. Cavalli et al. (3) conducted laboratory biodegradation studies of model iso-LAS compounds and observed that these species showed high levels of ultimate biodegradation. Ko¨lbener et al. (6, 10) followed the degradation of commercial LAS mixtures in a laboratory, flow-through, test system using immobilized activated sludge. These authors found that refractory organic carbon had arithmetically the same value as the level of impurities. Analysis by liquid chromatography with UV detection led the authors to deduce that these recalcitrants were primarily carboxylated metabolites of both DATS and alkyl branched LAS. Recently, laboratory simulations performed by Nielsen et al. (11) have confirmed that the microbial populations of domestic and industrial activated sludges, while very effective in the primary biodegradation of DATS and iso-LAS, are not capable of mineralizing most of the related metabolites. However, Cook et al. (12) claim that these metabolites cannot be considered refractory species as, under appropriate conditions, they can be utilized as a sulfur source for bacterial growth. Very recently, we have shown that LC/mass spectrometry (MS) with an electrospray (ES) ion source and a single quadrupole is a powerful technique for characterizing the structures of breakdown products originated from biotransformation of alkyl branched alcohol ethoxylate (13) and nonylphenol ethoxylate (14) surfactants. The purpose of this work has been that of looking in detail at the progression of the biodegradation of iso-LAS and DATS contained in a LAS mixture under laboratory conditions and using the same instrumentation reported above. Our efforts were particularly devoted to characterize structures of recalcitrant metabolites of LAS coproducts.

Experimental Section Reagents and Chemicals. C6-DATS (unknown purity), C12LAS (95% purity), and “Sirene-AlCl3”, a mixture containing both C10-C14 LAS and C6-C10 DATS, were kindly supplied by L. Cavalli (Enichem Augusta, Milano, Italy). As measured by us, the percent molar composition of LAS, DATS, and isoLAS in the Sirene-AlCl3 mixture was respectively 82, 10, and ca. 7%. The approximate iso-LAS estimation was based on a previous observation (4) that all the iso-LAS are eluted as an envelope of unresolved peaks just preceding those for positional isomers of the corresponding LAS homologues. C6-DATS was purified following a previously described procedure (4). n-C8-LAS was purchased from Aldrich (Milwaukee, WI) and was used as internal standard (i.s.). Sulfophenyl-3-propionic acid (SPA2C), sulfophenyl-4-butyric acid (SPA3C), sulfophenyl-5-valeric acid (SPA4C), sulfophenyl2-malonic acid (SPA1DC), and sulfophenyl-3-glutaric acid (SPA3DC) were synthesized and purified as reported elsewhere (15). Stock solutions (1 mg/mL) of the compounds

