biodegradation of coproducts of commercial linear alkylbenzene sulfonate

Biodegradation of Coproducts ofCommercial Linear AlkylbenzeneSulfonateA L L E N M . N I E L S E N , * L A R R Y N . B R I T T O N ,C H A R L E S E . B E A L L ,T I M O T H Y P . M C C O R M I C K , A N DG E O F F R E Y L . R U S S E L L

Research and Development Department, CONDEA VistaCompany, 12024 Vista Parke Dr., Austin, Texas 78726-4050

Dialkyltetralin sulfonate (DATS) and single methyl-branchedisomers of linear alkylbenzene sulfonate (iso-LAS) arecoproducts that together can range from 1 to 10% of com-mercial LAS depending on the manufacturing process.Biodegradation studies using radiolabeled DATS and iso-LAS showed mineralization by indigenous microbialpopulations in laboratory simulations of aquatic and soilenvironments. Half-lives ranged from 2 to 20 days, which israpid enough to suggest that accumulation would notoccur in these environments. Upon exposure to laboratoryactivated sludge treatment, most model iso-LAS compoundsshowed greater than 98% parent compound removal,extensive mineralization (>50%), and 79-90% ultimatebiodegradation (mineralization plus conversion to biomass).Activated sludge treatment of DATS and one of the iso-LAS isomers (methyl group attached to the benzylic carbonof the alkyl chain) resulted in >98% removal, 3-12%ultimate biodegradation and apparent formation of carboxyl-ated biodegradation intermediates that accounted for 88-97% of the original material. These DATS and iso-LASbiodegradation intermediates continued to mineralize insimulated receiving water and soil environments at ratessimilar to that of sulfophenyl carboxylate (SPC) intermediatesof a standard LAS.

IntroductionCommercial linear alkylbenzene sulfonate (LAS) is a widelyused synthetic surfactant in detergents and householdcleaning products. For example, consumption of LAS inEurope, North America, and Japan was approximately 950 000metric tons in 1994 (1). Numerous studies on the environ-mental fate and effects of LAS have been published (2).Extensive field monitoring studies in Europe and NorthAmerica as well as evaluations by several governmentalagencies have been completed (3-6). These studies confirmearlier conclusions that LAS is completely biodegradable;environmental concentrations of LAS are low, and the use ofLAS at current levels is protective of biological populationsin receiving environments (2, 7).

LAS is often represented as a linear alkyl chain attachedto a sulfonated benzene ring, as illustrated in Figure 1.Commercial LAS, however, is a blend of LAS molecules thatvary in terms of alkyl chain length, position of the benzenering along the alkyl chain, and concentrations of coproductscalled dialkyltetralin sulfonates and iso-LAS.

Dialkyltetralin sulfonates (DATS) and LAS with singlemethyl branching on the alkyl chains (iso-LAS) are minorcomponents in commercial LAS. Concentrations range from<1 to 8% for DATS and <l to 6% for iso-LAS depending onthe manufacturing process used. Because of the widespreadand high volume use of commercial LAS, significant quantitiesof LAS coproducts reach the environment. Consequently,several workers developed appropriate analytical methodsand monitored DATS and DATS biodegradation intermediates(DATSI). DiCorcia et al. (8) detected and Trehy et al. (9, 10)measured DATS and DATSI upstream and downstream ofdomestic wastewater treatment plants. Field and co-workers(11, 12) conducted monitoring of these compounds in asewage contaminated groundwater, and Tabor and Barber(13) found DATS in some bottom sediments of the MississippiRiver. Cavalli et al. (14) demonstrated the complete biode-gradability of model iso-LAS compounds in OECD screeningand continuous flow activated sludge tests. Recently, Kol-bener and his colleagues (15-17) have investigated the solubleorganic carbon fraction which remained after biologicaltreatment of commercial LAS in a laboratory, flow-through,test system using immobilized activated sludge. Theyconcluded that this fraction was “refractory” and wascomposed primarily of biodegradation intermediates of DATSand iso-LAS. In order to better understand the fate ofcommercial LAS coproducts in the environment, 14C-ring-labeled DATS and iso-LAS model compounds were synthe-sized and exposed to simulated activated sludge, soil, andreceiving water environments. Furthermore, the effluentscoming from activated sludge treatment, which containedbiodegradation intermediates, were exposed to simulatedreceiving water environments, and the fates of the radiola-beled chemicals were measured.

