Environ. Sci. Technol. 1902, 16, 433-436
Mineralization of Linear Alcohol Ethoxylates and Linear Alcohol Ethoxy Sulfates at Trace Concentrations in Estuarine Water Robert D. Vashon" and Burney S. Schwab
Environmental Safety Department, The Procter & Gamble Company, Ivorydale Technical Center, Cincinnati, Ohio 45217 with a previously synthesized ethoxylate chain. Purity was determined by radio TLC on silica gel and gas chromatography with FID and radiochemical detection. *C16E3 had a specific activity of 2.8 pCi/rng-l and a radiochemical purity >98%. C12*Eghad a specific activity of 3.78 pCi/mg-l and a radiochemical purity of 70-8070,with C12*E, as the major contaminant. Further details on the synthesis and purification of these compounds can be found elsewhere (12). Sulfation of LAE to form LAES was accomplished by reacting LAE with C1S03H. Following cleanup, the *C16EgShad a specific activity of 1.4 pCi/mg and a radiochemical purity of >95%. C16*E3Swas 85% radiochemically pure and had a specific activity of 2.2 pCi/mg. Biodegradation Assays. Estuarine water for biodegradation assays was collected from Escambia Bay, Florida and shipped to our laboratory in Cincinnati, Ohio. Samples of Escambia Bay water (EBW) vary in salinity from 22%0to 33%0depending on the relative contributions from the Escambia River and from Pensacola Bay. This proportion varies with tidal stage and river flow Introduction volume. The sample used in this work had a salinity of Primary biodegradation of linear alcohol ethoxylates 28%0.Analyses showed the concentration of total organic (LAE), a class of nonionic surfactants, has been demoncarbon to be less than 1mg/L, of total nitrogen to be less strated in both fresh water and sea water (1). Mineralithan 0.5 mg/L, and of total phosphate to be less than 20 zation (ultimate biodegradation) of the carbon in these pg/L. Asays began within 48 h of collection. EBW concompounds to C02 by bacteria in synthetic media occurs tained 5 X lo4 colony forming units/mL able to grow on as well (2,3).While both the ethoxylate and alkyl moieties nutrient agar of the same salinity as the water. are biodegradable, there is evidence that the rate of ethUltimate biodegradation was monitored by measureoxylate degradation is somewhat slower (4,5). The anionic ment of the 14C02produced from the 14C-labeledcomsurfactants, linear alcohol ethoxy sulfates (LAES), have pound. Test compounds were added to 200 mL of EBW been shown to degrade in laboratory media (6,7). Ultimate at the concentrations shown in Table I to give 5,50, and biodegradation of LAE or LAES in estuarine water by 500 dpm/mL in stoppered 500-mL flasks. Periodically, indigenous microorganisms has not been reported. the contents of the flasks were acidified (> S. The rate and extent of mineralization (ultimate biodegradation) of linear alcohol ethoxylates (LAE), a class of nonionic surfactants, and of linear alcohol ethoxy sulfates (LAES),a class of anionic surfactants, were measured in water from Escambia Bay, FL (EBW). Results indicate that mineralization of LAE and LAES trace concentrations in estuarine water is rapid and extensive. Studies were conducted on four pure chain length materials (*C16E3, C12*E9,C16E9S,C16*E3S)labeled with 14C either at the a-alkyl carbon or uniformly in the ethoxylate chain. The sulfate moiety had no effect on the rate or extent of mineralization of either the alkyl or the ethoxylate chains of LAES. Kinetics of mineralization of a-alkyl carbon were exponential (first order) over a concentration range 850 ng/L to 140 pg/L with a half-life for a-alkyl carbon of 2.1 days. Kinetics of mineralization of ethoxylate carbon were exponential at an initial concentration of 1 pg L or less of LAE or LAES, with a half-life of 6.3 days, ut linear or sigmoidal at higher concentrations.
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0013-936X/82/09 16-0433$01.25/0
0 1982 American Chemical Society
Environ. Sci. Technol., Vol. 16, No. 7, 1982 433
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Flgure 1. Kinetics of I4C loss during biodegradation of glucose. P [U-14C]glucose was added to EBW. Periodically, radioactivity of the solution was assayed by LSC. Initial concentrations of glucose were 10 mg/L (O), 1 mg/L (A),100 pg/L (A),10 pg/L (O),and 1 pg/L (0).
The models are not equivalent. For enzymatic activity, R,,/K, has the dimension of time-l and is analogous to a first-order rate constant, implying that R is proportional to S. For microbial growth, R,,/K, has the dimensions of concentration-l X time-l, analogous to a second-order rate constant. (It is the rate of change of the rate of growth that is proportional to substrate concentration.) These models imply that in the presence of sufficient enzymatic activity the rate of biodegradation should be first order with respect to substrate concentration at concentrations below those supporting microbial growth and mixed order at concentrations that support microbial growth (and a net increase in enzymatic activity). They further imply that when S >> K , for growth ( R = R,,), the biodegradation rate is constant (zero order). Examples of first-order (exponential), mixed-order (sigmoidal), and zero-order (linear) kinetics are given in the present study. Where first-order kinetics were observed, the data were analyzed by nonlinear regression to an exponential model (14). Values in Table I were derived from this analysis. No attempt was made to fit obviously nonexponential data to a model or to differentiate linear from sigmoidal biodegradation curves.
