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Degradation of some phthalic acid esters in soil
Environmental Pollution (Series A) 39 (1985) 1-7
Degradation of Some Phthalic Acid Esters in Soil
Rishi Shanker, C. Ramakrishna & Prahlad K. Seth* Industrial Toxicology Research Centre, Gheru Campus, Post Box 80, M.G. Marg, Lucknow--226 001, India
ABSTRACT The biodegradation of three phthalic acid esters (PAEs)---dimethyl phthalate (D MP), dibutyl phthalate (DBP) and di(2-ethyl hexyl)phthalate (DEHP)--was studied in a garden soil. The degradation rates of DMP and DBP were greater than that of DEHP under aerobic conditions. Anaerobiosis created by flooding greatly retarded the degradation of the three PAEs. The results suggest that microflora, especially bacteria, are actively involved in the degradation of the three phthalate esters.
INTRODUCTION Phthalic acid esters (PAEs) have a variety of industrial applications. Peakall (1975) estimated the world production of phthalates as being about 1.35 to 1.81 x 109kg per year. Di-n-alkyl phthalates are widely used as plasticisers to impart the desired flexibility to polyvinyl chloride plastics. The use of PAE-containing plastics is increasing rapidly. Due to their ubiquitous presence, PAEs have assumed significance as microchemical environmental pollutants (Giam et al., 1978). They have been detected in ground, river, drinking and open ocean water, urban, suburban and marine air, soil humates, lake and marine sediments, fish and crustaceans (Sullivan et al., 1982). The oceans and marine sediments appear to act as the final repositories of PAEs (Sullivan et al., 1982). A * To whom all correspondence should be addressed. 1
Environ. Pollut. Set. A. 0143-1471/85/$03.30© ElsevierApplied SciencePublishersLtd, England, 1985. Printed in Great Britain
2
Rishi Shanker, C. Ramakrishna, P. K. Seth
direct relationship between the production of phthalates and their accumulation in the sediments has also been reported (Peterson & Freeman, 1982). PAEs are generally considered to be substances of low order of acute toxicity. However, their widespread distribution and recent reports of their hepatotoxic (Seth, 1982), mutagenic (Kozumbo et al., 1982) and carcinogenic (Kluwe et al., 1982) effects have aroused great concern over their toxicogenic potential. At present, very little information is available on the fate of PAE in terrestrial systems, especially soil. Degradation of di-2-ethyl hexyl phthalate (DEHP) and butyl benzyl phthalate (BBP) in activated sludge (Graham, 1973) and river water (Saeger & Tucker, 1976) has been described. In the present study, evidence of microbial degradation of three extensively used phthalates--DMP, DBP and DEHP--in a garden soil is presented. MATERIALS AND METHODS Chemicals
Di-n-methyl, di-n-butyl and di-2-ethyl hexyl phthalates were obtained from BDH, Great Britain, and Ranbaxy Laboratories Ltd, India. Phthalic acid was obtained from IDPL, India. All other chemicals and solvents used were of analytical grade. Collection of soil samples
Small quantities of garden soil (alluvial, pH 8-2) were taken from the top (15 cm) layer and air-dried and sieved (2 mm). Glassware
The glassware used was meticulously cleaned to reduce any background contamination of phthalates. All chromic-acid washed glassware was placed in a 300 °C oven overnight. After cooling, the glassware was rinsed twice with acetone and petroleum ether and air-dried before use. Aerobic studies
Ten grams of dry and sieved garden soil were taken in 25-ml cotton plugged Erlenmeyer flasks and incorporated with any of the three
Degradation o f some phthalic acid esters in soil
3
phthalates, D M P , D B P and D E H P , in 0.1 ml methanol to give a final concentration of 500 #g of phthalate per gram of soil. The flasks were left unplugged overnight to evaporate the solvent carrier at room temperature. The soil was then mixed thoroughly and the moisture level of the soil was adjusted to 60 ~o of its water-holding capacity with sterile distilled water. The controls were run in parallel using autoclaved soil (autoclaved three times on alternative days at 121 °C at 7 kg pressure for 1 h). All flasks were incubated at 30 _+ 1 °C.
