Reduction of aquatic toxicity of linear alkylbenzene sulfonate (LAS) by biodegradation

I$ater Research Vol. 11. pp. 31 to 37. Pergamon Press 1977. Printed in Greal Britain.

REDUCTION OF AQUATIC TOXICITY OF LINEAR ALKYLBENZENE SULFONATE (LAS) BY BIODEGRADATION RICrtARD A. KIMERLE and R. D. SWISHER Monsanto Company, 800 N. Lindbergh Blvd., St Louis, MO 63166, U.S.A. (Received 3 June 1976)

Abstract--Partial biodegradation of LAS is shown to significantly reduce the specific toxicity (i.e. per unit weight) of the remaining LAS to Daphnia magna (water fleas) and Pimephates promelas (fathead minnows). This results from the fact that the longer homologs and more terminal isomers, which are the more toxic, are also the more rapidly degraded under bacterial action. The acute aquatic LCs0 of LAS may range from ~0.5 to ~50mg/1. depending mainly upon the chain length of the particular homolog. A high molecular weight commercial type LAS with LC~o around 2 mg/l. before biodegradation may show Daphnia LCso'Sof 30--40mg/l. for the LAS remaining after 80--85% degradation. A further contribution to this toxicity reduction may occur if the methylene blue analytical method is used to determine the amount of LAS remaining, since some of the biodegradation intermediates show methylene blue activity but no significant toxicity. For example, suifophenyhindeeanoate, a model of early intermediates, shows Daphnia and fatbead LCso's ~200 and ~75rag/1., respectively. Sulfophenylbutyrate, modeling somewhat later intermediates, gives LCso values around 5000--10,000rag/1. Dialkyl tetralin/indane sulfonates (the major non-linear components in commercial LAS) exhibit 1/2-1/10 the toxicity of the corresponding LAS homologs. These results re-emphasize that analysis simply for methylene blue active substances (MBAS) gives no basis for predicting the aquatic toxicity of an environmental sample. And furthermore, that meaningful water quality criteria and standards cannot be established in terms of MBAS content while based on toxicity studies on intact, undegraded LAS.

INTRODUCTION

of greater than 75 mg/1., much less toxic than an intact LAS of the same chain length. Kimerle et al. (1975) demonstrated by use of partition coefficient data that the bioaccumulation potential of LAS was markedly reduced when the alkyl chain of LAS was carboxylated as in the case of SOU. Divo (1976) coneluded that bemuse of the preferential faster rate of biodegradation of the more toxic LAS components that even partial biodegradation can significantly reduce the toxicity (per unit weight remaining) of a surface active agent such as LAS. Direct examination of partially degraded LAS systems by Dolan & Hendricks (1975a, b) has borne out this expectation. Degradation of an LAS molecule begins with carboxylation of the terminal methyl group. At this point LAS loses most of its sufactancy and toxicity, with a significant reduction in MBAS activity. Biodegradation continues at a rapid rate on these long chain carboxylated intermediates with the cleavage of carbon pairs from the alkyl chain until only the benzene ring and a few carbons are left. Ultimate biodegradation occurs when the remaining ring and short chain carboxylates are converted to CO2 and/or bacterial protoplasm (Swisher, 1970). The term "partial biodegradation" as applied to LAS may include two aspects. First, some of the original LAS may have disappeared, but some of it may still remain. Since LAS may contain up to 15-25 isomers and homologs which degrade at different rates, their proportions in

Linear alkylbenzene sulfonate (LAS) is a commonly used surfactant in detergents sold in the United States. Even though intact LAS is toxic to aquatic organisms (LCso values 3-7 mg/l.) it does not create any significant threat to the life of aquatic organisms because LAS is readily degraded by bacteria in waste treatment facilities (GledhiU, 1975). Biodegradation rate and acute toxicity are both very much related to the chain length and phenyl position on the alkyl chain. Biodegradation of LAS with mixed chain lengths and isomers proceeds at a faster rate on the longer chain lengths and more terminal isomers (Huddleston & Allred, 1963; Swisher, 1963; Tarring, 1965). The longer chain lengths and more terminal phenyl isomers also have been shown to be the more toxic components of LAS (Hirsch, 1963, Swisher et al., 1964; Borstlap, 1967; Marchetti, 1965, Divo, 1976; Kimerle et al., 1975). Swisher et al. (1964) demonstrated that the toxicity of C12 and C14 pure homolog LAS samples was greatly reduced by biodegradation in laboratory activated sludge units, the LC~o values of 3 rag/1, and 0.6 rag/1., respectively, becoming greater than 100 mg/l. (based on initial LAS content). It was thus shown that no significant amounts of toxic biodegradation intermediates accumulated in the waste water; further, a model biodegradation intermediate, sulfophenylundecanoic acid (SOU), had an LCso value 31

