Volume 1, Issue 2, 2007 Pulp mill effluent is a source of environmental estrogens on Alabama’s Coosa River Quentin Felty, Assistant Professor, Florida International University: Department of Environmental & Occupational Health Science, Feltyq@fiu.edu Abstract Alkylphenols are degradation by-products of detergents used to disperse tacky deposits on equipment in the paper industry. β-sitosterol is a phytosterol from pine trees used in the pulping industry. Both of these compounds are reported to be endocrine disrupting chemicals (EDCs). The goal of this study was to monitor a combination of EDCs in pulp mill effluent. Water samples collected from a pulp mill were derivatized and analyzed for trimethylsilyl derivatives by GC/MS. Mean concentrations of 4-tertoctylphenol, 4-nonylphenol, and β-sitosterol in the effluent were 3.45, 6.62, and 19.92μg/L, respectively. We detected a 1.4-fold higher level of 4-t-OP and showed β-sitosterol to be 2.8-fold higher than reports in the literature. The presence of alkyphenols and β-sitosterol in pulp mill effluent show that Alabama’s water sources are being contaminated by EDCs. The concentrations we detected are individually capable of inducing an adverse effect which indicates a need for a human risk assessment. Introduction Synthetic and natural chemicals that mimic endogenous hormones can interfere with the normal growth and development of wildlife and humans. The presence of endocrine disrupting chemicals (EDCs) in the environment is alarming. Evidence in further support of this view comes from the observation of adverse health effects associated with EDCs in various wildlife species (1). The health concern over environmental estrogens is partly based on the pivotal role that natural estrogens such as 17β-estradiol play in reproduction and development. The ability of EDCs to mediate cell signaling pathways during critical periods in vertebrate development may have profound adverse effects on exposed wildlife and humans. Considerable attention has been focused on compounds known as alkylphenols. Alkylphenols originate from a class of nonionic surfactants known as alkylphenol ethoxylates (APEs). APEs have been used in cleaning products and industrial processes for over 40 years. Environmental concentrations of alkylphenols in sewage effluents and rivers mainly refer to nonylphenol and octylphenol; and their concentrations are reported to range from <0.1 to 369 µg/L in US wastewater effluents (2). In addition to these synthetic compounds, exposure to the phytosterol β-sitosterol has also been implicated in reproductive dysfunction seen in wildlife (3, 4). Since pine trees are used in the pulping industry, high levels of β-sitosterol enter the aquatic environment via discharged effluent. β-sitosterol concentrations in pulp mill effluent were reported to be 280µg/L (5). The high levels of β-sitosterol released into the environment are of concern because, it has been demonstrated to promote estrogen-dependent processes such as uterine growth in various mammals and vitellogenin production (5). It is clear that alkylphenols and β-sitosterol are in the aquatic environment at levels which have been experimentally demonstrated to cause estrogenic effects (2, 4). Recognition of the potential for adverse effects in wildlife and humans suggests a need to measure these chemicals in lakes and rivers used as sources of drinking water. Because there is a lack of data on the presence or absence of these chemicals in Alabama’s water sources, we measured the level of the following EDCs: 4-nonylphenol (4-NP), 4-tertoctylphenol (4-t-OP), 4-octylphenol (4-OP), and β-sitosterol. Water samples were collected from an Alabama pulp mill by grab sampling in front of a discharge pipe which transports wastewater from a settling pond to the Coosa River. Following liquid-liquid extraction, concentrated extracts were derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide, and analyzed for trimethylsilyl (TMS) derivatives by GC/MS. Materials And Methods
