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Biodegradation of 4-nonylphenol in seawater and sediment
Environmental Pollution 79 (1993) 59-61
BIODEGRADATION OF 4-NONYLPHENOL IN SEAWATER A N D SEDIMENT R. Ekelund, ./k. Granmo, K. Magnusson, M. Berggren The Swedish Environmental Protection Agency, Kristineberg Marine Biological Station, PI. 2130 S-450 34 Fiskebdckskil, Sweden
&
,/k. Bergman Environmental Chemistry, Wallenberg Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden (Received 27 July 1991; accepted 8 October 1991)
rapid and, after the hydraulic residence time of 8 h in the testing system, 20--40% of the tritium from the labelled N P was found in 3H20. The degradation of N P under conditions more relevant to a temperate marine environment has been studied in seawater in the laboratory at 11°C by means of t4C-labelled NP.
Abstract
Biodegradation of ~4C-labelled nonylphenol at the concentration 11 tzg litre-J in seawater has been estimated by collection and quantification of the formed labelled carbon dioxide. Initially degradation was very slow but when the microorganisms had become adapted, after four weeks at 11°C, the degradation rate increased rapidly and after 58 days about 50% of 14Cfrom NP was found in the COe fraction. In the presence of sediment the initial degradation rate was high and did not increase after longer incubation. Lack of oxygen reduced the degradation rate by half in the presence of sediment.
MATERIALS AND M E T H O D S 4-Nonyl-I [14C]-phenol Q ~ - N P ) was synthetized from uniformly labelled p4C]-phenol and unlabelled commercial nonene which consists of a mixture of different branched isomers (Ekelund et al., 1990). To a number of 2-1itre Erlenmeyer flasks was added either only 1 litre of seawater or both 1 litre seawater and 50 ml of sieved (1 mm) soft bottom sediment. The seawater used was collected below the halocline in a less polluted coastal area. Four flasks containing seawater were supplied with 5.45 ml of concentrated formalin. Half of the flasks containing seawater and sediment were bubbled with nitrogen gas for 15 min. An amount of 0.06 /zCi 14C-NP (143 558 dpm, 11 p,g) dissolved in acetone was added to each of a number of small glass plates and the solvent was evaporated at room temperature. One glass plate with 14C-NP was added to each of the prepared E-flasks which were sealed and shaken vigorously. Incubation occurred at I 1° + 2oC in darkness with shaking twice a week for one minute. The N2-bubbled flasks were not shaken. Samples were taken after 1, 2, 4 and 8 weeks and moreover for flasks without sediment after 16 weeks, four replicates of each kind, and the t4CO2 formed, was collected and estimated. For this purpose the contents in the flasks were acidified to pH 3 with sulphuric acid, 1 ml of cod liver oil was added to prevent N P from evap-orating and a jar containing 3.5 ml of 2-5 M K O H (flasks without sediment) or 10 ml of 5 M K O H (flasks with sediment) was hung in each flask which was sealed and shaken for 24 h at room temperature. The jar with K O H was then taken out and it was provided with a piece of ice and a smaller jar containing 0.7 ml of 6 M H2SO4 (without-sediment jar) or 2 ml of concentrated H2SO4
INTRODUCTION Nonylphenol etholxylates (NPE) are widely used as surfactants in industries and in households. In Sweden about 5000 tonnes of nonylphenol (NP) are used in ethoxylates per year and a significant part of this may reach the sea. The original compounds are known to disappear rather rapidly in treatment plants or the environment (Giger 1984; Ahel, 1987; G r a n m o et al., 1991). However, the intermediates formed, shortchained NPE and especially free NP, are degraded slowly (Rudling & Solyom, 1974). The degradation of N P has been studied in only two cases in freshwater (Sundaram & Szeto, 1981; Ahel, 1987) and once under sewage plant conditions (Kravetz et al., 1982). In the two former cases no conclusions about the biodegradability of N P can be drawn as considerable losses of N P occurred even from the sterilized samples. In the latter case N P 9 EO labelled with tritium only in the N P part of the molecule was used, so it was possible to follow the degradation of that part separately by determination of the 3H20 formed and the soluble 3Hlabelled degradation products. Under these conditions, which were more optimal for degradation than normally in the field, the degradation o f N P was very