32

RICHARD A. K1MERLEand R. D. SWISHER and ~ii) comparing the acute toxicity of intact LAS to that of the MBAS contents of effluent from a laboratory continuous flow activated sludge unit which was degrading LAS. Only Daphnia magna were used in effluent toxicity tests because of the limited quantity of effluent available compared to the larger quantities needed for fish bioassays.

the remaining LAS changes as the degradation proceeds. Second, LAS has all gone, but some residual material may remain in the form of intermediate biodegradation products, not yet fully converted to bacterial protoplasm or carbon dioxide. These intermediates would be principally the carboxylates mentioned above. The concentration of LAS in waste water effluents and surface waters is most frequently estimated by either the "Standard Method" (APHA, 1971) or the "'Hellige Method" (Swisher et al., 1964). Both methods depend upon the reaction of an anionic surfactant with methylene blue and extraction into chloroform. This reaction is subject to interferences by non-LAS methylene blue active substances in the water sample. These interferences fall into several categories, including (i) other anionic surfactants, natural or man-made, {ii) long chain carboxysulfonates such as SOU, formed as early intermediates in the biodegradation of LAS and other anionic surfactants, (iii) inorganic ions such as chloride, nitrate, etc. By performing the prescribed backwashes in the "Standard Method" the second and third types are minimized to some extent. Some, but not all, interferences of the first type may be removed by preliminary hydrolysis, but this still does not allow unequivocal identification of the remainder as LAS. The more sophisticated desulfonation and gas chromatographic method enables quantitation of the LAS present as well as the relative concentration of each of the chain lengths and isomers. Given the facts that acute toxicity can vary greatly with chain 'length and isomer position, one cannot deduce the toxicity of any particular LAS mixture from its MBAS analysis. Toxicity estimates on the basis of MBAS values are even more ambiguous when dealing with environmental or other partially degraded samples. All the MBAS is not necesarily LAS, and the LAS content itself will have been partially degraded with resulting further changes in homolog/isomer distribution favoring lower toxicity. It was the purpose of this study to confirm and extend the above findings by (i) determining the acute toxicity to Daphnia maona and fathead minnows (Pimephales promelas) of intact LAS components and synthesized models of biodegradation intermediates,

MATERIALS AND METHODS

Materials Linear alkylbenzene sulfonate. LAS samples were prepared by sulfonation of the corresponding alkylbenzenes in the usual manner, neutralizing the sulfoni¢-sulfuric acid mixture in 80~o isopropyl alcohol with NaOH. filtering out the precipitated sodium sulfate and drying the filtrate. This product is approximately 99% pure LAS, the remaining 1°~ being mainly sodium sulfate and moisture. Composition of the commercial type (high molecular weight) LAS and pure homolog samples used in this study are summarized in Table I. Figure I shows a typical LAS molecule. Dialkyl tetralin-lndane Sulfonate (Cto, Ct2 and Ct4 DTIS). These were prepared by alkylation of benz~ne with the Ct0, C1~ and C14 a,t~ dienes, (Phillips Petroleum Co., Chemical Samples Co.) respectively, using AICI3 catalyst under conditions similar to those reported by Wulf et al. (1969). Fractional distillation yielded heart cuts having 9~/~ DTI (by GLC) in 20--35~°~of theoretical amounts. Sulfonation, neutralization, alcohol dcsalting and drying of the sodium sulfonates were done similar to the LAS, above. Gas chromatography of the original hydrocarbons before sulfonation showed 6, 8 and 10 major peaks, respectively, for the C1o, Ct2 and C ~ derived material, the same as the theoretical numbers of isomers. Small samples of the unsulfonated DTI hydrocarbons (a few drops in the bottom of stoppered test tubes) underwent profound changes upon standing several months. The original material gave clean chromatograms, but the aged samples showed reduction or disappearance of some peaks and formation of many new ones. This was probably the result of autoxidation by the large excess of atmospheric air in the test tubes. The dried sulfonates also changed upon prolonged standing, going from tan powders to brown resinous masses. Aqueous solutions of the original sulfonates did not darken significantly. Sulpophenylundecanoic acid disodiura salt (SOU). This was obtained in a similar manner by sulfonation of phenylundecanoic acid (Eastman 5352). The five components of this material separated by gas chromatography of the methyl esters (Swisher et al. 1964) have subsequently been identified as the 6-, 7-, 8-, 9- and 10-phenyl isomers (Zeman et al., 1969). Mcthylation-gas chromatography showed that the 1975 sample of Eastman 5352 contained about equal