and mixtures of the compounds reported above were prepared by dissolving them in methanol. Various working standard solutions were also prepared by suitably mixing the various analytes. For LC, distilled water was further purified by passing it through the Milli-Q Plus apparatus (Millipore, Bedford, MA). Acetonitrile “Plus” of gradient grade was obtained from Carlo Erba, Milan, Italy. All other solvents and chemicals were of analytical grade (Carlo Erba) and they were used as supplied. Experimental samples of solid-phase extraction (SPE) cartridges, referred to as “Carboprep” and filled with 0.5 g of Carbograph 4, were kindly supplied by Restek, Bellefonte, PA. Carbograph 4 is an example of graphitized carbon black with a surface area of 210 m2/g. The design of these SPE cartridges is similar to that used in a previous work (16) and allows analyte back-elution to be carried out. After fitting this device into a sidearm filtering flask, it was washed with 10 mL of the eluent phase for eluting analytes (see below), followed by 5 mL of methanol, and 10 mL of distilled water. Liquids were forced to pass through the cartridge by the aid of a vacuum from a water pump. Biodegradation Assay. Biodegradation experiments of LAS and coproducts in the sirene-AlCl3 mixture were conducted under the same previously reported conditions (13, 14), that is according to the OECD screening test 301 B (17). A 2-L test solution containing the necessary organic nutrients and inorganic salts was prepared. Half a milliliter of a filtered fresh effluent of an activated sludge sewage treatment plant was added per liter of test solution as the source of microorganisms. Initial concentrations of LAS, DATS, and iso-LAS in the test solution were 24, 3.0 and 2.1 µmol/L, respectively. The bioassay was conducted under continuous stirring in a constant-temperature room (21 ( 2 °C) and using 12-h dark-light cycle. Fifty-milliliter samples were withdrawn at intervals and analyzed in duplicate by the procedure reported below. When not immediately analyzed, samples were stored at 4 °C, after the addition of 2% formalin to inhibit bacterial activity. Sample Preparation. Analytes were extracted from 10 mL of the biodegradation test solution. Extraction and elution procedures of the analytes were carried out by suitably modifying previously reported procedures (13, 18). After passage of the samples through the SPE cartridge, this was washed with 50 mL of water followed by 3 mL of methanol. After eliminating the last drops of methanol by further decreasing the pressure inside the vacuum flask, the cartridge was turned upside down, and analytes were back-eluted by 12 mL of CH2Cl2/CH3OH (80:20, v/v) basified with 5 mmol/L NaOH. The eluate was collected in a 1.4 cm i.d. glass vial with a conical bottom. The extract was first neutralized by adding 0.12 mL of 1 mM HCl and then taken to dryness in a water bath at 40 °C, under a gentle stream of nitrogen. After drying, carboxylic groups of the LAS and DATS intermediates were methylated by adding 1 mL of methanol acidified with HCl, 60 mmol/L, and heating this solution in a GC oven at 100 °C for 20 min, after sealing the vial with a Teflon lined screw cap. After methanol was removed by evaporation, the residue was reconstituted with 200 µL of a water/methanol solution (50:50, v/v) containing 5 ng/µL of the i.s., and 20 µL of the final extract was injected into the LC column. LC/ES/MS Analysis. The LC/ES/MS apparatus and several experimental conditions were the same as reported elsewhere (13). The analytes were chromatographed on an “Alltima” 25 cm × 4.6 mm i.d. column filled with 5-µm C-18 reversed phase packing (Alltech, Sedriano, Italy). For fractionating the analytes, the phase A was acetonitrile and the phase B was water. Both solvents contained ammonium acetate, 0.2 mmol/L. The mobile phase composition was 10% A, which was first linearly increased to 60% in 31 min and then to 100% in 5 min. The flow rate of the mobile phase was 1 VOL. 33, NO. 22, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

4113

mL/min, and 50 µL/min of the column effluent was diverted to the ES source. The MS was operated in the negative-ion mode by applying to the capillary a voltage of 3 kV. Unless otherwise specified, LC/MS chromatograms were obtained by scanning the quadrupole from 220 to 450 m/z with a 2-s scan and setting the skimmer cone voltage at 30 V. Quantitation. Calibration curves of C12-LAS, C10-DATS, methyl esters of the available SPAC and SPADC standards and of the i.s. (C8-LAS) were constructed by flow injection analysis (FIA). The electrosprayed solution was composed of water/acetonitrile (80:20, v/v) and contained 0.2 mmol/L ammonium acetate. These curves showed that the response of the ES/MS detector was linearly related to injected amounts of all the above compounds up to 2 µg. Moreover, molar response factors for all the above compounds did not differ significantly from each other. Thus, we reasonably assumed that all the LAS and DATS intermediates for which standards were not available gave the same molar response factors as that of C8-LAS. The ES/MS detector response is known to be dependent on the water content in the electrosprayed solution. Still using FIA, the extent of this effect was evaluated by increasing gradually the acetonitrile percentage in the electrosprayed solution from 20 to 60% by 10% increments. At each solvent composition, constant amounts of the i.s. were repeatedly injected. Although to a not dramatic extent, the response factor for the i.s. steadily increased with increasing the organic modifier percentage. As reported above, mass chromatograms were generated by solvent gradient elution. When performing analyte quantitation (see below), the analyte peak area to i.s. peak area ratio was thus suitably corrected by a factor that took into account the fact that the analyte and the IS could leave the LC column and enter the ES ion source dissolved in a solvent mixture of different composition. Quantitation of each analyte was performed by extracting its ion chromatographic profile from the total ion current chromatogram, measuring the peak areas of all related isomers as a total, and relating this area to that of the i.s.

