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River-water biodegradation of surfactants in liquid detergents and shampoos
War. Rex. Vol. 25, No. l l, pp. 1425-1429, 1991 Printed in Great Britain. All fights reserved
0043-1354/91 $3.00+0.00 Copyright © 1991 PergamonPress pie
RIVER-WATER BIODEGRADATION OF SURFACTANTS IN LIQUID DETERGENTS AND SHAMPOOS G. C. OKPOKWASILIand A. O. OLISA Department of Microbiology, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Nigeria (First received April 1988; accepted in revised form April 1991)
Abstract--Biodegradabilitiesof surfactants in four detergents namely SDS, Teepol, Apollo, Spencer and Triton X-100, and two shampoos, Flex and Rainbow, were assessed using the river-water die-away method. The shampoos underwent a more rapid primary biodegradation than the liquid detergents. The ease of degradation over a 12-day period followed the order--SDS (97%), Rainbow (85%), Flex (79%), Spencer (79%), Apollo (77%), Triton X-100 (63%) and Teepol (59%) and appears to be related to their sulphate concentrations. A microbial consortium comprising the following genera--Vibrio, Flavobacterium, Klebsiella, Pseudomonas, Enterobacter, Bacillus, Escherichia, Shigella, Citobacter, Proteus and Anaebena--were found to effect the degradation. The detergents and shampoos supported microbial growth in the following decreasing order; Teepol, Spencer, Flex, Rainbow, Apollo and Triton X-100. It is concluded that the detergent or shampoo that supports the most microbial growth is not necessarily the most easily degraded. Key words--primary biodegradation, surfactant, shampoo, detergent, river-water, microbial consortium
INTRODUCTION The need for biodegradability studies comes especially as a result of consumer use and disposal patterns of detergent chemicals prior to discharge into rivers and estuaries. Though these chemicals are presumed to undergo degradation as a result of the metabolic activities of aquatic microbial communities, the occurrence and overall importance of the degradation of xenobiotic organic compounds in the Nigerian environment has not received any appreciable attention in the literature, particularly for chemicals used in synthetic detergent products (Okpokwasili and Nwabuzor, 1988), This paucity of attention is surprising considering that the aquatic environment is not a limitless dump and that certain chemicals bioaccumulate in the food chain. Larson and Payne (1980) have observed that considerable biodegradation activity resides in surface water environments for some detergent chemicals. Detergent formulations for personal and domestic use (e.g. laundry detergents, hair shampoos, washingup and dishwashing liquids) are designed to combine good foaming properties with low skin irritancy (Shore and Berger, 1976). Important surfactant components of such products include alcohol ether sulphate and alkyibenzene sulphonates. Griffiths et al. (1986) have reported that sodium dodecyltriethoxy[35S]sulphate (SDTES), either pure or as a component of commercial detergent mixtures underwent rapid primary biodegradation by mixed bacterial cultures in screen and river-water die-away tests. Whenever [35S]SDTES was a component of a commercial mixture, there was complete mineralization to 35SO[- within a month (Griffiths et al., 1986).
Biodegradability tests for assessing the environmental acceptability of synthetic compounds usually employ mixed cultures of various microorganisms. These tests are known to fall into two groups (Gilbert and Watson, 1977; Gilbert, 1979); biodegradability potential tests, which indicate the susceptibility of the chemical to microbial degradation; and simulation tests (e.g. river-water die-away tests), which provide information about rates of biodegradation under relevant environmental conditions (Swisher, 1987). The present study was undertaken to extend the earlier work with powdered detergents (Okpokwasili and Nwabuzor, 1988) and especially to determine whether the suffactant components of liquid detergents and shampoos exhibit the same persistence tendencies in river-water die-away conditions. MATERIALS AND METHODS
Sources of samples The river-water sample used in this study was obtained from the New Calabar river located about 200 m west oftbe University of Port Harcourt, Choba Park. Tap-water samples were taken from the laboratory. The liquid detergents were obtained from the following sources: Teepol was a kind donation from National Oil and Chemical Marketing Company; the other liquid detergents--Apollo, Spencer, Triton X-100, and shampoos, Flex and Rainbow--were purchased from the store. Sodium dodecyl sulphate (SDS), obtained from Sigma Chemical Company, St Louis, Me.. U.S.A., was employed as standard. Bacterial counts Samples of the river- and tap-water were serially diluted before inoculating them onto nutrient agar plates in quadruplicates. The plates were then incubated at room temperature for 48 h before total aerobic beterotrophic bacterial counts were taken.
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G.C. OKPOKWASILIand A. O. OL|SA
The numbers of bacteria in New Calabar river- and tap-water samples that could grow on an agar medium containing a test liquid detergent or shampoo as sole carbon source were investigated. The agar medium contained per litre of distilled water: NaC1, 5.0g; KCI, 0.6g; MgSO4.7H~O, 7.0 g; NH4NO3, 1.0g; detergent (or shampoo), 10.0 ml; agar, 20.0 g. Serial dilutions of the river-water samples were obtained and 0.1 ml of the dilutions were plated out in quadruplicate onto the detergent (or shampoo) agar medium. The same procedure was used for tap-water samples. The plates were then incubated for 48 h at room temperature. Organisms growing on the plates were counted, isolated and then stored for characterization and identification.