Table 1. Chemical composition of LAS samples used in toxicity tests

Commercial type (high tool. wt.) Pure homologs Cto C~1 C12 C13 C~4

og Homolog distribution

Average chain length

Cl0

Clt

Ct:

C,3

Ct4

20

30

40

13.3

--

1

8

52

39

16

14

15

10.0 11.0 12.0 13.0 14.0

99 1 ----

. 99 2 ---

--99 1

---99

24 18 15 19 17

25 20 17 15 16

23 21 18 15 15

.

. -98 1 --

°o Isomer distribution

.

50

60

70

--

--

55 27 40 17 17 16

32

-33 36

Linear alkylbenzene sulfonate (LAS) by biodegradation

CH3-C ~

H2-CH~CH~CI'Lz-CH;t-CH~ CH2-CH2-CH2"CH3

SO,No+ Fig. 1. Typical LAS Molecule, 3-phenyltridecane sulfonate. (Commercial LAS is a mixture of 15-25 such isomers and homologs with alkyl chain length ranging from 10 to 14 carbons, phenyl groups distributed approx evenly among all positions along the chain except very few at the two ends, and sulfonate group predominantly in the para position). amounts of the five isomers in contrast with the skewed distribution (6-phenyl lowest with progressively increasing amounts up to the 10-phenyL highest) of the 1964 sample. The characteristic isomer distributions of the two samples were retained upon sulfonation and desulfonation. The current sample of disodium salt was of somewhat lower purity than the 1964 sample, showing only about 889~ (Weatherbum-Epton titration). The LCso values are for this product as is, without taking into account any toxic contributions by the 10--t2~ of other material present. (This toxic contribution appears to be quite substantial in the light of subsequent work, to be reported later. Further purification of this lot of SOU has yielded a major fraction of SOU having the same isomeric composition but much lower toxicity, along with small fractions of impurities with very much higher toxicity (Swisher et al., 1976).) 3-su!fophenylbutyric acid, 4sulfophenylvaleric acid. These two model short chain biodegradation intermediates were obtained as the d i ~ i u m salt monohydrates synthesized by the procedure described by Hwa and Fleming (1957) for 2-sulfophenyl butric acid. The free acids, 3-phenylbutyric (Aldrich) and 4-phenylvalcric (Chemical Procurement Laboratory) were converted to the ethyl esters, sulfonated with 95-98% sulfuric acid, diluted with water and extracted with benzene to remove unsulfonated material. Ethanol was added to the remaining aqueous phase, precipitating the product, which was filtered out and.then purified by several steps of dissolving in water and precipitating witth acetone. Yields of purified product were 60 and 309'0, respectively.

Siphon

J .... Effluent

~

st~;m

,-

[,~t~m t)

Pol~fl~/lene

fil?ereondle

/

J Aird Fig. 2. Laboratory continuous flow activated sludge unit with polyethylene porous filter candle. w.n. 11 l l - - . c

33

The products were characterized by NMR, i.r., and thermogravimetric analysis. Alkalimetric titration of the free acids, obtained by passage through an ion-exchange column, showed two breaks, the first around pH 3.5 at the end of the sulfonate acidity, the second around pH 8 at the end of the carboxylate acidity. In the case of the 3-sulfophenylbutyrate, the strong acidity was about 13°o higher than theoretical, suggesting the presence of a few percent of sodium sulfate. Details of the syntheses and characterizations will be published elsewhere later.

