Environ. Sci. Technol. 1983, 17, 268-272
Electroanalytical Determination of the Biodegradation of Nonionic Surfactants Zlata Kozarac, Dubravka Hrgak, and BoZena 6osov16" Rudjer BogkoviC Insitute, Center for Marine Research Zagreb, Zagreb, Yugoslavia
Jasmlna VrZlna
Institute of Public Health of the City of Zagreb, Zagreb, Yugoslavia The biodegradation of different types of nonionic surfactants was studied by the electrochemical method based on the measurement of adsorption effects at the mercury electrode and by the modified Wickbold method. The proposed electrochemicalmethod is simple and rapid and applicable to direct determination of the loss of surfactant activity in the biodegradation testing. The biodegradation degree of nonionic surfactants obtained by the electrochemical method is lower than by the Wickbold method because the latter method does not determine some surface-active intermediates of biodegradation. The applicability of different types of surfactants in commercial detergents is generally considered today on the basis of technical, economical, and ecological aspects. The increased production and use of nonionic surfactants in the last decade raised a need for development of suitable methods for their determination, especially of trace residues of nonionic surfactants in natural waters. Surfactant content of natural waters depends to a great extent on the degree of biodegradability of surfactants. The biodegradability of about 80% for both anionic and nonionic surfactants in detergents is regulated in most developed countries. According to the test method proposed by the Organizationfor Economic Cooperation and Development (OECD) the biodegradation of anionic surfactants is determined by measuring the methylene blue active substances (MBAS) decrease and in the case of nonionic surfactants the bismuth-active substances (BAS) decrease (Wickbold method). It is, however, supposed that by both methods specifically the loss of surface-active substances is measured during the course of the biodegradationtesting (1).
Some specific problems still remained unsolved in the analysis of nonionic surfactants; for example, most of the methods including the Wickbold method are not applicable for the determination of polyethylene glycols and nonionic surfactant with low degrees of ethoxylation. The latter substances are, however, very probably the intermediate products of microbial surfactant degradation (2). Although the increasing importance and greater possibilities were indicated by high-pressure liquid chromatography, mass spectrometry, and nuclear resonance spectroscopy (3),there is still interest in developing simple techniques that indicate detailed, logical, and systematic relationships between structure and biodegradability. Besides, they will be suitable for official test stations as well as for development work by industry. Among simple techniques, the electroanalytical methods, based on the measurement of adsorption phenomena of surfactants at charged electrodes, are widely used for determination of surface-active substances in aquatic solutions and natural aquatic systems (4-10). Some of them seem to be promising for biodegradationtesting procedures (4-6).
The electrochemical method, based on the measurement of the capacity current at the mercury electrode using the Kalousek switch, was proposed as a simple and direct 268
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method for the determination of the biodegradability of alkylbenzenesulfonates (11). Very good agreement between this electrochemical method and the standard MBAS method was achieved in biodegradability tests of linear alkylbenzenesulfonate (LAS) and tetrapropylene alkylbenzenesulfonate (TBS). Nonionic surfactants have already been studied by different electroanalytical techniques, for example, by tensametry (4,121, by measuring polarographic maxima of oxygen (5,13) and of mercury (14), and by the Kalousek commutator technique (6, 14). The aim of this work was to evaluate the advantages and limitations of the Kalousek commutator technique in comparison with the modified Wickbold method for determination of different nonionic surfactants in biodegradation testing. Materials and Methods Surfactants. tert-Octylphenol ethoxylates (Triton-X series) and tert-nonylphenol ethoxylates (Triton-N series) were obtained courtesy of Rohm and Haas (Milano). Primary linear Clbm alcohol ethoxylate with 15 ethylene oxide (EO) groups (LPAE) is a commercial product (Tenzinat 75AA, Chromos, Zagreb) used in washing powders and various detergents. Polyethylene glycols (PEGS) are pure chemicals from Kemika, Zagreb. Microbial Culture. A sample