Comparative biodegradability of anionic surfactants in synthetic and natural test systems

Chemos.phere, Voi.22, Nos.9-10, Printed in Great Britain

pp 873-880,

1991

0045-6535/91 $3.00 + 0.00 Pergamon Press plc

COMPARATIVE BIODEGRADABILITY OF ANIONIC SURFACTANTS IN SYNTHETIC AND NATURAL TEST SYSTEMS W. E. Gledhill*, M. L. Trehy and D. B. Carson Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO, USA

ABSTRACT

Studies on the biodegradability of representative anionic surfactants using the Modified OECD Screening Test indicated deficiencies in the test for these materials. Biodegradation studies in natural ecosystems employing realistic substrate concentrations were found to contribute to a better understanding of the actual disappearance in the environment. INTRODUCTION Results from microbial degradation studies are being used to a much greater extent than previously in the international regulation of industrial chemicals. Significant impact on commercialization and use of specific chemicals is based on the interpretation of relatively simplistic biodegradation tests such as the OECD ready biodegradation tests 1. Currently, a vast amount of such biodegradation data is being generated and concern exists that these data are not properly being used in the assessment of environmental safety2 .

The literature is replete with data concerning the rate and extent of surfactant biodegradation in a wide variety of test systems 3.

Conclusions are drawn as to the biodegradability of chemicals in nature often without

knowledge of the limitations of the various biodegradation tests. For synthetic test systems (e.g. OECD ready biodegradation tests), these limitations include nutrient deficiencies, artificially high substrate concentrations, substrate toxicity, low microbial populations, and lack of natural environmental compartments. Technical weaknesses for synthetic test systems have been documemted 4,5,6.

While certain of these limitations can

occur for natural test systems, the main criticism of such systems is for the lack of inter-laboratory reproducibility. Understanding these potential limitations is the key to the design of environmentally relevant biodegradation test procedures. Some reports have shown that laboratory biodegradation results can differ from field results 7; however, many studies have established that relatively simple biodegradation test systems which contain natural water and/or

873

874

sediments yield environmentally relevant kinetic data 2. For example, Spain et al 8 found that biodegradation of p-nitrophenol in laboratory test systems containing pond water and sediment was very similar to that generated in actual ponds. While many biodegradation studies have been reported in natural waters, the role of sediments in these systems has become recognized as important. Sediments can either increase, decrease or not alter rates of microbial biodegradation 9. The present study was conducted to establish and document reasons for the variability in the rate and extent of anionic surfactant biodegradability in synthetic (specifically the Modified OECD Screening Test) test systems and to compare these data to biodegradation results in natural (river water/sediment) test systems. MATERIALS AND METHODS Test Materials Commercial linear alkylbenzenesulphonate (LAS) products obtained from Monsanto were A-215 LAS, A-227 LAS and A-230 LAS (average carbon chain lengths of 11.4, 12.3 and 13.2, respectively). 14C-benzene ring labelled sodium n-dodecylbenzene sulfonate (sp. act. 26.8 mCi/mmol) and 14C-radiolabelled glucose (sp. act. 3.7 mCi/mmol) were used for mineralization (14CO 2-

evolution) studies in natural water/sediment systems.

Other anionic surfactants including tallow methyl ester sulfonate (MES), alcohol ethoxysulfate, C14_16olefin sulfonate, lauryl sulfate, 1-octane sulfonate and lignosulfate were obtained as research samples from Stepan Chemical Company and not characterized further. Glucose, sodium acetate and p-chlorophenol were obtained from Sigma Chemical Company. Biode~radation Test Procedures The Modified OECD Screening test followed the protocol of OECD Method 301E.:} Duplicate flasks containing 1 L of synthetic medium and A-227 LAS or MES at concentrations of 5, 20 and 40 mg/L as dissolved organic carbon (DOC) were inoculated with 0.5 mL effluent from a laboratory semi-continuous activated sludge unit receiving natural sewage.

Primary biodegradation was assessed by MBAS analysis, while ultimate

biodegradation was determined by DOC analysis following centrifugation using a OI Corporation Model 700 total organic carbon analyzer. For microcosm studies, river water and sediment (top 2 cm) were collected from the Illinois River at a site about 10 miles north of St. Louis. Water was passed through a 5 um Nucleopore filter and sediment through a 1.7 mm steel mesh screen. Some sediments were sterilized via autoclaving for 1 hr. Microcosms consisted of 50 mL Erlenmeyer flasks sealed with a rubber septum containing a glass center well with 0.3 mL 2.0 N KOH to trap14CO2 . River water (20 mL) alone or with sediment (1.0 g dry wt.) was added to the flask along with either 100 or 1150 ug/L radiolabeled C12LAS or 180 ug/L radiolabeled glucose. Flasks were incubated in the dark at 22°C on a rotary shaker at 100 rpm. Triplicate flasks were sacrificed and analyzed for LAS and LAS biodegradstrm intermediates over a 2 week period.