Environ. Pollut. 0269-7491/92/$05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain 59
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R. Ekelund, ,~. Granmo, K. Magnusson, M. Berggren, A. Bergman
(with-sediment jar). The KOH jar was placed in a bigger glass jar together with a scintillation vial containing 1.5 ml (without-sediment jar) or 4 ml (with sediment jar) of Carbosorb (Packard). The glass jar was closed air-tight and shaken at 10°C for 4 h~. Then 15 ml of Permaflour V (Packard) were added to each scintillation vial and the samples were scintillation counted and corrected for quenching using an Hnumber quench monitor (Beckman LS 5000 TD). To determine the recovery of ,4CO2, about 0-004 p.Ci (7816 dpm) of ~4C-carbonate were added to each of five E-flasks containing l litre of seawater and five E-flasks with seawater and sediment and the samples were treated as described above. The exchange of 14CO2 was also determined from E-flasks which had been incubated for 8 weeks at +1 l°C in darkness after the addition of ~4C-carbonate. After 14CO: had been collected from the NP flasks incubated for 8 weeks, 15 ml of n-hexane:diethyl ether (1:1) were added to each flask, which was shaken for 30 min. The organic phase was saved and the water was shaken with another portion of hexane:diethyl ether (10 ml) and the combined organic phases were evaporated. Scintillation cocktail was added (15 ml Packard toluene scintillator POPOP 0.1 g litre-~, PPO 5 g litre-l) and the samples were measured to determine radioactivity remaining in the hydrophobic fraction. RESULTS AND DISCUSSION The recovery of labclled carbon dioxide which was expelled and collected immediately after the addition of 14C-carbonatc to the flasks with seawater, was about 78% and from the flasks with sediment in addition it was about 100%. The corresponding recovery at collection 8 weeks after the carbonate addition was 94% and 80%, respectively. When using the method described above, no radioactivity was obtained from flasks which
had been incubated for 8 weeks after the addition of formalin besides 14C-NP. This indicates that no carbon dioxide is formed from the labelled NP when living microorganisms are absent and like the samples at the start it shows that the unchanged NP does not contribute to the radioactivity in the final sample which is measured. All the radioactivity trapped in the Carbosorb does not necessarily represent CO2. Other volatile metabolites may also be present but in every case all the radioactivity should correspond to transformation or degradation products of NP. The amount of oxygen in a sealed flask (1 litre of air) is more than enough for complete degradation of the organic material in the water and in addition for degradation of 10% of the organic material in the sediment. The results from the whole test are shown in Fig. 1 where the degradation is expressed as percent radioactivity in the CO2 fraction per radioactivity of the added NP. The degradation in the absence of sediment was very slow during the first few weeks (about 0.06% of NP degraded day-l) but increased after 28 days to about 1% day-l. Evidently the microorganisms were then adapted to the added substrate. Probably the concentration of NP should be above a certain critical level before any adaptation of the microorganisms occurs, as has been shown for other pollutants (Boethling & Alexander, 1979; Spain & Van Veld, 1983). The critical level mentioned should lie somewhere between the original NP concentration in the used natural seawater and the concentration 11 /zg NP litre-,, which was used in the degradation test. The most relevant degradation rate with regard to field conditions with limited pollution by nonylphenol should therefore be the lower rate which prevails before the adaptation, and this is much lower than the one reported by Sundaram & Szeto (1981) and Kravetz et al. (1982). The variation between replicates is most extensive in this kind of sample, where a substrate adaptation is evident. This
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Days Fig. 1. Biodegradation of 14C-labelled nonylphenol (NP) in seawater, estimated by quantification of liberated labelled carbon dioxide. Degradation is expressed as a percentage of the originally added radioactivity. Explanations: x = NP + seawater, © -- NP + seawater + sediment, • = NP + seawater + sediment + nitrogen bubbled, • = NP + seawater + formalin. Confidence intervals (95%) are given as vertical bars.
