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The environmental fate of the primary degradation products of alkylphenol ethoxylate surfactants in recycled paper sludge
Chemosphere, Vol.
39, No. 5, pp. 745-752, 1999 © 1999 Elsevier Science Ltd. All rights reserved 0045-6535/99/$ - see front matter
Pergamon PII: S0045-6535(99)00010-7
The Environmental Fate of the Primary Degradation Products of Alkylphenol Ethoxylate Surfactants in Recycled Paper Sludge M. Hawrelak, E. Bennett and C. Metcalfe* Environmental and Resource Studies, Trent University, Peterborough, Ontario, K9J 7B8 CANADA (Received in Germany 4 September 1998; accepted 9 December 1998)
ABSTRACT
Alkylphenol ethoxylates (APEOs) are a group of non-ionic surfactants that are degraded microbially into more lipophilic degradation products with estrogenic potential, including nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), octylphenol (4-tOP) and nonylphenol (4-NP). Nonylphenol ethoxylates are used in paper recycling plants for de-inking paper and have the potential to be released into the environment through spreading ofwastewater treatment sludge for soil amendment. Three samples of recycled paper sludge were collected fi'om farmers' fields and analyzed for concentrations ofNP1EO, NP2EO, 4-NP and 4-tOP. Each sample differed in the amount of time elapsed since the sludge was placed on farmers' fields. Primary degradation products of APEOs were present at low ~tg/g concentrations in the sludge samples. Differences in the concentrations of these analytes in sludge samples indicated that APEO concentrations declined by 84% over a period of 14 weeks on farmers' fields. Changes in the chromatographic patterns of acetylated 4-NP indicated that there is a group of recalcitrant nonylphenol isomers that degrades more slowly than other isomers. These data indicate that microbial degradation may reduce the risk of environmental contamination by these compounds, but more work is required to assess the toxic potential of APEOs in sludges used for soil amendment. ~) 1999 Elsevier Science Ltd. All rights reserved
INTRODUCTION
Alkylphenol ethoxylates (APEOs) surfactants are used in a variety of industrial applications, such as in the manufacturing of pulp and paper, textiles, paints, adhesives, leather products, rubber and plastics. Annual global production of APEOs is over 500,000 tonnes; consisting of approximately 80% nonylphenol ethoxylates (NPEOs), 15% octylphenol ethoxylates (OPEOs) and the remainder as dodecylphenol and dinonylphenol ethoxylates (1,2). Since these compounds are used in aqueous solutions, they primarily enter the environment through sewage and industrial wastewater treatment plants (3). 745
746 APEOs are relatively non-toxic, but several of their primary degradation products are considered estrogenic (3-5). The parent compounds are microbially biotransformed under either aerobic or anaerobic conditions to form more lipophilic compounds with estrogenic potential (6), including nonylphenol monoethoxylate (NP1EO) and diethoxylate (NP2EO) and the fully de-ethoxylated compound, 4-nonylphenol (4NP). The primary microbial degradation product of OPEOs is 4-tert-octylphenol (4-tOP). All of these estrogenic compounds have been detected in sewage sludges and effluents, groundwater, surface water and aquatic sediments in both Europe and North America (1,7-11). NPEO surfactants are used for de-inking in the production of recycled paper. The de-inking solution is typically an aqueous blend of NPEO and linear alcohol ethoxylates mixed in a ratio of up to 5% surfactant by weight with the paper to be de-inked (12,13 ). The NPEO content could be as high as 4% by weight of the paper to be processed, although concentrations may be less than one tenth of that amount (12-14). NPEO products with oxyethylene chain lengths between 6 and 9 units (i.e. NPEO~) are the best formulations for de-inking (13). Wastewaters from paper recycling plants located in Canada have been analyzed for standard wastewater parameters and acute toxicity (15), but there are no published data on the concentrations of APEOs and their primary degradation products in these effluent streams. In ,~heprimary wastewater treatment processes used in recycled paper mills, much of the APEOs probably partition into the inky sludge, which also contains a substantial amount of fine cellulose fibres. This primary sludge, along with the activated sludge accumulated in a secondary aerobic wastewater treatment facility is typically spread on farm fields as a soil amendment (15). In this study, we determined the concentrations of 4-NP, 4-tOP, NP1EO, and NP2EO in recycled paper sludge collected l~om farm fields at various points in the soil amendment process. It was anticipated that this study would indicate whether primary degradation products of APEOs are resistant to degradation. These estrogenic compounds could potentially leach into surface and groundwaters (1,16) or they could accumulate in crops planted on the amended soils. These results may also be useful for predicting the fate of APEOs in other wastewater sludges used for soil amendment.