of forest soil taken from the wider Zagreb area that is supposed to be free from the influence of synthetic chemicals was used as the source of microorganisms. The filtrate of soil was prepared according to the OECD method, screening test procedure (15). Synthetic Sewage. This was made according to the OECD method, confirmatory test procedure (15) containing the following (units of mg/L of tap water): Bacto Peptone (Difco), 160; meat extract, 110; urea, 30; NaC1, 7; CaC1p2H20, 4; MgS04.7H20,2; KH2P04,28; and appropriate surfactant concentration. BiodegradationTest Method. The continuous-flow biodegradation method that was used in the biodegradation experiment of anionic surfactants ( 1 1 , 16) was also applied in this work. The continuous flow unit in operation is given in Figure 1. The experiment began by culture adaptation to particular surfactants, Le., 6 weeks of intermittent feeding (24 h on, 24 h off) and 2 weeks of continuous cultivation. During the first 4 weeks of intermittent feeding the synthetic sewage containing 10 mg/L of the particular surfactant (T-X-45, T-X-100, T-X-165, and LPAE respectively) was introduced every second day at a flow rate of 100 mL/h (D= 0.025 h-l). During the remaining 2 weeks of intermittent feeding schedule the concentration of surfactants was increased to 20 mg/L. After that, for a further 2 weeks the culture adaptation was continued by coninuous cultivation at a dilution rate of 0.025 h-' and an initial surfactant concentration of 20 mg/L. Microbial cultures thus adapted to a particular surfactant were used
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Figure 1. Continuous flow unit in operation: A, storage vessel; B, doslng pump; C, aeration section; D, settling section; F, collectlng vessel; G, capillary; H, alr-flow meter.
for the biodegradation experiment. A biodegradation assay was performed in three subsequent series of experiments, with dilution rates of 0.05-0.1 h-’ and initial surfactant concentrations so = 10-100 mg/L. Between the first and the second experiment elapsed 4 weeks and between the second and the third experiment 8 weeks. During that time the cultures were maintained in 500-mL shaken flasks by successive passages every 2 weeks (20% of inoculum) in sterile synthetic sewage with 10 mg/L of the particular surfactant. The concentrations of surfactants in the synthetic sewage and in effluents were determined daily by two methods: (a) precipitation with Dragendorff reagent (modified Wickbold method); (b) the electrochemical method. Samples that were not analyzed within 3 h were preserved by adding 1% HgC12to give a concentration of 50 mg of HgC12/L. The addition of mercuric salt does not interfere with the analytical determination of surfactants by the electrochemical method and with precipitation by means of Dragendorff reagent. Modified Wickbold Method. Nonionic surfactants were isolated from samples by the solvent sublation technique (17) and the extracts reacted with the Dragendorff reagent. The resulting precipitate was filtered off through a sintered crucible (G-4) with glass fiber filter pads (Whatman G-F/A) and dissoved in ammonium tartarate. Bismuth was determined spectrophotometrically by reaction with EDTA according to the Wickbold-Longman procedure (18). Electrochemical Method. Electrochemical determination was performed by the polarographic method of discontinuouslychanging potential known as the Kalousek commutator technique. The basic principle of the method was described in more detail in our previous papers (9,19). Measurements were done by using the instrument for characterization of electrode processes (20) made in our laboratory in connection with a Hewlett-Packard x-y recorder Model 7035B. The commercial instrument is available from Metrohm, Switzerland. Measurements were carried out in a standard 50-mL cell equipped with a two-electrode system. A hanging mercury drop electrode was used as a working electrode. All potentials were referred to the saturated calomel electrode (SCE). The potential scan was applied after accumulation of surfactants at the starting potential (-0.6 V) for 5 min. The decrease of the charging current Ai at the potential -1.4 V vs. SCE was used for quantitative determination of the surfactant. Stirring was not used. Procedure. An aliquot amount of the solution prepared for biodegradability testing containing approximately 10-30 pg of surface-active substances was diluted with sodium chloride and redistilled water to a volume of 50
POTENTIAL / V VSSCE
Flgure 2. Charging current-potential curves of diferent concentrations of PEG 600 and PEG 4000 In 0.55 M NaCI; accumulation time 5 min at E = -0.6 V.