Materials and Methods14C-Radiolabeled Compounds. 14C-uniformly-labeled D-glucose (98% radiochemical purity) was purchased from NewEngland Nuclear, Boston, MA. [14C]benzene ring-labeled C12

linear alkylbenzene sulfonate (LAS) was synthesized by NewEngland Nuclear and was shown by autoradiography of thinlayer chromatograms (TLC) to be 97.5% radiochemically pure.The Procter and Gamble Company provided the following[14C]benzene ring-labeled single methyl branched alkylben-zene sulfonates (Figure 1): iso-LAS type IA (sodium 5-methyl-2-undecyl [14C]benzenesulfonate), iso-LAS type IB (sodium10-methyl-2-undecyl [14C]benzenesulfonate), iso-LAS type IIA(sodium 2-methyl-2-undecyl [14C]benzenesulfonate), and iso-LAS type IIB (sodium 5-methyl-5-undecyl [14C]benzene-sulfonate). The radiochemical purities of the iso-LAS com-pounds were determined by high-performance liquidchromatography (HPLC)/radiochemcical analysis. Puritieswere iso-LAS type IA, 97.8%; iso-LAS type IB, 77.6%; iso-LAStype IIA, 94.7%; and iso-LAS type IIB, 97.5%. Two [14C]benzenering-labeled C8 DATS samples were synthesized and used inthis study (Figure 1). Huntsman Corporation, Houston, TX,provided the first sample which was shown by HPLC/radiochemcial analysis to be 97.3% chemically pure and 92.7%radiochemically pure. The second sample was synthesizedby Wizard Laboratories, Davis, CA, under the direction ofCONDEA Vista Company. The radiolabeled dialkyltetralin(DAT) sample was analyzed at 98.9% chemical purity by gaschromatography (GC) and GC-mass spectrometry (MS).Following sulfonation, the radiolabled DAT sulfonic acid wasshown by TLC autoradiography to be 99.6% radiochemicallypure. The DAT sulfonic acid was neutralized before use.

Nonradioactive Compounds. CONDEA Vista Companysynthesized a C12-LAB which was determined by GC analysis

* Corresponding author telephone: 512-331-2461; fax: 512-331-2387.

Environ. Sci. Technol. 1997, 31, 3397-3404

S0013-936X(97)00023-0 CCC: $14.00 1997 American Chemical Society VOL. 31, NO. 12, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3397

to be >98% chemically pure. It was sulfonated and neutral-ized before use. A type I C12 iso-LAS (i.e., methyl branchingon the nonbenzylic carbon atoms at various positions on thealiphatic chain) was prepared by the alkylation of benzenewith an iso-alcohol in an excess of AlCl3. This iso-alcohol wasa mixture with hydroxyl groups alpha to methyl groups andwith each vicinal OH and CH3 pair at different positions alongthe carbon chain. In this process, some benzylic methyl-branched iso-LABs (type II) are inevitably formed. Analysisby GC, GC-MS, HPLC, and 1H and 13C nuclear magneticresonance (NMR) showed that the majority of the sample(70.5%) was type I iso-LAB; approximately one-fourth (23.3%)was iso-LAB type II and 6.2% was DAT. It was referred to asthe type I iso-LAS following sulfonation and neutralization.A second iso-LAB was synthesized and analyzed (GC and GC-MS) by CONDEA Vista Company. Since it was shown tocontain 90.0% type II iso-LABs, 5.6% C12-LAB, and a 4.4%mixture of DAT and indanes, it was referred to as the type IIiso-LAS sample after sulfonation and neutralization. A C8

DAT sample, 89.9% chemical purity by GC analysis, was alsosynthesized by CONDEA Vista Co. and designated as the C8

DATS sample after sulfonation and neutralization.

Sample Collection/Handling and Test System Setup.Surface soils were collected at the following locations: (1)“pristine” soil from McKinney Falls State Park, Austin, TX; (2)sludge-amended soil from a cornfield adjacent to the HornsbyBend Wastewater Treatment Plant, Austin, TX; and (3) graywater contaminated soil from the top of a percolation bedthat receives surface applications of laundry water from alaundromat at Summit Lake, WI (18). The samples werecollected, sealed in “zipper”-seal, plastic bags, stored at 4 °C,and used within 20 days of collection.