METHODS Samnles:
Recycled paper sludge samples were collected in February, 1998 from two locations in Brock Township, to the northeast of Toronto, Ontario, Canada. The sludge originated from a recycled paper mill operated by Atlantic Packaging Ltd. in Whitby, Ontario. Samples I and lI came from the southeast and northwest comers, respectively of a farm field located at the intersection of Highway 12 and 2nd Concession in Brock Township. Sample III was from a nearby farm field located at the intersection of the Tenth Concession and Thorah Side Road. Both of these farms were using the recycled sludge for soil amendment. The three samples differed in the amount of time elapsed since the sludge had been transported to the fields from the paper recycling plant. Sample I was collected 1 d after the sludge had been transported to the site from the mill and deposited on the field for
747 stockpiling. Sample II was collected 2 mo after stockpiling in the field and Sample HI was collected after the sludge had been stockpiled for 2 mo and then spread on the field for an elapsed period of 6 weeks. Grab samples were placed in solvent-washed glass jars and frozen at -20°C for a period of approximately 2 me. Prior to extraction, sludge samples were thawed, placed in acid washed crucibles and air dried in a fumehood. Replicate (n=3) subsamples of approximately I g dry weight were prepared for analysis.
Recycled paper sludge samples were extracted with distilled in glass dichloromethane (DCM) in a Soxhlet apparatus; essentially as described by Bennie et al. (9). Briefly, spproaimately 1 g of dried sludge was ground with sodium sulphate and placed in a cellulose extraction thimble, topped by approximately 1 g of sodium sulphate and glass wool and extracted with DCM for 6 h. The extract was rotary evaporated to a volume of 5 mL and then filtered through Celite (1 g) and transferred into a centrifuge tube for further concentration to 1 mL under a stream of UHP nitrogen gas. The extract was then split into two-500 ~L aliquots for: a) acetylation and analysis of acetylated 4-NP and 4-tOP by C-C-MS and, b) direct analysis of NPIEO and NP2EO by HPLC. The extract was acetylated for analysis of 4-NP and 4-tOP by adding 10 mg of 1% potassium carbonate and 0.1 mL of triply distilled acetic anhydride, followed by stirring for 45 min. After derivatization, 2 mL of 1% potassium carbonate was added and the sample was mixed by vortex for 15 sec. The organic layer was removed and dried by passing through a glass disposable Pasteur pipet (2 mL) packed with sodium sulphate and plugged with glass wool. The sample was rinsed with hexane into a centrifuge tube and concentrated to a volume of 0.5 mL under a stream of UHP nitrogen. Acetylated samples were cleaned-up by silica-gel column chromatography as described by Bennett and Metalfe (11). A fraction containing acetylated 4-NP and 4-tOP was collected in a glass centrifuge tube, solvent exchanged into iso-octane and evaporated to a volume appropriate for analysis.
Analysis:
The acetylated 4-NP and 4-tOP were analyzed by gas chromatograpby-mass spectrometry in selected ion mode (GC-MS-SIM) using a Hewlett-Packard Model 5890 series 11 gas chromatograph equipped with a 30 m DB-5 column and a Model 5971A mass selective detector, as described previously (I I). Acetylated 4-NP was quantified as the mean concentration calculated from separate calibrations for the 9 major peaks of 4-NP isomers in the samples. Acetylated 4-tOP consists era single isomer, so this compound was quantified from a single peak. Calibration standards were prepared by acetylating known amounts of 4-NP and 4-tOP (Aldrich Chemical, Milwaukee, Wisconsin, USA) dissolved in distilled in glass acetone. Calibration standards of I and I0/~g/mi 4NP and 0.1 and 1 ~g/ml 4-tOP were used for sample quantification. All final volumes were adjusted before analysis so that concentrations were bracketed within the linear two-point calibration curve. Procedural blanks were also prepared using unspiked sodium sulphate. Limits ofDe,tection (LODs) were calculated as 0.1 ~tg/g for 4-NP and 0.001 ~tg/g for 4t-OP.