Anaerobic studies The soil was taken in stoppered tubes, incorporated with phthalate esters and flooded with sterile water at least double the length of the soil column. The flooded heat-sterilised controls were run simultaneously. All tubes were incubated at 30 + I°C.
Extraction After incubation for desired periods of time, the soils were extracted three times with 15 ml of methanol. The methanol extracts were pooled, filtered and stored at 4°C until analysed but never for more than 48 h.
Analysis A Waters Associates 440 high performance liquid chromatograph equipped with a #-Bondapak C18 column and a uv detector fixed at 254 nm was used for analysis and estimation of phthalates and their metabolite(s). The solvent system was 60 ~o or 90 ~ aqueous methanol at a pumping rate of 1.5-3.0mlmin -1. The parent c o m p o u n d or metabolite(s) were estimated with the help of authentic standards. Soil without the addition of phthalates was used to determine baseline levels of the phthalates/metabolite(s) or interfering compounds.
RESULTS The results, summarized in Tables 1 to 3, show the degradation of di-nmethyl, di-n-butyl and di-2-ethyl hexyl phthalates to phthalic acid by the soil microflora. No significant degradation of any phthalate was seen
4
Rishi Shanker, C. Ramakrishna, P. K. Seth TABLE 1 Biodegradation of Di-n-Methyl Phthalate in a Garden Soil
Incubation time (days)
Micrograms of compound recoveredper gram of soil Aerobic Anaerobic DMP
0 5 10 15 20 30 (Autoclaved control)
468 _+ 16 180___11 43 + 9 0 0 0 465 ± 6
PA 0 9___0.5 8 + 0.5 0 0 0 Traces
DMP
PA
471 _+ 12 410_+8 376 -+ 6 302 _+ l0 245 _+6 178 _+2
0 8_+ l.l 10 _+0.5 24 _+ 1.7 9 -+ I. I 3 _+ 1.0
467 _+ 8
0
DMP, Di-n-methyl phthalate. PA, Phthalic acid. Each value is the mean _+ SE of triplicate samples.
TABLE 2 Biodegradation of Di-n-Butyl Phthalate in a Garden Soil
Incubation Micrograms of compound recoveredper gram of soil time (days) Aerobic Anaerobic DBP 0 5 10 15 20 30 (Autoclaved control)
472 _+ 14 110___13 40_+6 0 0 0 465 _+ 10
PA 0 8+0-6 6_+0-6 0 0 0 Traces
DBP 470 ± 17 402+9 348_+8 301 _+ 9 239 _+9 159_+4 463 _+9
DBP, Di-n-methyl phthalate. PA, Phthalic acid. Each value is the mean + SE of triplicate samples.
PA 0 12__.1-1 14_+2.9 29 _+ 3.5 22 _+ 2-3 15_+ 1.7 0
Degradation of some phthalic acid esters in soil
5
TABLE 3 Biodegradation of Di-2-Ethyl Hexyl Phthalate in a Garden Soil
Incubation Micrograms of compound recoveredper gram of soil time (days) Aerobic Anaerobic DEHP 0 5 10 20 30 (Autoclaved control)
480 + 9 430 + 8 320+11 120-1-4 40+8 471 ___4
PA 0 8 _ 1.1 7___1.1 11-t-0.6 5-t-0.6 0
DEHP
PA
478 -t- 9 460 _ 8 439+6 389+__5 318-t-7
0 Traces 2___0 8+1.1 11-t-0-6
478 _ 7
0
D E H P , Di-2-ethyl hexyl phthalate. PA, Phthalic acid. Each value is the mean + SE of triplicate samples.
when sterilised soil was used. As is evident from Tables 1 to 3, the rate of degradation of the three PAEs was different. The shorter chain PAEs, such as D M P or DBP, were degraded at a higher rate than the longer chain phthalate DEHP. The soil microflora were able to degrade actively up to 500pgg-~ concentration of various phthalates under aerobic conditions (Table 1 to 3). However, the rate of degradation was very low under anaerobic conditions. Significant amounts of DMP, DBP and D E H P were recovered from soil (Tables 1 to 3) even after 30 days of post inoculation in anaerobic conditions. The degradation appears to be via phthalic acid as small quantities of this substance were detected under both aerobic and anaerobic conditions. No further metabolites were detected.