875

Thymidin¢ uotake orocedure The Modified OECD Test medium and inoculum were added to 22 mL scintillation vials to assess toxicity of test materials. Glucose, p-chlorophenol and anionic surfactants were added at concentrations ranging from 10 ug/L to 500 mg/L. Sterile vials containing 2% formaldehyde were also included as controls. Methyl-tritiated Thymidine (43.5 Ci/mmol) was added at a final concentration of 15 nM. After 90 min. incubation at 22 °C, thymidine uptake was determined by the procedure of Gledhill 1°. EC-20, the concentration causing a reduction in thymidine uptake by 20%, was determined by regression analysis. Differences of 5 mg/L are considered significant. Analytical measurements The analytical procedure of Matthijs and DeHenaullwas used to examine the fate of LAS and LAS metabolic intermediates in sediment and water fractions. Water and water/sediment systems were centrifuged at 4*C in 30 mL Corex tubes. The supernatant was passed through an octyl (C8 ) reversed phase column (Analytichem). To elute LAS polar metabolites, the column was eluted with 5.0 mL water followed by 3.0 mL 40% methanol. These fractions were combined, evaporated to dryness, redissolved in 1.0 mL 30% acetonitrile and assayed for radioactivity via scintillation counting and for LAS by HPLC analysis. Intact LAS was eluted with 5.0 mL of 100% methanol and assayed similarly. The sediment pellet was resuspended in 20 mL methanol and sonicated for 24 h and centrifuged. The methanol extract was removed to a clean vial and the pellet rinsed with two 5.0 mL volumes of methanol. Methanol extracts were combined and passed through a preparative anion exchange resin (SAX - Analytichem). The SAX column was washed with 10 mL methanol and eluted with 2.0 mL 20% HC1 in methanol solution. Aliquots were assayed via scintillation counting. The acidic methanol eluate was diluted to 30 mL, neutralized and passed through a C 8 reversed phase column. The column was eluted as described above for the water samples. Residual 14C activity on sediments was assayed via combustion with a Packard Model 306 Tri-carb sample oxidizer followed by liquid scintillation counting of the evolved14CO2 . For some microcosm studies, LAS and metabolic intermediates were also assayed by an improved GC/MS procedure 12. The procedure involves a two-step derivitization process with PC1 and trifluoroethanol to form gas chromatographical derivatives of LAS and LAS metabolites (sulfophenyl carboxylates) which can be analyzed at low ug/L concentrations. RESULTS AND DISCUSSION Primary and ultimate biodegradation of LAS and MES are summarized in Tables 1 and 2. After 28 days, greater than 99% MBAS removal (primary biodegradation) was observed for MES at all test concentrations, while LAS displayed 95% MBAS removal only at the 5 mg/L DOC concentration. Little or no primary biodegradation was observed for LAS at the 20 and 40 mg/L DOC concentrations. Ultimate biodegradation (DOC removal) was not observed for either anionic surfactant at any test concentration.

The test was

876

considered valid since the positive control, sodium acetate, yielded 94% DOC removal. These two surfactants are known to be readily biodegradable in numerous other biodegradation tests13 and several explanations can be postulated for the poor biodegradation results in the modified OECD screening test.

However, since

surfactants interact with membrane surfaces and the Modified OECD Screening Test employs an extremely low inoculum, toxicity to the microorganisms in the inoculum by artificially high substrate concentrations specified by the test seemed the primary cause.

primary biodegradation

T A B L E 1. A n i o n i c s u r f a c t a n t Surfactant Initial conc. (mg/L as DOC) LAS, C12.3 avg. 5

MBAS conc., Day* 0

3

8.0

7.1

20 40

30.5 75.3

7

14

7.3

6.9

28.9 71.8

21

28

0.3

0.4

31.4 78.2

30.6 74.3

0.0 0.2 0.3

0.0 0.1 0.2

MES, tallow 5 20 40

8.1 28.9 78.0

7.9 28.3 75.2

3.5 15.6 58.3

0.0 0.1 1.1

*values represent average of duplicate flasks

T A B L E 2. A n i o n i c s u r f a e t a n t u l t i m a t e b i o d e g r a d a t i o n Surfactant Initial conc. (mg/L as DOC) Acetate