Results and Discussion Optimization of the Analytical Conditions. LC/MS analysis of the target compounds was initially performed according to conditions reported in a previous paper (19). These conditions involve the use of a LC mobile phase containing 5 mmol/L of both triethylamine and acetic acid. Sufficiently sharp and retained peaks for LAS and DATS breakdown products were obtained. In terms of absolute sensitivity, however, this condition was unfavorable to detect very low amounts of analytes in the full-scan acquisition mode. Presumably, the large salt concentration in the LC mobile phase was responsible for the low response of the ES/MS detector. In addition, the presence of abundant amounts of triethylammonium ions in the mobile phase shortened the life of the LC column by slow dissolution of the siliceous stationary phase. The aim of devising a sensitive and robust method capable of monitoring even analyte trace amounts in environmental samples (see ref 24) was reached by converting carboxylic groups of LAS and DATS breakdown products to their methyl esters. After this modification, the addition of only 0.2 mol/L ammonium acetate to the LC mobile phase sufficed to give sharp peaks for all the analytes. Under this new chromatographic condition, a 10 times enhancement of the sensitivity of the ES/MS detector was obtained as compared to that experienced following previously reported conditions (19). The rates of conversion of monocarboxylated and dicarboxylated analytes to their methyl esters were evaluated by derivatizing available SPAC and SPADC standards by the procedure reported in the Experimental Section. LC/MS analysis showed that the reaction was virtually complete. 4114

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 22, 1999

Analyte recoveries of those parent and intermediate compounds for which standards were available were assessed by a conventional procedure. A water sample having the same composition of organics and inorganic salts as the biodegradation test solution was spiked with analytes at the individual level of 100 ng/L. Then, four 10-mL aliquots of this water sample were analyzed as reported in the Experimental Section. Mean recovery of all the species mentioned above were better than 90% with relative standard deviations not larger than 5%. Analyte Characterization. The ES/MS system set in the negative-ion mode and at a low skimmer cone voltage (see the Experimental Section) offers the opportunity of detecting analytes as intact [M - H]- ions. Alternatively, structurally significant fragment ions (confirmatory ions) of the quasimolecular anions can be generated by the in-source collisioninduced decomposition (CID) process after suitably raising the skimmer cone voltage. Providing the analyte is chromatographically separated by other compounds, these insource CID spectra have the same confirmatory power than that obtained by the MS/MS technique. The concentrations of the large number of LAS and DATS intermediates in the biodegradation test solution were assessed by choosing the first option, as measuring the intensity of one single ion signal for each analyte greatly simplified the quantitation procedure. Vice versa, confirmatory ions of the analyte structures were obtained by the in-source CID spectra. These spectra were obtained by occasionally reanalyzing extracts of the test solution after increasing the skimmer lens voltage from 30 to 55 V and setting the lower mass scan limit at m/z 70. Exemplary CID spectra of some selected analytes are shown in Figures 2 and 3. The large number of homologues and isomers of LAS and DATS intermediates produced complex chromatograms with many peak overlappings. CID spectra shown here were taken from peaks for those isomers that appeared to be resolved from peaks relative to isomers belonging to different metabolite classes. Characterization of LAS Metabolites. The molecular ion at m/z 299 observed at low cone voltage (no CID process) was assigned to a methylated SPA6C ion after observing that the CID spectrum in Figure 2A contained an abundant ion signal at m/z 183 that could be generated by neutral loss of C4H9COOCH3. This ion is typically and abundantly produced by decomposition of LAS (20-22) and SPAC (19) molecules. The [M - H]- ion at m/z 357 in the spectrum shown in Figure 2B was postulated to belong to a dicarboxylated LAS intermediate by observing that the CID process of the above ion generated the fragment ion at m/z 285 corresponding to loss of a methyl acetate moiety and the ion at m/z 183 that could result by the additional loss of methylbutyrate. Although predicted, the existence of double carboxylated metabolites of LAS (and DATS, see below) resulting from bacterial attack at the opposite ends of the alkyl chains was till now never clearly demonstrated. Characterization of DATS Metabolites. The CID spectrum in Figure 3A was characterized by a signal at m/z 237, which could be produced by loss of methyl acetate from the quasimolecular ion at m/z 311. The ion at m/z 223 was presumably produced by loss of methyl formate, instead of methyl acetate, coupled to that of ethylene. These clues led us to assign the ion at m/z 311 to a STA3C isomer. We observed that the ions at m/z 223 and 209 invariably characterized a tetralinsulfonate moiety. Finally, The CID process of the [M - H]- anion at m/z 355 (spectrum in Figure 3B) caused loss of methylformate followed by loss of methylpropionate. These two neutral losses and the presence in the spectrum of the signals at m/z 223 and 209 indicated that a group of STA2DC isomers was formed by the ω/β oxidation mechanism of both free alkyl chains of DATS.