748 NP1EO and NP2EO were analyzed by HPLC using a Waters 600E HPLC equipped with a Basic Marathon autosampler, Waters 470 fluorescence detector (excitation at X = 230 nm; emission at ~, -- 300 nm) and a 5 ~tm Hypersil APS normal phase column (10 × 2.1 mm i.d.) eluted in isocratic mode at a flow rate of 0.3 mL/min in a mobile phase of hexane/2-propanol (98:2, v/v). NPEO analytes were quantified by calculating the concentration from a multi-point calibration curve with an external standard prepared from a technical APE mixture (POE 1-2, Chemservice) containing NP1EO, NP2EO and NP3EO. The concentrations of NPlEO and NP2EO in this standard were determined by comparing response factors to those of an analytical standard containing known amounts of APEOs (supplied by C. Naylor, Huntsman Chemical Co., Texas). Procedural blanks were prepared using unspiked sodium sulphate. LODs were calculated as 0.01 IJg/g for NP1EO and 0.005 ~tg/g for NP2EO.
Statistical Analvsis The mean concentrations and coefficients of variance were calculated for replicate analyses (n=3) of samples. For a few samples, it was not possible to determine the coefficients of variance because there were only two analytical replicates.
RESULTS AND DISCUSSION
The primary degradation products of APEOs, including 4-NP, 4-tOP, NPIEO, and NP2EO were detected at ~tg/g concentrations (dry weight) in all sludge samples (Table 1). Concentrations of all analytes were ranked in order (highest to lowest) of Sample I>Sample II>Sample HI, which is consistent with the elapsed time since these sludges had been deposited on the farm fields. However ~it cannot be ruled out that the differences in concentrations of analytes are due to variability in the APEO content of the original sludge brought to the farm fields, rather than degradation of the APEOs over time. The greatest differences in concentrations between samples were observed for NP1EO, which differed by close to two orders of magnitude between Sample I and Sample llI. Concentrations of all other analytes varied by less than one order of magnitude between samples. The coefficients of variation for replicate analyses were between 2.8%-41.2%; an indication that the distribution of analytes in sludge was relatively heterogeneous. Concentrations in the Sample I sludges were lower than the concentrations reported in the sludges of municipal sewage treatment plants in Canada, which are generally in the range of approximately 100-500 I~g/gfor NP and NP1EO, 5-150 I~g/g for NP2EO, and <20 gg/g for OP (9-11). Chromatograms of acetylated 4-NP and 4-tOP (Figure 1) show that 9 peaks representing different isomers of 4-NP were resolved under the chromatographic conditions used in this study. Comparisons between chromatographic patterns in the 4-NP standard (Figure la) and the samples (Figure lb-d) indicate that the intensities of most peaks declined in order of Samples I, II and III, but peak #5 (arrow) declined to a lesser degrce, resulting in "enrichment" of this peak relative to the others. Wheeler et al. (17) identified 22 para-isomers
749 of 4-NP by high-resolution gas chromatography that were divided into 5 distinct groups according to the degree and pattern of branching of the alkyl chain. Eluting in Peak #5 are 4-NP isomers in Group 1, (i.e. alpha-dimethyl) and in Group 2 (i.e. alpha-methyL alpha-ethyl, beta-primary). Isomer Group 1 consists of 10 possible isomers, while Group 2 consists of 4 possible isomers, making up 48.6% and 24~7%, respectively of the total isomers in 4-NP. The alkyl chains in Group I isomers are linear but the alkyi chains in Group 2 isomers are more highly branched. Since 4-NP isomers with branched alkyl chains are subject to slower rates of microbial degradation than isomers with linear alkyl chain (18,19), it is hypothesized that the changes in chromatographic patterns for 4-NP in sludge samples were due to slower rates of degradation of recalcitrant Group 2 isomers that elute in Peak #5. A decline in the concentrations of the primary degradation products of NPEO in sludge with time is consistent with a study by Hughes et al. (20) in which NPEO degradation in soil was studied in the laboratory under aerobic conditions. Total NPEO concentrations declined by 57% a/~er 64 days in natural soil spiked with a commercial NPEO formulation containing 76% NPEOg. Marcomini et al. (21) studied changes in the concentrations of4-NP, NPlEO, and NP2EO under aerobic conditions when sludges were added to natural soils in the laboratory. After the first month, total analyte concentrations declined by 80%, with further reductions equaling 90% afcer a period of one year.
Table 1: Mean concentrations (% coefficient of variation in brackets) of 4-NP, 4-tOP, NP1EO and NP2EO in replicate subsamples (n=3, except where specified) of sludge from a recycled paper plant applied to farmland as a soil amendment. * Replicates = 2.