Flgure 3. Charging current-potential curves of different concentrations of Trlton-N-150 and Triton-X-45 in 0.55 M NaCI; accumulation time 5 mln at E = -0.6 V.
mL and a final concentration of 0.55 M NaC1. The content of surfactants was determined from the electrochemical measurement on the basis of the correspondingcalibration curves. The same procedure was repeated for the determination of the remaining surfactants in the effluents collected over 24 h. Reproducibility of independent measurements was *5% at a concentration level of 0.5 mg/L. All measurements were done at room temperature.
Results and Discussion Electroanalytical Determination of Nonionic Surfactants in Aqueous Solutions. The adsorption of nonionic surfactants at the mercury electrode regardless of type and number of ethoxy groups occurs in a wide range of potentials, resulting in the decrease of the capacity current in comparison with the electrolyte without surfactant. Typical capacity current-potential curves without and in the presence of various nonionic surfactants are shown in Figures 2 and 3. The decrease of the capacity current Ai measured at the constant potential (deliberately chosen here as -1.4 V) is a function of the bulk concentration of a surface-active substance and the time of its accumulationat the electrode surface. The Ai vs. concentration plot, i.e., the apparent adsorption isotherm for a chosen accumulation time, can be used as the calibration curve for analytical determination of a surfactant. Typical calibration curves for three Environ. Sci. Technoi., Vol. 17, No. 5, 1983
269
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Table I. Nonionic Detergents Investigated by the Electrochemical Method
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investigated tensides
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different types of nonionic surfactants, obtained with an accumulation period of 5 min controlled by diffusion, are presented in Figure 4. For quantitative determination of the surfactant concentration in solution only the rising part of the calibration curve can be used, which corresponds to approximately 0.1-1 mg of surfactant/L. However, if longer accumulation times are used the calibration curves are shifted toward lower concentrations and for shorter accumulation times, toward higher concentrations. In the case of detergents analysis of effluents and in polluted waters as well as in the course of the biodegradation testing, a 5-min accumulation seems to be a satisfactory procedure for selective determination of a surfactant in the presence of other organic substances that can be themselves adsorbed at the electrode surface only at high concentrations (6, 11,211. Although practically all types of widely used nonionic surfactants can be determined by the proposed electroanalytical method, we have observed certain differences in the sensitivity of the measurement depending on the type of surfactant molecule and the degree of polymerization. The dependencies of the sensitivity of the method on the number of ethoxy groups per molecule for two different series of tensides are given in Figure 5 by plotting the concentration of tenside needed to give the Ai value of 20 pA for the Triton-X series and 10 mA for the PEG series. The adsorbability of Triton-X compounds at the mercury electrode and thus the sensitivity of the electroanalytical determination described herein decrease with increasing number of ethoxy groups per molecule up to 30. Four times higher concentration of T-X-305 is needed to 270
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polyethylene glycols PEG-200 PEG-400 PEG-600 PEG-4000 PEG-10000 tert-octylphenol ethoxylates Triton-X-15 Triton-X-35 Triton-X-45 Triton-X-114 Triton-X-100 Triton-X-102 Triton-X-165 Trito n-X-30 5 Triton-X-405 Triton-X-705 tert-nonylphenol ethoxylates Triton-N-42 Triton-N-60 Triton-N-101 Triton-N-150 primary linear C,,-,,alcohol ethoxylate
no. of ethoxy groups
concn range, mg/L
4-5 9 13-14 90 227
0.5-100 0.05-50 0.05-2 0.1-2 0.2-10
1 3 5 7 -a 9-10 12-13 16 30 40 70
4 6 9-10 15 15
0.05-2
0.1-10 0.1-10
0.05-2 0.1-10
