Environ. Sci. Technol. 1989, 23,951-961
Michael, A,; Carlson, G. H. J. Am. Chem. Soc. 1935, 57, 1268-76. Lucas, G. R.; Hammett, L. P. J. Am. Chem. Soc. 1942,64, 1928-37. Csizmadia, V. M.; Houlden, S. A.; Koves, G. J.; Boggs, J. M.; Csizmadia, I. G. J . Org. Chem. 1973, 38,2281-7. Dixon, W. B.; Wilson, E. B. J . Chem. Phys. 1961,35,191-8. Harris, L. E. J . Chem. Phys. 1973,58, 5615-26. Calvert, J. G.; Pith, J. N., Jr. Photochemistry; Wiley: New York, 1966; p 454. Maria, H. J.; McDonald, J. R.; McGlynn, S. P. J. Am. Chem. SOC.1973, 95, 1050-6. Johnston, H. S.; Chang, S.-G.;Whitten, G. J . Phys. Chem. 1974, 78, 1-7. Gray, P.; Rogers, G. T. Trans. Faraday Soc. 1954,50,28-36. Gray, J. A.; Style, D. W. G. Trans. Faraday Soc. 1953,49, 52-7. Rebbert, R. E. J . Phys. Chem. 1963,67,1923-5. Renlund, A. M.; Trott, W. M. Chem. Phys. Lett. 1984,107, 555-60. Demerjian, K. L.; Schere, K. L.; Peterson, J. T. Adv. Environ. Sei. Technol. 1980, 10, 369-459. Atkinson, R. Chem. Rev. 1986,86, 69-201. Atkinson, R. Int. J . Chem. Kinet. 1987,19,799-828. Atkinson, R.; Lloyd, A. C. J . Phys. Chem. Ref. Data 1984, 13, 315-444. Moortgat, G. K.; Burrows, J. P.; Schneider, W.; Tyndall, G. S.; Cox, R. A. In Physico-Chemical Behavior of Atmospheric Pollutants; D. Reidel: Dordrecht, The Netherlands, 1986; pp 271-81.
Kurylo, M. J.; Dagaut, P.; Wallington, T. J.; Neuman, D. M. Chem. Phys. Lett. 1987,139,513-8. Jenkin, M. E.; Cox, R. A.; Hayman, G. D.; Whyte, L. J. J . Chem. Soc., Faraday Trans. 2 1988,84,913-30. Basco, N.; Parmar, S. S. Int. J. Chem. Kinet. 1985, 17, 891-900. Wallington, T. J.; Dagaut, P.; Kurylo, M. J. Photochem. Photobiol. 1988, 42, 173-85. Lin, X.; Trainer, M.; Liu, S. C. J . Geophys. Res. 1988,93, 15879-88. Atherton, C. S.; Penner, J. E. J. Geophys. Res., submitted. Atherton, C. S. Geophys. Res. Lett., submitted. Penkett, S. A.; Brice, K. A. Nature 1986, 319, 655-7. Stephens, E. R. Ado. Environ. Sei. 1969, 1, 119-46. Senum, G. 1.; Fajer, R.; Gaffney, J. S. J . Phys. Chem. 1986, 90,152-6. Singh, H. B. Environ. Sei. Technol. 1987,21, 320-7. Singh, H. B.; Kasting,J. F. J. Atmos. Chem. 1988,7,261-85. Ciccioli,P.; Brancaleoni,E.;DiPalo, V.; Liberti, M.; DiPalo, C. Acqua Aria 1986, 7, 675-83.
Received for review August 29,1988. Accepted March 10,1989, This work was supported by the Officeof Health and Environmental Research (DOE) and performed under the auspices of the US.Department of Energy, under Contract No. DEACO2-76CHOOOl6. By acceptance of this article, the publisher andlor recipient acknowledges the US.Government's right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.
Biotransformation of Halogenated and Nonhalogenated Octylphenol Polyethoxylate Residues under Aerobic and Anaerobic Conditions Harold A. Ball, Martln Reinhard,' and Perry L. McCarty
Environmental Engineering and Science, Department of Civil Engineering, Stanford University, Stanford, California 94305 Halogenated and nonhalogenated residues of octylphenol polyethoxylate (OPEO) surfactants, including octylphenol polyethoxycarboxylates(OPEC), were subjected to biotransformation under aerobic and anaerobic conditions. Under both conditions, relatively recalcitrant intermediates were formed within 10 days. Under aerobic conditions, degradation was complete within 127 days. Under anaerobic conditions, there was partial transformation to octylphenol and recalcitrant OPEC compounds over a 190-day period. Halogenated residues, which can be formed during wastewater chlorination, were transformed to recalcitrant metabolites such as halogenated octylphenol diethoxycarboxylate and halogenated octylphenol. The activity of an anaerobic methanogenic consortium was inhibited by halogenated OPEC at a concentration of 135 pM (approximately 60 mg/L).