MBAS determination and primary biodegradation test The method of determining the concentration of methylene blue active substances (MBAS) in the detergents and shampoos was that adapted from Standard Methods for the Examination of Water and Wastewater (APHA, 1985). This involved the preparation of a series of ten separatory funnels for each of the test detergents and shampoos. Each series of funnels contained different volumes, 1.0, 3.0, 5.0, 7.0, 9.0, 11.0, 13.0, 15.0 and 20.0ml of solutions of the test liquid detergents and shampoos each made up to 100ml with deionized water such that, with the exception of Teepol, the concentrations of detergents and shampoos in the above solutions were 0.76, 2.28, 3.8, 5.32, 6.84, 8.36, 9.88, 11.4 and 15.2#g/ml respectively. In the case of Teepol and SDS, dilutions were prepared such that the concentrations of detergent in the resultant solutions after making up to 100ml with deionized water corresponded to 0.05, 0.15, 0.25, 0.35, 0.45, 0.55, 0.65, 0.75 and 1.0 gg/ml respectively. The lower Teepol concentration was because it contained more surfactant than the other detergents under test. The tenth funnel in each series contained no detergent or shampoo and served as control. The solutions of detergents in each series of separatory funnels were made alkaline by adding 1 N NaOH, using 1 drop of 1% phenolphthalein solution as indicator to obtain a change in colour from colourless to pink. Then I N H~SO4 was added in drops to make the solution acidic thereby reverting the colour from pink to colourless. Thereafter, 10ml of chloroform and 25 ml of methylene blue reagent were added to the funnel after which each funnel was shaken vigorously for 30 s for the contents to mix. The flasks were then kept quiescent for 30 rain for the phases to separate. The chloroform layer was drawn off into a 100ml Erichmeyer flask. Extraction was performed three times employing I0 ml of chloroform each time. All extracts were pooled in the 100 ml Erlenmeyer flask. Extracts collected were later transferred back to the separatory funnels and 50 ml wash solution were added to each funnel. The funnels were vigorously shaken for 30 s after which they were allowed 30 min to settle before the chloroform layer was drawn off through glass wool into 50 ml volumetric flasks. The chloroform extracts were finally shaken to ensure uniform mixing. Absorbance measurements of the extracts was done using a Hitachi Model 100--20u.v.-visible spectrophotometer (Ogawa Seiki Co. Ltd, Japan) set at 652 nm wavelength against a blank of chloroform. The concentrations of the surfactants present in the test liquid detergents and shampoos in terms of methylene blue active substances (MBAS) were then plotted against the absorbance readings of the various extracts to obtain a calibration curve for each test detergent or shampoo. The results obtained with SDS served as standard. To determine primary biodegradation of the liquid detergents and shampoos in the river-water die-away tests, six Erlenmeyer flasks each holding 1000 ml of freshly collected New Calabar river-water containing 15.2 #g/ml in the case of Apollo, Spencer, Triton X-100, Flex and Rainbow, and 1.0 #g/ml in the case of Teepol and SDS were set up. The
solutions were placed in plugged 2-1. Erlenmeyer flasks which were thereafter left quiescent under room temperature (28 + 2°C). One hundred millilitres were drawn from each flask as described above. The residual surfactant concentration in terms of MBAS for each test detergent or shampoo was read off the calibration curve for the respective detergent or shampoo and used to construct the degradation time courses presented in Fig. 1.
Microbial growth in river-water containing liquid detergents and shampoos Samples were taken from the Erlenmeyer flasks containing suspensions of the liquid detergents or shampoos in river-water for measurement of pH, using a pH meter, and total aerobic viable counts after serial dilution, spread-plating on nutrient agar and incubation at room temperature for 48 h.
Determination of chemical oxygen demand (COD), phosphate (P034 - ) and sulphate (SO~- ) in the liquid detergents and shampoos The method used for the COD determination was the dichromate reflux method adapted from Standard Methods .for the Examination of Water and Wastewater (APHA, 1985) after appropriate dilutions of the test products. Phosphate (PO] - ) and sulphate (SO~ - ) determinations of the detergents and shampoos followed the stannous chloride and turbidimetric methods respectively contained in Water Technology Manual (1975).
Characterization and identification of detergent- and shampoo-utilizing isolates Bacterial isolates were characterized and identified after studying their Gram-staining reaction and cell microIc0 90 8O
~7o E a/_
60.
< 50 r'o :2
40 o
30.
m 20 I0 0
o
~
& T~me
6
,'z
(days)
Fig. 1. Time courses for the degradation of surfactants in liquid detergents and shampoos: Triton X-100 (O); Teepol (O); Apollo (V); Spencer (~); Rainbow (1); Flex (i-q) and SDS (x). The degradation test was carried out at room temperature (28 + 2°C). The points represent means of duplicate determinations and the initial concentrations of MBAS were (in #g/ml): Triton X-100, 1.00; Teepol, 2.00; Apollo, 1.00; Spencer, 1.20; Rainbow, 1.00; Flex, 1.50 and SDS, 4.00.
