Environ. Sci. Technol. 1982, 16, 483-488
Fate and Distribution of a Quaternary Ammonium Surfactant, Octadecyltrimethylammonium Chloride (OTAC), in Wastewater Treatment Larry M. Games and James E. King
Packaged Soap & Detergent Division, The Procter & Gamble Company, Cincinnati, Ohio 45217 Robert J. Larson"
Environmental Safety Department, The Procter & Gamble Company, Cincinnati, Ohio 45217 The distribution and transformation of a quaternary ammonium surfactant, octadecyltrimethylammonium chloride (OTAC), were studied in laboratory-scale activated-sludge systems a t various initial concentrations. OTAC was removed and extensively biodegraded when tested at concentrations ranging from 0.1 to 20 mg/L. Quantitative adsorption data indicated that greater than 99% of OTAC was adsorbed to wastewater solids within 30 min of initial exposure. Biodegradation of sorbed OTAC was slower than adsorption and followed apparent first-order kinetics. The half-life for OTAC primary biodegradation was about 2.5 h, based on the first-order rate constant. Mineralization of 14C-labeledmethyl and C1alkyl carbons occurred at somewhat slower rates, with half-lives of 28 and 40 h, respectively.
Introduction Usage of quaternary ammonium compounds (QAC's), a class of commercially important chemicals commonly used in fabric softeners, cosmetics, shampoos, and a variety - of other products, has increased significantly in recent years. In 1978, over 86 X lo6 pounds of QAC's were sold in the United States, double the amount sold in 1971 (I). For many of the more common QAC's, entry to the environment is preceded by wastewater treatment. Consequently, several studies have focused on the treatability of QAC's in laboratory-scale wastewater treatment systems, by using nonspecific analytical techniques to monitor removal (2-4). While the techniques used in these studies were capable of distinguishing primary from ultimate biodegradation, they could not separate the amount of removal due to ultimate biodegradation (mineralization) from that due to adsorption. Difficulties in assessing the ultimate biodegradability of QAC's are mainly due to the adsorptive nature of these materials. QAC's strongly sorb to glass surfaces of test containers, to natural solids such as clay, to bacterial cell walls, and to humic materials (5-7). Therefore, it is difficult to distinguish removal due to adsorption from that due to biodegradation, a situation that may account for the wide range of biodegradabilities reported (0-100%) for structurally similar QAC's (5). In this report we describe the results of studies designed to determine the distribution and biodegradation of a model QAC, octadecyltrimethylammonium chloride (OTAC), in laboratory-scale wastewater treatment systems. Quantitative equilibrium adsorption data and rates of biodegradation were measured for radiolabeled materials by using techniques applicable to highly sorptive materials. Experimental Section Materials. Octadecyltrimethylammonium chloride (OTAC) synthesized in our laboratories had a purity of 91%, as measured by TLC and IR. The remaining impurities were other alkyl chain lengths (4%) plus water and 0013-936X/82/0916-0483$01.25/0
nonionic organic impurities. The linear alkylbenzenesulfonate (LAS) used in conjunction with biodegradability testing was a commercial c11.6LAS paste which was 42.5% LAS with the remainder water and sodium sulfate. The D-glucose used as a positive control in degradation studies was reagent grade with an assumed purity of 100%- All test concentrations were normalized for percent purity and were tested on a 100% active basis. Methyl-labeled [14C]OTACwas prepared by reaction of octadecyldimethylamine with [14C]CH31followed by ion exchange to form the chloride. Chemical and radiochemical purity were both greater than 98% as determined by TLC and HPLC. The remaining impurities were predominantly c16 and shorter chain length quaternaries, and the specific activity was 5.4 mCi/mmol. The [14C]alkyl OTAC was prepared from [14C]C1-octadecylbromideand trimethylamine. After conversion to the chloride, purity was checked by TLC and HPLC. Chemical purity of the alkyl-labeled OTAC and 95%, and radiochemical purity was 95%, with the remaining 5% impurity divided between cl6 and shorter chain length quaternaries. The specific activity was 4.3 mCi/mmol. The various test materials used are shown in Figure 1. Analyses. Radiolabel present in volatile (14C02)and nonvolatile fractions was quantitated after acidification (pH 2) by standard liquid scintillation techniques (8). Radioactivity measurements were made with an Isocap scintillation counter (Nuclear Instrument Co., Chicago, IL) with automatic quench correction. Enumeration of bacterial numbers was performed on nutrient agar spread plates as previously described (8). Two types of DBAS (disulfine blue active substance) analyses were used in biodegradation (SCAS) and adsorption studies. The DBAS procedure is one of a number of nonspecific dye complex methods that have been used previously to quantitate cationic surfactants (9). In the SCAS (semicontinuous activated sludge) studies (IO),the total colorimetric response for cationic surfactants was measured in settled effluent and activated-sludge mixed liquors. In the adsorption experiments, the standard DBAS procedure was followed with quantitative TLC to remove positive interferences (DBAS-TLC). Thin-layer chromatography was done on silica gel with chloroform/ methanol/water (75:25:3). Further details of our version of both methods are available (16) and will be published separately. OTAC was removed from solids in both types of DBAS analysis by extraction with methanolic HC1. Biodegradation Assays. Biodegradation and removal studies were conducted in both semicontinuous activated sludge (SCAS) and carbon dioxide evolution ((20,) test systems ( I O ) . In SCAS studies, activated sludge (3000 mg/L of total suspended solids) from a domestic wastewater-treatment facility was exposed to 20 mg/L of OTAC for several days. At 24-h intervals, sludge solids were allowed to settle, the settled effluent was removed, and fresh nutrient and OTAC were added. The settled effluent