DISCUSSION PAEs are known to be degraded in river water and marine regions (Saeger & Tucker, 1976; Taylor et al., 1981). Mathur (1974) provided respirometric evidence for the degradation of di-2-ethyl hexyl phthalate and di-n-octyl phthalate by soil microflora. Our data suggest the presence of micro-organisms in soil capable of degrading D M P , DBP and DEHP. This was further confirmed by the isolation of bacteria from the soil
6
Rishi Shanker, C. Ramakrishna, P. K. Seth
capable ofutilising the three PAEs as the sole carbon source (unpublished data). The strains isolated varied in characteristics and were both grampositive or gram-negative, oxidase and catalase positive, could reduce nitrate, and most could grow at 45 °C. Our findings complement those of others that PAEs are biodegradable, and the accumulation reported in the environment (Giam et al., 1978; Giam & Atlas, 1980) might possibly be due to various physical and chemical factors and the behaviour of the chemical in that environment may limit its biodegradability.
ACKNOWLEDGEMENTS The authors are grateful to Dr P. K. Ray, Director, Industrial Toxicology Research Centre, Lucknow, India, for his advice and encouragement and to Dr C. R. Krishna Murti, former Director, ITRC, Lucknow, India, for his valuable suggestions in the initiation of these studies. Research support provided by the Department of the Environment, Government of India, is gratefully acknowledged.
REFERENCES Giam, C. S. & Atlas, E. L. (1980). Accumulation ofphthalate ester plasticizers in Lake Constance sediments. Naturwissenschaften, 67, 508. Giam, C. S., Chan, H. S., Neff, C. S. & Atlas, E. L. (1978). Phthalate ester plasticizers: A new class of marine pollutant. Science, N.Y., 199, 419-21. Giam, C. S., Atlas, E. L., Chan, H. S. & Neff, G. S. (1980). Phthalate esters, PCB and DDT residues in the Gulf of Mexico atmosphere..4 tmos. Environ., 14, 65-9. Graham, P. R. (1973). Phthalate ester plasticizers: Why and how they are used. Environ. Hlth Perspectives, 3, 3-12. Kluwe, W. M., McConnell, E. E., Huff, J. E., Haseman, J. K., Douglas, J. F. & Hartwell, W. V. (1982). Carcinogenicity testing of phthalate esters and related compounds by the National Toxicology Programme and the National Cancer Institute. Environ. Hlth Perspectives, 45, 129-33. Kozumbo, W. J., Kroll, R. & Rubin, R. J. (1982). Assessment ofmutagenicity of phthalate esters. Environ. Hlth Perspectives, 45, 103-10. Mathur, S. P. (1974). Respirometric evidence of utilization of di-octyl and di-2ethyl hexyl phthalate plasticizers. J. environ. Qual., 3, 207-9. Peakall, D. B. (1975). Phthalate esters: Occurrence and biological effects. Residue Rev., 54, 141.
Degradation of some phthalic acid esters in soil
7
Peterson, J. C. & Freeman, D. H. (1982). Phthalate ester concentration variations in dated sediment cores from Chesapeake Bay. Environ. Sci. Technol., 16, 464-9. Saeger, V. W. & Tucker, E. S. (1976). Biodegradation of phthalate acid esters in river water and activated sludge. Appl. environ. Microbiol., 31, 29-34. Seth, P. K. (1982). Hepatic effects of phthalate esters. Environ Hlth Perspectives, 45, 27-34. Sullivan, K. F., Atlas, E. L. & Giam, C. S. (1982). Adsorption of phthalic acid esters from sea water. Environ. Sci. Technol., 16, 428-32. Taylor, B. F., Curry, R. W. & Corcoran, E. F. (1981). Potential for biodegradation of phthalic acid esters in marine regions. Appl. environ. Microbiol., 42, 590-5.