Biodegradation of fiquid detergents and shampoos morphology. Other tests performed include spore formation, citrate utilization, oxidase and catalase production, methyl red-Voges Proskauer reaction, indole production. oxidative--fermentative utilization of glucose, and appearance on thiosulphate citrate bile salts sucrose (TCBS) agar. The blue-green algae were observed after observation of their wet mounts. These tests were according to the methods of Gerhardt et aL (1981). Schemes for identification of the isolates followed those contained in Bergey's Manual of Systematic Bacteriology (1984). RESULTS
AND
1427
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The number of bacteria in river- and tap-water samples that can utilize the liquid detergents and shampoos as sole carbon sources are presented in Table 1. The numbers indicate that the higher percentage of the heterotrophic population can utilize Teepol, Spencer and Flex in both river- and tap-water samples examined. Rainbow, Apollo and Triton X100 supported lower numbers of microorganisms. The a m o u n t of microbial numbers growing on the detergent agar media may be indicative of either the ease o f biodegradation of the detergent components or the efficacy of the biocides incorporated in the detergent fomulations rather than bioavailability of the surfactant components per se. Table 2 shows the changes in p H and cell numbers as the microbial inoculum in river-water utilized the detergents and shampoos for growth. The total viable counts increased fastest with Tcepol and slowest with Triton X-100. Considering the fact that Triton X-100 is a pure single species product, this result suggests that Triton X-100 (known in the U.K. to be a polyoxyethylene p-t-octyl phenol) is not a readily utilizable carbon source for the microorganisms in the river-water. While the p H of the cultures containing Triton X-100, Apollo, Spencer and Flex fell generally over the 12-day monitoring period, those of cultures containing Teepol and Rainbow showed initial increases before falling. The culture containing the shampoo, Rainbow, actually exhibited p H
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Table 1. Mean counts of detergent- and shampoo-utilizingbacteria in colony forming units per ml River-water Tap-water (total (total heterotrophs heterotrophs Substrate 2.25 x l0s) % 5.0 × 10 4) % Apollo 2.0 x 10~ 8.9 3.3 x 10z 0.7 (I.7-2.5 x 10~) (3.0-3.6 x 10:) Triton X-100 1.0 x l0~ 4.4 6.2 x l0~ 1.2 (0.8-1.3 x 10~) (5.8-6.4 x 10:) Teepol 1.3 x 105 57.8 1.0 x 103 2.0 (1.1-1.6 x 105) (0.9-1.1 x 103) Spencer 1.0 x 10s 44.4 8.3 x 102 1.7 (0.7-1.2 x l0s) (7.8-8.6 x l02) Flex 9.7 x l04 43.1 1.2 x l0 ~ 2.4 (8.5-10.1 x l0~) (0.9-1.4 x l0a) Rainbow 4.0 x l0~ 17.1 5.0 x l02 1.0 (3.6-4.5 x 10~) (4,6-5.5 x 10~) SDS 8.5 x 104 0.4 4.0 x 10~ 0.8 (7.8-9.2 x 10~) (3.6-4.2 x 10~) Percent of production-utilizers/heterotrophic population (heterotrophic population-100%). The data in parentheses represent the ranges of the bacterial counts.
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1428
G . C . OKPOKWASILIand A. O. OLISA
fluctuations. It was possible that the degradation of Triton X-100, Apollo, Spencer and Flex resulted in the production of acidic metabolites. However, the initial breakdown of SDS, Teepol and Rainbow probably resulted in the generation of alkaline intermediates which may have accounted for the initial pH rises. Table 3 shows the COD as well as levels of PO~--P and inorganic SOl- in the test liquid detergents and shampoos. Triton X-100 and Apollo had the highest COD values, Teepol and Flex had intermediate levels, and Rainbow and Spencer the lowest. Teepol contained the lowest amount of phosphate followed in ascending order by Rainbow, Flex, Apollo, Spencer and Triton X-100. SOl- content was highest in the two shampoos, Rainbow and Flex, with lower concentrations in Apollo, Spencer and Triton X-100. No inorganic sulphate was detected in Teepol. The river-water degradation curves for the surfactant components (in terms of methylene blue active substances, MBAS) of the liquid detergents and shampoos tested are presented in Fig. 1. This figure shows that Teepol and Triton X-100 underwent the slowest primary biodegradation, being only about 60% degraded in 12 days while Apollo, Spencer and Flex were about 90% degraded in 12 days. SDS proved the most degradable with about 97% degradation in 12 days. Our success in obtaining methylene blue reaction with Triton X-100 indicates that the product as marketed in Nigeria contains an anionic surfactant. The detergents supporting the highest numbers of bacteria would have been expected to degrade fastest. Though Teepol supported the greatest amount of growth in terms of microbial numbers (Table 1), its rate of biodegradation is low (Fig. 1). On the other hand, Triton X-100 was the poorest supporter of microbial growth and was also very poorly degraded. Hales et al. (1986) in studying the biodegradation of sodium dodecyltriethoxy sulphate (SDTES) by four detergent-degrading bacterial species observed that liberation of SOl- directly from SDTES by two isolates was significant and contributed 30--40% of primary biodegradation of that anionic surfactant. The increase in cell numbers as the natural microbial communities in the river-water samples utilized the detergents and shampoos (Table 2) may be due to the utilization of the surfactants as sole carbon and Table 3. Chemical oxygen demand (COD), phosphatephosphorus (PO]--P) and inorganic sulphate (SO~-) concentrations of the liquid detergents and shampoos Test compound Apollo Triton X-100 Teepol Spencer Flex Rainbow SDS*
COD (mg/l)
POI- -P (,ug/l)
SOl (mg/l)
300,000 760,000 228,000 132,000 172,000 120,000 542,000
42.7 169.0 4.0 95.0 40.0 21.0 ND
3350 750 ND 2350 3450 5450 1250
ND = not detected; *mg/kg.