Introduction Alkylphenol polyethoxylates (APEO) are nonionic surfactants commonly used for industrial and agricultural purposes. Approximately 1.56 X lo8 kg of APEO was produced in the United States in 1985 (1). APEO compounds typically consist of hydrophobic branched nonyl (NP) or octyl (OP) phenol attached to a hydrophilic polyethylene glycol chain having from 1to 30 ethoxy (EO) units. As shown in Figure 1, we denote the multiple OPEO homologues by a number within the acronym (i.e., OPnEO) to indicate the number (n)of EO units attached to the octylphenoxy moiety. In OPnEC, n indicates the number of unaltered EO units plus the terminal OCHzCOOHunit. 0013-936X189/0923-0951$01.50/0
In this paper, chlorinated and brominated OPnEO are denoted ClOPnEO and BrOPnEO, respectively. At one time, surfactant biodegradability was demonstrated by primary transformation or loss of the parent compound and loss of surfactant properties such as foaming (2,3).However, such an approach ignores the possibility that ecologically significant bioresistant intermediates may be formed from the parent surfactant. Many studies have shown that biotransformation products of APEO are ubiquitous in the environment. APEO metabolites, often including the carboxylated APEC, have been found in wastewaters (4-81, sewage sludges (9), surface waters (10-12), and in municipal landfii leachate (13). A chlorinated OPEO was found in river water samples (14), while chlorination of wastewaters was found to result in the formation of both chlorinated and brominated OPEO and OPEC (7,15), with halogenated OP2EC a predominant product. A wide range of halogenated OPEO residues was present in the municipal secondary wastewater treatment plant effluent at total concentrations up to 51 pg/L, while nonhalogenated OPEO residues were present at a total concentration from 36 to 112 pg/L (7). In addition, chlorination at a drinking water treatment plant treating river water containing NPEO and NPEC residues resulted in the formation of brominated NPEO and NPEC products that were detected in tap water (12). Not only the disappearance of parent APEO compounds, but also the fate of APEO metabolites formed needs to be better understood. Toward this end, the biological transformation of halogenated and nonhalogenated OPEO metabolites was examined in this study. Potential bio-
0 1989 American Chemical Society
Environ. Sci. Technol., Vol. 23, No. 8, 1989
951
OPnEO H3C-CFCH, IH-CH23
~ C CH, Q
- ~
~
~
p
2
~
~
H
,
~
~
X
XOP2EC Figure 1. Example structures and nomenclature for octylphenol polyethoxylates. In this study, the ethoxy chain length ( n ) ranged from 1 to 6.
transformation pathways of APEO can initiate at the akyl substituent, and/or the ethoxy chain, and ultimate degradation must include cleavage and degradation of the aromatic ring. Carboxylation and rapid biotransformation of the alkyl substituent occurs when the substituent has few or no branches (16). However, APEO compounds typically have highly branched alkyl substituents, which prevent the oxidation pathway effective for straight-chain aliphatic compounds (17). The primary aerobic biotransformation pathway observed for branched APEO is through shortening of the EO chain by hydrolysis or an oxidative-hydrolytic mechanism (2, 16, 18, 19). Several investigators have found that APEO degrades to a relatively recalcitrant metabolite with two ethoxy units remaining on the intact aromatic ring with its alkyl substituent (3,16,20). This phenomenon suggests hindered biotransformation of the shortened EO chain. Alternatively, a different mechanism or a different responsible organism may be involved in the biotransformation of APEO residues with two or fewer ethoxy units. As part of the overall objective of this research, the biotransformation potential of halogenated and nonhalogenated OPEO compounds under various aerobic and anaerobic conditions was studied, and the influence of halogenation on biotransformation pathways was evaluated. Six OPEO mixtures were used: OPnEO and OPnEC, chlorinated OPnEO and OPnEC, and brominated OPnEO and OPnEC. OPnEO was subjected to aerobic activated sludge inoculum in order to assess the potential distribution of metabolites found in secondary effluents. Nonhalogenated and halogenated OPEO and OPEC were individually subjected to aerobic primary sewage inoculum and to an anaerobic methanogenic consortium in order to evaluate the biodegradative potential under different environmental conditions. In addition, the Anaerobic Toxicity Assay (ATA) (21) was used to evaluate toxic and inhibitory effects of halogenated OPEC on anaerobic microorganisms.