Biodegradation o f 4-nonylphenol in seawater and sediment variation might be due to the random occurrence in time of the relevant mutations o f bacteria in the separate flasks. The degradation rate in the presence of sediment and oxygen was high from the beginning (about 1.2% o f N P degraded day -1) and was half as rapid at very low concentrations of oxygen. The higher rate in the presence of sediment is due probably to the larger number of microorganisms, which increases the probability that bacterial cells able to degrade N P are present. That rate did not increase with time and this lack of substrate adaptation may be explained by the richer supply of other carbon sources and by decreased availability of N P due to adsorption (Neilson, 1989; Shimp & Pfaender, 1985) down to a level where no adaptation is induced. As log of the partition coefficient of N P between organic carbon and water (Koc) is about 4, most of it in the studied system (50 g sediment with 3% organic carbon content in one litre of water) is estimated to be adsorbed to particles. No transformation of N P should have occurred in the flasks with added formalin, and from these samples 84% of the added radioactivity was extracted by hexane:diethyl ether. From the samples without formalin and sediment, 64% of the activity was recovered, 44% in the CO2 fraction and 20% in the organic solvent. The remaining 36% of the radioactivity not recovered, may have existed partially as N P metabolites with high water solubility. Whether the nonylphenol molecule is attacked in the nonyl or the phenol part, carboxylic groups should be formed (Von Schrberl et al., 1981) and the reaction will then give products which are more water-soluble than the original compound. Inability to recover all the added radioactivity is a common observation in degradation tests (Jensen et al., 1988). From the flasks with sediment, 49% of the added activity was regained, 46% in the CO2 fraction and 3% in the organic solvent. It is possible that a significant part of the undegraded N P was not extracted as it had been adsorbed to the sediment particles for a long period of time. In this case part of the lost radioactivity may similarly have occurred in water-soluble intermediates. Thus a larger proportion of nonylphenol may have been transformed or degraded than is indicated by the radioactivity in the carbon dioxide fraction. ACKNOWLEDGEMENTS Thanks are due to the Kerstin Wijk-Brostr6m foundation for financial support and to the Royal Academy of Sciences for providing laboratory facilities. This
61
work was done within a research contract from the Swedish Environmental Protection Agency, contract No. 5312149-7. REFERENCES Ahel, M. (1978). Biochemical behaviour of alkylphenol polyethoxylates in the aquatic environment. Doctoral thesis. University of Zagreb, Yugoslavia. Boethling, R. & Alexander, M. (1979). Microbial degradation of organic compounds at trace levels. Environ. Sci. TechnoL, 13, 989-91. Ekelund, R., Bergman, A., Granmo, A. & Berggren, M. (1990). Bioaccumulation of 4-nonylphenol in marine animals---a re-evaluation. Environ. Pollut. (Ser. A), 64, 107-20. Giger, W. (1984). Das Verhalten organischer Waschmittelchemikalien in der Abwasserreinigung und in den Gewgtssem. EA WAG-News, 18, 1-7. Granmo, A., Ekelund, R., Magnusson, K. & Berggren, M. (1989). Lethal and