FIGURE 2. CID spectra of two metabolites of LAS-type molecules.

FIGURE 3. CID spectra of two metabolites of DATS. Biodegradation Pathway of LAS and Coproducts. Concentration changes vs time plot for LAS, iso-LAS, and their intermediates is visualized in Figure 4, while that for DATS and their breakdown products is shown in Figure 5. After 2 days of lag phase, LAS entered the first biodegradation cycle. At day 5, all of the initial mass of LAS (24 µmol/L) was consumed. At this time, the test solution contained 8.6 µmol/L of SPAC and SPADC breakdown products and a residual isoLAS concentration of about 1.3 µmol/L (initial concentration ) 2.1 µmol/L). Assuming that 0.8 µmol/L of the intermediates had arisen from biotransformation of iso-LAS, it follows that 68% of LAS compounds were mineralized and/or converted to biomass in few days. All of the iso-LAS compounds disappeared by day 10. At this time, the total molar concentration of LAS and iso-LAS breakdown products was 2.8 µmol/L. After 7 days, this concentration decreased to 2.2 µmol/L and did not vary significantly over the next 15 days.

This concentration was very similar to that of iso-LAS and seemed to indicate that, right from day 17, persistent sulfophenylcarboxylated metabolites were essentially those coming from iso-LAS biotransformation. From day 32 to the end of the biodegradation experiment, the total concentration of these metabolites slowly decreased with time and stabilized to 0.74 µmol/L. Considering that the initial molar iso-LAS concentration was ca. 2.1 µmol/L, it follows that 35% of the initial iso-LAS was not mineralized. On the basis of results from a previous study (11), residual iso-SPACs should be mainly of type II. Unfortunately, the considerations made above are not supported by sound experimental evidence, as linear SPACs, iso-SPACs of both types I and II, could not be distinguished from each other by their CID spectra. According to a previous observation (11), DATS appeared to be more resistant to primary biodegradation than isoLAS, as more than 8% of the initial mass of the former VOL. 33, NO. 22, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

4115

FIGURE 4. Concentration vs time plot of LAS, iso-LAS, and their intermediates.

FIGURE 5. Concentration vs time plot of DATS and their intermediates. compounds was still present at the time that the latter compounds were no longer detected in the test solution. Primary biodegradation of DATS was completed by day 17. At this time, the total molar concentration of the related intermediates was 1.4 µmol/L, which amounted to 47% of the initial mass of DATS. This percentage very slowly decreased over the next 4 months and stabilized at 40%. The relative amount of DATS that was not mineralized was similar to that of iso-LAS. This finding contrasts with the results of a previous study (11), indicating that DATS are much more recalcitrant to ultimate biodegradation than iso-LAS. Compared to total SPACs, which can be formed by biotransformation of LAS and iso-LAS, the yield of formation of SPADCs was rather low. At day 6, the SPADC total concentration represented less than 10% of the initial mass of the parent compounds. Probably, SPADCs originate 4116