Experimental Section Starting Materials and Reagents. Laboratory studies were conducted with a technical-grade mixture of tertoctylphenol polyethoxylate (3 mol of ethylene oxide) (SU 144, Chem Service, West Chester, PA). Gas chromatographic analysis indicated the following composition of SU 144 on a molar percent basis: OPlEO (13%), OP2EO (40%), OP3EO (29%), OP4EO (14%), OP5EO (4%). OPEO was converted from the alcohol to the corresponding carboxylic acid (OPEC) by oxidation with Jones reagent (0.4 mol of potassium dichromate in 700 mL of 2 M sulfuric acid) (15). Halogenation of the aromatic ring of OPEO and OPEC was carried out in aqueous bromine or chlorine solutions as described previously (22). Chemical transformation of the starting OPEO mixture did not 952
Environ. Sci. Technol., Vol. 23, No. 8, 1989
~
appreciably change the relative distribution of homologues in the final products except that halogenated and nonhalogenated OPlEC decreased to -1.5% of the final mixture. The molecular mass of the nonhalogenated products and metabolites studied ranged from 206 g/mol for octylphenol (OP) to 498 g/mol for OPGEC. Activated Sludge Inoculation. A 1-L grab sample of ~ mixed liquor was collected from the activated sludge aeration basin of a municipal wastewater treatment plant where OPEO residues were previously detected (7). The suspended solids concentration (23) was 720 mg/L. The suspended solids were “washed” to remove surfactants or surfactant residues. Here, the suspended solids were allowed to settle for 20 min, -90% of the supernatant was withdrawn with a vacuum aspirator, 900 mL of aerated “biochemical oxygen demand“ (BOD) dilution water (23) was added, and the sample was thoroughly agitated. This procedure was repeated twice. The final 1 L of washed mixed liquor was serially diluted with aerated BOD dilution water to obtain two 1-L samples, each containing -180 mg/L suspended solids. Each 1-L sample was transferred into a separate 2-L open-mouthed beaker mixed with 100 rpm synchronized mechanical paddle stirrers (Model 7790-300, Phipps and Bird, Richmond, VA). OPEO (500 pg) in 50 pL of methanol was added to one beaker and an equal quantity of CHBOHalone was added to the other or control beaker. This OPEO concentration is considered representative of typical APEO concentrations in sewage treatment plants (8). The reactors were kept at ambient temperature (20 f 1“C), and sampling began after 1h and continued over a 24-h period. Primary Sewage Inoculation. Aerobic primary sewage inoculation experiments followed the standard biochemical oxygen demand (BOD) procedure, which simulates conditions found in natural water bodies (24). Surfactant [final concentration 10 mg/L (approximately 25 pM)] was transferred individually to a series of open glass bottles filled with 2 L of BOD dilution water that had been inoculated with 20 mL of fresh settled primary effluent from a municipal wastewater treatment plant. This OPEO concentration is much higher than that found in wastewater treatment plants; however, it was anticipated that possible transformation intermediates would be easier to detect with a high initial OPEO concentration. Each bottle was stored in the dark at ambient temperature (20 f 1 “C). A control bottle, without the surfactant, was also evaluated. Anaerobic Bioassay. A procedure for measuring biochemical methane potential (BMP) (21) was used in the anaerobic biotransformation experiments. Here, a defined anaerobic medium was prepared and seeded with anaerobic organisms maintained on a mixture of primary and waste activated sludge from a municipal wastewater treatment plant. Inoculated media (50 mL) were spiked with approximately 25 pM of each surfactant and added to individual 60-mL serum bottles, which were then stoppered with black butyl rubber stoppers, capped with aluminum crimp-on seals, and placed in an environmental chamber maintained at 35 “C. Eight replicates of each sample and eight controls with no added surfactant were prepared. Surfactant transformation was evaluated over time by sacrificing one of the replicate sample bottles and analyzing for reactants and products. Anaerobic Toxicity Assay. Toxic or inhibitory effects of halogenated OPECs to anaerobic biological processes were assessed by the Anaerobic Toxicity Assay (ATA) (21). The defined anaerobic media with seed organisms used in the anaerobic bioassay were spiked with halogenated OPEC, 48-mL aliquots were placed in 150-mL serum