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Sample collection and extraction: Water samples were collected in the state of Alabama by grab sampling in front of a discharge pipe which transports wastewater from a pulp mill settling pond to a river. A total of 6 samples were collected on two separate visits in the fall season of 2004. Pre-cleaned 1 liter glass bottles with tin foil covered Teflon caps were used to collect the water samples. All samples were refrigerated until time of extraction. EDCs were extracted from wastewater samples using a modified procedure presented by Stephanou and Giger (6). Water samples of 200 mL were transferred to a glass amber bottle with a magnetic stir bar, spiked with 2.5 μg of the internal standard equilenin, and 20 mL of methylene chloride. The sample was placed on a magnetic stir plate and stirred for 15 minutes. Afterwards, the sample was transferred to a 500 mL separatory funnel in which aqueous and organic layers were allowed to separate for 10 minutes before the organic extract was collected. The extraction procedure was repeated two more times and the combined extract was dried by passing it through a glass column packed with sodium sulfate. A Kuderna-Danish evaporator set underneath a nitrogen stream was used to concentrate the extract to 1-2 mL. Derivitization: To determine the levels of 4-NP, 4-t-OP, 4-OP, and β-sitosterol, a method presented by Porubek and Nelson was employed (7). The alkylphenols and phytosterol in a given sample from the above procedure were derivatized by adding 50 μL of BSTFA and 5 μL of pyridine to the dry extracts. The reaction vials were then capped, gently vortexed, and heated at 80°C for 30 minutes. This mixture formed the TMS derivative of the previously mentioned alkylphenols and phytosterol to be analyzed by GC/MS the same day. GC/MS analysis: The TMS derivatives of 4-NP, 4-t-OP, 4-OP, β-sitosterol, and the internal standard equilenin were analyzed and quantified using a HP 5890 gas chromatograph (GC) connected to a HP 5970 mass spectrometer (MS). The derivatized extract was injected as 1 μL aliquots in the splitless mode using helium (5 mL/min.) as the carrier gas. The GC contained a HP-5MS fused silica capillary column (0.25 μm film thickness, 30m X 0.25 inner diameter). The GC was programmed as described by Porubek and Nelson (7). The injector temperature was set at 280°C. An initial heating rate of 30°C/min. for 4 minutes was followed by a second heating rate of 5°C/min. until 280°C was reached and then held at 280°C for 10 minutes. The interface temperature between the GC and MS was 280°C. The MS was operated in the select ion monitoring mode (SIM) mode with ionization energy of 70 eV. The fragment ions and retention time used to identify the TMS derivatives of the compounds are shown in Table 1. Table 1. Mass spectra of TMS derivatives. Compound Retention Time (min.) 4-tert-octylphenol 5.35 4-nonylphenol 5.90-6.23 4-octylphenol 6.45 Equilenin 17.20 β-sitosterol 31.70
m/z 207, 208, 278 179, 193, 207, 221, 263, 292 179, 180, 278 282, 295, 338 129, 255, 357, 396 (8)
Standard curves and sample recovery: Standard curves using TMS derivatized 4-NP, 4-t-OP, 4-OP, βsitosterol, and the internal standard equilenin were prepared using the previously described method. Standard solutions were prepared in ethanol ranging from 0.1 μg/mL to 200 μg/mL. Sample recovery efficiency was assessed by spiking 200 mL of distilled water with 2.5 μg of each standard and performing the previously described extraction method. Standard curves of peak area ratio versus weight ratio were constructed by holding constant the amount of internal standard (equilenin) at 2.5μg and varying the amount of each target compound from 0.1 μg/mL to 200 μg/mL. The peak area ratio represents the compound’s peak area divided by the internal standard’s peak area. The weight ratio represents the weight of each compound (μg) divided by the weight of the internal standard (2.5 μg). Standard curves of each target analyte are shown in Fig. 2. The corresponding linear regression parameters for each compound are in Table 2. The slope and y-intercept from each standard curve was used to calculate the unknown concentration of target analytes recovered from effluent.
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Fig. 2
A
Peak Area Ratio
4-tert-octylphenol
4-nonylphenol
2.000
B
Peak Area Ratio
3
2
1.000
1
0.000 0 0.5 We ight Ratio 1 1.5
0 0 0.5 Weight Ratio 1 1.5
4-octylphenol
Beta Sitosterol
C
Peak Area Ratio
4 3 2 1
0 0 0.5 1 1.5
D
Peak Area Ratio
4 3 2 1
0 0 2 4 6 8 10
Weight Ratio
Equilenin
6 5
Weight Ratio
E
Peak Area Ratio
4 3 2 1 0 0 1 2 3 4 5 Weight Ratio
Figure 2. Standard Curves of weight ratio versus peak area ratio: (A) 4-tert-octylphenol, (B) 4nonylphenol (C) 4-octylphenol (D) β-sitosterol (E) equilenin. Internal standard (equilenin) was held constant at 2.5 μg.