Samples were screened through no. 4 (4.7 mm opening)and no. 14 (1.4 mm opening) sieves to remove vegetation,rocks, and debris. The moisture content was maintainedbetween 59 and 94% of water field capacity, and samples (90g) were mixed with 50% by volume perlite to promote aerationand to minimize clumping and compaction.

Each soil sample was mixed thoroughly with 10 mL of amineral salts medium (19) containing the test surfactant andthen placed in a 500 mL Gledhill flask (Ace Glass Inc., Vineland,NJ). Internal CO2 traps received 5 mL of 0.5 N KOH with0.0002% thymolphthalein pH indicator. Units were sealed,flushed with 70% O2/30% N2 for 3 min at one L/min, andincubated at 23-25 °C. The KOH trapping solution wasperiodically replaced, and trapped 14CO2 determined by liquidscintillation counting (LSC) in Ultima Gold XR scintillationfluid, Packard Instruments, Meriden, CT, in a PackardInstruments Model 2550 TR analyzer.

Sediment samples were collected from the upper inch ofa small stream (Lake Creek, Austin, TX), which receivedeffluent from a domestic wastewater treatment plant. Sedi-ments were transported and held in plastic buckets at 4 °Cand were used within 24 h of collection. Sediment samples(50 or 98 g) with 100 mL of creekwater were placed in duplicate500 mL Gledhill flasks and spiked with two mL of the testsurfactant in the mineral salts medium. The testing was doneas described above except that incubation was with shakingat 70 rpm on a gyrotary shaker.

Periphyton samples were collected as rocks (approximately1 inch diameter) coated with heavy growth from the samestream locations as were the water and sediment samples.The rocks were carefully placed in “zipper”-seal bags tominimize disturbance of the periphyton layer, transported at

FIGURE 1. Chemical structures of [14C]benzene, uniformly ring-labeled test compounds. The 2-phenyl isomer of the C12 LAS homolog is shown.

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4 °C and used the same day. Four to five rocks were placedin duplicate flasks, covered with 100 mL of overlying water,and spiked with the test compound in mineral salts medium.Incubations and sampling were the same as for the sediment/water samples.

Each test system consisted of duplicate test flasks and acontrol flask which was identical except for the addition ofHgCl2 at 1.0 g/flask in waters or 2.5 g/flask in soils.

Tests lasted at least 30 days. Radiochemical recoveries of14C in solids, liquids, and CO2 (including 14CO3

2- and H14CO3-)

were done at the end of each test and ranged from 65 to107%; most were >90%.

Porous Pot Biodegradation Test System. The porous potmethod for assessing biodegradation of the test compoundsin a simulated wastewater activated sludge was a modificationof ASTM test method E1798-96. The porous pot systemconsisted of cylindrical glass containers with porous (>65 µpore size) high-density polyethylene (HDPE) candles insertedinto the glass cylinders (Figure 2). The candles held 282 mLof activated sludge, and a glass aeration tube to the bottomof the candle provided mixing and aeration at a 0.5 standardft3/h flow rate. Sewage feed was pumped into the units froma 10 L carboy container through Teflon tubing by means ofModel QG6 laboratory metering pumps (F. M. I., Inc., OysterBay, NY). Test chemicals were fed via Teflon tubing withModel 22 syringe pumps manufactured by Harvard Apparatus(South Natick, MA). Effluents from the porous pot units werecollected in 2 L vacuum flasks which were manifolded to thelaboratory vacuum system. The CO2 trapping system was aseries of three 500 mL gas collection bottles, each with 300

mL 2 N KOH. A comparison between the performance of thelaboratory scale porous pot biodegradation test system andconventional activated sludge treatment in the U.S. is shownin Table 1.