bottles, and 2 mL of a propionate solution was added prior to sealing with black butyl rubber stoppers and capping with aluminum crimp-on seals. The propionate solution contained 41 mg/mL sodium propionate in deionized water and served as a source of substrate for the organisms. Three different concentrations of BrOPEC and ClOPEC (1, 18, and 135 pM) were evaluated. The sealed bottles were placed in a 35 "C environmental chamber. Duplicate samples and two controls with no OPEC were included. Total gas production and methane and carbon dioxide gas composition were analyzed over a period of 32 days. Sample Analysis. Liquid samples were extracted in (120-mL) bottles sealed with Teflon-lined screw caps. p-Bromophenylacetic acid (BPA) and 3,6-dimethylphenanthrene (DMPh) were used as surrogate standards for the acid fraction and neutral fraction, respectively. The samples (100 mL) from the primary sewage inoculation and activated sludge inoculation studies were spiked with BPA (1.25 and 2.00 mg/L, respectively) and DMPh (0.34 and 0.67 mg/L, respectively) prior to extraction. Approximately 2 g of NaCl was added to each bottle to control emulsions, followed by 10 drops of sulfuric acid to lower the pH to 1.5. Diethyl ether (ether) (20 mL) was added to each bottle, which was then placed on a shaker table and agitated vigorously for at least 20 min. The ether extract was then removed, dried over anhydrous sodium sulfate (Na804),and filtered through a coarse paper filter. Samples were extracted three times in this manner. The combined extracts were concentrated by rotoevaporation to approximately 5 mL under reduced pressure in a 30 "C water bath, evaporated to dryness under a purified nitrogen stream, and redissolved in l mL of a 1:l ethermethanol mixture. Carboxylic acids were methylated with diazomethane (15). Methylated extracts were evaporated under purified nitrogen, redissolved in 1mL of dichloromethane, and spiked with 200 pg/L of the internal standard anthracene-dlo. The final volume of each extract was 1 mL. The anaerobic bioassay samples (50 mL) were spiked with BPA (2.00 mg/L) and DMPh (0.67 mg/L) and extracted as above. Extracts were purified by silica gel chromatography (15). Compounds of interest were eluted with CH2C12 and CHsOH and combined into a single fraction. As described above, the final fraction was concentrated, evaporated, redissolved in 1 mL of a 1:l ether-methanol mixture, methylated, redissolved in 1mL of CH2C12,and spiked with 400 pg/L of the internal standard anthracene-dIo. A detailed discussion of the protocols used for GC/MS analysis of OPEO compounds is presented in a previous article (22) where the organic synthesis procedures are reviewed, mass spectral features and tentative fragmentation mechanisms presented, and quantitation method and precision discussed. Briefly, a Finnigan mass spectrometer (Model 4000, upgraded with a Model 4500 ion source; Finnigan, Sunnyvale, CA) with the INCOS data system was used. A Finnigan Model 9610 gas chromatograph was connected to the mass spectrometer and equipped with a fused-silica capillary column (DB-5, 30 m X 0.33 mm i.d.; film thickness 0.25 pm; J&W, Rancho Cordova, CA) coupled directly to the ion source. Helium was used as the carrier gas with an inlet pressure of 0.5 bar. A 1-pL sample of extract was injected splitless for 30 s. The gas chromatograph temperature program was as follows: 70 "C isothermal for 1min, increasing from 70 to 300 "C at 3 "C/min. The ionization conditions were as follows: ionization energy, 70 eV; electron multiplier voltage, 1700 V; ionizer temperature, 140 "C; mass range,
Table I. Activated Sludge Inoculationf Transformation of OPEO
starting compds: OPlEO OP2EO OP3EO OP4EO OP5EO
subtotal products: OP OPlEC OP2EC OP3EC total
Oh
2h
concn, NM 6 h 12h
0.18 0.54 0.40 0.18 0.10 1.4
0.05 0.20 0.06
0.03 0.14 0.02
0.31
1.4
tr 0.26 0.61 0.19 1.4
-
18h
24h
0.02 0.03 -
0.01 0.01 -
-
tr tr -
0.19
0.05
0.02
tr
tr 0.17 0.39 0.12 0.87
tr 0.19 0.50 0.08 0.82
tr 0.23 0.85 0.20 1.3
tr 0.14 0.72 0.15 1.0
-
-
not detected; tr, trace, detected at a concentration C0.005 NM.
45-500 m/z; scan time, 2 s. Overall efficiency of the three ether extractions was near complete as judged from peak height ratios of surrogate standards to analyte peaks. The sensitivity of the method was greater than 0.005 pM for all compounds. Characteristic ions were chosen for quantitation to ensure selective detection of individual compounds (22). Analysis of C02 and CHI was performed on a gas partitioner (Model 25V, Fisher Scientific, Pittsburgh, PA) equipped with standard dual HMPA on Columpak/molecular sieve columns (Fisher Scientific, Pittsburgh, PA), with helium as the carrier gas.