0 1982 American Chemical Society
Environ. Sci. Technol., Vol. 16, No. 8, 1982
483
A
,
S 0 3 r Nas
C, LAS . The 1 1 6 refers to the average alkyl chain length All chainlengths from C,, to C,, are present as are the various phenyl isomers of each
where y = evolved 14C02(% of theoretical), z = time, a = upper asymptote (% of theoretical C02),k = rate constant (time-l), and c = lag time (time). Exponential decay model y = So
for z Ic
+
y = (So- a)(e-k(x-c)) a Octadecyltrimethyl ammonium chloride Methyl l 4 C label.
CH3 CIC
I
CH3-(CH,)j,-Ns-CH3
I
* CH3
CH,
Cl's
Octadecyltrimethyl ammonium chloride . C, alkyl l4C label.
CH3 Flgure 1. Structures of the test materials and the position of the "C label In the two OTAC materials tested.
was analyzed for dissolved organic carbon (DOC) and compared with unexposed controls to determine removal. Carbon analyses were performed with a Beckman 915 carbon analyzer equipped with an 865 nondispersive infrared analyzer. In addition to DOC measurements, DBAS measurements were also made on settled effluent and mixed-liquor suspended solids to determine primary removal and primary biodegradation, respectively. The DBAS levels in test SCAS units were corrected for background via suitable controls. In the C02 studies, influent wastewater was used as the microbial inoculum. The wastewater was allowed to settle for 30 min, and 5% by volume of the settled supernatant was added to Erlenmeyer flasks containing test material (10 or 20 mg/L) as the sole added carbon and energy source. Two concentrations were tested to verify the kinetics of biodegradation. At the lowest concentration tested (10 mg/L) COz production from test units was approximately 5 times higher than that in inoculated controls containing no test material. Flasks were aerated with COz-free air, and the rate and extent of COP evolution were measured for 25 days. So that the fate of the quaternary nitrogen could be determined, biodegradation of two radiolabeled materials, methyl- and CI-alkyl-labeled OTAC, was measured in a modified SCAS system. The suspended solids level was decreased 3-fold to 1000 mg/L, and the exposure period was increased to 172 h. The activated sludge was not acclimated to OTAC in the laboratory prior to testing, and radiolabel was quantitated in three fractions: as 14C02,in solution as nonvolatile carbon, and in biomass. Statistical Analysis of Data. All biodegradation data were analyzed by nonlinear regression models to estimate the rate constants for and extent of ultimate biodegradation. The models, which were used to analyze both C02 production data (eq 1)or 14Cremoval data (eq 2), are given as follows: Exponential product formation model y=O
y = a(1 484
forxIc for x
>c
Environ. Scl. Technol., Vol. 16, No. 8, 1982
(1)
for z > c
(2)
where y = 14Cactivity remaining in solution (% of initial), z = time, So = initial concentration (% of nominal), a = lower asymptote (% of initial concentration), k = rate constant (time-I), and c = lag time (time). Adsorption. The rate and extent of adsorption of OTAC to wastewater solids was measured by using [14C]alkyl-labeledOTAC. Raw wastewater and activated-sludge samples were collected from a domestic wastewater-treatment plant and tested within 24 h of collection at ambient laboratory temperatures. The activated sludge was treated with 30 mg/L of mercuric chloride to prevent biodegradation of the test material. Adsorption to glassware was negligible in studies in which solids were present. Mixtures of test chemical and wastewater were placed in 500-mL Erlenmeyer flasks and then agitated to maintain a uniform suspension of solids. At various intervals, aliquots were removed and centrifuged for 30 min at 39 OOOg. Radioactivity in the supernatant was counted directly by liquid scintillation. Radioactivity'or OTAC on the solids were determined in one of three ways. In preliminary experiments, the amount of OTAC present on the solids was calculated by subtracting the radioactivity in solution from the total amount added. In later experiments, the solids were combusted and the evolved 14C02was trapped and used to calculate the amount of OTAC present. Combustion was carried out at 800 OC in a tube furnace containing CuO/Cu catalyst, and the 14C02evolved was collected in a 50-mL glass trap containing 1:7 monoethanolamine/ethylene glycol monomethyl ether. Finally, in a few experiments, the solids were also analyzed directly for OTAC by using the DBAS-TLC method. Adsorption Coefficient. Adsorption results are reported as K d , the solid/solution partition coefficient,which is defined by eq 3 Kd
=
Caolids/ Csolution
(3)
where Cs0~,= pg/g on the solids and Cso~ution = pg/mL in solution. Its applicability in our studies was confirmed by varying solute concentrations over a 1000-fold Concentration range and finding that K d remained constant.