sublethal toxicity of 4-nonylphenol to the common mussel (Mytilus edulis). Environ. Pollut. ( Ser. A), 59, 115-27. Granmo, A., Kollberg, S., Berggren, M., Ekelund, R., Magnusson, K., Renberg, L. and Wahlberg, C. (1991). Bioaccumulation of nonylphenoi in caged mussels in an industrial coastal area on the Swedish west coast. In Organic Micropollutants in the Aquatic Environment, ed. G. Angeletti & A. Bjorseth. Proceedings of the sixth European symposium held in Lisbon, Portugal, May 22-24, 1990. Jensen, K., Albrechtsen, H.-J., Nielsen, J. & Kruse, B. (1988). The use of ecocores to evaluate biodegradation in marine sediments. Water, Air and Soil Pollution, 39, 89-99. Kravetz, L., Chung, H., Guin, K. F., Shebs, W. T., Smith, L. S. & Stupel, H. (1982). Ultimate biodegradation of an alcohol ethoxylate and a nonylphenol ethoxylate under realistic conditions. Household Pers. Prod. Ind., 19, 62-70. Neilson, A. H. (1989). Factors determining the fate of organic chemicals in the environment: The role of bacterial transformations and binding to sediments. In Chemicals in the Aquatic Environment, ed. L. Landner. Springer Verlag Berlin. Rudling, L. & Solyom, P. (1974). The investigation of biodegradability of branched nonyl phenol ethoxylates. Water Res., 8, 115-19. Shimp, R. & Pfaender, F. P. (1985). Influence of naturally occurring humic acid on biodegradation of monosubstituted phenols by aquatic bacteria. Appl. Environ. Microbiol, 49, 402-7. Spain, J. C. & Van Veld, P. A. (1983). Adaptation of natural microbial communities to degradation of xenobiotic compounds: Effects of concentration, exposure time, inoculum and chemical structure. Appl. Environ. Microbiol., 40, 726-34. Sundaram, K. M. S. & Szeto, S. (1981). The dissipation of nonylphenol in stream and pond" water under simulated field conditions. J. Environ. Sci. Health, B 16, 767-76. Von Sch6berl, P., Kunkel, E. & Espeter, K. (1981). Vergleichende Untersuchungen tiber den mikrobiellen Metabolismus eines Nonylphenol--und eines oxoalcohol-ethoxylates. Tenside Detergents, 18, 64-72.
BIODEGRADATION OF 4-NONYLPHENOL IN SEAWATER A N D SEDIMENT R. Ekelund, ./k. Granmo, K. Magnusson, M. Berggren The Swedish Environmental Protection Agency, Kristineberg Marine Biological Station, PI. 2130 S-450 34 Fiskebdckskil, Sweden
&
,/k. Bergman Environmental Chemistry, Wallenberg Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden (Received 27 July 1991; accepted 8 October 1991)
rapid and, after the hydraulic residence time of 8 h in the testing system, 20--40% of the tritium from the labelled N P was found in 3H20. The degradation of N P under conditions more relevant to a temperate marine environment has been studied in seawater in the laboratory at 11°C by means of t4C-labelled NP.
Abstract
Biodegradation of ~4C-labelled nonylphenol at the concentration 11 tzg litre-J in seawater has been estimated by collection and quantification of the formed labelled carbon dioxide. Initially degradation was very slow but when the microorganisms had become adapted, after four weeks at 11°C, the degradation rate increased rapidly and after 58 days about 50% of 14Cfrom NP was found in the COe fraction. In the presence of sediment the initial degradation rate was high and did not increase after longer incubation. Lack of oxygen reduced the degradation rate by half in the presence of sediment.