Table 4. Major taxa of detergent- and shampoo-utilizing bactcrial isolates from river-water
Genus
Frequency of isolation (%)
Vibrio Flavobacterium Klebsiella Pseudomonas Enterobacter Bacillus Shigella Citrobacter Escheriehia Proteus
43.3
Actinomycete
3.3
I 0.0
10.0 6.7 6.7 6.7
3.3 3.3
3.3 3.3
energy sources for growth and metabolic processes. Builders such as sodium tripolyphosphate in the detergents and shampoos may also serve as sources of nutrients for microbial growth. Builders act as water softeners and are an aid to detergent action (Giddings, 1973). Though phosphorus present in the form of sodium tripolyphosphates in heavy-duty liquid detergents may be necessary for growth, the knowledge of the chemical formulation of a detergent or shampoo product may not actually tell if microbial growth and degradation would be expected. Thus, the overall increase in microbial numbers in the riverwater/detergent or shampoo cultures may be attributed to the availability of carbon source for growth as well as the concentration of sulphates in the various detergents and shampoos. Hence, Rainbow, Flex, Spencer and Apollo with high SO~concentrations exhibited higher percentage degradation. This supports the observations of Higgins and Burns (1975) who stated that the relationship between surfactants and microbes is complex and involves factors other than biodegradation and that under appropriate conditions, surfactants can act as bactericides and bacteriostats. However, the ability of a surfactant to be bactericidal depends largely on the microbial species, size of the hydrophobic portion of the surfactant molecule, purity of the water sample in terms of organic matter such as sewage and the presence of divalent metal ions (Higgins and Burns, 1975). The microorganisms found growing on the liquid detergents and shampoos were mainly Gram-negative rods suggesting that they are more tolerant to the surfactant concentrations present in the isolation medium than the Gram-positive organisms, Bacillus and actinomycetes. It has been reported by Higgins and Burns (1975) that many Gram-positive bacteria are noticeably affected by surfactant concentrations of 10-20 ppm while several thousand ppm may be without effect on Gram-negative organisms. The microbial isolates from the river-water capable of utilizing the liquid detergents and shampoos were Vibrio, Flavobacterium Klebsiella, Pseudomonas, Enterobaeter, Bacillus, Eseheriehia, Shigella, Citrobaeter, Proteus, Anaebena and an actinomycete (Table 4). Some of these genera have been reported as capable of assimilating portions of pure anionic surfactant
Biodegradation of liquid detergents and shampoos molecules (Gledhill, 1974) and surfactant components of powdered detergents (Okpokwasili and Nwabuzor, 1988). The isolation of Vibrio as the predominant microorganism capable of utilizing the liquid detergents and shampoos goes to support the increasing role of this bacterial genus in organic molecule biodegradation (West et al., 1984). It is concluded, from the results of this study, that the detergent that supports the highest microbial numbers does not necessarily contain the most readily biodegradable surfactants. Acknowledgements--We would like to thank L. O. Odokuma for technical assistance and T. Jeremiah for typing the manuscript. This work was supported by the University of Port Harcourt Senate Research Grant Awarded to the senior author. REFERENCES
APHA (1985) Standard Methods for the Examination of Water and Wastewater, 16th edition. American Public Health Association, New York. Bergey' s Manual of Systematic Bacteriology (1984) (Edited by Krieg N. R. and Holt J. G.). Williams & Wilkins, Baltimore, Md. Gerhardt P., Murray R. G. E., Costilow R. N., Nester E. W., Wood W. A., Krieg N. R. and Phillips G. B. (1981) Manual of Methods for General Bacteriology. American Society for Microbiology, Washington, D.C. Giddings J. C. (1973) Chemistry, Man and Environmental Change: An Integrated Approach. Canfield Press, San Francisco. Gilbert P. A. (1979) Biodegradability and the estimation of
1429
environmental concentration. Ecotoxicol. envir. Safety 3, 111-115. Gilbert P. A. and Watson G. K. (1977) Biodegradability testing and its relevance to environmental acceptability. Tenside Deter. 11, 171-177. Gledhill W. E. (1974) Linear alkylbenzene sulphonate biodegradation and aquatic interactions. Adv. appl. Microbiol. 17, 265-284. Griffiths E. T., Hales S. G., Russcl N. J., Watson G. K. and White G. F. (1986) Metabolite production during the biodegradation of the surfactant sodium dodecyltriethoxy sulphate under mixed culture die-away conditions. J. gen. Microbiol. 132, 963-972. Hales S. G., Watson G. K., Dodgson K. S. and White G. F. (1986) A comparative study of the biodegradation of the surfactant sodium dodccyltriethoxy sulphate by four detergent-degrading bacteria. J. gen. Microbiol. 132, 953-961. Higgins I. J. and Burns R. G. (1975) The Chemistry and Microbiology of Pollution. Academic Press, London. Larson R. J. and Payne A. G. (1980) Fate of the benzene ring of linear alkylbenzene sulfonate in natural waters. Appl. envir. Microbiol. 41, 621~27. Okpokwasili G. C. and Nwabuzor C. N. (1988) Primary biodegradation of anionic surfactants in laundry detergents. Chemosphere 17, 2175-2182. Shore S. and Berger D. (1976) Alcohol and ether alcohol sulphates. In Surfactant Science Series, Vol. 7, Anionic Surfactants, Part I (Edited by Linfield W. M.), pp. 135-217. Dekker, New York. Swisher R. D. (1987) Surfactant Biodegradation, 2nd edition. Dekker, New York. Water Technology Manual (1975) Milton Roy, New York. West P. A., Okpokwasili G. C., Brayton P. R., Grimes D. J. and Colwell R. R. (1984) Numerical taxonomy of phenanthrene-degrading bacteria isolated from Chesapeake Bay. Appl. envir. Microbiol. 48, 988-998.