A 21 day acclimation phase was initiated by feeding settleddomestic sewage [adjusted to a chemical oxygen demand(COD) of 400 mg/L] and the nonradioactive analogs of thetest compounds. The units were operated with the followingparameters: nonradioactive chemical feed concentrations:C12 LAS, 5 mg/L; C8 DATS, 500 µg/L; iso-LAS, 250 µg/L;hydraulic retention time (HRT), 0.25 days; sludge retentiontime (SRT), 10 days (DATS units were 20 days); activated sludgeconcentration, ∼2000 mg/L volatile suspended solids (VSS).Steady state conditions, which were defined as a period of 7days, in which COD removal was >90% and HRT dailyvariation was <5%, were obtained and maintained over thefinal week of acclimation.

During the 15 day test phase, the radioactivities in CO2,liquids and solids, and effluent total suspended solids (TSS)and COD were determined each day, and the removal of testcompound from the feed by activated sludge treatment wasmeasured once. During the last 10 days, remaining effluentswere collected and frozen until use in the die-away tests.Appropriate amounts of sludge were wasted each day in orderto maintain the designated sludge retention time (SRT).Radiochemical recoveries for the porous pot test werecalculated by adding the total radioactivity recovered in CO2,solids, and liquids during the test. Corrections for radiola-

FIGURE 2. Porous pot biodegradation test system apparatus.

TABLE 1. Operation of Porous Pot Systems and Comparison to Conventional Activated Sludgea

HRT(h)

SRT(days)

MLVSS(mg/L)

effluent TSS(mg/L)

BOD5(% removal)

COD(% removal)

% removalof test compounds

porous pot 6.1 ( 0.1 10 2038 ( 216 <1 97.2 ( 2.7 91.2 ( 2.2 99.1 ( 0.5U.S. ASU (typical) 4-8 5-15 1500-3000 20-30 85-95 60-70 >99 (LAS)

a HRT ) hydraulic retention time; MLVSS ) mixed liquor volatile suspended solids; BOD5 ) 5 day biochemical oxygen demand; COD ) chemicaloxygen demand; ASU ) activated sludge unit. Mean ( 1 std. dev. are shown. Data for typical ASU are from ref 20.

VOL. 31, NO. 12, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3399

beled carbonates and bicarbonates were included. Totalrecoveries ranged from 95-102%.

Die-Away Tests with Porous Pot Effluents. The combinedeffluents from individual units were tested for mineralizationof radiolabeled parent and intermediate compounds in 2 LGledhill flasks as described above except that 1 L of combinedporous pot effluent and 10-15 periphyton covered rocks or250 g of sediment were used. In the soil die-away with DATSand LAS, 400 g of sludge amended soil, and 27 mL of effluentwere used. All tests were run at least 30 days, and anabiological control for each duplicate was prepared by adding1 g of HgCl2. The radioactivities at the end of each test weremeasured in the solids, liquids, and CO2 (including correctionsfor radioactive bicarbonate and carbonates) and were com-bined to give the radiochemical recoveries. Total recoveriesranged from 67.5 to 100%.

Analysis of Porous Pot Effluents and Porous Pot Die-Away Samples. Approximately 1 L of the remaining watercolumn samples of the porous pot effluents and porous poteffluent die-aways were filtered through 0.22 µ Millipore filters.These filtrates were concentrated from 35 to 93-fold in a Roto-Vap system at 90 °C. Pretests demonstrated that no radio-activity was lost in the overhead fraction. A chromatographicHPLC-ultraviolet fluorescence (UVF) analysis was done onthe concentrates to increase sensitivity and improve chro-matographic peak shapes over the ion-pair method used toanalyze the purity of the test materials. The column usedwas a Dionex AS11, 4× 250 mm, P/N 44076. Eluent flow ratewas 1 mL/min, and 100 µL was injected. Eluent componentswere A, 0.2 N NaOH in H2O; B, 95/5 acetonitrile/H2O; and C,distilled water. The gradients were initial A, 5%; B, 10%; C,85%; 0 to 10 min linear gradient to A, 40%; B, 10%; C, 50%;10 to 20 min hold at A, 40%; B, 10%; C, 50%; 20 to 30 minlinear gradient to A, 40%; B, 60%; and C, 0%; 30 to 40 min holdat A, 40%; B, 60%; C, 0%; step change to A, 5%; B, 10%; C, 85%then hold to 55 min. UVF excitation was 225 nm, and emissionwas at 295 nm. HPLC effluent fractions were collected everyminute for the first 40 min after injection except that withDATS only 10 fractions were collected. Radioactivity in theeach fraction was determined by liquid scintillation counting(LSC). Radiochemical recoveries were the ratios of totalradioactivity of all fractions in a sample to the unfractionatedsample radioactivity. Recoveries ranged from 83.3 to 108.2%;most were >95%.