Results and Discussion Glucose Mineralization. In preliminary experiments with [U-14C]glucose(Amersham) designed to measure the microbioal activity in EBW, we found that first-order &e., exponential) kinetics for loss of [14C]glucosefrom solution occurred at initial concentrations ranging from 1 to 100 pg/L (Figure 1). At concentrations 1 mg/L and above, degradation kinetics were linear (i.e., zero order with re434
Environ. Sci. Technol., Vol. 16, No. 7, 1982
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Flgure 2. Kinetics of CO, evolution during biodegradation of *C,,E, a-alkyl carbon in EBW. Initial concentrations of LAE were 68 pg/L (O), 6.6 pg/L (A),and 0.85 pg/L (M).
spect to substrate). This activity is low compared to laboratory media inoculated with wastewater-treatment-plant bacteria or river water, where glucose degradation is exponential at 20 mg/L (14). Furthermore, the lack of any increase in biodegradation rates over the course of the experiment indicates that growth was not carbon limited. It was important, therefore, to measure biodegradation at concentrations well below the saturation level for LAEor LAES-degrading activity in EBW in order to predict the half-life of these compounds in the estuarine environment. LAE Mineralization. Rapid mineralization of the a-alkyl carbon of *C16E3LAE occurred in Escambia Bay water (EBW) (Figure 2). The kinetics of 14C02evolution were first order with respect to concentration. There were no significant differences among the rate constants for biodegradation at the initial concentrations tested (Table I). Based on the average rate constant for these three experiments, the half-life for ultimate biodegradation of the alkyl-chain carbon in EBW was 2.3 days. The kinetics of ethoxylate-chain mineralization were more complex (Figure 3). A t an initial concentration of 420 ng/L, 14C02evolution with time was exponential (first order). However, at 3.9 pg/L the rate of mineralization increased with time until 30% of the theoretical 14C02had been recovered and then decreased through the 30th day of the experiment. When the initial concentration was 31.2 pg/L, biodegradation was linear through day 30. These results indicate first that the available enzymatic activity is approaching saturation at 3.9 pg/L. Second, the increasing rate of degradation indicates an initial increase
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Table I. Rate and Extent of Mineralization of LAE and LAES concn compd (label position) *C,,E, (C,-alkyl) *C 16E3(C ,-a1ky 1 ) *C ,,E, (C,-alkyl) C,,*E, (U-ethoxylate) C,,*E, (U-ethoxylate) C,,*E, (U-ethoxylate) *C,,E,S ((2,-alkyl) *C,,E,S (C,-alkyl) *C,,E,S ((2,-alkyl) C,,*E,S (U-ethoxylate) C,,*E,S (U-ethoxylate) C,,*E,S (U-ethoxylate)
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rate constant, days‘’ t 1 sd
asymptote, % TCO,
182 17.5 2.3 53 6.7 0.79 199 19.3 2.0 266 25.9 3.20
68 6.6 0.850 31.2 3.9 0.420 140 13.6 1.43 113 10.9 1.21
0.22 ? 0 . 0 7 0.34 * 0.12 0.33 * 0.11 lineara linear 0.12 r 0.03 0.39 f 0.04 0.31 r 0.03 0.32 f 0.04 linear linear 0.10 f 0.02
86.3 87.2 79.3 82.4 82.4 96.7 92.4 74.5
a Rate constants were not evaluated for nonexponential curves. “Linear” is meant t o include those curves that may in fact be sigmoidal.
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Figure 3. Kinetics of CO, evolution during biodegradation of CI2*Es ethoxylate carbon in EBW. Initial concentrations of LAE were 31.2 pg/L (O),3.9 pg/L (A),and 0.42 pg/L (a).
in the enzymatic activity through growth or induction. Finally, the capacity to increase this activity is limited, as degradation at 31.2 pg/L is close to zero order. The rate of biodegradation in estuaries is known to be limited by the low concentration of available nitrogen (15). Based on the first-order rate constant for biodegradation at an environmentally relevant concentration (420 ng/L), the half-life for LAE ethoxylate carbon in EBW was 5.8 days. Recovery of 14C02from both the e;thoxylate-labeled and the a-alkyl-labeled LAE was extensive ( 4 0 % theoretical I4CO2). Little carbon from these compounds was assimilated, therefore, by the microorganisms responsible for biodegradation of either moiety. This low ratio of assi-
Time (days)
Figure 4. Kinetics of C 0 2 evolution during biodegradation of *C,BEsS a-alkyl carbon in EBW. Initial concentrations of LAES were 140 pg/L (01, 13.6 pg/L (A),and 1.43 pg/L (a).