Degradation of Some Phthalic Acid Esters in Soil
Rishi Shanker, C. Ramakrishna & Prahlad K. Seth* Industrial Toxicology Research Centre, Gheru Campus, Post Box 80, M.G. Marg, Lucknow--226 001, India
ABSTRACT The biodegradation of three phthalic acid esters (PAEs)---dimethyl phthalate (D MP), dibutyl phthalate (DBP) and di(2-ethyl hexyl)phthalate (DEHP)--was studied in a garden soil. The degradation rates of DMP and DBP were greater than that of DEHP under aerobic conditions. Anaerobiosis created by flooding greatly retarded the degradation of the three PAEs. The results suggest that microflora, especially bacteria, are actively involved in the degradation of the three phthalate esters.
INTRODUCTION Phthalic acid esters (PAEs) have a variety of industrial applications. Peakall (1975) estimated the world production of phthalates as being about 1.35 to 1.81 x 109kg per year. Di-n-alkyl phthalates are widely used as plasticisers to impart the desired flexibility to polyvinyl chloride plastics. The use of PAE-containing plastics is increasing rapidly. Due to their ubiquitous presence, PAEs have assumed significance as microchemical environmental pollutants (Giam et al., 1978). They have been detected in ground, river, drinking and open ocean water, urban, suburban and marine air, soil humates, lake and marine sediments, fish and crustaceans (Sullivan et al., 1982). The oceans and marine sediments appear to act as the final repositories of PAEs (Sullivan et al., 1982). A * To whom all correspondence should be addressed. 1
Environ. Pollut. Set. A. 0143-1471/85/$03.30© ElsevierApplied SciencePublishersLtd, England, 1985. Printed in Great Britain
2
Rishi Shanker, C. Ramakrishna, P. K. Seth
direct relationship between the production of phthalates and their accumulation in the sediments has also been reported (Peterson & Freeman, 1982). PAEs are generally considered to be substances of low order of acute toxicity. However, their widespread distribution and recent reports of their hepatotoxic (Seth, 1982), mutagenic (Kozumbo et al., 1982) and carcinogenic (Kluwe et al., 1982) effects have aroused great concern over their toxicogenic potential. At present, very little information is available on the fate of PAE in terrestrial systems, especially soil. Degradation of di-2-ethyl hexyl phthalate (DEHP) and butyl benzyl phthalate (BBP) in activated sludge (Graham, 1973) and river water (Saeger & Tucker, 1976) has been described. In the present study, evidence of microbial degradation of three extensively used phthalates--DMP, DBP and DEHP--in a garden soil is presented. MATERIALS AND METHODS Chemicals
Di-n-methyl, di-n-butyl and di-2-ethyl hexyl phthalates were obtained from BDH, Great Britain, and Ranbaxy Laboratories Ltd, India. Phthalic acid was obtained from IDPL, India. All other chemicals and solvents used were of analytical grade. Collection of soil samples
Small quantities of garden soil (alluvial, pH 8-2) were taken from the top (15 cm) layer and air-dried and sieved (2 mm). Glassware
The glassware used was meticulously cleaned to reduce any background contamination of phthalates. All chromic-acid washed glassware was placed in a 300 °C oven overnight. After cooling, the glassware was rinsed twice with acetone and petroleum ether and air-dried before use. Aerobic studies
Ten grams of dry and sieved garden soil were taken in 25-ml cotton plugged Erlenmeyer flasks and incorporated with any of the three
Degradation o f some phthalic acid esters in soil
3
phthalates, D M P , D B P and D E H P , in 0.1 ml methanol to give a final concentration of 500 #g of phthalate per gram of soil. The flasks were left unplugged overnight to evaporate the solvent carrier at room temperature. The soil was then mixed thoroughly and the moisture level of the soil was adjusted to 60 ~o of its water-holding capacity with sterile distilled water. The controls were run in parallel using autoclaved soil (autoclaved three times on alternative days at 121 °C at 7 kg pressure for 1 h). All flasks were incubated at 30 _+ 1 °C.