SAMPLE
DESCRIPTION
CONCENTRATIONS (~tg/g dry weight) 4-NP
4-tOP
NP lEO
NP2EO
Sample I
Stockpiled, 1 d
4.61(9.6)
0.18 (22.6)
1.21 (30.4)
0.39 *
Sample II
Stockpiled, 2 mo
3.98 (17.6)
0.14 (41.2)
0.17 *
0.14 *
2.35 (13.1)
0.05 (34.9) 0.07(30.0)
Sample III Stockpiled 2 mo, 6 wk post-application
0.08(25.2)
The results in this study are consistent with a decline in total NPEO analyte concentrations in recycled paper sludge by 84% alter 14 weeks on farm fields. It is not clear whether conditions for microbial degradation in the sludge were aerobic or anaerobic in this case. Conditions within the core of the stockpiled material may have been anaerobic. However, sludge subsequently spread on the land after stockpiling would be exposed to the air. As stated previously, because of deficiencies in the study design, it cannot be stated with certainty that degradation was responsible for differences in NPEO concentrations in the 3 samples, despite the weight of evidence.
750
B Sample I
A Analytical Standard
OP
i D Sample III
C Sample II
l J
o.,
. . . . ' " '6 ' ~ 0 ' ' . . . . " ' " " "
~
"
.... ' .... "'
" I ~
Figure 1: Total ion chromatograms of acetylated 4-tert-octylphenol (OP) and multiple isomers of 4-nonylphenol (NP) in: a) an analytical standard, b) Sample I, c) Sample H and d) Sample II]. The arrow indicates the position of a recalcitrant peak representing co-eluting isomers of 4-NP.
CONCLUSIONS Primary degradation products of APEOs, including 4-NP, 4-tOP, NP1EO, and NP2EO were present at low I~g/g concentrations in the sludge from a paper recycling plant. Differences in the concentrations of these analytes in sludge samples used for soil amendment indicate that concentratigns of APEOs have the potential to decline by 84% over a period of 14 weeks on farm fields. Changes in the chromatographic patterns of acetylated 4-NP indicate that a group of recalcitrant nonylphenol isomers degraded more slowly than other isomers. It is dii~cult to speculate on the potential for NPEO contamination of surface water and groundwater and agricultural crops in areas where recycled paper sludges are used for soil amendment, but it appears that mineralization of NPEOs through microbial degradation can significantly reduce the risk of environmental contamination. More work is needed to evaluate the fate of NPEOs in sludges and to identify degradation products that may persist,
751 Acknowledgments: Sludge samples were collected by Maureen Riley and Larry Hoover. We thank Carter Naylor for providing us with an analytical standard of NPEOs.
REI~RENCES I. Ahel, M., C. Scha~er and W. Giger. Behaviour of a l k y l p ~ l polyethoxylate suffactants in the aquatic environment-re. Occurrence and elimination of their persistent metabolites during infdtration of river water to groundwater. War.Res. 30, 37-46 (1996). 2. Naylor, C.G. Environmental fate of alkylphenol ethoxylates. Soap Chemical Specialties 68, 27-32 (1992). 3. Jobling, S., D. Sheahan, J.A. Osborne, P. Matthiessen and J.P. Sumpter. Inhibition of testicular growth in rainbow trout (Oncorh~chns mykiss) exposed to estrogenic alkylphenolic chemicals. Emqro~ Toxlcol. Chem.
I5, 194-202 (1996). 4. White, R., S. Joblin8, S.A. Hoare, J.P. Sumpter and M.G. Parker. Environmentally persistent alkylphenol compounds are estrogenic. Endocrinol. 135, 175-182 (1994). 5. Gray, MA. and C.D Metcalfe. Induction of testis-ova in Japanese medaka (Oryzias/at/pes) exposed to pnonylphenol. Environ. Toxicol.Chem. 16, 1082-1086 (1997). 6. Nimrod, A.C.and WH. Benson. Environmental estrogenic effects of alkylphenol ethoxylates. CriticalRev. ToxicoI. 26, 355-364 (1996). 7. Abel, M., W, Giger and M. Koch. Behaviour of alkylphenol polyethoxylates in the aquatic environment--I. Occurrence and transformation in sewage treatment. War.Res. 28, 1131-1142 (1994). 8. Naylor,C.G. Environmentalfate and safety ofnonytphenol ethoxylates. Surfact. Wastewat. 27, 29-33 (1995). 9. Bennie DT., C.A. Sullivan, H-B. Lee, TE. Peart and R.J. Maguire. Occurrence of alkylphenols and alkylphenol mono- and diethoxylates in natural waters. Sci. TotalEnviron. 193,263-275 (1996). 10. Lee, H-B,, TE. Pearl D.T. Beanie and RJ. Maguire. Determinationofnonylphenol polyethoxylates and their carboxylic acid metabolites in sewage treatment plant sludge by supercritical carbon dioxide extraction. J. Chromatogr. A, 785, 385-394 (1997). 11. Bennett, E.R. and C.D. Metcalfe. Distribution of alkylphenol compounds in Great Lakes sediments; United States and Canada. Environ. Toxicol. Chem. 16,1230-1235 (1998). 12. Hipolet, K.J. ChemicalProcessingAids in Papenna~ng'.4 Practical Guide. Tappi Press, Atlanta, GA, USA, 223p. (1992). 13. McDonald, M Paper Recycling and the Use of Chemicals. Noyes Data Corp., Park Ridge, NJ, USA, 124p. (1971). 14. Turvery, 1%W.Chemical use in recycling. In: Technology of Paper Recycling, Edited by R.W.J. McKinney, pp. 34-45, Blackie Academic Press, London, UK. (1994).