Kinetic Analysis. Data for CO2 evolution were analyzedusing nonlinear regression models, a technique which hasbeen successfully applied to several chemicals which manifestfirst order kinetics (21). The half-life and asymptote werecalculated using Statgraphics Plus 5.2 software. These wereobtained by minimizing the residual sum of squares using asearch procedure suggested by Marquardt (22). All CO2 datawere corrected for CO2 released in the HgCl2 controls beforeuse in kinetic analysis.

Results and DiscussionMineralization of Model Compounds in Aquatic and SoilEnvironments. Since commercial LAS is primarily discardeddown-the-drain with or without sewage treatment, the initialobjective was to evaluate the potential ultimate biodegrada-tion of DATS and iso-LAS in receiving environmental com-partments. A typical receiving water (Lake Creek) was thefirst environmental compartment tested. Long lag times (>10days) and very slow rates were observed which did not fitfirst-order kinetics. Therefore, no attempt was made tocalculate rate constants for DATS, iso-LAS, and standard LASin Lake Creek water. This was probably due to the very lowlevel of biodegradable organic compounds (BOD5 < 2 mg/L)which resulted in a low population of bacteria (<15 000 cfu/mL) in the water.

By adding biomass to the creekwater with either per-iphyton-covered rocks or sediment, rapid microbial miner-

alization occurred (Table 2). The rates for LAS and most ofthe iso-LAS isomers were biphasic. The first-order initial rateswere at least twice as fast as the apparent zero-order finalrates. Explanations on the mechanism or significance of thefinal rate are lacking. The initial mineralization rate maycorrespond to both mineralization and incorporation of theradiolabeled substrate carbon into cell components. Theslower, final rate may reflect the turnover of the incorporatedcarbon, or it may represent the formation of more biologicallystable catabolites. Static conditions of the experimentalsystem resulting in depletion of cosubstrates, essentialnutrients, etc., could also be an explanation. Since theradiolabel is in the benzene ring and assuming that, like LAS,the benzene ring is the last point of catabolic attack, theevolution of CO2, regardless of amounts, during breakdownof the DATS and iso-LAS intermediates, is evidence that stablecatabolites probably are not responsible for the slower finalrate. Only the initial rates and the extents of mineralizationafter 30 days are reported in Table 2. In all cases, themineralization after 30 days had reached a final, slow rate.Mineralization continued (as illustrated by typical curvesshown in Figure 3) until termination of the experiments, whichfor some incubations was 120 days. The rate and extent dataare consistent with the interpretation that accumulationwould not occur in environments that receive treatedwastewater

The large-scale application of wastewater effluents andsludges to soils has led to concerns about the potentialaccumulation of surfactants in soil environments. Since alarge portion of sewage sludges is applied to land in Europeand North America, soil biodegradation should be animportant process for removing surfactant residues from theterrestrial environment (25). Gray water contaminated soilsand sludge-amended soils offer an opportunity to studybiodegradation of DATS and iso-LAS in soils which have beenexposed to commercial LAS and should have microbialpopulations that are acclimated to these coproducts. Thelaundromat site at Summit Lake, WI, offers an ideal locationfor such samples and has been extensively characterized andevaluated (18, 24). Surface samples were obtained from thetop of the percolation bed which was exposed directly towashing machine effluent. Both DATS and LAS mineraliza-tion began immediately and continued in typical curvilinearfashion until the test was terminated after 45 days (Table 3).The mineralization rates and extents of both DATS and iso-LAS in sludge-amended soil are also shown in this table.