Table 2. Linear regression parameters. 4-t-OP 4-NP 4-OP Equilenin β-sitosterol 1.2616 1.5451 2.3162 1.3437 0.4276 Slope 0.0479 0.0167 0.0914 0.0613 -0.0284 Y-int. 0.9878 0.9733 0.9936 0.9983 0.9995 r Statistical methods: Linear regression was used to determine the limit of detection (LOD) for each compound from the standard curves. To determine recovery efficiency of the extraction method, 200 mL distilled water was spiked with a 2.5 μg per compound mixture of standards. The percent recovery of each standard was calculated as the mean of three separate experiment recoveries. The student’s t-test
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was used to determine the significant differences in means between the wastewater sample and tap water. Results Wastewater Samples: Sample recovery was 69% ± 36% for all of the compounds investigated. In Table 3, detection limits and levels of each compound detected in effluent are given. Wastewater results indicate mean levels of 3.45 μg/L for 4-t-OP, 6.62 μg/L for 4-NP, and 19.92 μg/L for β-sitosterol. The compound 4-octylphenol was below detection limits. Table 3. Compounds detected in effluent Compound LOD SD Level in Effluent (μg/L) (μg/L) 4-t-OP 0.020 3.45 * 1.24 4-NP 0.027 6.62 * 2.38 4-OP 0.046 Not detected 1.25 19.92 * 7.17 β-sitosterol (*) Levels detected in effluent are expressed as mean of six separate experiments. Chemical in effluent significantly different from tap water P<0.05) The total ion current (TIC) chromatogram of the TMS derivatives of 4-tert-octylphenol (retention time 5.35 min.), 4-nonylphenol (retention time 5.90-6.23 min.), and β-sitosterol (retention time 31.73 min.) found in wastewater are shown in Fig. 3. In addition, corresponding mass spectrums of each individual compound are shown. Fig. 3A. TIC of investigated compounds in pulp mill effluent
Fig. 3A TIC Chromatogram
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Fig. 3B-3D. Individual mass spectrums of investigated compounds measured in effluent:
Fig. 3B Mass spectrum of 4-tert-octylphenol
Fig. 3C Mass spectrum of 4-nonylphenol
Fig. 3D Mass spectrum of β-sitosterol
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Discussion The goal of this study was to measure a combination of environmental estrogens in pulp mill effluent: 4nonylphenol, 4-tert-octylphenol, 4-octylphenol, and β-sitosterol. Mean effluent concentrations of 4-t-OP, 4-NP, and β-sitosterol were 3.45 μg/L, 6.62 μg/L, and 19.92 μg/L, respectively. Rudel et al. published a similar derivitization and GC/MS method in which they monitored over 20 estrogen-like chemicals in wastewater, septage, and groundwater(9). Although some of the analytes we monitored overlapped with the compounds reported by Rudel et al., there were some differences. In particular, our study identified 4tert-octylphenol and the plant sterol β-sitosterol. Rudel et al., reported recoveries for 4-nonylphenol and 4octylphenol in the range of 66% to 107% which concur with our recovery of 69%. Rudel et al.,’s method detection limits for 4-NP ranged from 0.01 to 0.02 μg/L and from 0.005 to 0.02 μg/L for 4-OP. Compared to our detection limits for the same compounds, 0.027 μg/L for 4-NP and 0.046 μg/L for 4-OP, our detection limits were also similar to Rudel’s. The presence of APE degradation by-products and β-sitosterol in the water samples is an indication that pulp mill effluent is contaminating the river with environmental estrogen-like chemicals. Currently, the United States has no national criteria for the regulation of nonionic surfactants, their degradation byproducts, or β-sitosterol. Since rivers are used as sources of drinking water in the state of Alabama, the possibility does exist for drinking water to become contaminated with these pollutants. For example, 20 different types of alkylphenolic compounds were detected in New Jersey drinking water (10) and more recently a study in Cape Cod, Massachusetts, reported bisphenol A along with some alkylphenol ethoxylates in drinking water wells(9) Although our study detected environmental estrogen-like compounds in wastewater from a pulp mill, we emphasize that pulp mill effluent may not be the only source of these compounds entering the aquatic environment. A large variety of household products such as laundry detergents and surface cleaners also contain nonionic surfactants which are used everyday and disposed of in public sewer systems. Since nonionic surfactants break down to alkylphenols in the environment, sewage treatment plant (STP) effluent which is discharged into surface waters poses as another potential source of drinking water contamination. Levels of 4-NP and 4-t-OP in STP effluent have been reported in the range of 2.8-30 μg/L and 0.12-2.5 μg/L, respectively (11). Further inputs of estrogenic compounds to surface water may also occur from agriculture where estrogenic pesticides, such as atrazine, are used (12). As discussed previously, alkylphenol concentrations in the range of 30-1000 μg/L have experimentally been shown to cause adverse effects. Although these concentrations may rarely exist in the aquatic environment, the lipophillic