DOC c o n c . , D a y * 0

3

7

14

21

28

20

19.6

1.7

2.8

1.7

1.6

1.2

LAS, C12.3 avg. 5 20 40

3.6 14.5 20.9

3.5 14.3 26.1

3.5 14.8 26.7

3.7 14.4 25.6

3.1 13.7 25.1

2.5 13.8 23.4

MES, tallow 5

2.0

2.4

2.6

3.4

3.4"*

3.1 **

20 40

6.8 10.8"*

6.0 12.1

7.7 13.0

11.1 20.6

10.7 20.0

8.8 19.1

*values represent average of duplicate flasks **values represent measurementfrom single flask TO examine the possibility of substrate toxicity, the effect of 10 anionic surfactants and 2 reference compounds to thymidine uptake by microorganisms in the OECD inoculum was studied. Table 3 presents EC-20's for these materials and indicates the C12" 3 and C13" 2 average chain length LAS to be the most toxic. These EC-20 values corresponds to a substrate concentration of approximately 12-13 m g / L as DOC and may indicate

877

microbial toxicity of LAS to be responsible for poor biodegradation results in the Modified OECD Screening Test. Concentrations of test materials effecting thymidine uptake did not correlate with those concentrations effecting LAS and MES biodegradation. Thymidine may not be the most sensitive indicator of microbial toxicity.

Jonas et al 14 found thymidine uptake to be more sensitive than viable counts for metal and

organometal toxicity to estuarine microorganismss; however, Pettibone and Cooney15found thymidine uptake to be 2-7 times less sensitive than colony forming ability in measuring methyltin toxicity sediment microorganisms from Boston Harbor. T A m , E 3. Effect of anionic sur~ctants on thymidine uptake by

microorganisms in Modified OECD screening test medium

Chemical LA.S, Cll.4 avg. LAB, C12.3 avg. LAS, C13.2 avg. M:ES, tallow MES, coconut Lignosulfonate Alcohol ethoxysulfate C 14-16 olefin sulfonate Lauryl Sulfate 1-Octane sulfonate Glucose p-Chlorophenol

Concentration, mg/L EC20 39 23 21 38 41 28 65 74 63 140 > 180 26

In addition to biodegradation screening tests in synthetic test systems, biodegradation studies can be conducted in natural ecosystems (microcosms) which may provide results more comparable to those in the environment. 14C-radiolabeled C12 LAS biodegradation was studied in a sediment/water system that might simulate the sediment/water interface of a river or lake. The system contained 20 mL river water, 1 gm sterile sediment and 100 ppb LAS. Sterile sediment was added to provide a surface for microbial activity. The Matthijs and DeHenau 11procedure was used to assess LAS and LAS metabolite transformation in the system. Tables 4 and 5 summarize the results for the sterile control and biologically active systems, respectively. Overall radioactivity recovery averaged 97.7% for both systems and the values in the Tables are expressed as the percentage of that radioactivity recovered. In the sterile control (Table 4) LAS partitioned primarily to the sediment (93-95%), with about 3% associated with the water columns and the remainder as apparent sediment metabolites or non extractable residue. In the biologically active system (Table 5), both sediment and water levels of LAS declined rapidly with a half life of 1-3 days. Polar metabolites, which represent both LAS transient biodegradation intermediates and cellular metabolites, appeared in the water column. The sediment residue fraction increased slightly throughout the study and CO 2 evolution proceeded to about 34% over the 12 day period. A similar system containing 180 ppb radiolabeled glucose yielded 64.5 %14CO 2 evolution over the same time period. The study was concluded at this point, but biodegradation of LAS was still continuing.