752 15. M_cChfl~Consultants. Best AvailableTechnologyfor the Ontario Pulp and Paper Industry. Prepared for the
Ontario Ministry ofthe Environment, Water Resources Branr~ Toronto, Ontario, Canada, 186p. (1992). 16. Rudel, R.A., S.J. Melly, P.W. Geno, G. Sun and I.G. Brody. Identification of alkylphenols and other estrogenicphenoliccompoundsin wastewa~, septage and groundwater on Cape Cod Massachusetts. Environ. Sci. Technol. 32,861-869 (1998.).
17. Wheeler, T.D., J.R. Heim, M.R. LaTorre and A.B. Janes. Mass spectral characterization ofp-nonylphenol isomers usin8 high-resolution capillaryGC-MS. J. Chromatogr. Sci. 35,19-30 (1997). 18. Kravetz,L. Biodegradationofnonionic sm'factants:Alcohol ethoxylates vs. nonylphenol ethoxylates.Amer. Assoc. Textile Chemists 15, 23-28 (1983).
19. Swisher, R.D. Chemical structure and primm7 biodegradation VI. Ethoxylate nonionics. In: Surfactant Biodegradation~ 2ndEditio~ pp. 487-497, Marcel Dekker, New York, NY (1987).
20. Hughes, A.I., Fisher, J. and E. Brumbaugh. Biode~adation of ArPE in Soil Alkylphenols& Alkylphenol Ethoxylates Review 1,17-22 (1998). 21. Marcomini, A., P.D. Capel, W. Giger and H. Hani. Residue8 of detergent-derived organic pollutants and polychlorinatedbiphenyisin sludge-amended soil. Naturwissenschaflen 75,460-462 (1988).
39, No. 5, pp. 745-752, 1999 © 1999 Elsevier Science Ltd. All rights reserved 0045-6535/99/$ - see front matter
Pergamon PII: S0045-6535(99)00010-7
The Environmental Fate of the Primary Degradation Products of Alkylphenol Ethoxylate Surfactants in Recycled Paper Sludge M. Hawrelak, E. Bennett and C. Metcalfe* Environmental and Resource Studies, Trent University, Peterborough, Ontario, K9J 7B8 CANADA (Received in Germany 4 September 1998; accepted 9 December 1998)
ABSTRACT
Alkylphenol ethoxylates (APEOs) are a group of non-ionic surfactants that are degraded microbially into more lipophilic degradation products with estrogenic potential, including nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), octylphenol (4-tOP) and nonylphenol (4-NP). Nonylphenol ethoxylates are used in paper recycling plants for de-inking paper and have the potential to be released into the environment through spreading ofwastewater treatment sludge for soil amendment. Three samples of recycled paper sludge were collected fi'om farmers' fields and analyzed for concentrations ofNP1EO, NP2EO, 4-NP and 4-tOP. Each sample differed in the amount of time elapsed since the sludge was placed on farmers' fields. Primary degradation products of APEOs were present at low ~tg/g concentrations in the sludge samples. Differences in the concentrations of these analytes in sludge samples indicated that APEO concentrations declined by 84% over a period of 14 weeks on farmers' fields. Changes in the chromatographic patterns of acetylated 4-NP indicated that there is a group of recalcitrant nonylphenol isomers that degrades more slowly than other isomers. These data indicate that microbial degradation may reduce the risk of environmental contamination by these compounds, but more work is required to assess the toxic potential of APEOs in sludges used for soil amendment. ~) 1999 Elsevier Science Ltd. All rights reserved
INTRODUCTION
Alkylphenol ethoxylates (APEOs) surfactants are used in a variety of industrial applications, such as in the manufacturing of pulp and paper, textiles, paints, adhesives, leather products, rubber and plastics. Annual global production of APEOs is over 500,000 tonnes; consisting of approximately 80% nonylphenol ethoxylates (NPEOs), 15% octylphenol ethoxylates (OPEOs) and the remainder as dodecylphenol and dinonylphenol ethoxylates (1,2). Since these compounds are used in aqueous solutions, they primarily enter the environment through sewage and industrial wastewater treatment plants (3). 745
746 APEOs are relatively non-toxic, but several of their primary degradation products are considered estrogenic (3-5). The parent compounds are microbially biotransformed under either aerobic or anaerobic conditions to form more lipophilic compounds with estrogenic potential (6), including nonylphenol monoethoxylate (NP1EO) and diethoxylate (NP2EO) and the fully de-ethoxylated compound, 4-nonylphenol (4NP). The primary microbial degradation product of OPEOs is 4-tert-octylphenol (4-tOP). All of these estrogenic compounds have been detected in sewage sludges and effluents, groundwater, surface water and aquatic sediments in both Europe and North America (1,7-11). NPEO surfactants are used for de-inking in the production of recycled paper. The de-inking solution is typically an aqueous blend of NPEO and linear alcohol