property of these chemicals may allow them to bioaccumulate to biologically significant levels after long-term exposure. In surface waters of the Glatt Valley, Switzerland, NP concentrations in fish ranged from <0.03-1.6 mg/kg dry weight (13). In a single mallard duck, concentrations were reported to be similar to those found in fish. Alkylphenols are in the aquatic environment at levels which have been experimentally proven to cause estrogenic effects. Since APEs have been in use for over 40 years, situations of endocrine disruption may already exist in wildlife and humans. In addition to synthetic compounds, exposure to natural compounds such as β-sitosterol have already been implicated in reproductive dysfunction seen in wildlife (3). In conclusion, we detected a 1.4-fold higher level of 4-t-OP when compared to previous reports in effluent (2) and our detected level of 4-NP was consistent with the literature. As for the level of β-sitosterol in pulp mill effluent, the average concentration was 2.8-fold higher than what has been reported to be 7.2 μg/L from pulp mill effluent (4). The presence of alkyphenols and β-sitosterol in pulp mill effluent show that Alabama’s water sources are being contaminated by environmental estrogens. This is a major concern since they are reported to cause adverse effects in aquatic organisms such as fish and mollusks. The concentrations we detected are individually capable of inducing an adverse effect (14), however, the potential for EDCs to act additively at environmentally relevant concentrations indicates a need for a comprehensive human risk assessment. In the future, a study of river bottom sediment around this pulp mill discharge site is recommended to further explore environmental levels of estrogen-like compounds. Because these compounds are highly lipophillic, we expect a high amount of alkylphenols to be present in the sediment where they may be available to benthic organisms and fish. Based on the levels of these compounds in the river sediment, a sample of freshwater mussels, minnows, and darters may also be
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gathered and analyzed for the presence of these compounds. Further investigation may provide insight to the bioavailability and bioaccumulation of these compounds in local wildlife and humans. Reference List 1 Roy,D., Palangat,M., Chen,C.W., Thomas,R.D., Colerangle,J., Atkinson,A. and Yan,Z.J. Biochemical and molecular changes at the cellular level in response to exposure to environmental estrogen-like chemicals, J.Toxicol.Environ.Health, 50: 1-29, 1997. 2 Ying,G.G., Williams,B. and Kookana,R. Environmental fate of alkylphenols and alkylphenol ethoxylates--a review, Environ.Int., 28: 215-226, 2002. 3 Denton,T.E., Howell,W.M., Allison,J.J., McCollum,J. and Marks,B. Masculinization of female mosquitofish by exposure to plant sterols and Mycobacterium smegmatis, Bull.Environ.Contam Toxicol., 35: 627-632, 1985. 4 Fernandez,M.P., Ikonomou,M.G. and Buchanan,I. An assessment of estrogenic organic contaminants in Canadian wastewaters, Sci.Total Environ., 373: 250-269, 2007. 5 MacLatchy,D.L. and Van Der Kraak,G.J. The phytoestrogen beta-sitosterol alters the reproductive endocrine status of goldfish, Toxicol.Appl.Pharmacol., 134: 305-312, 1995. 6 Stephanou,E. and Giger,W. Persistent organic chemicals in sewage effluents. 2. Quantitative determinations of nonylphenols and nonylphenol ethoxylates by glass capillary gas chromatography., Environ Sci Technol., 16: 800-805, 1982. 7 Porubek,D.J. and Nelson,S.D. A gas chromatographic/mass spectrometric assay for catechol estrogens in microsomal incubations: comparison with a radiometric assay, Biomed.Environ Mass Spectrom., 15: 157-161, 1988. 8 Gavaler,J.S., Rosenblum,E.R., Van Thiel,D.H., Eagon,P.K., Pohl,C.R., Campbell,I.M. and Gavaler,J. Biologically active phytoestrogens are present in bourbon, Alcohol Clin.Exp.Res., 11: 399-406, 1987. 9 Rudel,R.A., Melly,S.J., Geno,P.W., Sun,G. and Brody,J.G. Identification of alkylphenols and other estrogenic phenolic compounds in wastewater, septage, and groundwater on Cape Cod, Massachusetts., Environ.Sci.Technol., 32: 861-869, 1998. 10 Naylor,C.G. Environmental fate of alkylphenol ethoxylates, Soap Cosmet.Chem.Spec, 68: 27, 1992. 11 Lee,H.B. and Peart,T.E. Determination of 4-nonylphenol in effluent and sludge from sewage treatment plants, Anal.Chem., 67: 1976-1980, 1995. 12 Hayes,T.B., Collins,A., Lee,M., Mendoza,M., Noriega,N., Stuart,A.A. and Vonk,A. Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses, Proc.Natl.Acad.Sci U.S.A, 99: 5476-5480, 2002. 13 Sharpe,R.M., Fisher,J.S., Millar,M.M., Jobling,S. and Sumpter,J.P. Gestational and lactational exposure of rats to xenoestrogens results in reduced testicular size and sperm production, Environ Health Perspect., 103: 1136-1143, 1995. 14 Brian,J.V., Harris,C.A., Scholze,M., Kortenkamp,A., Booy,P., Lamoree,M., Pojana,G., Jonkers,N., Marcomini,A. and Sumpter,J.P. Evidence of estrogenic mixture effects on the reproductive performance of fish, Environ Sci Technol., 41: 337-344, 2007.
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