MATERIALS AND M E T H O D S 4-Nonyl-I [14C]-phenol Q ~ - N P ) was synthetized from uniformly labelled p4C]-phenol and unlabelled commercial nonene which consists of a mixture of different branched isomers (Ekelund et al., 1990). To a number of 2-1itre Erlenmeyer flasks was added either only 1 litre of seawater or both 1 litre seawater and 50 ml of sieved (1 mm) soft bottom sediment. The seawater used was collected below the halocline in a less polluted coastal area. Four flasks containing seawater were supplied with 5.45 ml of concentrated formalin. Half of the flasks containing seawater and sediment were bubbled with nitrogen gas for 15 min. An amount of 0.06 /zCi 14C-NP (143 558 dpm, 11 p,g) dissolved in acetone was added to each of a number of small glass plates and the solvent was evaporated at room temperature. One glass plate with 14C-NP was added to each of the prepared E-flasks which were sealed and shaken vigorously. Incubation occurred at I 1° + 2oC in darkness with shaking twice a week for one minute. The N2-bubbled flasks were not shaken. Samples were taken after 1, 2, 4 and 8 weeks and moreover for flasks without sediment after 16 weeks, four replicates of each kind, and the t4CO2 formed, was collected and estimated. For this purpose the contents in the flasks were acidified to pH 3 with sulphuric acid, 1 ml of cod liver oil was added to prevent N P from evap-orating and a jar containing 3.5 ml of 2-5 M K O H (flasks without sediment) or 10 ml of 5 M K O H (flasks with sediment) was hung in each flask which was sealed and shaken for 24 h at room temperature. The jar with K O H was then taken out and it was provided with a piece of ice and a smaller jar containing 0.7 ml of 6 M H2SO4 (without-sediment jar) or 2 ml of concentrated H2SO4
INTRODUCTION Nonylphenol etholxylates (NPE) are widely used as surfactants in industries and in households. In Sweden about 5000 tonnes of nonylphenol (NP) are used in ethoxylates per year and a significant part of this may reach the sea. The original compounds are known to disappear rather rapidly in treatment plants or the environment (Giger 1984; Ahel, 1987; G r a n m o et al., 1991). However, the intermediates formed, shortchained NPE and especially free NP, are degraded slowly (Rudling & Solyom, 1974). The degradation of N P has been studied in only two cases in freshwater (Sundaram & Szeto, 1981; Ahel, 1987) and once under sewage plant conditions (Kravetz et al., 1982). In the two former cases no conclusions about the biodegradability of N P can be drawn as considerable losses of N P occurred even from the sterilized samples. In the latter case N P 9 EO labelled with tritium only in the N P part of the molecule was used, so it was possible to follow the degradation of that part separately by determination of the 3H20 formed and the soluble 3Hlabelled degradation products. Under these conditions, which were more optimal for degradation than normally in the field, the degradation o f N P was very
Environ. Pollut. 0269-7491/92/$05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain 59
60
R. Ekelund, ,~. Granmo, K. Magnusson, M. Berggren, A. Bergman
(with-sediment jar). The KOH jar was placed in a bigger glass jar together with a scintillation vial containing 1.5 ml (without-sediment jar) or 4 ml (with sediment jar) of Carbosorb (Packard). The glass jar was closed air-tight and shaken at 10°C for 4 h~. Then 15 ml of Permaflour V (Packard) were added to each scintillation vial and the samples were scintillation counted and corrected for quenching using an Hnumber quench monitor (Beckman LS 5000 TD). To determine the recovery of ,4CO2, about 0-004 p.Ci (7816 dpm) of ~4C-carbonate were added to each of five E-flasks containing l litre of seawater and five E-flasks with seawater and sediment and the samples were treated as described above. The exchange of 14CO2 was also determined from E-flasks which had been incubated for 8 weeks at +1 l°C in darkness after the addition of ~4C-carbonate. After 14CO: had been collected from the NP flasks incubated for 8 weeks, 15 ml of n-hexane:diethyl ether (1:1) were added to each flask, which was shaken for 30 min. The organic phase was saved and the water was shaken with another portion of hexane:diethyl ether (10 ml) and the