Biodeoradation Biodegradation studies were conducted using the commercial LAS blend and the C13 pure homolog in a 300 ml continuous flow activated sludge unit with a 6 hr retention time (Fig. 2). Suspended solids were maintained between 2500 and 3500 mg/l. The unit was equipped with a porous polyethylene candle filter to separate the siphoning effluent liquid from the sludge solids. The nutrient feed was that recommended by the OECD (19711. The activated sludge was acclimated to degrade the LAS by starting at 20 mg/l. in the feed with subsequent increases in the LAS concentration as primary biodegradation took place. Foam problems were minimized by passing an air stream over the surface of the mixed liquor. Over a I yr period the LAS feed concentrations were varied up and down between 20 and 200 mg/1. It was during periods just before upsetting that partially degraded LAS was obtained of sufficient MBAS concentration for toxicity testing. On some occasions when the influent LAS was at 160-200rag/1. and the effluent MBAS was higher than desired, samples of the effluent were placed on a shake table to allow further degradation to proceed under control to levels suitable for the toxicity tests.

Analytical Analyses of MBAS concentrations in effluent samples and during toxicity tests were made using the Hellige Method (Swisher et al., 1964). During the toxicity study several effluent samples were analyzed by desulfonation-gas chromatography for actual LAS content and the relative concentrations of the various chain lengths and isomers. The technique described earlier (Swisher 1966; Sullivan & Swisher 1969) was modified by use of three new internal standards instead of Ca LAS. The new standards were 1-phenyl C1o, C11 and C12 LAS, obtained by sulfonation of the corresponding l-phenylalkanes (Humphrey-Wilkinson Co.). The sodium salts were purified by dissolving in hot water and cooling to room temperature, whereupon the sparingly soluble sodium salts crystallize out and can be filtered, washed and dried. Stock solutions were made at 0.1 mg/ml using 50% methanol-water to minimize crystallization upon standing. These three standards were added to the sample before desulfonation at one tenth the MBAS level e.g. 10#g of each to a sample containing 100 #g of MBAS. Little if any of the 1-phenyt isomers is present in commercial type LAS. Their peaks appear in the chromatogram between the 3- and 2-phenyl isomers of the next higher homolog. The 10#g amounts were sufficient to give full scale peaks when the desulfonate was made up to a volume of 50 ), and a 1 ). sample injected with a split ratio of 10:1. A 150' x 0.02" DC550 column was used at 170°, 20-40psig He pressure, in a HewlettPackard model 5710A gas chromatograph with flame ionization detector, attenuation 10x. Retention times were around 8, 12 and 18 min for the three standards. Interferences sometimes arose from the alkyl indanones and alkyl tetralones formed by eyclization of certain of the carboxylated biodegradation intermediates during the analytical desulfonation (Swisher, 1963). These can usually be distinguished by their asymmetric peaks, which rise more sharply and fall off more slowly than the more symmetrical peaks from the LAS components. Their identity can be

34

RICHARD A. KIMERLEand R. D. SWISHER ever, by use of the unit shown in Fig. 2, LAS ted at concentrations up to 150 mg/l. underwent primary biodegradation quite easily. It was necessary to gradually acclimate the microbial population in the unit to successively higher concentrations of LAS, 20-50 mg/1. increments once each week, over a period of 3-4 weeks. Just after spiking the unit to a higher concentration it would either completely upset, begin to upset and recover yielding partially degraded LAS for a few days, or acclimate and completely degrade the LAS. Daphnia toxicity tests were performed when the LAS was partially degraded. If the unit completely upset the feed rate was decreased back to 20mg/1. allowing the activated sludge unit to recover. If the unit successfully acclimated to the spiking, the feed rate was increased once again on the following week. By going through this process of starting at a low LAS feed level of 20mg/l. and working up to 200 mg/l. a number of times during the year it was possible to obtain enoug h partially degraded LAS effluent samples to describe the relationship between MBAS toxicity and degree of biodegradation. Analyses of some effluent samples from the laboratory activated sludge unit receiving the commercial LAS or pure C~3 homotog are shown in Table 2. The typical homolog/isomer distribution of the intact LAS samples in the feed can be compared to that in the effluent samples at various degrees of partial biodegradation. There was a loss of the 2-, 3- and 4-phenyl isomers relatively early in the biodegradation process. In the case of the commercial LAS there was also a shift in the homolog distribution with a significant loss of longer chain lengths after 90°o biodegradation. In addition to the homolog/isomer distribution change, semi-quantification of the actual

verified by use of a coupled gas chromatograph-mass spectrometer.