Results Activated Sludge Inoculation. Data from 24-h activated sludge inoculation appear in Table I with starting materials in the top half of the table and products in the lower half. Ether cleavage of longer chain length OPnEO homologues ( n > 2) to those with n = 1-3, followed by oxidation of the terminal ethylene glycolic alcohol, resulted in a rapid (within 12 h) transformation to the carboxylated OPnEC homologues ( n = 1-3), with OP2EC the predominant product formed. The proportional composition of OPEC homologues in the final sample was very similar to that of the nonhalogenated homologues found in wastewater treatment plant effluents (7). No OPEO or OPEO residues were found in the control, indicating that products observed in the experimental systems were not desorbed from the seed inoculum. These data confirm that OPEO surfactants could be a precursor to the OPEC found in wastewater treatment plant effluents (7, 8, 11) and indicate that OPEO compounds may be rapidly transformed to OPEC under these conditions. In addition, the OPnEC (n = 1-3) metabolites formed by activated sludge inoculum were slower to degrade. This is indicated by the relatively good mass balance of total concentration, indicating little, if any, transformation to mineralized end products. If formed in wastewater treatment plants, such carboxylated intermediates may therefore be available for halogenation upon effluent chlorination to products found in treatment plant effluents (7). Primary Sewage Inoculation. The results from biotransformation of OPEO and OPEC by primary sewage inoculum appear in Table 11, parts A and B, respectively. No transformation of halogenated and nonhalogenated OPEO and OPEC was observed for 52 days in bottles Environ. Sci. Technol., Vol. 23, No. 8, 1989
953
Table 11. Primary Sewage Inoculationa day 0
day 2
day 5
concn, uM day 17
day 36
day 64
day 127
A. Transformation of OPEO starting compds: OPlEO OP2EO OP3EO OP4EO OP5EO subtotal products: OP OPlEC OP2EC OP3EC total
3.6 11
8.0 3.7 0.88 27
27
2.3 19 0.58 tr -
2.9 21 0.33 tr -
0.13 26 0.19 -
5.1 0.03 -
-
-
-
22
24
26
5.1
0.04
0.03 tr
0.01 0.07 0.13 24
0.03 5.2
-
22
0.01 0.03 0.03 26
-
0.01 2.8 1.3 0.01
-
-
0.04
-
0.02 0.06
0.03 0.03
B. Transformation of OPEC starting compds: OPlEC OP2EC OP3EC OP4EC OP5EC OP6EC subtotal products: OP OPlEO OP2EO OP3EO total O-,
0.32 9.2 7.7 4.3 2.0 0.69 24
24
0.35 6.6 5.5 2.6 1.0 0.99 17
0.32 7.6 6.5 3.4 1.3 0.92 20
tr 0.05 6.3 3.4 1.2 1.1 12
5.5 3.4 1.3 0.38 11
0.02 0.03 0.01 -
0.01 0.03 0.01
17
20
0.01 0.20 0.19 tr 12
0.01 0.02 0.40 0.03 11
-
4.1 0.01
D1
not detected; tr, trace, detected at a concentration C0.005 pM.
where biotransformation was inhibited by high methanol concentration (data not shown). Hence, abiotic transformations were deemed insignificant. Ether cleavage of longer EO chain length OPnEO homologues ( n 2 3) occurred within 2 days, resulting in accumulation of OP2EO. Transformation of OPlEO required an adaptation time of -5 days while that of OP2EO was -17 days. No significant intermediate products from these two homologues were identified. Some oxidation of OPEO to OPEC did occur; however, it appears that carboxylation of OPnEO with n = 3-5 was a minor route of OPEO transformation since no OP5EC or OP4EC was detected, and OPSEC occurred at very low concentration. If formed, these OPEC would have been detected as they exhibited long adaptation periods prior to transformation (Table IIB). Table IIB data indicate that, if formed from OPEO, OPEC metabolites can be transformed by primary sewage inoculum. OPnEC (n I 3) requires longer adaptation times (>36 days) than for OP2EC and OPlEC. This is just the opposite of that found for OPnEO homologues. Halogenated OPEO were not completely degraded by primary sewage inoculum (Tables IIIA and IVA); however substantial and corresponding transformation, primarily to XOPPEC, did take place for both the brominated and chlorinated compounds. Ether cleavage of the longer EO chain length XOPnEO homologues ( n 1 3) to XOP2EO took place within 5 days, with transformation halted at XOP3EO for some homologues. After day 5, there appeared to be some slow transformation to carboxylates, with XOP2EC the predominant product after 127 days, as shown for BrOPEO in Figure 2. Halogenated phenols were detected at low concentration. Although no dehalogenated products were observed from BrOPEO, some 954
-
Environ. Sci. Technol., Vol. 23, No. 8 , 1989