combined organic phases were evaporated. Scintillation cocktail was added (15 ml Packard toluene scintillator POPOP 0.1 g litre-~, PPO 5 g litre-l) and the samples were measured to determine radioactivity remaining in the hydrophobic fraction. RESULTS AND DISCUSSION The recovery of labclled carbon dioxide which was expelled and collected immediately after the addition of 14C-carbonatc to the flasks with seawater, was about 78% and from the flasks with sediment in addition it was about 100%. The corresponding recovery at collection 8 weeks after the carbonate addition was 94% and 80%, respectively. When using the method described above, no radioactivity was obtained from flasks which
had been incubated for 8 weeks after the addition of formalin besides 14C-NP. This indicates that no carbon dioxide is formed from the labelled NP when living microorganisms are absent and like the samples at the start it shows that the unchanged NP does not contribute to the radioactivity in the final sample which is measured. All the radioactivity trapped in the Carbosorb does not necessarily represent CO2. Other volatile metabolites may also be present but in every case all the radioactivity should correspond to transformation or degradation products of NP. The amount of oxygen in a sealed flask (1 litre of air) is more than enough for complete degradation of the organic material in the water and in addition for degradation of 10% of the organic material in the sediment. The results from the whole test are shown in Fig. 1 where the degradation is expressed as percent radioactivity in the CO2 fraction per radioactivity of the added NP. The degradation in the absence of sediment was very slow during the first few weeks (about 0.06% of NP degraded day-l) but increased after 28 days to about 1% day-l. Evidently the microorganisms were then adapted to the added substrate. Probably the concentration of NP should be above a certain critical level before any adaptation of the microorganisms occurs, as has been shown for other pollutants (Boethling & Alexander, 1979; Spain & Van Veld, 1983). The critical level mentioned should lie somewhere between the original NP concentration in the used natural seawater and the concentration 11 /zg NP litre-,, which was used in the degradation test. The most relevant degradation rate with regard to field conditions with limited pollution by nonylphenol should therefore be the lower rate which prevails before the adaptation, and this is much lower than the one reported by Sundaram & Szeto (1981) and Kravetz et al. (1982). The variation between replicates is most extensive in this kind of sample, where a substrate adaptation is evident. This
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Days Fig. 1. Biodegradation of 14C-labelled nonylphenol (NP) in seawater, estimated by quantification of liberated labelled carbon dioxide. Degradation is expressed as a percentage of the originally added radioactivity. Explanations: x = NP + seawater, © -- NP + seawater + sediment, • = NP + seawater + sediment + nitrogen bubbled, • = NP + seawater + formalin. Confidence intervals (95%) are given as vertical bars.
Biodegradation o f 4-nonylphenol in seawater and sediment variation might be due to the random occurrence in time of the relevant mutations o f bacteria in the separate flasks. The degradation rate in the presence of sediment and oxygen was high from the beginning (about 1.2% o f N P degraded day -1) and was half as rapid at very low concentrations of oxygen. The higher rate in the presence of sediment is due probably to the larger number of microorganisms, which increases the probability that bacterial cells able to degrade N P are present. That rate did not increase with time and this lack of substrate adaptation may be explained by the richer supply of other carbon sources and by decreased availability of N P due to adsorption (Neilson, 1989; Shimp & Pfaender, 1985) down to a level where no adaptation is induced. As log of the partition coefficient of N P between organic carbon and water (Koc) is about 4, most of it in the studied system (50 g sediment with 3% organic carbon content in one litre of water) is estimated to be adsorbed to particles. No transformation of N P should have occurred in the flasks