0043-1354/91 $3.00+0.00 Copyright © 1991 PergamonPress pie
RIVER-WATER BIODEGRADATION OF SURFACTANTS IN LIQUID DETERGENTS AND SHAMPOOS G. C. OKPOKWASILIand A. O. OLISA Department of Microbiology, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Nigeria (First received April 1988; accepted in revised form April 1991)
Abstract--Biodegradabilitiesof surfactants in four detergents namely SDS, Teepol, Apollo, Spencer and Triton X-100, and two shampoos, Flex and Rainbow, were assessed using the river-water die-away method. The shampoos underwent a more rapid primary biodegradation than the liquid detergents. The ease of degradation over a 12-day period followed the order--SDS (97%), Rainbow (85%), Flex (79%), Spencer (79%), Apollo (77%), Triton X-100 (63%) and Teepol (59%) and appears to be related to their sulphate concentrations. A microbial consortium comprising the following genera--Vibrio, Flavobacterium, Klebsiella, Pseudomonas, Enterobacter, Bacillus, Escherichia, Shigella, Citobacter, Proteus and Anaebena--were found to effect the degradation. The detergents and shampoos supported microbial growth in the following decreasing order; Teepol, Spencer, Flex, Rainbow, Apollo and Triton X-100. It is concluded that the detergent or shampoo that supports the most microbial growth is not necessarily the most easily degraded. Key words--primary biodegradation, surfactant, shampoo, detergent, river-water, microbial consortium
INTRODUCTION The need for biodegradability studies comes especially as a result of consumer use and disposal patterns of detergent chemicals prior to discharge into rivers and estuaries. Though these chemicals are presumed to undergo degradation as a result of the metabolic activities of aquatic microbial communities, the occurrence and overall importance of the degradation of xenobiotic organic compounds in the Nigerian environment has not received any appreciable attention in the literature, particularly for chemicals used in synthetic detergent products (Okpokwasili and Nwabuzor, 1988), This paucity of attention is surprising considering that the aquatic environment is not a limitless dump and that certain chemicals bioaccumulate in the food chain. Larson and Payne (1980) have observed that considerable biodegradation activity resides in surface water environments for some detergent chemicals. Detergent formulations for personal and domestic use (e.g. laundry detergents, hair shampoos, washingup and dishwashing liquids) are designed to combine good foaming properties with low skin irritancy (Shore and Berger, 1976). Important surfactant components of such products include alcohol ether sulphate and alkyibenzene sulphonates. Griffiths et al. (1986) have reported that sodium dodecyltriethoxy[35S]sulphate (SDTES), either pure or as a component of commercial detergent mixtures underwent rapid primary biodegradation by mixed bacterial cultures in screen and river-water die-away tests. Whenever [35S]SDTES was a component of a commercial mixture, there was complete mineralization to 35SO[- within a month (Griffiths et al., 1986).
Biodegradability tests for assessing the environmental acceptability of synthetic compounds usually employ mixed cultures of various microorganisms. These tests are known to fall into two groups (Gilbert and Watson, 1977; Gilbert, 1979); biodegradability potential tests, which indicate the susceptibility of the chemical to microbial degradation; and simulation tests (e.g. river-water die-away tests), which provide information about rates of biodegradation under relevant environmental conditions (Swisher, 1987). The present study was undertaken to extend the earlier work with powdered detergents (Okpokwasili and Nwabuzor, 1988) and especially to determine whether the suffactant components of liquid detergents and shampoos exhibit the same persistence tendencies in river-water die-away conditions. MATERIALS AND METHODS
Sources of samples The river-water sample used in this study was obtained from the New Calabar river located about 200 m west oftbe University of Port Harcourt, Choba Park. Tap-water samples were taken from the laboratory. The liquid detergents were obtained from the following sources: Teepol was a kind donation from National Oil and Chemical Marketing Company; the other liquid detergents--Apollo, Spencer, Triton X-100, and shampoos, Flex and Rainbow--were purchased from the store. Sodium dodecyl sulphate (SDS), obtained from Sigma Chemical Company, St Louis, Me.. U.S.A., was employed as standard. Bacterial counts Samples of the river- and tap-water were serially diluted before inoculating them onto nutrient agar plates in quadruplicates. The plates were then incubated at room temperature for 48 h before total aerobic beterotrophic bacterial counts were taken.
1425
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G.C. OKPOKWASILIand A. O. OL|SA
The numbers of bacteria in New Calabar river- and tap-water samples that could grow on an agar medium containing a test liquid detergent or shampoo as sole carbon source were investigated. The agar medium contained per litre of distilled water: NaC1, 5.0g; KCI, 0.6g; MgSO4.7H~O, 7.0 g; NH4NO3, 1.0g; detergent (or shampoo), 10.0 ml; agar, 20.0 g. Serial dilutions of the river-water samples were obtained and 0.1 ml of the dilutions were plated out in quadruplicate onto the detergent (or shampoo) agar medium. The same procedure was used for tap-water samples. The plates were then incubated for 48 h at room temperature. Organisms growing on the plates were counted, isolated and then stored for characterization and identification.