ethoxylates mixed in a ratio of up to 5% surfactant by weight with the paper to be de-inked (12,13 ). The NPEO content could be as high as 4% by weight of the paper to be processed, although concentrations may be less than one tenth of that amount (12-14). NPEO products with oxyethylene chain lengths between 6 and 9 units (i.e. NPEO~) are the best formulations for de-inking (13). Wastewaters from paper recycling plants located in Canada have been analyzed for standard wastewater parameters and acute toxicity (15), but there are no published data on the concentrations of APEOs and their primary degradation products in these effluent streams. In ,~heprimary wastewater treatment processes used in recycled paper mills, much of the APEOs probably partition into the inky sludge, which also contains a substantial amount of fine cellulose fibres. This primary sludge, along with the activated sludge accumulated in a secondary aerobic wastewater treatment facility is typically spread on farm fields as a soil amendment (15). In this study, we determined the concentrations of 4-NP, 4-tOP, NP1EO, and NP2EO in recycled paper sludge collected l~om farm fields at various points in the soil amendment process. It was anticipated that this study would indicate whether primary degradation products of APEOs are resistant to degradation. These estrogenic compounds could potentially leach into surface and groundwaters (1,16) or they could accumulate in crops planted on the amended soils. These results may also be useful for predicting the fate of APEOs in other wastewater sludges used for soil amendment.
METHODS Samnles:
Recycled paper sludge samples were collected in February, 1998 from two locations in Brock Township, to the northeast of Toronto, Ontario, Canada. The sludge originated from a recycled paper mill operated by Atlantic Packaging Ltd. in Whitby, Ontario. Samples I and lI came from the southeast and northwest comers, respectively of a farm field located at the intersection of Highway 12 and 2nd Concession in Brock Township. Sample III was from a nearby farm field located at the intersection of the Tenth Concession and Thorah Side Road. Both of these farms were using the recycled sludge for soil amendment. The three samples differed in the amount of time elapsed since the sludge had been transported to the fields from the paper recycling plant. Sample I was collected 1 d after the sludge had been transported to the site from the mill and deposited on the field for
747 stockpiling. Sample II was collected 2 mo after stockpiling in the field and Sample HI was collected after the sludge had been stockpiled for 2 mo and then spread on the field for an elapsed period of 6 weeks. Grab samples were placed in solvent-washed glass jars and frozen at -20°C for a period of approximately 2 me. Prior to extraction, sludge samples were thawed, placed in acid washed crucibles and air dried in a fumehood. Replicate (n=3) subsamples of approximately I g dry weight were prepared for analysis.
Recycled paper sludge samples were extracted with distilled in glass dichloromethane (DCM) in a Soxhlet apparatus; essentially as described by Bennie et al. (9). Briefly, spproaimately 1 g of dried sludge was ground with sodium sulphate and placed in a cellulose extraction thimble, topped by approximately 1 g of sodium sulphate and glass wool and extracted with DCM for 6 h. The extract was rotary evaporated to a volume of 5 mL and then filtered through Celite (1 g) and transferred into a centrifuge tube for further concentration to 1 mL under a stream of UHP nitrogen gas. The extract was then split into two-500 ~L aliquots for: a) acetylation and analysis of acetylated 4-NP and 4-tOP by C-C-MS and, b) direct analysis of NPIEO and NP2EO by HPLC. The extract was acetylated for analysis of 4-NP and 4-tOP by adding 10 mg of 1% potassium carbonate and 0.1 mL of triply distilled acetic anhydride, followed by stirring for 45 min. After derivatization, 2 mL of 1% potassium carbonate was added and the sample was mixed by vortex for 15 sec. The organic layer was removed and dried by passing through a glass disposable Pasteur pipet (2 mL) packed with sodium sulphate and plugged with glass wool. The sample was rinsed with hexane into a centrifuge tube and concentrated to a volume of 0.5 mL under a stream of UHP nitrogen. Acetylated samples were cleaned-up by silica-gel column chromatography as described by Bennett and Metalfe (11). A fraction containing acetylated 4-NP and 4-tOP was collected in a glass centrifuge tube, solvent exchanged into iso-octane and evaporated to a volume appropriate for analysis.