Toxicity tests Acute static toxicity tests on intact LAS and LAS components were conducted following the general procedures of the Environmental Protection Agency (1975). Daphnia tests were performed in 250 ml beakers with 200 ml of well water of approximately 250 mg/l. hardness. Ten Daphnia less than 18 hr old were placed in each of 3 replicate beakers. Five concentrations were tested. No food was added. Fathead minnow toxicity tests were conducted in 5 1. of 100 mg/l. hardness water using 5 fish per concentration. LCso values and 959/0 confidence limits were calculated using a logit transformation computer program following the method of Litchfield & Wilcoxon (1949). The toxieities of effluent samples from the continuous flow activated sludge unit were determined using Daphnia in static tests. The daily effluent volumes of approximately 1.21. were analyzed for Hellige MBAS and diluted with well water to several appropriate MBAS concentrations. Twenty-four hr Lcso values were calculated on the basis of initial MBAS concentration without regard for possible further reduction by biodegradation during the test. RESULTS AND

DISCUSSION

LAS biodegradation Complete and partial primary biodegradation of LAS at feed concentrations up to 200mg/1. was achieved in the laboratory continuous flow activated sludge unit. Effective separation of liquid effluent from suspended solids, at high LAS feed rates, was made possible with the aid of the polyethylene porous candle filter. When a conventional settler type activated sludge unit was tried, one that depends on gravity separation of solids and liquid instead of filtration by the porous candle, frequent upsets were encountered with loss of sludge in the effluent. How-

Table 2. Comparison of homolog/isomer distribution in LAS and percent LAS in MBAS before and after partial biodegradation

Sample Commercial type (high tool. wt.) Influent undegraded Effluent 87~o degraded Effluent 90% degraded Pure homolog Ct3 Influent undegraded Effluent 700/0 degraded Effluent 80,0/0 degraded Effluent 84---9090 degraded Effluent 979/0 degraded * Calibration standard.

9,o Homolog distribution

°o Isomer distribution

Influent MBAS m&/l.

Effluent MBAS mg/1.

C 11

C 12

C 13

C14

20

30

40

5-70

'!o LAS in hellige MBAS

160

--

1

8

52

39

16

14

15

55

100*

160

21

4

18

61

18

0

0

2

98

30-37

160

16

10

26

43

20

0

0

0

100

20-35

100

--

17

l4

l5

54

100"

100

-.

0

0

13

87

65-75

100

--

0

0

2

98

60-80

.

.

.

.

.

50

15

--

--

200

40

....

100

t0-16

--

--

100

0

0

5

95

15-20

100

3

--

--

100

0

0

0

100

3-5

Linear alkylbenzene sulfonate (LAS) by biodegradation

35

Table 3. Acute toxicity of original materials (LC50, rag/1.)

Daphnia magna Average chain length Commercial type (high tool. wt.) Individual homologs LAS C1o ell C12 C13 Cl4 Non-linear L A S components (DTIS) C1o C12

C1, Model biodegradation intermediates C, (S¢~ Butyrate) C5 (SO Valerate) C11 (SOU)*

13.3 10 11 12 13

24 hr 2.6 ± 0.1 53.1 ± 15.8 ± 10.7 ± 2.7 ±

0.4 3.0 1.6 0.4

Fathead minnow

48 hr

24 hr

48 hr

2.3 ± 0.1

1.9

1.7

2.6 0.6 1.0 0.3

48.0 17.0 4.7 1.7

43.0 16.0 4.7

12.3 ± 5.7 ± 3.5 ± 2.0 ±

14

1.2 ± 0.2

0.7 ± 0.2

0.6

0.4 0.4

I0 12

106.0 ± 27.0 55.1 ± 9.1

98.0 ± 21.3 34.1 ± 5.1

87.0 ± 7.5 24.8 ± 5.8

86.1 ± 15.0 21.5 ± 5.5

14

12.4 ± 1.4

10.0 ± 1.0

8.1 ± 5.1

5.3 ± 3.9

4

~ 12,000 ~ 12,000 355 ± 150"

~ 6000 ~ 5000 208 ± 85*

~ 10,000 ~ 100(30 85.9 + 5.1"

5 11

~ 10,000 ~ 10,000 76.6 ± 12.4"

* Subsequent repurification of this sample yielded a product with the same isomeric composition but with LOs0 values over 1000 mg/l. for both daphnids and fatheads (Swisher et al, 1976). LAS contained in the Hellige MBAS shows that a major portion of the MBAS can result from non-LAS methylene blue active substances. These estimates were derived from the response of the internal standards added in the desulfonation/gas chromatographic analysis.