Results and Riscussion C 0 2Tests. Data from the C02-production tests using dilute (5%), settled raw wastewater as the microbial inoculum are given in Table I. When OTAC was tested at 20 mg/L, no C02 evolution was observed over the 25-day test period since OTAC was toxic to the test microorganisms at this concentration. Endogenous C02 production was inhibited (negative value in Table I), and only one bacterial strain from the original mixed inoculum survived OTAC exposure for the 25-day test period. The strain isolated, a gram-negative rod that formed smooth, round, white colonies on nutrient agar, was tentatively identified as a pseudomonad. These results are similar to those of Mackrell and Walker (II), who found that pseudomonads capable of tolerating, but not degrading, hexadecyltrimethylammonium bromide (HTAB) were selected when mixed bacterial cultures were exposed to HTAB at concentrations up to 1 mM.
S0=19.2mg/L a=3.6mg/L k:: 0.28/h~ c=O hr
Table I. CO, Production during Degradation of OTAC and LAS as Sole Carbon and Energy Sources obsd est concn, CO,, CO rate const k, test material mg/L %a %bz' days-'
20 -24.1 d 20 72.8 79.1 0.13 (0.10-0.16) 5 + 5e 78.3 82.1 0.18 (0.14-0.21) 10 + 10 81.3 83.2 0.19 (0.17-0.22) 20 + 20 80.5 80.7 0.20 (0.18-0.23) 20 92.2 88.9 0.40 (0.31-0.49) Amount of CO, evolved after 25 days relative to the theoretical amount possible based on molecular structure. The a constant estimated by eq 1. The k constant estimated by eq 1;values in parentheses are the 95% confidence intervals of the parameter estimates. Could not be determined due t o toxicity; initial number of bacteria added was 2.8 x lo5 CFU mL-'. e Equal weight is essentially equal molar.
OTAC LAS OTAC t LAS OTAC + LAS OTAC t LAS D-glUCOSe
I l
0
0
IC*---,---------------------*
Table 11. Adsorption of OTAC to Activated-Sludge Solids Treated with 30 mg/L of Mercuric Chloride OTAC concn on spiked concn solids solids, (14C), level, pg/g of dry OTAC concn mg/L mg/L solidsa in soln, mg/L 10-4Kd
3.5 2.8 X 1.8 1895 0.0068 40.1 2 x 10-3 2.0 1895 0.078 430 1.8 X lo-' 2.4 1895 0.84 1140 2.3 X lo-' 4.9 1.1 948 4380 1.8X lo-' 2.4 1895 8.5 a Solids were combusted to measure the 14C present. Concentration in solution remained constant for 24 h with no CO, formation.
.. 0
= A
b
4.0
8.0
i2.0 i6.0 TIME (days)
120.0
k.o
I
280
Figure 2. Kinetics of COP evolution during biodegradation of 20 mg/L of glucose (U),20 mg/L of OTAC 20 mg/L of LAS (A),and 20 mg/L of LAS (e), Blodegradabllity data have been analyzed by eq 1 and parameter estimates are given In Table I. The dotted contours represent the 95% confidence Intervals of the true mean.