878

TABLE 4. Fate of LAS and LAS metabolites in sterile sediment/water system % Radioactivity Recovered Day 0 Water Polar metabolites LAS Sediment MeOH extract Residue

3

12

0.9

0.9

0.8

2.4

2.0

1.7

94.7

92.6

93.6

2.0

4.5

3.9

100.0

100.0

100.0

CO 2 Total

Avg. of duplicate samples TABLE 5. Fate of LAS and LAS metabolites in sediment/water system % Radioactivity Recovered*

Water Polar metabolites LAS Sediment MeOH extract Residue CO 2 Total

0

1

Day 3

1.0 2.0

21.5 2.0

37.0 1.1

38.0 0.8

41.0 1.9

93.6 3.4

70.7 4.3

39.0 8.3

26.1 9.1

13.8 9.4

0.0

1.5

14.6

26.0

33.9

100.0

100.0

100.0

6

12

1 0 0 . 0 100.0

Avg. 3 replicates Even with the use of radiolabeled compounds and non specific chemical analyses, questions can still arise about the completeness of biodegradation. For instance in this study, it appears that polar metabolites are formed in the aqueous phase and remain at constant levels for the duration of the study. Actually with the analytical procedure used, differentiation between LAS biodegradation intermediates and cellular metabolites cannot be made.

Therefore, a new analytical technique for simultaneous measurement of both LAS and LAS

biodegradation intermediates (sulfophenyl carboxylates) was used to address this issue~2 The test system was similar to that previously described with the exceptions that biologically active sediment and 1.15 ppm C12LAS were employed. Figure 1 summarizes the results and indicates I_AS to biodegrade rapidly with a half life in the system of about 2 days. C4_ 7

sulfophenyl carboxylates appeared as transient intermediates as LAS

879

and then degraded to the limit of detection by 7 days. Longer chain length intermediates were undoubtedly also formed; however, there appearance was masked by the relatively high LAS levels.

Carbon dioxide

evolution was not followed in this study. Several hypotheses can be presented to explain differences between results in Table 5 and Figure 1. Among these include use of sterile versus non sterile sediment, a ten fold difference in LAS concentration and use of specific versus non specific (extraction/radiochemical) chemical analyses. It was not the purpose of this study to elucidate reasons for such differences, only that biodegradation testing can be a complex issue. moan

15 \

u0m

O12LASu

31

~ 105 ~ f o p h e n y l e a r b o x y l a t e s ( u g )

0

2

4

6 8 T]lVlE (days)

-1~'2 !

10

12

4

Fig. 1. Biodegradation of C12 LAS in River Sediment/Water Environment In summary, our results have documented that there are limitations to all biodegradation tests. Quite often standardized laboratory tests rely on unrealistic conditions (nutrients, substrate concentration, etc.) and, thus, may provide a unrealistic picture of a chemicals biodegradation characteristics in nature. For surfactants, which are known to affect cell membranes at relatively low concentrations, concentrations required by standardized biodegradation tests may elicit toxic responses by the microbes used as the inoculum for these tests. These limitations must be recognized, especially if these data are to be used for regulatory purposes. It must be realized that biodegradation tests which simulate more realistic environmental conditions in terms of ecosystem compartments and test substance concentration can provide a more clear picture of a chemicals' "real world" behavior. However, as was demonstrated in this study, questions concerning the completeness and rate of biodegradation can also be raised in natural test systems. For such situations, proper analytical tools must be brought to bear on the problem. REFERENCES 1.

OECD (1981). Guidelines for Testing of Chemicals. Section 3 - Degradation and Accumulation. Test

Guidelines Nos. 301 A,B,C,D.E; 302 A,B,C; 303 A; 304 A. 2.

Shimp, R. J., Larson, R. J. and Boethling, R. S. (1990). Use of biodegradation data in chemical

assessment. Environ. Sci. Technol. (in press).

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3.

Swisher, R. D. (1987). Surfactant Biodegradation. Marcel Dekker, Inc., New York and Basel.

4.

Means, J. L. and Anderson, S. J. (1981).

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ECETOC (1983). Technical Report No. 8. Biodegradation Testing: An assessment of the present status.

6.

Alexander, M. (1981). Biodegradation of chemicals of environmental concern. Science, 211, 132.

7.

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Proceedings of the Workshop: Microbial Degradation of Pollutants in the Marine

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nitrophenol biodegradation in field and laboratory test systems. Appl. Environ. Microbiol., 48, 944. 9.

Van Loosdrecht, M. C. M., Lyklema, J., Norde, W., and Zehnder, A. J. B. (1988). Influence of interfaces

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Swisher, R. D. 1987. Surfactant Biodegradation. Marcel Dekker, Inc. New York, NY.

14.

Jonas, R. B., Gilmour, C. C., Stoner, D. L., Weir, M. M. and Tuttle J. H. (1984). Comparison of methods

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Pettibone, G. W. and Cooney, J. J. (1988). Toxicity of methyltins to microbial populations in estuarine

sediments. J. Ind. Microbiol., 2, 373. (Received in Germany 22 February1991;

accepted 30 ~ril

1991)