TABLE 2. Mineralization of Radiolabeled Compounds in AquaticEnvironments

ratea

[t1/2 (days)]extenta

[%T 14CO2 (30 days)]b

Creek Water and Periphyton-Covered RocksLAS 3.4; 4.6 60; 76DATS 14.2; 18.2 19; 19iso-LAS type IA 3.1 71

IB 3.2 70IIA 4.0 63IIB 8.6 47

glucose 3.7; 2.6 61; 72

Creek Water and SedimentsLAS 4.2; 12.4 56; 63DATS 10.8; 21.0 27; 30iso-LAS type IA 3.6 50

IB 5.1 52IIA 5.4 50IIB 12.1 39

glucose 5.4; 3.6 52; 54a More than one value indicates results of separate experiments.

b Percent theoretical carbon dioxide production from radiolabeledsubstrate at 30 days.


The mineralization of iso-LAS (and standard LAS forcomparison) was immediate and rapid, whereas DATSmineralization proceeded at a slower rate. Even though DATSmineralization rates are slower than LAS rates in this side-

by-side comparison, the rates for DATS are within the rangeof those reported for LAS in other sludge-amended soils (26).

Although not shown in Table 3, the mineralization of DATS(over 40% within 30 days) was observed in a “pristine” soil.Because of no known agricultural or industrial activity orrecent habitation at this site, the microbial populationpresumably has never been exposed to commercial LAS. Theseresults suggest that the microbial capacity to mineralize DATSis ubiquitous in soils as has been reported for LAS (23). Inthis pristine soil, as well as occasionally observed in othersoils, the extent of mineralization of LAS and the iso-LAS andDATS coproducts exceeded that of the reference glucose.Unexpectedly low mineralization of radiolabeled glucose insoil has also been observed by Sharabi and Bartha (27). Theyattribute this to unavailability due to adsorption or alterna-tively to the possiblility that nongrowing steady-state mi-crobial communities may turn over glucose very differentlyfrom a community that is growing in response to significantsubstrate addition. The latter implies that the type andconcentrations of test substrates, relative to soil organicmatter, may affect mineralization. In any event the data fromTable 3 should not be interpreted as indicating that themineralization of LAS and coproducts is greater than glucose.

FIGURE 3. Mineralization of radiolabeled test compounds in creek water with sediment.

TABLE 3. Mineralization of Radiolabeled Compounds in Soils

ratea


[%T 14CO2 30 days)b

Gray Water Contaminated Soil; Summit Lake, WILAS 1.8 50DATS 7.2 33glucose 1.0 31

Sludge Amended Soil; Austin, TXLAS 1.8; 2.3 53; 57DATS 9.8; 17.7 30; 42iso-LAS type IA 1.6 48

IB 1.6 56IIA 2.8 60IIB 5.0 64

glucose 0.8; 3.2 37; 41a More than one value indicates results of separate experiments.

b Percent theoretical carbon dioxide production from radiolabeledsubstrate at 30 days.


Simulated Activated Sludge Treatment and Die-AwayTests. An important consideration of any down-the-drainchemical is its fate in domestic wastewater treatment systems.Therefore, the fate of the model compounds was examinedin the porous pot biodegradation system which simulatesthe most common treatment system-activated sludge. Ac-tivated sludge treatment (Table 4) effectively removes thecoproducts from wastewater as indicated by biologicalremoval values approaching 99%. Like LAS, most of the iso-LAS isomers underwent extensive mineralization (>50%) andultimate biodegradation (79-90%) but released some (10-20%) of their carbon as water soluble intermediates. Activatedsludge treatment of DATS and iso-LAS type IIB results in nearlycomplete removal (>98%) of the test surfactant, some ultimatebiodegradation (3-12%) and a concurrent release of apparentcarboxylated biodegradation intermediates (88-97%). Theseintermediates could be characterized as (1) having intactaromatic rings which demonstrate UV fluorescence; (2) having

increased polarity compared to the parent compounds; and(3) eluting on the HPLC-UVF chromatograms in the sameregion as with SPC biodegradation intermediates of LAS.Confirmatory evidence comes from the recent work of Cavalliet al. (14) and Kolbener et al. (16, 17) and the monitoringwork of Trehy et al. (10) in which carboxylated intermediateswere found to be the major intermediates remaining afterbiological treatment of commercial LAS.