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 22, 1999

primarily from the most internal isomers of LAS, as these compounds contain two relatively long alkyl chains at both sides of the aromatic ring. The “distance principle” states that the biotransformation rate increases with increased distance between the sulfonate group and remote alkyl chain end (7). Only 4% of the initial DATS mass was biotransformed to STADC. The major difficulty in producing dicarboxylated intermediates from DATS by enzymatic reactions could be traced to the fact that DATS have shorter linear alkyl chains than LAS. The fate of STADCs was rather different from that of SPADCs. About one-half of the total STADC mass was recalcitrant to further biotransformation, while only 3% of the initially formed SPADC persisted in the test solution. Variations of the homologue distributions of the four classes of metabolites with time are shown in Figure 6. For

FIGURE 7. Changes with time of the isomeric composition of SPA9C.

FIGURE 6. Changes of the homologue distributions with time of the four intermediate classes. each class, the average number of alkanoyl carbons in the residual alkyl chain at selected times was obtained by using the weighted average, after measuring the relative molar concentration of each homologue. At the initial stage of the biodegradation process, the SPAC metabolite class showed the largest fluctuations in the homologue distribution. These fluctuations reflected the fact that in the first part of the biodegradation process three different batches of SPACs were generated. At day 2, only small amounts of SPAC arising exclusively from LAS external isomers were formed. These SPACs had rather long alkyl chains, as they were the product of the initial step of the ω/β-oxidation process. According to the distance principle, these compounds were then rapidly shortened by C2 unit cleavage. After 0.5 day, SPA3C and SPA4C were the most abundant homologues. Following the first batch of SPACs, a second one formed at day 5, as the result of extensive degradation of LAS internal isomers. Still according to the distance principle, internal SPAC isomers are expected to be less prone to progressive chain shortening than external ones. In fact, SPA5C-SPA8C species largely prevailed over the other homologues. Finally at day 10, each SPAC homologue contained a remarkable fraction of isomers arising from degradation of iso-LAS. At this time, SPA7CSPA9C were the most abundant homologues. Assuming reasonably that, as occurs with iso-LAS and LAS, iso-SPACs are eluted before SPACs, Figure 7 gives a picture of the sequence of events occurring during the first part of the biodegradation process. Last formed SPACs contain a methyl branch located on one of the carbon atoms of the alkyl chain. Willets (23) observed that the biodegradation rates of methyl branched alkyl chains were characterized by slow phases after which chain shortening proceeded with splitting of one propionate moiety instead of an acetate one. Swisher (7) suggested that these pauses serve to cells for changing their enzymes as needed. The pauses each time needed for producing suitable enzymes able to catabolize the different isomers of each isoSPAC homologue could explain the slow but steady shift of the SPAC homologue distribution to short-chain species. At

the end of the biodegradation experiment, we observed that the total molar concentration of SPA2C-SPA4C homologues amounted to 47% of the refractory SPACs. STACs have shorter alkyl chain than SPACs. Nevertheless, the β-oxidation mechanism was still active and enriched progressively the test solution with short-chain STACs. As an example, the relative abundance of STA1C was initially 5% and raised to 21% at the end of the biodegradation experiment. From day 10 onward, the SPADC homologue distribution did not change significantly, with SPA5DC and SPA6DC being constantly the most prominent species. This finding combined with the slow disappearance of SPADCs from the biodegradation test solution (see Figure 4) suggests that the above two compounds could be “key intermediates” in SPADC mineralization. Key intermediates is a term introduced by Swisher (7) to indicate those metabolites that persist until critical enzymes required for further degradation are produced. The situation encountered with SPADCs was not repeated with STADCs. Among STADCs, the initial relative abundances of STA0DC and STA4DC were respectively 8 and 25%. While the final relative abundance of the former homologue increased up to 22%, that of the latter one decreased down to 7%.

Acknowledgments We are indebted to ACEA (Azienda Comunale per l’Energia e l’Acqua) personnel (in particular to Dr. L. Capuani) for supplying fresh effluent samples of activated sludge sewage treatment plants.