produce the same adsorption effect at the electrode surface as is obtained with T-X-15. We have not observed further change in the sensitivity of the method for Tritons with more than 30 ethoxy groups. For polyethylene glycols there is a much more complicated dependence of the adsorbability at the electrode surface, Le., the sensitivity of the determination to the degree of polymerization. Starting with PEG-200, with 4-5 ethoxy groups the sensitivity increases and approaches the maximum value for PEG 600 with 13-14 groups. After that, there is a further decrease in the sensitivity for higher polyethylene glycoles. The list of nonionic surfactants that were investigated in this work is given in Table I, together with the corresponding concentration ranges suitable for surfactant determination by the proposed method. Biodegradation Study of Nonionic Surfactants, The applicability of the electrochemical method in the biodegradability testing of nonionic surfactanb was studied by using different types of surfactants such as (1)tertiary octylphenol ethoxylates with different ethoxy chain lengths (T-X-45, 5 EO; T-X-100,9-10 EO; T-X-165, 16 EO, (2) primary linear C16-20alcohol ethoxylate with 15 EO, and (3) polyethylene glycol (M, ~ 6 0 0 ) . For the comparison,the initial surfactant concentration in the in-flowing media and the residual surfactant concentration in effluents were also determined by the modified Wickbold method. Since the applicability of the Wickbold method is limited to alkylphenol ethoxylates and alcohol ethoxylates with a chain length between 6 and 30 ethylene oxide groups, the biodegradation of T-X-45 as well as of polyethylene glycol was followed only by the electrochemical method. Results obtained in the biodegradation study of tertoctylphenol ethoxylates with different ethoxy chain lengths are presented in Figure 6 and in Table 11. The biodegradation degree of T-X-45 achieved by the electrochemical method is 53 f 1% , as shown in Figure 6a and Table 11. In the biodegradation experiment of tert-octylphenol ethoxylates with a higher degree of ethoxylation (T-X-100
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Table 11. Biodegradation Degree of Nonionic Surfactants Determined b y the Electrochemical and the Modified Wickbold Methods Wickbold electrochemical method method biodegradation std biodegradation std surfactant degree,a % dev degree,a % dev T-X-45 52.6 1:l T-X-100 17.6 1: 1.3 44.8 21.5 T-X-165 46.2 t3.5 56.4 t4.2 PEG-600 94.6 kO.5 Tenzinat 75 AA I 57.9 i: 2.9 99.7 f 0.3 t0.3 1: 5.5 99.7 I1 35.8 99.7 t 0.3 I11 98.3 i: 0.9 a
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Figure 6. Biogradatlon of fed-octylphenol ethoxylates with different length of ethoxy chain determined by electrochemlcal and modified Wickbold method at given conditions (T-X-45, D = 0.05 h-l, s o = 20 mg/L; 1-X-100; D = 0.05 h-1, so = 10 mg/L; T-X-165, D = 0.1 h-', so = 10 mg/L).
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Flgure 7, Biodegradatlon of primary linear Cle-noalcohol ethoxylate determlned by eiectrochemlcal method In subsequent serles of experiments at given conditions: (I) D = 0.05 h-', s o = 20 mg/L; (11) D = 0.05 h-I, s o = 50 mg/L; (111) D = 0.1 h-l, s o = 100 mg/L. Biodegradatlon data for PEG 600 ('.-') obtained at D = 0.05 h-l and so = 10 mg/L in the third experiment are shown for the comparison.
and T-X-165) in which the disappearance of the test compound was followed by both methods (Figure 6, b and c), generally higher results were observed by the modified Wickbold method than by the electrochemical method. The biodegradation degree of 45 f 2% and 56 f 4% for T-X-100 and T-X-165, respectively, were obtained by the modified Wickbold method under the given experimental conditions. These results are in agreement with some already published data (22) in which 30-50% biodegradation of nonylphenol ethoxylate (9EO) was observed in the confirmatory test of the OECD method. On the contrary some recent papers (2,23)reported a much higher biodegradation degree of that nonionic surfactant in the activated sludge experiment (up to 90%). According to Swisher (24)the main underlying causes for the disagreement and contradiction in the assessment of the biodegradability of alkylphenol ethoxylates are (1) failure to make sufficient provision for bacterial adaptation and (2) lack of specificity of analytical techniques to respond to biodegradation intermediates that still show substantial foaming and other surface activity. If the culture adaptation is so important for the rate and extent of alkyphenol ethoxylates biodegradation, then the relatively low biodegradation degree of tert-octylphenol ethoxylates obtained in this work can be discussed as a failure in the culture adaptation.
Mean values of last 5 days data.