Analysis:
The acetylated 4-NP and 4-tOP were analyzed by gas chromatograpby-mass spectrometry in selected ion mode (GC-MS-SIM) using a Hewlett-Packard Model 5890 series 11 gas chromatograph equipped with a 30 m DB-5 column and a Model 5971A mass selective detector, as described previously (I I). Acetylated 4-NP was quantified as the mean concentration calculated from separate calibrations for the 9 major peaks of 4-NP isomers in the samples. Acetylated 4-tOP consists era single isomer, so this compound was quantified from a single peak. Calibration standards were prepared by acetylating known amounts of 4-NP and 4-tOP (Aldrich Chemical, Milwaukee, Wisconsin, USA) dissolved in distilled in glass acetone. Calibration standards of I and I0/~g/mi 4NP and 0.1 and 1 ~g/ml 4-tOP were used for sample quantification. All final volumes were adjusted before analysis so that concentrations were bracketed within the linear two-point calibration curve. Procedural blanks were also prepared using unspiked sodium sulphate. Limits ofDe,tection (LODs) were calculated as 0.1 ~tg/g for 4-NP and 0.001 ~tg/g for 4t-OP.
748 NP1EO and NP2EO were analyzed by HPLC using a Waters 600E HPLC equipped with a Basic Marathon autosampler, Waters 470 fluorescence detector (excitation at X = 230 nm; emission at ~, -- 300 nm) and a 5 ~tm Hypersil APS normal phase column (10 × 2.1 mm i.d.) eluted in isocratic mode at a flow rate of 0.3 mL/min in a mobile phase of hexane/2-propanol (98:2, v/v). NPEO analytes were quantified by calculating the concentration from a multi-point calibration curve with an external standard prepared from a technical APE mixture (POE 1-2, Chemservice) containing NP1EO, NP2EO and NP3EO. The concentrations of NPlEO and NP2EO in this standard were determined by comparing response factors to those of an analytical standard containing known amounts of APEOs (supplied by C. Naylor, Huntsman Chemical Co., Texas). Procedural blanks were prepared using unspiked sodium sulphate. LODs were calculated as 0.01 IJg/g for NP1EO and 0.005 ~tg/g for NP2EO.
Statistical Analvsis The mean concentrations and coefficients of variance were calculated for replicate analyses (n=3) of samples. For a few samples, it was not possible to determine the coefficients of variance because there were only two analytical replicates.
RESULTS AND DISCUSSION
The primary degradation products of APEOs, including 4-NP, 4-tOP, NPIEO, and NP2EO were detected at ~tg/g concentrations (dry weight) in all sludge samples (Table 1). Concentrations of all analytes were ranked in order (highest to lowest) of Sample I>Sample II>Sample HI, which is consistent with the elapsed time since these sludges had been deposited on the farm fields. However ~it cannot be ruled out that the differences in concentrations of analytes are due to variability in the APEO content of the original sludge brought to the farm fields, rather than degradation of the APEOs over time. The greatest differences in concentrations between samples were observed for NP1EO, which differed by close to two orders of magnitude between Sample I and Sample llI. Concentrations of all other analytes varied by less than one order of magnitude between samples. The coefficients of variation for replicate analyses were between 2.8%-41.2%; an indication that the distribution of analytes in sludge was relatively heterogeneous. Concentrations in the Sample I sludges were lower than the concentrations reported in the sludges of municipal sewage treatment plants in Canada, which are generally in the range of approximately 100-500 I~g/gfor NP and NP1EO, 5-150 I~g/g for NP2EO, and <20 gg/g for OP (9-11). Chromatograms of acetylated 4-NP and 4-tOP (Figure 1) show that 9 peaks representing different isomers of 4-NP were resolved under the chromatographic conditions used in this study. Comparisons between chromatographic patterns in the 4-NP standard (Figure la) and the samples (Figure lb-d) indicate that the intensities of most peaks declined in order of Samples I, II and III, but peak #5 (arrow) declined to a lesser degrce, resulting in "enrichment" of this peak relative to the others. Wheeler et al. (17) identified 22 para-isomers