MBAS determination and primary biodegradation test The method of determining the concentration of methylene blue active substances (MBAS) in the detergents and shampoos was that adapted from Standard Methods for the Examination of Water and Wastewater (APHA, 1985). This involved the preparation of a series of ten separatory funnels for each of the test detergents and shampoos. Each series of funnels contained different volumes, 1.0, 3.0, 5.0, 7.0, 9.0, 11.0, 13.0, 15.0 and 20.0ml of solutions of the test liquid detergents and shampoos each made up to 100ml with deionized water such that, with the exception of Teepol, the concentrations of detergents and shampoos in the above solutions were 0.76, 2.28, 3.8, 5.32, 6.84, 8.36, 9.88, 11.4 and 15.2#g/ml respectively. In the case of Teepol and SDS, dilutions were prepared such that the concentrations of detergent in the resultant solutions after making up to 100ml with deionized water corresponded to 0.05, 0.15, 0.25, 0.35, 0.45, 0.55, 0.65, 0.75 and 1.0 gg/ml respectively. The lower Teepol concentration was because it contained more surfactant than the other detergents under test. The tenth funnel in each series contained no detergent or shampoo and served as control. The solutions of detergents in each series of separatory funnels were made alkaline by adding 1 N NaOH, using 1 drop of 1% phenolphthalein solution as indicator to obtain a change in colour from colourless to pink. Then I N H~SO4 was added in drops to make the solution acidic thereby reverting the colour from pink to colourless. Thereafter, 10ml of chloroform and 25 ml of methylene blue reagent were added to the funnel after which each funnel was shaken vigorously for 30 s for the contents to mix. The flasks were then kept quiescent for 30 rain for the phases to separate. The chloroform layer was drawn off into a 100ml Erichmeyer flask. Extraction was performed three times employing I0 ml of chloroform each time. All extracts were pooled in the 100 ml Erlenmeyer flask. Extracts collected were later transferred back to the separatory funnels and 50 ml wash solution were added to each funnel. The funnels were vigorously shaken for 30 s after which they were allowed 30 min to settle before the chloroform layer was drawn off through glass wool into 50 ml volumetric flasks. The chloroform extracts were finally shaken to ensure uniform mixing. Absorbance measurements of the extracts was done using a Hitachi Model 100--20u.v.-visible spectrophotometer (Ogawa Seiki Co. Ltd, Japan) set at 652 nm wavelength against a blank of chloroform. The concentrations of the surfactants present in the test liquid detergents and shampoos in terms of methylene blue active substances (MBAS) were then plotted against the absorbance readings of the various extracts to obtain a calibration curve for each test detergent or shampoo. The results obtained with SDS served as standard. To determine primary biodegradation of the liquid detergents and shampoos in the river-water die-away tests, six Erlenmeyer flasks each holding 1000 ml of freshly collected New Calabar river-water containing 15.2 #g/ml in the case of Apollo, Spencer, Triton X-100, Flex and Rainbow, and 1.0 #g/ml in the case of Teepol and SDS were set up. The
solutions were placed in plugged 2-1. Erlenmeyer flasks which were thereafter left quiescent under room temperature (28 + 2°C). One hundred millilitres were drawn from each flask as described above. The residual surfactant concentration in terms of MBAS for each test detergent or shampoo was read off the calibration curve for the respective detergent or shampoo and used to construct the degradation time courses presented in Fig. 1.
Microbial growth in river-water containing liquid detergents and shampoos Samples were taken from the Erlenmeyer flasks containing suspensions of the liquid detergents or shampoos in river-water for measurement of pH, using a pH meter, and total aerobic viable counts after serial dilution, spread-plating on nutrient agar and incubation at room temperature for 48 h.
Determination of chemical oxygen demand (COD), phosphate (P034 - ) and sulphate (SO~- ) in the liquid detergents and shampoos The method used for the COD determination was the dichromate reflux method adapted from Standard Methods .for the Examination of Water and Wastewater (APHA, 1985) after appropriate dilutions of the test products. Phosphate (PO] - ) and sulphate (SO~ - ) determinations of the detergents and shampoos followed the stannous chloride and turbidimetric methods respectively contained in Water Technology Manual (1975).
Characterization and identification of detergent- and shampoo-utilizing isolates Bacterial isolates were characterized and identified after studying their Gram-staining reaction and cell microIc0 90 8O
~7o E a/_
60.
< 50 r'o :2
40 o
30.
m 20 I0 0
o
~
& T~me
6
,'z
(days)
Fig. 1. Time courses for the degradation of surfactants in liquid detergents and shampoos: Triton X-100 (O); Teepol (O); Apollo (V); Spencer (~); Rainbow (1); Flex (i-q) and SDS (x). The degradation test was carried out at room temperature (28 + 2°C). The points represent means of duplicate determinations and the initial concentrations of MBAS were (in #g/ml): Triton X-100, 1.00; Teepol, 2.00; Apollo, 1.00; Spencer, 1.20; Rainbow, 1.00; Flex, 1.50 and SDS, 4.00.