dehalogenated products (OP2EO and OP2EC) were detected from ClOPEO; however, the extent f this transformation was very limited. Under aerobic conditions, dehalogenation of aryl halides typically occurs after ring cleavage or loss of aromaticity (25). Therefore, dehalogenation of ClOPEO prior to transformation of the aromatic ring under these experimental conditions was an unexpected finding. Chlorinated and brominated OPEC (Tables IIIB and IVB) showed only limited transformation under aerobic conditions. XOPlEC, XOP2EC, and XOPSEC were not transformed during this experiment. However, longer chain length homologues, XOP4EC and XOPBEC, were gradually transformed, with complete disappearance of XOPBEC and 90% transformation of XOP4EC by day 127. XOPBEC increased in concentration over the course of the experiment suggesting that aerobic biotransformation, when it did take place, had XOP2EC as a recalcitrant end product. Although a good mass balance for some analyses was not maintained, qualitative observation of the reconstructed ion chromatograms for the final samples showed that transformation of XOPnEO (n > 2) to the carboxylated products did occur with XOP2EC being the major peak, as shown in Figure 2. The apparent lack of a mass balance in some experiments was due to some large measured concentrationsfor OPEC and XOPEC. Review of recovery data indicates that this result can be partially explained by inconsistent recovery of the acid surrogate standard, BPA. Anaerobic Bioassay. Data on OPEO biotransformation under anaerobic conditions (Table VA) indicate substantial transformation took place. Ether cleavage of the longer EO chain length homologues ( n 1 2) was rapid
100
1 J
6
A
\
60
-
40
-
-=-
50
20
7
-
Id,
c
0 0
E
BrOPEC 18 U M R
--cj
Control
+ BrOPEC 135 uWL
/
-c
BrOPEC 1 uM/L
A --
I
I
I
I
10
20
30
40
E 0
z
9
100,
1
l7
? i B e
2
(I;
50
80
-
40
-
20
---!-
-,
ClOPEC 1 UWL
-A-,
ClOPEC 18 uM/L
*
ClOPEC 135 uMR Control
0 0
10
20
30
40
Time (days) 1640
50 00
33 20
TIME
66 40
(mln)
Figure 2. GUMS reconstructed ion chromatograms of samples from aerobic primary sewage inoculum biotransforming B r W O on day 17 (chromatogram A) and day 127 (chromatogramB) showing slow terminal alcohol oxidation of BrOPnEO (n = 1-3) to the corresponding carboxylates with BrOP2EC the predominant end product. Peaks are labeled as follows: 1, BPA 2, anthracene-dlo; 3, DMph; 4, BrOPlEO 5, BrOPlEC; 6, BrOP2EO; 7, BrOP2EC; 8, BrOP3EO; 9, BrOP3EC.
(within 10 days) with near quantitative conversion to OPlEO. OPlEO was then gradually transformed with OP a likely intermediate, as it accumulated and was the most significant product after 190 days. Some OPEC metabolites were detected, indicating that terminal oxidation of the alcohol group was slow. Under anaerobic conditions (Table VB) OPEC homologues with 2 In I 4 were not transformed. However, OPlEC was rapidly transformed and not detected in samples taken after day 0. XOPEO mixtures (Tables VIA and VIM) were primarily transformed under anaerobic conditions by ether cleavage to XOPSEO, followed by oxidation to XOPZEC. This result was the same as for aerobic XOPEO transformation. However, unlike aerobic transformation, the data suggest that slow conversion of XOPBEO to XOPlEO was possible and that XOPlEO underwent slow transformation to the halogenated phenol (XOP). Under anaerobic conditions, the halogenated OPEC compounds did not degrade, except XOPlEC and possibly XOPBEC (Tables VIB and VIIB). Qualitative comparison of successive data indicates no significant change in the concentration of the XOPBEC, XOPBEC, and XOP4EC homologues, although the data for the anaerobic OPEC compounds were somewhat erratic primarily due to inconsistent recovery of the BPA standard. Anaerobic Toxicity Assay. Figure 3 contains plots of cumulative gas production over time for BrOPEC and CIOPEC. The maximum rate ratio (MRR) of metabolism was computed as the ratio between the rate of gas pro-
Figure 3. Cumulative aas Droduction from anaerobic Droolonate degradation in the preselce of different XOPEC concentradons. Note inhibkion of gas production at BrOPEC and CiOF‘EC concentrations of 135 HM.
duction from a sample and that of the average of the controls. MRR was calculated after 7 days. The MRRs for the low- and medium-dose samples after 7 days were within 6% of each other and the control. Methane to carbon dioxide ratios after 32 days were on the order of 1.5 with no significant differences between samples and the control. However, the MRR values for the high-dose samples, 135 pM BrOPEC and ClOPEC, were found to be approximately 0.5 when a MRR of “less than 0.9 suggests significant inhibition” (21). After 10 days, the 135 pM BrOPEC and ClOPEC samples had exerted 48% and 55% of their total gas production, respectively. Over the same time period, the low- and medium-dose BrOPEC and ClOPEC samples had exerted 90% of their total gas production. Although the rate of gas production was significantly affected at high XOPEC doses, the final cumulative gas production and overall produced gas composition after 32 days was not significantly different from the low- and medium-dose samples or controls.