The differences between our results obtained by the modified Wickbold method and the electrochemical method that were observed for both substances T-X-100 and T-X-165 resulted from the different specificities of these methods. The electrochemical method is sensitive to all types of nonionic surfactants regardless of the ethoxylate chain length. Therefore, if the alkylphenol ethoxylates with a lower chain length down to 1-3 EO are the most abundant intermediates formed in the biodegradation of high chain length alkylphenol ethoxylates (23, 24), it is reasonable to obtain lower results of biodegradation by the electrochemical method than by the modified Wickbold method. The failure of the Wickbold method to measure surface-active intermediates formed in the biodegradation of nonionic surfactants was also evident from the experiment with the primary linear Cle2,, alcohol ethoxylate with 15 EO. The biodegradation testing was performed in three subsequent series of experiments with a gradual increasing of the initial surfactant concentration and the dilution rate. The results obtained (Figure 6 and Table 11) show that in all experiments (from the first to the eighth day) nearly complete (- 100%) biodegradation of LPAE was obtained by modified Wickbold method. On the contrary, in the first experiment only 58 i 3% biodegradation of LPAE was determined by the electrochemical method. In the second experiment with the increased surfactant concentration from 20 to 50 mg/L and at the same dilution rate of 0.05 h-l an even lower biodegradation degree (36 f 6%) was achieved by the electrochemical method. Finally in the third experiment a complete biodegradation of LPAE (98 f 0.9%)was obtained by both methods. It has been already established that a rapid disappearance of the original nonionic compound and the liberation of the ethoxylate portion of the molecule as free polyethylene glycol (PEG) (24) characterize the biodegradation of linear primary alcohol ethoxylate. It is also known that the PEGS as intermediates can accumulate after primary LPAE biodegradation when the rate of their formation is higher than their biodegradation rate. This was very probably the case in our first two experiments, presented in Figure 6. Since PEGS were not determined by the modified Wickbold method but by the electrochemical method, we observed a lower degree of LPAE biodegradation by the electochemical method than by the modified Wickbold method. A nearly complete biodegradation of LPAE in the third experiment (Figure 6) was obtained even from the electrochemical measurement after a gradual culture adaption on PEGS that were formed during the primary LPAE biodegradation. The high degree of biodegradation (95%) Envlron. Sci. Technoi., Vol. 17, No. 5, 1983 271
Envlron. Sci. Technol, 1903, 17, 272-277
of PEG 600 that we obtained in the separate experiment using the culture adapted to LPAE (the same as in the third experiment) supports the above explanation. During the biodegradation experiments of tert-octylphenol ethoxylates, the highest biodegradation degree that could be ascribed to gradual culture adaptation was not observed. Conclusions (1) The biodegradation test of nonionic surfactant is significantly simplified by introducing direct electrochemical determination of surfactants in synthetic sewage and in effluents. (2) The proposed electrochemical method is very sensitive and selective to surfactants contained in detergents and can be used for their direct determination in the presence of organic matter in synthetic sewage and in effluents. (3) Unlike many other methods, the electrochemical method is applicable to the determination of different types of nonionic surfactants regardless of the number of ethylene oxide groups including polyethylene glycols and biodegradation intermediates with a low degree of ethoxylation. (4) The biodegradation degree of nonionic surfactants obtained by the electrochemical method is generally lower than by the modified Wickbold method. Registry No. Polyethylene glycol, 25322-68-3;polyethylene glycol tert-octylphenyl ether, 9036-19-5; polyethylene glycol tert-nonylphenyl ether, 37281-58-6; polyethylene glycol p-tertoctylphenyl ether, 9002-93-1.
(5) Linhart, K. Tenside Deterg. 1972, 9, 241. (6) Kozarac, Z.; ZutiE, V.; CosoviE, B. Tenside Deterg. 1976, 13, 260. (7) Kozarac, Z.; ZvonariE, T.; iutiE, V.; CosoviE, B. Thalassia Jugosl. 1977, 13, 109. (8) CosoviE, B.; ZutiE, V.; Kozarac, A. Croat. Chem. Acta 1977, 50, 229. (9) Kozarac, A.; eosoviE, B.; Branica, M. J. Electroanal. Chem. 4976, 68, 75. (10) CosoviE, B.; VojvodiE, V. Limnol. Oceanogr. 1982,27,361. (11) CosoviE, B.; Hrlak, D. Tenside Deterg. 1979, 16, 262. (12) Rosen, M. J.; Hua, X.; Bratin, P.; Cohen,A. W. Anal. Chem. 1981, 53, 232, (13) ZvonariE, T.; ZutiE, V.; Branica, M. Thalassia Jugosl. 1973, 9, 65. (14) Kozarac, A.; iutib, V.; CosoviE, B. Fourth Yugoslav Symposium on Surface Active Substances, Dubrovnik 1977,
Yugoslavia.