Anaerobic studies The soil was taken in stoppered tubes, incorporated with phthalate esters and flooded with sterile water at least double the length of the soil column. The flooded heat-sterilised controls were run simultaneously. All tubes were incubated at 30 + I°C.
Extraction After incubation for desired periods of time, the soils were extracted three times with 15 ml of methanol. The methanol extracts were pooled, filtered and stored at 4°C until analysed but never for more than 48 h.
Analysis A Waters Associates 440 high performance liquid chromatograph equipped with a #-Bondapak C18 column and a uv detector fixed at 254 nm was used for analysis and estimation of phthalates and their metabolite(s). The solvent system was 60 ~o or 90 ~ aqueous methanol at a pumping rate of 1.5-3.0mlmin -1. The parent c o m p o u n d or metabolite(s) were estimated with the help of authentic standards. Soil without the addition of phthalates was used to determine baseline levels of the phthalates/metabolite(s) or interfering compounds.
RESULTS The results, summarized in Tables 1 to 3, show the degradation of di-nmethyl, di-n-butyl and di-2-ethyl hexyl phthalates to phthalic acid by the soil microflora. No significant degradation of any phthalate was seen
4
Rishi Shanker, C. Ramakrishna, P. K. Seth TABLE 1 Biodegradation of Di-n-Methyl Phthalate in a Garden Soil
Incubation time (days)
Micrograms of compound recoveredper gram of soil Aerobic Anaerobic DMP
0 5 10 15 20 30 (Autoclaved control)
468 _+ 16 180___11 43 + 9 0 0 0 465 ± 6
PA 0 9___0.5 8 + 0.5 0 0 0 Traces
DMP
PA
471 _+ 12 410_+8 376 -+ 6 302 _+ l0 245 _+6 178 _+2
0 8_+ l.l 10 _+0.5 24 _+ 1.7 9 -+ I. I 3 _+ 1.0
467 _+ 8
0
DMP, Di-n-methyl phthalate. PA, Phthalic acid. Each value is the mean _+ SE of triplicate samples.
TABLE 2 Biodegradation of Di-n-Butyl Phthalate in a Garden Soil
Incubation Micrograms of compound recoveredper gram of soil time (days) Aerobic Anaerobic DBP 0 5 10 15 20 30 (Autoclaved control)
472 _+ 14 110___13 40_+6 0 0 0 465 _+ 10
PA 0 8+0-6 6_+0-6 0 0 0 Traces
DBP 470 ± 17 402+9 348_+8 301 _+ 9 239 _+9 159_+4 463 _+9
DBP, Di-n-methyl phthalate. PA, Phthalic acid. Each value is the mean + SE of triplicate samples.
PA 0 12__.1-1 14_+2.9 29 _+ 3.5 22 _+ 2-3 15_+ 1.7 0
Degradation of some phthalic acid esters in soil
5
TABLE 3 Biodegradation of Di-2-Ethyl Hexyl Phthalate in a Garden Soil
Incubation Micrograms of compound recoveredper gram of soil time (days) Aerobic Anaerobic DEHP 0 5 10 20 30 (Autoclaved control)
480 + 9 430 + 8 320+11 120-1-4 40+8 471 ___4
PA 0 8 _ 1.1 7___1.1 11-t-0.6 5-t-0.6 0
DEHP
PA
478 -t- 9 460 _ 8 439+6 389+__5 318-t-7
0 Traces 2___0 8+1.1 11-t-0-6
478 _ 7
0
D E H P , Di-2-ethyl hexyl phthalate. PA, Phthalic acid. Each value is the mean + SE of triplicate samples.
when sterilised soil was used. As is evident from Tables 1 to 3, the rate of degradation of the three PAEs was different. The shorter chain PAEs, such as D M P or DBP, were degraded at a higher rate than the longer chain phthalate DEHP. The soil microflora were able to degrade actively up to 500pgg-~ concentration of various phthalates under aerobic conditions (Table 1 to 3). However, the rate of degradation was very low under anaerobic conditions. Significant amounts of DMP, DBP and D E H P were recovered from soil (Tables 1 to 3) even after 30 days of post inoculation in anaerobic conditions. The degradation appears to be via phthalic acid as small quantities of this substance were detected under both aerobic and anaerobic conditions. No further metabolites were detected.
