Biodegradation of Linear Alcohol Ethoxylates in Natural Waters Robert J. Larson* and Larry M. Games Environmental Safety Department, The Procter & Gamble Company, lvorydale Technical Center, Cincinnati, Ohio 4521 7
Kinetic and thermodynamic parameters for biodegradation of linear alcohol ethoxylates (LAE's), a class of nonionic surfactants used in detergent products, were measured in Ohio River water'(0RW). Biodegradation studies were conducted with two pure-chain-length LAE materials (ClzEg and C16E3) uniformly labeled with 14C in the ethoxylate chain or at the a carbon in the alkyl chain. At concentrations ranging from 1to 100 pg/L, degradation of both LAE's was first order with respect to concentration, and neither'.alkyl nor ethoxylate chain length had a significant effect on the rate or extent of LAE degradation. The kinetics of degradation of a longermixed-chain-length LAE, a C17E22 material, were also comparable to the kinetics for the shorter-chain-length LAE's. The effect of temperature on the rate of ethoxylate degradation was described by the Arrhenius equation, with freeenergy requirements typical of those expected for biological svstems.
Introduction Linear alcohol ethoxylates (LAE's) are mixed-chain-length nonionic surfactants of considerable commercial importance (1).The primary biodegradability of linear alcohol ethoxylates has been well established by colorimetric techniques (2-7). Linear alcohol ethoxylates have also been shown to undergo ultimate degradation in laboratory-scale wastewater treatment systems and in biodegradability screening studies (2, 7-10). However, there have been conflicting reports in the literature about the biodegradability of the ethoxylate portion of LAE's. Some investigators have reported that degradation of alkyl and ethoxylate chains occurs rapidly and is correlated with the loss of colorimetric response (11, 12). Others have indicated that loss of colorimetric response and degradation of the alkyl chain occur much more rapidly than degradation of the ethoxylate chain (13, 14). To provide additional perspective about the fate of LAE's in the environment, the present study was conducted to measure the ultimate biodegradability (mineralization) of LAE's a t ppb levels in Ohio River water. Previous studies have not addressed the ultimate biodegradability of LAE's in surface waters by naturally occurring river-water microorganisms, nor have they been conducted at environmentally relevant test concentrations. The objectives of our study were twofold: (1)to measure the rate and extent of mineralization (14C02evolution) of both alkyl and ethoxylate chains of LAE's and (2) to determine the effect of temperature on the rate of ethoxylate degradation.
Experimental Section
Chemicals. Two pure-chain-length linear alcohol ethoxylates, uniformly labeled with 14C in the ethoxylate chain or, separately, a t the a carbon in the alkyl chain, were used for the majority of biodegradation studies. Commercial nonionic surfactants are composed of mixtures of LAE's which range from c6 to cl6 in the alkyl chain and from E l to E20 in the ethoxylate chain ( I ) . The two materials studied, CH~(CHZ)~OCH~(~CHZCHZ)~OH (C12Ed and CH3( C H ~ ) ~ & H ~ ( O C H Z C H (C16E3) ~ ) ~ O Hcover the low to middle range of ethoxylate chain lengths and the middle to high range of alkyl chain lengths. was prepared by condensing The ethoxylate-labeled hexadecanol with [14C]ethyleneoxide in the presence of sodium metal with diglyme as a solvent. The alkyl-labeled 1488
Environmental Science & Technology
*C16E3 was prepared by reacting C1-labeled [14C]hexadecanol with a previously synthesized E3 ethoxylate chain. Purification was achieved by preparative TLC on silica gel with methyl ethyl ketone/water (95:5) followed by methyl ethyl ketone/ hexane (50:50) on a reverse phase. Final radiochemical and chemical purity was checked by gas chromatography with FID and radiochemical detection (Figure 1A) ( 1 5 ) .Gas chromatography (GC) was done on a P.E. 3920B which had been modified to split its effluent between an FID (20%) and an on-line radioactivity detector (80%). The radioactivity detector (RAD) consisted of a combustion furnace (810 "C, CuO) which converted the material to COz before counting in a 20-mL flow-through Geiger-Muller tube with propane quench gas added. Sensitivity was -1000 dpm. The gas chromatography was done on a 6-ft glass column (0.25-in. o.d., 2-mm i.d.) packed with SP-2100 on 8O/lOO mesh Supelcoport. Chromatographic conditions were as follows: injection port, 290 OC; FID, 325 "C; column held at 190 "C for 4 min and then programmed a t 8 "C/min to 290 "C. The two C12Eg labeled compounds were prepared in a manner similar to that for C&3 materials by using labeled and unlabeled dodecanol, an E9 ethoxylate chain, and [14C]ethylene oxide. Purity was determined by radio-TLC on silica gel (Figure 1B). The shoulder on the ethoxylate-labeled material is presumed to be C12*EB since, as Figure 1B indicates, synthesis of Clz ethoxylates leads to a complex mixture of materials, only a minor fraction of which is C12*E9. The final purity and specific activity for each of the four synthesized materials are given in Table I. T o study the biodegradability of LAE's with longer chain lengths, an unlabeled LAE (TAE22) was used in one study. The TAE22 material was a tallow alcohol condensed with an average of 22 mol of ethylene oxide. Elemental analysis of the TAE22 was consistent with a molecular formula of C18H370(CHzCHz0)2zH, and NMR spectra indicated that 21.5 ethoxylate groups were present if the alkyl chain was CIS. The range of ethoxylate groups for the unlabeled TAE22 was not established, but it was assumed to be broad with a maximum at E21.5Biodegradation Assays. River water for all biodegradation studies was collected from the Ohio River -1 mi below the discharge of a municipal wastewater treatment plant (Muddy Creek, Cincinnati, OH). Raw wastewater to the Muddy Creek plant was predominately domestic (daily flow = 8 X 106 gal), and dilution of the effluent upon discharge was -10 0OO:l. River water contained an average of 4 X lo5 colony-forming units/mL (CFU/mL) when assayed by tube dilution (100 mM phosphate buffer, pH 7.2) on nutrient agar spread plates. River water samples were collected in 1-gal plastic containers, immediately refrigerated (4 "C), and used within 48 h of collection. Samples were collected over the period June 1979July 1980,during which time water temperatures ranged from 0 to 24 "C. Suspended-solid levels were relatively constant in the water samples tested, averaging (fstandard deviation) 54 f 12 mg/L, and the pH ranged from 6.8 to 7.2. Biodegradation assays were conducted in a closed shakeflask system as described previously (16). Various concentrations of LAE's (1-100 pg/L, depending on specific activity) were added to flasks which were incubated a t 3,14,25, or 34 OC in constant-temperature rooms ( f 2 "C). At various intervals, 10-mL samples were collected and filtered through a 0.45-pm metricel filter (Gelman Instrument Co.) to remove particulate matter. The solids were washed once with an ad-
0013-936X/81/0915-1488$01.25/0
@ 1981 American Chemical Society
ditional5 mL of deionized water (18-Ma resistivity), and the 15-mL filtrate was added to 250-mL biometer flasks (17).The filtrate was acidified with 1mL of concentrated HC1 to release 14C02,and the 14C02was trapped in 2 mL of 1.5 N KOH in the biometer side arm. After 24 h, 10- and 1-mL aliquots were taken from the water and base fractions, respectively. Water and base fractions, and filters after air drying overnight, were
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counted by liquid scintillation techniques (16).Owing to the carbonate buffering capacity of ORW, most of the 14COZ produced was trapped in the river water and