with added formalin, and from these samples 84% of the added radioactivity was extracted by hexane:diethyl ether. From the samples without formalin and sediment, 64% of the activity was recovered, 44% in the CO2 fraction and 20% in the organic solvent. The remaining 36% of the radioactivity not recovered, may have existed partially as N P metabolites with high water solubility. Whether the nonylphenol molecule is attacked in the nonyl or the phenol part, carboxylic groups should be formed (Von Schrberl et al., 1981) and the reaction will then give products which are more water-soluble than the original compound. Inability to recover all the added radioactivity is a common observation in degradation tests (Jensen et al., 1988). From the flasks with sediment, 49% of the added activity was regained, 46% in the CO2 fraction and 3% in the organic solvent. It is possible that a significant part of the undegraded N P was not extracted as it had been adsorbed to the sediment particles for a long period of time. In this case part of the lost radioactivity may similarly have occurred in water-soluble intermediates. Thus a larger proportion of nonylphenol may have been transformed or degraded than is indicated by the radioactivity in the carbon dioxide fraction. ACKNOWLEDGEMENTS Thanks are due to the Kerstin Wijk-Brostr6m foundation for financial support and to the Royal Academy of Sciences for providing laboratory facilities. This
61
work was done within a research contract from the Swedish Environmental Protection Agency, contract No. 5312149-7. REFERENCES Ahel, M. (1978). Biochemical behaviour of alkylphenol polyethoxylates in the aquatic environment. Doctoral thesis. University of Zagreb, Yugoslavia. Boethling, R. & Alexander, M. (1979). Microbial degradation of organic compounds at trace levels. Environ. Sci. TechnoL, 13, 989-91. Ekelund, R., Bergman, A., Granmo, A. & Berggren, M. (1990). Bioaccumulation of 4-nonylphenol in marine animals---a re-evaluation. Environ. Pollut. (Ser. A), 64, 107-20. Giger, W. (1984). Das Verhalten organischer Waschmittelchemikalien in der Abwasserreinigung und in den Gewgtssem. EA WAG-News, 18, 1-7. Granmo, A., Ekelund, R., Magnusson, K. & Berggren, M. (1989). Lethal and sublethal toxicity of 4-nonylphenol to the common mussel (Mytilus edulis). Environ. Pollut. ( Ser. A), 59, 115-27. Granmo, A., Kollberg, S., Berggren, M., Ekelund, R., Magnusson, K., Renberg, L. and Wahlberg, C. (1991). Bioaccumulation of nonylphenoi in caged mussels in an industrial coastal area on the Swedish west coast. In Organic Micropollutants in the Aquatic Environment, ed. G. Angeletti & A. Bjorseth. Proceedings of the sixth European symposium held in Lisbon, Portugal, May 22-24, 1990. Jensen, K., Albrechtsen, H.-J., Nielsen, J. & Kruse, B. (1988). The use of ecocores to evaluate biodegradation in marine sediments. Water, Air and Soil Pollution, 39, 89-99. Kravetz, L., Chung, H., Guin, K. F., Shebs, W. T., Smith, L. S. & Stupel, H. (1982). Ultimate biodegradation of an alcohol ethoxylate and a nonylphenol ethoxylate under realistic conditions. Household Pers. Prod. Ind., 19, 62-70. Neilson, A. H. (1989). Factors determining the fate of organic chemicals in the environment: The role of bacterial transformations and binding to sediments. In Chemicals in the Aquatic Environment, ed. L. Landner. Springer Verlag Berlin. Rudling, L. & Solyom, P. (1974). The investigation of biodegradability of branched nonyl phenol ethoxylates. Water Res., 8, 115-19. Shimp, R. & Pfaender, F. P. (1985). Influence of naturally occurring humic acid on biodegradation of monosubstituted phenols by aquatic bacteria. Appl. Environ. Microbiol, 49, 402-7. Spain, J. C. & Van Veld, P. A. (1983). Adaptation of natural microbial communities to degradation of xenobiotic compounds: Effects of concentration, exposure time, inoculum and chemical structure. Appl. Environ. Microbiol., 40, 726-34. Sundaram, K. M. S. & Szeto, S. (1981). The dissipation of nonylphenol in stream and pond" water under simulated field conditions. J. Environ. Sci. Health, B 16, 767-76. Von Sch6berl, P., Kunkel, E. & Espeter, K. (1981). Vergleichende Untersuchungen tiber den mikrobiellen Metabolismus eines Nonylphenol--und eines oxoalcohol-ethoxylates. Tenside Detergents, 18, 64-72.