Biodegradation of fiquid detergents and shampoos morphology. Other tests performed include spore formation, citrate utilization, oxidase and catalase production, methyl red-Voges Proskauer reaction, indole production. oxidative--fermentative utilization of glucose, and appearance on thiosulphate citrate bile salts sucrose (TCBS) agar. The blue-green algae were observed after observation of their wet mounts. These tests were according to the methods of Gerhardt et aL (1981). Schemes for identification of the isolates followed those contained in Bergey's Manual of Systematic Bacteriology (1984). RESULTS
AND
1427
L) X¢'4
X¢',l x r - -
X~
XO'~
DISCUSSION xrq X~ Xr ~ X~ xos c ~ o "~ "r-.eiv~r-i
The number of bacteria in river- and tap-water samples that can utilize the liquid detergents and shampoos as sole carbon sources are presented in Table 1. The numbers indicate that the higher percentage of the heterotrophic population can utilize Teepol, Spencer and Flex in both river- and tap-water samples examined. Rainbow, Apollo and Triton X100 supported lower numbers of microorganisms. The a m o u n t of microbial numbers growing on the detergent agar media may be indicative of either the ease o f biodegradation of the detergent components or the efficacy of the biocides incorporated in the detergent fomulations rather than bioavailability of the surfactant components per se. Table 2 shows the changes in p H and cell numbers as the microbial inoculum in river-water utilized the detergents and shampoos for growth. The total viable counts increased fastest with Tcepol and slowest with Triton X-100. Considering the fact that Triton X-100 is a pure single species product, this result suggests that Triton X-100 (known in the U.K. to be a polyoxyethylene p-t-octyl phenol) is not a readily utilizable carbon source for the microorganisms in the river-water. While the p H of the cultures containing Triton X-100, Apollo, Spencer and Flex fell generally over the 12-day monitoring period, those of cultures containing Teepol and Rainbow showed initial increases before falling. The culture containing the shampoo, Rainbow, actually exhibited p H
o
o ao
._.q
I
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rr
== o, ~
==
,~ ~ o, ~,~ ~" ,, ~"
"r. x~
x~.
x~.
xo.
x~.
~ ~-4"~
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Table 1. Mean counts of detergent- and shampoo-utilizingbacteria in colony forming units per ml River-water Tap-water (total (total heterotrophs heterotrophs Substrate 2.25 x l0s) % 5.0 × 10 4) % Apollo 2.0 x 10~ 8.9 3.3 x 10z 0.7 (I.7-2.5 x 10~) (3.0-3.6 x 10:) Triton X-100 1.0 x l0~ 4.4 6.2 x l0~ 1.2 (0.8-1.3 x 10~) (5.8-6.4 x 10:) Teepol 1.3 x 105 57.8 1.0 x 103 2.0 (1.1-1.6 x 105) (0.9-1.1 x 103) Spencer 1.0 x 10s 44.4 8.3 x 102 1.7 (0.7-1.2 x l0s) (7.8-8.6 x l02) Flex 9.7 x l04 43.1 1.2 x l0 ~ 2.4 (8.5-10.1 x l0~) (0.9-1.4 x l0a) Rainbow 4.0 x l0~ 17.1 5.0 x l02 1.0 (3.6-4.5 x 10~) (4,6-5.5 x 10~) SDS 8.5 x 104 0.4 4.0 x 10~ 0.8 (7.8-9.2 x 10~) (3.6-4.2 x 10~) Percent of production-utilizers/heterotrophic population (heterotrophic population-100%). The data in parentheses represent the ranges of the bacterial counts.
o
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¢q
1428
G . C . OKPOKWASILIand A. O. OLISA
fluctuations. It was possible that the degradation of Triton X-100, Apollo, Spencer and Flex resulted in the production of acidic metabolites. However, the initial breakdown of SDS, Teepol and Rainbow probably resulted in the generation of alkaline intermediates which may have accounted for the initial pH rises. Table 3 shows the COD as well as levels of PO~--P and inorganic SOl- in the test liquid detergents and shampoos. Triton X-100 and Apollo had the highest COD values, Teepol and Flex had intermediate levels, and Rainbow and Spencer the lowest. Teepol contained the lowest amount of phosphate followed in ascending order by Rainbow, Flex, Apollo, Spencer and Triton X-100. SOl- content was highest in the two shampoos, Rainbow and Flex, with lower concentrations in Apollo, Spencer and Triton X-100. No inorganic sulphate was detected in Teepol. The river-water degradation curves for the surfactant components (in terms of methylene blue active substances, MBAS) of the liquid detergents and shampoos tested are presented in Fig. 1. This figure shows that Teepol and Triton X-100 underwent the slowest primary biodegradation, being only about 60% degraded in 12 days while Apollo, Spencer and Flex were about 90% degraded in 12 days. SDS proved the most degradable with about 97% degradation in 12 days. Our success in obtaining methylene blue reaction with Triton X-100 indicates that the product as marketed in Nigeria contains an anionic surfactant. The detergents supporting the highest numbers of bacteria would have been expected to degrade fastest. Though Teepol supported the greatest amount of growth in terms of microbial numbers (Table 1), its rate of biodegradation is low (Fig. 1). On the other hand, Triton X-100 was the poorest supporter of microbial growth and was also very poorly degraded. Hales et al. (1986) in studying the biodegradation of sodium dodecyltriethoxy sulphate (SDTES) by four detergent-degrading bacterial species observed that liberation of SOl- directly from SDTES by two isolates was significant and contributed 30--40% of primary biodegradation of that anionic surfactant. The increase in cell numbers as the natural microbial communities in the river-water samples utilized the detergents and shampoos (Table 2) may be due to the utilization of the surfactants as sole carbon and Table 3. Chemical oxygen demand (COD), phosphatephosphorus (PO]--P) and inorganic sulphate (SO~-) concentrations of the liquid detergents and shampoos Test compound Apollo Triton X-100 Teepol Spencer Flex Rainbow SDS*
COD (mg/l)
POI- -P (,ug/l)
SOl (mg/l)
300,000 760,000 228,000 132,000 172,000 120,000 542,000
42.7 169.0 4.0 95.0 40.0 21.0 ND
3350 750 ND 2350 3450 5450 1250
ND = not detected; *mg/kg.