milative to respiratory catabolism of LAE has been shown to occur in fresh water as well (12). Apparently the qinimal energy requirement for cell maintenance is higher relative to the total energy available from sources at trace concentrations than from those in typical bacterial media. This was true for glucose in our preliminary experiments as well. The high rate of a-alkyl mineralization compared to ethoxylate chain mineralization supports the theory that hydrolysis of the ether linkage occurs prior to further degradation (16).That the pathways for biodegradation of these chains diverge after they are cleaved is further indicated by the lower concentration of LAE needed to Environ. Sci. Technol., Voi. 16, No. 7, 1982
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Conclusion Alkyl- and ethoxylate-chain carbon of LAE is rapidly and extensively mineralized a t trace concentrations in estuarine water. The sulfate moiety of LAES has no effect on the mineralization of either alkyl or ethoxylate carbon. The relatively higher mineralization rate of a-alkyl carbon is consistent with hydrolysis of the ether linkage between alkyl and ethoxylate chains prior to mineralization of either chain. The rate of mineralization of LAE or LAES a t concentrations below saturation for biodegradation activity and microbial growth is first order with respect to concentration. There is no evidence for a concentration threshold below which these compounds will not degrade, a t initial concentrations as low as 7.9 X M.
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at the ether linkage of these compounds is the first step of the degradative pathway. However, the similarity in biodegradation of ethoxylate chains from LAE and LAES indicates either that the sulfate group is removed first or that the sulfate group simply does not affect the mechanism of ethoxylate chain degradation. The former explanation is most likely in view of the published mechanism of poly(ethy1ene glycol) degradation, which shows that both ends of this compound are degraded at the same time (I 7). If the sulfate group was not removed prior to degradation, one would expect ethoxylate chains from LAE to be mineralized at a somewhat higher rate than those from LAES.
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Flgure 5. Kinetics of COPevolution during biodegradation of C,B*E,S ethoxylate carbon in EBW. Initial concentration of LAES were 113 pg/L (O), 10.9 pg/L (A),and 1.21 pg/L (U).
saturate the ethoxylate-degrading activity than the alkyl-degrading activity. Effect of 050,. The presence of sulfate had virtually no effect on the rate of extent of mineralization of the alkyl or ethoxylate chains of LAES. The rate of 14C02evolution from * C 1 M LAES was proportional to concentration over an inital concentration of 1.43-140 pg/L (Figure 4). The half-life for a-alkyl carbon in EBW was 2.1 days based on the average rate constant in these three experiments. Mineralization at the ethoxylate chain from CI6*E3SLAES is remarkably similar to that of the ethoxylate chain from CI2*E, LAE (Figures 2 and 5). There is no significant difference in the rate constants for mineralization of these compounds a t the lowest concentration tested, and ethoxylate-degrading activity was saturated at comparable concentrations. Thus, neither chain length (within the limits studied here) nor the presence of OSO, affected the mineralization of ethoxylate chains in EBW. It is not surprising that the alkyl chains from LAE and LAES degrade at the same rate, particularly if hydrolysis
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(1) Schoberl, P.; Mann, H. Arch. Fisch. Wiss. 1976,29,149-158. (2) Strum, R. N. J. Am. Oil Chem. SOC.1973, 50, 154-167. (3) Larson, R. J. App. Environ. Microbiol. 1979,38,1153-1161. (4) Tobin, R. S.; Onuska, F. I.; Brownlee, B. G.; Anthony, D. H. J.; Comba, M. E. Water Res. 1976, 10, 529. ( 5 ) Tobin, R. S.; Onuska, F. I.; Anthony, D. H. J.; Comba, M. E. Ambio 1976, 5, 30. (6) Itoh, S.; Setsuda, S.; Utsunomiya, A.; Naito, S. Yukagaku 1979,29, 199-204. (7) Muira, K.; Yamanaka, K.; Sangai, T.; Yoshimura, K.; Hayashi, N. Yukagu 1979,28, 351-355. (8) Maki, A. W. Proc. 14, Znt. Marine Biol. Symp., in press. (9) Boethling, R. S.;Alexander, M. Environ. Sci. Technol. 1979, 13,989-991. (10)Alexander, M. ASM News 1980,49, 35-38. (11) Michaelis, L.; Menten, M. L. Biochem. 2.1913,49,333-369. (12) Larson, R. J.; Games, L. M. Environ. Sci. Technol. 1981, 15, 1488-1492. (13) Monod, J. Ann. Rev. Microbiol. 1949, 3, 371-394. (14) Larson, R. J. App. Environ. Microbiol. 1979,38,1153-1161. (15) Atlas, R. M.; Bartha, R. Biotech. Bioeng. 1972,14,309-317. (16) Patterson, S. J.; Scott, C. C.; Tucker, K. B. E. J . Am. Oil Chem. SOC.1970,47, 37-41. (17) Kravetz, L. J. Am. Oil Chem. SOC.1981, 58, 58A-65A. (18) Kawai, F.; Kimura, T.; Fukaya, M.; Tani, Y.; Koichi, 0.; Ueno, T.; Fukami, T. Appl. Environ. Microbiol. 1978,35, 679-684.
Received for review October 6, 1981. Accepted March 30, 1982.