Acute toxicity of LAS components and model intermediates The acute toxicity data for all the original materials tested are shown in Table 3. The LCs0 concentrations for the commercial LAS, pure homologs and dialkyl tetralin-indane sulfonates (DTIS) ranged from less than 1 mg/l. to 100 mg/L and for the model biodegradation intermediate samples from 100 to 12000 rag/1. Differences within a series of compounds can be attributed primarily to chain length; the longer the chain the more toxic the compound. Figure 3 shows the nearly straight line relationship when the log of the 24 hr LCso values are plotted against the carbon chain lengths of the LAS and DTIS samples. Comparison of the LCs0 values obtained for the same compound using Daphnia and fathead minnows indicates the minnows to be slightly more sensitive to the LAS compounds than Daphnia. The range in toxicity values for the pure homolog LAS samples demonstrates the importance of knowing the composition of an LAS product. In terms of environmental implications, it is even more important to understand the role of biodegradation in reducing LAS toxicity. The first step in the biodegradation of an LAS is carboxylation at the terminal methyl group. The toxicity data on synthesized models of biodegradation intermediates, Table 3, demonstrates the effect of carboxylation, or primary biodegradation, on reducing LAS toxicity. The 24 hr Daphnia

LCso value for the C11 increased from .15.8 (LAS to 355 mg/l. (SOU). As biodegradation continues down the chain with removal of 2 carbons at each step there is a further reduction in toxicity. The 4 and 5 carbon short chain carboxylates had 24 hr Daphnia LCso concentration of ~12,000mg/l. These data clearly confirm that biodegradation reduces the inherent toxicity of LAS by the formation of intermediates of less toxicity than intact LAS. IOC

IC

\

L

"\ % %

E

\

\

%

o.

% 0% %'%%

i,¢ --

o Daphnia • Fotheod minnow

x

\x xe

DTIS ....

o,

,b

LA S

i

,L

Carbon chain length

Fig. 3. Acute toxicity of linear alkylbenzene sulfonate (LAS), individual chain lengths C10 through C1,, and nonlinear components in commercial LAS, the dialkyl tetralin/ indane sulfonates, C1o, C12, and C~, to Daphnia moona and fathead minnow.

36

RICHARD A. KIMERLEand R. D. SWlSlaER 1O© - -

90

/

80

,io;X'-.

"

70

It a ,"

6O c o 5o o o',

~

so

en 30

20

--

5

I0

15 24h,

2O t.c~o,

25

40

rng t -~

Fig. 4. Acute toxicity to Daphnia magna of effluent from laboratory continuous flow activated sludge unit being fed commercial LAS mixed bomologs • and C t a LAS pure homolog <3 biodegraded 41)-87% below initial Hellig¢ MBAS concentration. Commercial LAS usually contains a few percent of non-linear compounds with carbon chains in the same range as LAS. Table 3 and Fig. 3 show these dialkyl tetralin/indane sulfonates (DTIS) to be less toxic than LAS of the same chain length.

Acute toxicity of partially degraded LAS Approximately 100 acute Daphnia toxicity tests were performed on effluent samples from the laboratory continuous flow activated sludge unit. Most of these samples were degraded beyond the point of having enough toxicity to produce significant mortalities and LCso estimates. However, it was possible to obtain approximately 35 valid Daphnia LCso estimates by diluting effluent samples with well water to several concentrations of Hellige MBAS. In Fig. 4 the black squares clearly show that the toxicity of partially degraded mixed homolog/isomer commercial LAS is a function of the extent of biodegradation. The toxicity of the original LAS, in effluent from the control unit, yielded- Daphnia LCso concentrations of approximately 3 mg/l. Upon partial biodegradation to 5000 of initial MBAS concentration the LCso values increased to 6--8 mg/1. Further biodegradation of the LAS. 80-90% below initial concentration, resulted in LCso values being increased to 20-35 mg, 1. There were 3 major reasons for this reduction in toxicity: (i) there was a complete loss in the more toxic 2-, 3- and 4-phenyl isomers at 85~'~obiodegradation, (ii~ after 80-90% biodegradation there was a significant reduction in the more toxic longer alkyl chain lengths, and (iii) only one-third of the measured