Literature Cited (1) Berth, P.; Heidrich, J.; Jakobi, G. Tenside Deterg. 1980,
(15) Pollution by detergents; Determination of the biodegradability of anionic surface active agents; Publications de l’OCDE, Paris 1972. (16) Hrlak, D.; Bolnjak, M.; Johanides,V. Tenside Deterg. 1981, 18, 137. (17) Wickbold, R. Tenside Deterg. 1973, 10, 179. (18) Longman, G. F. ”The Analysis of Detergents and Detergent Products”;Wiley: New York, 1975; p 513. (19) CosoviE, B.; Branica, M. J. Electroanal. Chem. 1973,46, 63. (20) Radej, J.; RuiiE, I; Konrad, D.; Branica, M. J. Electroanal. Chem. 1973,46,261. (21) CosoviE, B.; Batina, N.; Kozarac, Z. J. Electroanal. Chem. 1980, 113, 239. (22) Gerike, P.; Schmid. R. Tenside Deterg. 1973, 10, 186. (23) Schoberl, P.; Kunkel, E.; Espeter, K. Tenside Deterg. 1981, 2, 64. (24) Swisher,R. D. “SurfactantBiodegradation”;Marcel Dekker: New York, 1970.
17, 228. (2) Schoberl, P.; Bock, K. J. Tenside Deterg. 1980, 17, 262. (3) Kinkel, E. Tenside Deterg. 1980, 17, 247. (4) Jehring, H. “Electrosorptionsanalyse mit der Wechselstrompolarographie”; Akademie-Verlag: Berlin, 1974.
Received for review May 6, 1982. Accepted December 13,1982. This work was supported by the Self-management Authority for the Scientific Research in SR Croatia and by Grant NBSI 6-261 from the National Bureau of Standards, Washington, D.C.
Mutagenicity of Municipal Sewage Sludges of American Cities John G. Babish,*t Brian E. Johnson,? and Donald J. Llskt Department of Preventive Medicine, NYS College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, and Toxic Chemicals Laboratory, NYS College of Agriculture and Life Sciences, Cornell University, Ithaca, New York 14853 ~_____
Dichloromethane extracts of sludge samples from 34 cities were tested for mutagenicity by using the Salmonella/mammalian microsome assay. Of the 34 samples, 33 demonstrated a dose-related increase in revertants in at least 1 of the 5 tester strains; of these 33 samples, 12 were positive with 2 or more strains. Seventy-six percent of the positive samples required metabolic activation to demonstrate mutagenicity, while 18% were mutagenic with and without the S-9 fraction, and 6% were mutagenic only in the absence of any metabolic activation. No association existed between the independent variables percent industrialization, wastewater treatment scheme, and chemical additives when tested individually against mutagenicity. Introduction Approximately 8 X loe gallons of municipal waste containing some 17 000 dry tons of sediments (sludges) are t Department of
Preventative Medicine.
t Toxic Chemical Laboratory. 272
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produced daily in the United States (1). These sludges are generated from residential, commercial, and industrial sources, and may contain human excreta, pathogenic bacteria (2),viruses (3))and a myriad of chemicals ( 4 , 5 ) . Disposal methods have included incineration, fresh water dilution, ocean dumping, disposal in landfills, and limited use on lawns, ornamentals, forests, and agricultural land (6, 7).
Estimating the potential hazards from these waters requires that their toxic properties be determined. Once these characteristics are known, environmentally sound methods of disposal may be developed and employed. As part of our studies on ambient exposures to carcinogenic and mutagenic compounds, we evaluated the mutagenicity of organic extracts of sewage sludges from 34 American cities. Experimental Section In 1980, a letter and questionnaire describing our proposed study was sent to 65 cities. They were requested to participate by returning a representative sample of their
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