DISCUSSION PAEs are known to be degraded in river water and marine regions (Saeger & Tucker, 1976; Taylor et al., 1981). Mathur (1974) provided respirometric evidence for the degradation of di-2-ethyl hexyl phthalate and di-n-octyl phthalate by soil microflora. Our data suggest the presence of micro-organisms in soil capable of degrading D M P , DBP and DEHP. This was further confirmed by the isolation of bacteria from the soil
6
Rishi Shanker, C. Ramakrishna, P. K. Seth
capable ofutilising the three PAEs as the sole carbon source (unpublished data). The strains isolated varied in characteristics and were both grampositive or gram-negative, oxidase and catalase positive, could reduce nitrate, and most could grow at 45 °C. Our findings complement those of others that PAEs are biodegradable, and the accumulation reported in the environment (Giam et al., 1978; Giam & Atlas, 1980) might possibly be due to various physical and chemical factors and the behaviour of the chemical in that environment may limit its biodegradability.
ACKNOWLEDGEMENTS The authors are grateful to Dr P. K. Ray, Director, Industrial Toxicology Research Centre, Lucknow, India, for his advice and encouragement and to Dr C. R. Krishna Murti, former Director, ITRC, Lucknow, India, for his valuable suggestions in the initiation of these studies. Research support provided by the Department of the Environment, Government of India, is gratefully acknowledged.
REFERENCES Giam, C. S. & Atlas, E. L. (1980). Accumulation ofphthalate ester plasticizers in Lake Constance sediments. Naturwissenschaften, 67, 508. Giam, C. S., Chan, H. S., Neff, C. S. & Atlas, E. L. (1978). Phthalate ester plasticizers: A new class of marine pollutant. Science, N.Y., 199, 419-21. Giam, C. S., Atlas, E. L., Chan, H. S. & Neff, G. S. (1980). Phthalate esters, PCB and DDT residues in the Gulf of Mexico atmosphere..4 tmos. Environ., 14, 65-9. Graham, P. R. (1973). Phthalate ester plasticizers: Why and how they are used. Environ. Hlth Perspectives, 3, 3-12. Kluwe, W. M., McConnell, E. E., Huff, J. E., Haseman, J. K., Douglas, J. F. & Hartwell, W. V. (1982). Carcinogenicity testing of phthalate esters and related compounds by the National Toxicology Programme and the National Cancer Institute. Environ. Hlth Perspectives, 45, 129-33. Kozumbo, W. J., Kroll, R. & Rubin, R. J. (1982). Assessment ofmutagenicity of phthalate esters. Environ. Hlth Perspectives, 45, 103-10. Mathur, S. P. (1974). Respirometric evidence of utilization of di-octyl and di-2ethyl hexyl phthalate plasticizers. J. environ. Qual., 3, 207-9. Peakall, D. B. (1975). Phthalate esters: Occurrence and biological effects. Residue Rev., 54, 141.
Degradation of some phthalic acid esters in soil
7
Peterson, J. C. & Freeman, D. H. (1982). Phthalate ester concentration variations in dated sediment cores from Chesapeake Bay. Environ. Sci. Technol., 16, 464-9. Saeger, V. W. & Tucker, E. S. (1976). Biodegradation of phthalate acid esters in river water and activated sludge. Appl. environ. Microbiol., 31, 29-34. Seth, P. K. (1982). Hepatic effects of phthalate esters. Environ Hlth Perspectives, 45, 27-34. Sullivan, K. F., Atlas, E. L. & Giam, C. S. (1982). Adsorption of phthalic acid esters from sea water. Environ. Sci. Technol., 16, 428-32. Taylor, B. F., Curry, R. W. & Corcoran, E. F. (1981). Potential for biodegradation of phthalic acid esters in marine regions. Appl. environ. Microbiol., 42, 590-5.