MBAS was LAS and two-thirds was presumably from non-LAS products of biodegradation. It can be assumed that some of the non-LAS Hellige MBAS consisted of long chain carboxylates which are much less toxic than the intact LAS. In Fig. 4 the circles represent data points when the feed LAS was the pure homolog of 13 carbons. The observed shift in the L¢5o estimates, from approximately 3 mg/l. for the original to 9-14 mg/l. for 85~o biodegradedCt3 LAS, are not as dramatic as the LCso values derived from mixed homolog commercial LAS. The reason is that the differences in toxicity between the isomers of a given chain length of LAS are much less than the differences in toxicity from one chain length to another. The LAS remaining after partial degradation of the C~3 pure homolog is still C13 chain length, albeit with a preponderance of the somewhat less toxic isomers. In contrast, the partially degraded commercial product may contain a preponderance of the much less toxic lower homoiogs as well. Gas chromatographic data in Table 2 show that the loss of the 2-, 3- and 4-phenyl isomers takes place prior to a major shift in the homolog distribution. Figure 4 demonstrates the toxicological effect of this shift. The LCso values for the 50-85°0 biodegraded pure and mixed homolog LAS ovdrlap each other with the pure homolog LC~o values tending to be on the lower side of the mixed homolog LCso values. At 85°,0 biodegradation they do not overlap. The toxicity of the mixed homolog LAS has been reduced to a much greater extent because of the shift toward shorter, less toxic chain lengths becoming significant with greater than 85% biodegradation. SUMMARY

AND

CONCLUSIONS

A homologous series of linear alkylbenzene sulfonates (LAS) with individual carbon chain lengths of 10 to 14, three non-linear components of commercial LAS (dialkyl tetralin-indane sulfonates Clo, C12 and C14), a commercial LAS blend with an average carbon chain length of 13.3, and three synthesized models of carboxylated biodegradation intermediates with carbon chain lengths of 4, 5 and 11 were tested for toxicity to Daphnia magna and fathead minnows. The toxicity of partially degraded LAS, obtained from a laboratory continuous flow activated sludge unit, was also tested for its toxicity to Daphnia ma#na. (1) The most important factor influencing the acute toxicity of intact LAS samples was the length of the alkyl carbon chain. The 24 hr Daphnia and fathead minnow tCso values ranged from around 1 to around 50mg/l. for the Ct4 to the Cto pure homotogs respectively. (2) The non-linear, dialkyl tetralin-indane sulfonate components of commercial LAS showed only 1,2-1/10 the toxicity of LAS samples of the same carbon chain length. (3) The acute toxicity of synthesized models of biodegradation intermediates (long and short chain car-

Linear alkylbenzene sulfonate (LAS) by biodegradation boxylates) was significantly less than ilatact LAS. The 24 hr Daphnia LCs0 concentrations were 355 mg/1. and around 12,000 mg/l. for the Cla and Ca intermediates respectively. Fathead minnow results yielded similar ranges in toxicity. (4) Partially biodegraded LAS was obtained in the effluent from a laboratory continuous flow activated sludge unit operated under stress loading conditions. Desulfonation/gas chromatography of the effluent confirmed that the longer chain homologs and more terminal isomers were the first constituents of the LAS mixture to degrade. The 2-, 3- and 4-phenyl isomers were generally absent in 50--80% degraded samples, while the homolog distribution underwent a sitmificant shift to lower chain lengths only in samples degraded above 80%. (5) Daphnia acute toxicity tests on partially degraded LAS demonstrated that on the basis of Hellige MBAS the 24 hr LCso values had increased from 3 mg/l. to approx 35 rag/1, with 87% degraded LAS. It was possible to attribute the loss in per unit weight MBAS toxicity to the preferential faster rate of biodegradation of the more toxic longer chain lengths and more terminal isomers and to the presence of non-LAS MBAS. (6) Because of their non-specific nature, MBAS analytical methods cannot be used as a basis in establishing water quality criteria and standards relating to LAS. First, toxic limits determined with LAS samples of differing homolog distribution may differ markedly even though their MBAS content may be the same. Second, MBAS analysis of environmental samples is irrelevant not only with respect to LAS homolog distribution, but also with respect to LAS content since many other materials of widely differing aquatic toxicity do give MBAS response. Acknowledgements--The authors wish to express their appreciation to Dr. S. G. Clark, J. F. Schejbal and J. B. HiUard for providing the samples needed to conduct this study; ~md to R. M. Schroeder-Comotto, A. F. Werner, L. K. Reiner, and I~1.A. Ferrell for assistance in the biological work.

REFERENCES

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