Since the rate-limiting and last steps in SPC biodegradationare desulfonation, ring cleavage, and further oxidation of thering-cleavage products (28), it is reasonable to assume thatanalagous biochemical pathways occur during the completebiological destruction of DATS and iso-LAS. Therefore, thecomplete biodegradation of these coproducts can be assessedby following the oxidation of the ring-labeled compounds inthe porous pot effluents as summarized in Table 5.

No kinetics were calculated for the mineralization ofporous pot effluents in the creekwater alone. However,significant mineralization, which followed first-order kinetics,occurred if biomass in the form of periphyton-covered rocks,sediments, or sludge amended soils were added. Further-more, the mineralization rates for the DATS and iso-LAScarboxylated intermediates were similar to those for the LASSPCs.

Table 6 presents the radiochemical mass balances at thetemination of the die-away experiments with creekwater plusperiphyton and creekwater plus sediments. Total CO2

production was significant for all the intermediates duringthe 77 or 37 day incubations except for the DATS catabolicintermediates (DATSI) in the water/sediment incubations.In contrast, the total CO2 from DATSI incubations in soil was35% during the 37 day incubation (data not shown). There-fore, it appears that mineralization of DATSI can vary withthe environmental conditions and presumably the microbialpopulations.

The transient nature of these carboxylated biodegradationintermediates was demonstrated by HPLC-UVF/radiochemi-cal detection. Water soluble fractions were analyzed of theporous pot feeds, effluents, effluent/sediment, and effluent/periphyton die-aways, and the results are displayed in Figures4, 5, and 6. The typical chromatographic pattern of C12-LASis shown in Figure 4. The parent compound is almostcompletely removed during porous pot activated sludgetreatment, and about 14% of it is released into the effluentas many water-soluble biodegradation intermediates. Duringthe effluent/sediment die-away, 47% of the remainingradioactivity was mineralized, and only 3.3% remained insolution after 77 days of exposure. Only the results for LASare shown since the HPLC-UVF/radiochemical patterns foriso-LAS types IA, IB, and IIA were the same as those for LAS.Furthermore, very little (6% or less) of these starting radio-activities remained after the die-away.

The biodegradation pattern for iso-LAS type IIB is shownin Figure 5. It is clear that one major and several minor water-soluble SPCs are formed during activated sludge treatment.Cavalli (14) also found that one major and several minor SPCswere formed during activated sludge treatment of another

TABLE 4. Fate of LAS Coproducts in Porous Pot ActivatedSludge Treatmenta

parentremoval

(%)

mineral-ization

(%14CO2)

percentin

biomass

ultimatebiodegradation

(%)

residual inliquids

(%)

LAS 98.4 57.5 28.6 86.1 13.9DATS 98.6 0.8 1.8 2.6 97.4iso-LAS IA 99.7 53.0 26.2 79.2 20.8

IB 99.3 58.2 31.5 89.7 10.3IIA 99.5 51.6 27.8 79.4 20.6IIB 98.5 7.6 4.2 11.8 88.2

a Values normalized to 100%; actual 14C recoveries were 95-102%.Ultimate biodegradation ) mineralization + radioactivity in biomass.

TABLE 6. Radiochemical Mass Balances for Porous Pot Effluent Die-Awaysa

water/periphyton water/sediment

actual recovery (%) CO2b solubleb solidsb actual recovery (%) CO2

b solubleb solidsb

LAS 91.4 48.1 43.1 8.8 100.3 46.9 23.6 29.5DATS 67.5 29.2 62.5 8.3 89 11 75.8 13.1iso-LAS IA 90.2 59.5 11.2 29.3 93.7 70.3 5.2 24.5

IB 92.6 45.3 24.9 29.8 87.9 57.1 17.2 25.7IIA 89.2 51.1 32.6 16.3 94.3 56.4 7.1 36.5IIB 88.4 60.8 31.7 7.5 96.7 50.3 24.1 25.6

a Die-away incubations in creek water plus periphyton or creek water plus sediments were 77 days except for DATS which was 37 days. b Percentagerecoveries of CO2, soluble fraction and solids fraction are normalized to 100%. Actual recoveries are given.