Literature Cited (1) Ainsworth, S. J. J. Chem. Eng. News 1996, 74, 32-54. (2) Trehy, M. L.; Gledhill, W. E.; Orth, R. G. Anal. Chem. 1990, 62, 2581-2586. (3) Cavalli, L.; Cassani, G.; Lazzarin, M.; Maraschin, C.; Nucci, G.; Berna, J. L.; Bravo, J.; Ferrer, J.; Moreno A. Environ. Toxicol. Chem. 1996, 54, 167-186. (4) Crescenzi, C.; Di Corcia, A.; Marchiori E.; Marcomini, A.; Samperi, R. Water Res. 1996, 30, 722-730. (5) Trehy, M. L.; Gledhill, W. E.; Mieure, J. P.; Adamove, J. E.; Nielsen, A. M.; Perkins, H. O.; Eckhoff, W. S. Environ. Toxicol. Chem. 1996, 15, 233-240. (6) Ko¨lbener, P.; Baumann, U.; Leisinger, T.; Cook, A. M. Environ. Toxicol. Chem. 1995, 14, 571-577. (7) Swisher, R. D. Surfactant Biodegradation, 2nd ed.; Marcel Dekker: New York, 1987. VOL. 33, NO. 22, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

4117

(8) Scho¨berl, P. Tenside Deterg. 1989, 26, 86-94. (9) Field, J. A.; Leenheer, K. A.; Thorn, K. A.; Barber, L. B.; Rostad, C.; Macalady, D. L.; Daniel, S. R. J. Contam. Hydrol. 1992, 9, 55-77. (10) Ko¨lbener, P.; Baumann, U.; Leisinger, T.; Cook, A. M. Environ. Toxicol. Chem. 1995, 14, 561-569. (11) Nielsen, A. M.; Britton, L. N.; Beall, C. E.; McCormick, T. P.; Russell, G. L. Environ. Sci. Technol. 1997, 31, 3397-3404. (12) Mampel, J.; Hitzler, T.; Ritter, A.; Cook, A. M. Environ. Toxicol. Chem. 1998, 17, 1960-1963. (13) Di Corcia, A.; Crescenzi, C.; Marcomini, A.; Samperi, R. Environ. Sci. Technol. 1998, 32, 711-718. (14) Di Corcia, A.; Costantino, A.; Crescenzi, C.; Marinoni, E.; Samperi, R. Environ. Sci. Technol. 1998, 32, 2401-2409. (15) Marcomini, A.; Di Corcia A.; Samperi, R.; Capri, S. J. Chromatogr. 1993, 59, 644-651. (16) Di Corcia, A.; Marcomini, A.; Samperi, R.; Stelluto, S. Anal. Chem. 1993, 65, 907-912. (17) OECD (Organization for Economic Cooperation and Development). Test Guidelines for ready biodegradability: modified Sturm Test; Document 301 B, 1980.

4118

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 22, 1999

(18) Di Corcia, A.; Marcomini, A.; Samperi, R. Environ. Sci. Technol. 1994, 28, 850-858. (19) Gonzalez-Mazo, E.; Honing, M., Barcelo´, D.; Gomez-Parra, A. Environ. Sci. Technol. 1997, 31, 504-510. (20) Weber, R.; Levsen, K.; Louter, G. J.; Boerboom, A. J. H.; Haverkamp, Anal. Chem. 1982, 54, 1458-1464. (21) Lyon, P. A.; Stebbings, W. L.; Crow, F. W.; Tomer, K. B.; Lipstrue, D. L.; Gross, M. L. Anal. Chem. 1984, 56, 8-16. (22) Field, J. A.; Barber, L. B.; Thurman, E. M.; Moore, B. L.; Lawrence, D. L.; Peake, D. L. Environ. Sci. Technol. 1992, 26, 1140-1148. (23) Willets, A. J. Antonie van Leeuwenhoek 1974, 40, 561-574. (24) Di Corcia, A.; Capuani, L.; Casassa, F.; Marcomini, A.; Samperi, R. Environ. Sci. Technol. 1999, 33, 4119-4125.

Received for review May 24, 1999. Revised manuscript received August 30, 1999. Accepted August 30, 1999. ES9905952