749 of 4-NP by high-resolution gas chromatography that were divided into 5 distinct groups according to the degree and pattern of branching of the alkyl chain. Eluting in Peak #5 are 4-NP isomers in Group 1, (i.e. alpha-dimethyl) and in Group 2 (i.e. alpha-methyL alpha-ethyl, beta-primary). Isomer Group 1 consists of 10 possible isomers, while Group 2 consists of 4 possible isomers, making up 48.6% and 24~7%, respectively of the total isomers in 4-NP. The alkyl chains in Group I isomers are linear but the alkyi chains in Group 2 isomers are more highly branched. Since 4-NP isomers with branched alkyl chains are subject to slower rates of microbial degradation than isomers with linear alkyl chain (18,19), it is hypothesized that the changes in chromatographic patterns for 4-NP in sludge samples were due to slower rates of degradation of recalcitrant Group 2 isomers that elute in Peak #5. A decline in the concentrations of the primary degradation products of NPEO in sludge with time is consistent with a study by Hughes et al. (20) in which NPEO degradation in soil was studied in the laboratory under aerobic conditions. Total NPEO concentrations declined by 57% a/~er 64 days in natural soil spiked with a commercial NPEO formulation containing 76% NPEOg. Marcomini et al. (21) studied changes in the concentrations of4-NP, NPlEO, and NP2EO under aerobic conditions when sludges were added to natural soils in the laboratory. After the first month, total analyte concentrations declined by 80%, with further reductions equaling 90% afcer a period of one year.
Table 1: Mean concentrations (% coefficient of variation in brackets) of 4-NP, 4-tOP, NP1EO and NP2EO in replicate subsamples (n=3, except where specified) of sludge from a recycled paper plant applied to farmland as a soil amendment. * Replicates = 2.
SAMPLE
DESCRIPTION
CONCENTRATIONS (~tg/g dry weight) 4-NP
4-tOP
NP lEO
NP2EO
Sample I
Stockpiled, 1 d
4.61(9.6)
0.18 (22.6)
1.21 (30.4)
0.39 *
Sample II
Stockpiled, 2 mo
3.98 (17.6)
0.14 (41.2)
0.17 *
0.14 *
2.35 (13.1)
0.05 (34.9) 0.07(30.0)
Sample III Stockpiled 2 mo, 6 wk post-application
0.08(25.2)
The results in this study are consistent with a decline in total NPEO analyte concentrations in recycled paper sludge by 84% alter 14 weeks on farm fields. It is not clear whether conditions for microbial degradation in the sludge were aerobic or anaerobic in this case. Conditions within the core of the stockpiled material may have been anaerobic. However, sludge subsequently spread on the land after stockpiling would be exposed to the air. As stated previously, because of deficiencies in the study design, it cannot be stated with certainty that degradation was responsible for differences in NPEO concentrations in the 3 samples, despite the weight of evidence.
750
B Sample I
A Analytical Standard
OP
i D Sample III
C Sample II
l J
o.,
. . . . ' " '6 ' ~ 0 ' ' . . . . " ' " " "
~
"
.... ' .... "'
" I ~
Figure 1: Total ion chromatograms of acetylated 4-tert-octylphenol (OP) and multiple isomers of 4-nonylphenol (NP) in: a) an analytical standard, b) Sample I, c) Sample H and d) Sample II]. The arrow indicates the position of a recalcitrant peak representing co-eluting isomers of 4-NP.
CONCLUSIONS Primary degradation products of APEOs, including 4-NP, 4-tOP, NP1EO, and NP2EO were present at low I~g/g concentrations in the sludge from a paper recycling plant. Differences in the concentrations of these analytes in sludge samples used for soil amendment indicate that concentratigns of APEOs have the potential to decline by 84% over a period of 14 weeks on farm fields. Changes in the chromatographic patterns of acetylated 4-NP indicate that a group of recalcitrant nonylphenol isomers degraded more slowly than other isomers. It is dii~cult to speculate on the potential for NPEO contamination of surface water and groundwater and agricultural crops in areas where recycled paper sludges are used for soil amendment, but it appears that mineralization of NPEOs through microbial degradation can significantly reduce the risk of environmental contamination. More work is needed to evaluate the fate of NPEOs in sludges and to identify degradation products that may persist,
751 Acknowledgments: Sludge samples were collected by Maureen Riley and Larry Hoover. We thank Carter Naylor for providing us with an analytical standard of NPEOs.
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