TABLE 5. Fate of Biodegradation Intermediates in Aquatic andSoil Environments

ratea


[%T 14CO2 (30 days)]

Creekwater and SedimentLAS SPC 5.3; 12.1 14; 32DATS intermediates 6.8 9iso-LAS type IA 9.3 53

IB 15.0 35IIA 34.4 22IIB 23.2 33

Creekwater and Periphyton-Covered RocksLAS SPC 3.9; 11.9 23; 34DATS intermediates 2.4 18iso-LAS SPC type IA 4.0 44

IB 9.4 31IIA 13.7 28IIB 27.1 30

Sludge-Amended SoilLAS SPC 7.8 22DATS intermediates 6.2 34

a More than one value indicates results of separate experiments.


iso-LAS type II isomer of the same homolog. This isomer,however, showed a higher level (∼87%) of ultimate biodeg-radation than the type IIB during activated sludge treatment.

Even though these SPC intermediates formed by activatedsludge treatment of iso-LAS type IIB make up most (88%) ofthe starting radioactivity, they are not recalcitrant sinceexposure to biomass on sediments during the 77 day die-away mineralized all the minor SPCs and over 50% of themajor intermediate.

A very similar pattern was observed for the DATS bio-degradation as illustrated in Figure 6, except that almost all(97%) of the intact DATS were converted to water solubleintermediates (DATSI) in the porous pot treatment. TheseDATSI were metabolized by the presumably different mi-crobiological populations found in the sediments and per-iphyton. The end result of the DATSI die-aways in water/periphyton was removal of 56% of the soluble radioactivity(97% or original radiolabel at beginning minus 41% remaining)during the 37 days.

Kolbener and co-workers have concluded from theirstudies on the biodegradation of commercial LAS in alaboratory “trickling filter” test system using immobilizedactivated sludge that from 3.2 to 13.6% of the commercialLAS carbon is refractory (16). These researchers haveextended their studies and have reported that major com-ponents of this refractory portion are carboxylated biodeg-radation intermediates of DATS and iso-LAS (17). Theirconclusion was that many of the coproducts of commercialLAS are recalcitrant.

The porous pot results of this paper and field monitoringresults of Trehy et al. (10) are consistent with the observationsof Kolbener et al. (15-17). It is clear from these studies, aswell as other unpublished observations, that the microbialpopulations of domestic and industrial activated sludge andtrickling filter wastewater treatment plants are not capable

FIGURE 4. HPLC-UVF/radiochemical analysis of C12LAS in porouspot feed (top panel), effluent (middle panel), and effluent/creekwater+ sediment die-away (bottom panel). Results (not shown) for thecreekwater + periphyton die-away experiment are comparableexcept that the amounts in each fraction are slightly greater.

FIGURE 5. HPLC-UVF/radiochemical analysis of iso-LAS type IIB inporous pot feed (top panel), effluent (middle panel), and effluent/creekwater + sediment die-away (bottom panel). Results (not shown)for the creekwater + periphyton die-away experiment are com-parable except that the amounts in each fraction are slightly greater.

FIGURE 6. HPLC-UVF/radiochemical analysis of C8DATS in porouspot feed (top panel), effluent (middle panel) and effluent/creek water+ periphyton die-away (bottom panel). Results (not shown) for thecreek water + sediment die-away experiment are comparable exceptthat the amounts in each fraction are slightly greater.


of the complete mineralization of all the DATS and iso-LAShomologs and isomers found in commercial LAS even thoughnearly complete primary biodegradation of these coproductsoccurs. The new information provided in this paper is thatthese DATS and iso-LAS biodegradation intermediates pro-duced during wastewater treatment could mineralize whenexposed to receiving water environments or soils that containa more diverse population of degraders.

AcknowledgmentsWe thank John Lin (CONDEA Vista Company), James Innis(The Procter and Gamble Company), and Paul Sieving (WizardLaboratories, Inc.) for synthesis of test compounds; BruceLeach (formerly of CONDEA Vista Company) for assistancewith kinetic calculations; and Paul Filler (CONDEA VistaCompany) for technical assistance and John Heinze (Councilfor LAB/LAS Environmental Research) for helpful discussions.

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Received for review January 10, 1997. Revised manuscriptreceived August 11, 1997. Accepted September 13, 1997.X

ES970023M

X Abstract published in Advance ACS Abstracts, October 15, 1997.


biodegradation of coproducts of commercial linear alkylbenzene sulfonate

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