+
Owing to their net positive charge, it has been suggested that quaternary ammonium surfactants in wastewater tend to complex with anionic surfactants such as LAS which are present (3, 11). This complexation capability was investigated in our study to determine if the microbial toxicity of OTAC could be mitigated, thereby allowing biodegradation to occur. b u d weights of OTAC and LAS (1:l molar ratio) were tested at various concentrations, and the amount of COz evolved from the mixtures was measured for 25 days. The results are shown in Table I and Figure 2. COz evolution from several OTAC/LAS mixtures was comparable to that observed for LAS alone (Table I). After a 2-3-day lag period the rate of COz evolution for the mixture was actually somewhat higher than the rate for LAS degradation (Figure 2), approaching the rate of degradation of the glucose control. The lag periods for LAS and OTAC degradation, which are typically observed in the COz test system (IO),indicate that the number of microorganisms capable of degrading these materials are initially rate-limiting at the substrate concentrations tested. However, after an increase in degradation capability due to enzyme synthesis and/or microbial growth, degradation of the OTAC/LAS mixture followed apparent first-order kinetics and was directly proportional to sur-
factant concentration over the range tested. These results indicate that OTAC is rapidly degraded in the presence of LAS when microbial toxicity is mitigated. SCAS Tests. During seven consecutive 24-h test periods, removal of 20 mg/L of OTAC in SCAS test units exceeded 99%, based on DOC analyses of centrifuged effluents. Kinetic studies, using DBAS analyses to determine OTAC distribution between solution and solids phases, indicated that OTAC removal in SCAS tests was a function of both adsorption and biodegradation. Adsorption was the more rapid of the two processes, with greater than 98% of the DBAS reactive material being removed from solution after 90 min of incubation (Figure 3). Biodegradation of OTAC on sludge solids also occurred fairly rapidly, however, following an exponential decay function with a first-order rate constant of 0.28/h (Figure 3). The half-life for OTAC in the SCAS system, based on the first-order biodegradation rate constant, was about 2.5 h. This rate of biodegradation is rapid enough to support extensive biodegradation of sorbed OTAC by activated sludge, since the solids retention time in conventional activated-sludge treatment plants is typically several days (12). Adsorption. The results of the adsorption tests in activated sludge and in raw wastewater using [14C]OTAC are shown in Tables I1 and 111. The Ka's shown are equilibrium adsorption coefficients, and kinetic studies consistently showed that equilibrium was reached within 30 min in both matrices. This rapid attainment of equilibrium was consistent with the results of the previously mentioned biodegradation studies (Figure 3). In adsorption studies with varying OTAC concentrations, Kd values Environ. Sci. Technol., Vol. 16, No. 8, 1982 485
Table 111. Adsorption of OTAC in Raw Wastewater OTAC concn wastewater on solids ('*C), OTAC concn OTAC initial OTAC solids concn, pg/g of in soln ( W ) , remaining, concn, mg/L mg/L dry solids mg/L total % 10-3~~ Aa 0.01 155 ND 4.5 x 10-3 100 5.6 B 0.1 155 ND 6.9 x 10-3 100 3.0 C 0.1 74 ND 4.7 x 10-3 100 8.9 D 0.1 105 ND 5.8 x 10-3 100 7.1 E 1.0 155 ND 7.0 X lo-' 100 2.8 F 1.0 74 ND 5.7 x lo-' 100 9.0 G 1.0 105 ND 5.5 x lo-' 100 8.6 Hb 0.01 65 15 3.0 x 10-3 40' 5.0 I 0.1 73 170 4.1 X lo-* 53 4.2 J 1.0 47 2412 5.1 X 10" 63 4.9 a For A-G, final K d ' s are averages of 0-6-h incubation time data; concentration on solids was calculated by subtracting radioactivity in solution from total amount added, 100% is considered remaining because the concentration on the solids was calculated by difference. For H-J,incubation time was 96 h. This is obtained by adding the total OTAC in solution and on solids and comparing this total to the OTAC added initially.
remained essentially constant over a broad concentration range, much broader than expected for typical wastewater treatment plants (Table 11). This indicates that a simple partition coefficient can be used to accurately describe OTAC adsorption to wastewater solids. During the adsorption studies using raw wastewater solids, evolution of 14C02from [14C]0TACwas observed in raw wastewater after 8 h. In most testa (Table 111,A-G), the Kd's were calculated during the 6-h period before biodegradation began by averaging 3-4 measurements of radioactivity in solution. However, several longer experiments using raw wastewater were used to quantify the distribution of OTAC during biodegradation (Table 111, H-J). In H-J,Kd was determined by measuring radioactivity both in the solution and on the solids. These K