Table 4. Major taxa of detergent- and shampoo-utilizing bactcrial isolates from river-water
Genus
Frequency of isolation (%)
Vibrio Flavobacterium Klebsiella Pseudomonas Enterobacter Bacillus Shigella Citrobacter Escheriehia Proteus
43.3
Actinomycete
3.3
I 0.0
10.0 6.7 6.7 6.7
3.3 3.3
3.3 3.3
energy sources for growth and metabolic processes. Builders such as sodium tripolyphosphate in the detergents and shampoos may also serve as sources of nutrients for microbial growth. Builders act as water softeners and are an aid to detergent action (Giddings, 1973). Though phosphorus present in the form of sodium tripolyphosphates in heavy-duty liquid detergents may be necessary for growth, the knowledge of the chemical formulation of a detergent or shampoo product may not actually tell if microbial growth and degradation would be expected. Thus, the overall increase in microbial numbers in the riverwater/detergent or shampoo cultures may be attributed to the availability of carbon source for growth as well as the concentration of sulphates in the various detergents and shampoos. Hence, Rainbow, Flex, Spencer and Apollo with high SO~concentrations exhibited higher percentage degradation. This supports the observations of Higgins and Burns (1975) who stated that the relationship between surfactants and microbes is complex and involves factors other than biodegradation and that under appropriate conditions, surfactants can act as bactericides and bacteriostats. However, the ability of a surfactant to be bactericidal depends largely on the microbial species, size of the hydrophobic portion of the surfactant molecule, purity of the water sample in terms of organic matter such as sewage and the presence of divalent metal ions (Higgins and Burns, 1975). The microorganisms found growing on the liquid detergents and shampoos were mainly Gram-negative rods suggesting that they are more tolerant to the surfactant concentrations present in the isolation medium than the Gram-positive organisms, Bacillus and actinomycetes. It has been reported by Higgins and Burns (1975) that many Gram-positive bacteria are noticeably affected by surfactant concentrations of 10-20 ppm while several thousand ppm may be without effect on Gram-negative organisms. The microbial isolates from the river-water capable of utilizing the liquid detergents and shampoos were Vibrio, Flavobacterium Klebsiella, Pseudomonas, Enterobaeter, Bacillus, Eseheriehia, Shigella, Citrobaeter, Proteus, Anaebena and an actinomycete (Table 4). Some of these genera have been reported as capable of assimilating portions of pure anionic surfactant
Biodegradation of liquid detergents and shampoos molecules (Gledhill, 1974) and surfactant components of powdered detergents (Okpokwasili and Nwabuzor, 1988). The isolation of Vibrio as the predominant microorganism capable of utilizing the liquid detergents and shampoos goes to support the increasing role of this bacterial genus in organic molecule biodegradation (West et al., 1984). It is concluded, from the results of this study, that the detergent that supports the highest microbial numbers does not necessarily contain the most readily biodegradable surfactants. Acknowledgements--We would like to thank L. O. Odokuma for technical assistance and T. Jeremiah for typing the manuscript. This work was supported by the University of Port Harcourt Senate Research Grant Awarded to the senior author. REFERENCES
APHA (1985) Standard Methods for the Examination of Water and Wastewater, 16th edition. American Public Health Association, New York. Bergey' s Manual of Systematic Bacteriology (1984) (Edited by Krieg N. R. and Holt J. G.). Williams & Wilkins, Baltimore, Md. Gerhardt P., Murray R. G. E., Costilow R. N., Nester E. W., Wood W. A., Krieg N. R. and Phillips G. B. (1981) Manual of Methods for General Bacteriology. American Society for Microbiology, Washington, D.C. Giddings J. C. (1973) Chemistry, Man and Environmental Change: An Integrated Approach. Canfield Press, San Francisco. Gilbert P. A. (1979) Biodegradability and the estimation of
1429
environmental concentration. Ecotoxicol. envir. Safety 3, 111-115. Gilbert P. A. and Watson G. K. (1977) Biodegradability testing and its relevance to environmental acceptability. Tenside Deter. 11, 171-177. Gledhill W. E. (1974) Linear alkylbenzene sulphonate biodegradation and aquatic interactions. Adv. appl. Microbiol. 17, 265-284. Griffiths E. T., Hales S. G., Russcl N. J., Watson G. K. and White G. F. (1986) Metabolite production during the biodegradation of the surfactant sodium dodecyltriethoxy sulphate under mixed culture die-away conditions. J. gen. Microbiol. 132, 963-972. Hales S. G., Watson G. K., Dodgson K. S. and White G. F. (1986) A comparative study of the biodegradation of the surfactant sodium dodccyltriethoxy sulphate by four detergent-degrading bacteria. J. gen. Microbiol. 132, 953-961. Higgins I. J. and Burns R. G. (1975) The Chemistry and Microbiology of Pollution. Academic Press, London. Larson R. J. and Payne A. G. (1980) Fate of the benzene ring of linear alkylbenzene sulfonate in natural waters. Appl. envir. Microbiol. 41, 621~27. Okpokwasili G. C. and Nwabuzor C. N. (1988) Primary biodegradation of anionic surfactants in laundry detergents. Chemosphere 17, 2175-2182. Shore S. and Berger D. (1976) Alcohol and ether alcohol sulphates. In Surfactant Science Series, Vol. 7, Anionic Surfactants, Part I (Edited by Linfield W. M.), pp. 135-217. Dekker, New York. Swisher R. D. (1987) Surfactant Biodegradation, 2nd edition. Dekker, New York. Water Technology Manual (1975) Milton Roy, New York. West P. A., Okpokwasili G. C., Brayton P. R., Grimes D. J. and Colwell R. R. (1984) Numerical taxonomy of phenanthrene-degrading bacteria isolated from Chesapeake Bay. Appl. envir. Microbiol. 48, 988-998.