Discussion The above experiments do not simulate the range of environmental conditions found in nature, and transformation rates or persistence of recalcitrant intermediates may be different under other conditions. The study results can give qualitative information on possible biotransformation pathways and potential recalcitrance of various halogenated and nonhalogenated OPEO metabolites that may be encountered in nature. For example, the proportional composition of OPEO metabolites in the final activated sludge inoculation sample was very similar to that of nonhalogenated metabolites found in actual wastewater treatment plant effluents (7) even though the laboratory experiment was carried out with dilute microbial populations in synthetic media under nondynamic, Environ. Sci. Technol., Voi. 23, No. 8, 1989
955
Table 111. Primary Sewage Inoculation" day 0
day 2
day 5
concn, r M day 17
day 36
day 64
day 127
A. Transformation of BrOPEO
starting compds: BrOPlEO BrOP2EO BrOP3EO BrOP4EO BrOP5EO subtotal products: BrOP BrOPlEC BrOP2EC BrOP3EC BrOP4EC total
2.9 10 7.9 2.8 0.60 24
24
1.9 5.9 4.0 1.5 tr
1.8 16 1.2 -
2.1 18 2.3 0.13 -
1.8 15 2.3 -
2.0 16 2.0 -
1.8 14 2.0 -
13
19
23
19
20
18
tr 0.02 0.02 13
-
0.01 0.19 2.5 0.35 0.04 26
0.02 0.57 5.9 0.68 0.04 26
-
-
35
3.9 39 2.4 0.09 63
0.13 19
1.4 13 0.96 -
B. Transformation of BrOPEC starting compds: BrOPlEC BrOP2EC BrOP3EC BrOP4EC BrOP5EC subtotal products: BrOP BrOPlEO BrOP2EO BrOP3EO total
0.20 7.0 6.1 3.5 1.5 18
0.23 5.9 4.7 2.0 0.82 14
0.29 7.3 6.3 3.1 1.5 18
0.30 7.0 6.0 3.2 1.5 18
0.28 8.0 6.3 2.8 0.73
0.33 11 6.4 1.9 0.11
0.62 15 6.9 0.44 -
18
20
23
tr tr -
-
-
tr -
-
tr 0.06 0.06 -
14
18
18
tr tr 0.44 0.21 19
18
0.08 0.51 0.01 20
0.08 0.34 23
* -, not detected; tr, trace, detected at a concentration 2) to OP2EO without the formation of acid intermediates (Table IIA) 956
Environ. Sci. Technol., Vol. 23, No. 8, 1989
indicates that terminal alcohol oxidation was not necessary for ether cleavage to occur. The accumulation of acid intermediates in some systems, and the resistance to transformation of OPEC under both aerobic and anaerobic conditions, may suggest that terminal alcohol oxidation tends to hinder ether cleavage and further biotransformation of this metabolite. However, some OPEO metabolites from OPEC biotransformation were detected at low concentration. In the primary sewage inoculation experiment, the appearance of increased concentration of OPlEO corresponded with the onset of OP2EC disappearance. Likewise, increased concentrations of OP2EO and the appearance of OP3EO corresponded with the initiation of OPGEC transformation. This result suggests that pathways for OPEC biotransformation include ether cleavage of the longer chain length OPEC homologues to shorter chain length OPEO molecules. Some systems tended to show more terminal alcohol oxidation than others, such as the aerobic activated sludge inoculation experiment, and aerobic and anaerobic studies on XOPEO. Our data are consistent with sequential loss of monomer EO units from the end of the EO chain as reported previously (17). Many features of the aerobic XOPEO/XOPEC biotransformation pathway are similar to aerobic OPEO/ OPEC transformation. As in the biotransformation of OPEC, the appearance of BrOP2EO and BrOPlEO corresponded with the initiation of BrOP5EC and BrOP4EC transformation, suggesting that a pathway for aerobic XOPEC biotransformation involves ether cleavage of longer EO chain length XOPEC homologues to shorter chain length XOPEO molecules. Overall, halogenated OPEO are subject to slower transformation rates than nonhalogenated OPEO, with some steps, ether cleavage
Table IV. Primary Sewage Inoculation' day 0
day 2
day 5
concn, pM day 17
day 36
day 64
day 127
1.9 0.36 15 1.1 2.8 2.2 0.05 -
1.9 0.31 18 1.1 1.7 2.0
1.8 0.41 15 1.2 1.8 2.1 -
1.1 0.68 7.2 0.87 0.64 1.0 -
23
25
22
11
0.02 0.38 3.4 0.09 0.37 0.09 0.17 0.10 30
0.03 0.54 6.7 0.40 0.44 0.31 0.14 0.24 31
0.04 1.5 23 2.1 1.3 1.0 0.02 1.2 42
12 3.1 8.5 2.5 1.9
0.15 15 4.2 5.7 2.0 0.13 -
28
27
A. Transformation of ClOPEO starting compds: ClOPlEO ClzOPlEO ClOP2EO ClzOP2EO ClOP3EO C120P3EO ClOP4EO ClOP5EO subtotal products: clop ClOPlEC ClOP2EC C120P2EC ClOP3EC Cl2OP3EC OP2EO OP2EC total
3.0 0.61 9.1 1.8 6.2 1.0 2.5 0.45 25
0.94 0.17 2.9 0.47 2.0 0.33 0.73 0.18 7.7 tr
-
17
-
-
-
tr
-
-
-
0.02 0.14 0.98 0.10
0.04
0.25 tr
0.28 0.04
25
1.4 0.25 11 0.77 2.0 1.6
1.8
0.02
tr
17
25
-
B. Transformation of ClOPEC starting compds: ClOPlEC ClOP2EC C120P2EC ClOP3EC C120P3EC ClOP4EC ClOP5EC subtotal products: clop ClOPlEO ClOP2EO C120P2EO ClOP3EO total a-,
0.21 5.5 1.6 4.2 1.6 2.4 0.97 16
16
0.32 4.5 1.4 3.7 1.6 2.4 1.8 16
0.32 5.3 1.6 4.3 1.8 2.6 1.7 18
0.10 0.04 0.02 -
0.05 0.04 0.02
-
16
18
0.18 5.6 1.6 5.4 2.1 3.5 2.4
21
0.04 0.05
-
-
21
7.8 1.9 6.0 2.0 2.4 0.33 20 0.04 0.09 0.13 0.13 0.08 21
-
0.08 0.11 0.39
0.03 tr 0.15 0.61
-
-
29
28
not detected: tr. trace, detected at a concentration 2) was transformed by ether cleavage to XOP2EO as a predominant intermediate. It appears that further ether cleavage of XOP2EO was restricted, whereupon the oxidation route to XOP2EC was favored. Terminal alcohol oxidation was a favored pathway even in the absence of oxygen, as observed in the anaerobic bioassay (Tables VIA and VIIA). Although OP2EC was ultimately degradable (Table IIB), XOP2EC was recalcitrant under the conditions studied. The rate and pathway of XOPnEO and XOPnEC transformation appeared to be affected by the presence of the halogen only when the EO chain length was short (n I 3). Under aerobic conditions, ether cleavage of XOPnEO (n I 2) and XOPnEC (n I3) was apparently precluded. It is possible that the enzymes mediating the cleavage of the ether bonds in proximity to the aromatic ring were sterically hindered by the presence of the bulky halogen atom. Ethoxylate Chain. Transformation of the EO chain of OPEO can be compared to transformation of poly-
ethylene glycol. The EO chain is made up of a string of ethylene oxide (ethylene glycol) monomers which, as a separate molecule, is often referred to as polyethylene glycol (PEG). The ability to biologically attack the ethylene glycol monomer is very common (18).In general, increased PEG molecular weight hinders biotransformation and biological growth (26). Aerobic and anaerobic biotransformation pathways for PEG are consistent with stepwise removal of monomer units from the end of the polymer (18,27), while internal attack of the PEG has not been observed (18). Under aerobic conditions, the EO chain is shortened by (1) hydrolysis of the ether bond (17, 27,B)and (2) oxidation of the terminal EO group followed by its hydrolysis from the polymer (18,26).This mechanism was observed in pure cultures isolated from aerobic activated sludge where polyglycols were carboxylated at one or both ends of the molecule (resulting in the ROCHzCOOH end group) (26).Under anaerobic conditions, hydrolysis of the polyglycol is a major pathway leaving a terminal alcohol on the polyglycol chain (18,29). There is no evidence in the literature on PEG biotransformation that resolves which conditions favor the hydrolysis pathway and which promote the initial oxidation prior to the hydrolysis. The above PEG transformation pathways are consistent with the OPEO transformations observed in this study. Environ. Sci. Technol., Vol. 23, No. 8 , 1989 057
Table V. Anaerobic Bioassay" day 0
day 10
day 23
concn, pM day 46
day 66
day 116
day 190
A. Transformation of OPE0 starting compds: OPlEO OP2EO OP3EO OP4EO OP5EO subtotal products: OP OPlEC OP2EC OP3EC OP4EC total
3.8 11 8.3 3.8 0.91
26 0.23 0.26 tr 0.37
18 0.03 -
-
0.51 -
-
-
-
-
28
27
18
2.8
0.51
0.15
tr
0.19 tr 1.0 0.72 -
1.1 0.39 0.85 0.44 -
2.2 0.91 0.56 0.13
5.1 0.89 0.48 0.17
2.7 1.1 0.33 0.17
2.2 0.82 0.11 0.06
29
21
6.6
7.2
4.5
3.2
12 7.7 2.6
28
2.8 -
-
0.15
-
tr -
-
B. Transformation of OPEC starting compds: OPlEC OP2EC OP3EC OP4EC OP5EC OP6EC subtotal products : OP OPlEO total
0.34 9.7 8.1 4.6 2.1 0.73
9.8 7.8 2.8
8.7 6.6 1.9
-
-
-
7.9 5.1 1.5 I -
9.4 7.0 2.2 I -
-
-
8.6 6.2 2.2 I -
26
20
17
17
15
19
22
26
0.27 0.24 21
0.19 0.13 18
0.19 0.11 17
0.19 0.07 15
0.17 0.05 19
0.17 0.04 23
I
I
I
-
" -, not detected; tr, trace, detected at a concentration
