Degradation of Five Selected Endocrine-Disrupting Chemicals in

Environ. Sci. Technol. 2003, 37, 1256-1260

Degradation of Five Selected Endocrine-Disrupting Chemicals in Seawater and Marine Sediment GUANG-GUO YING* AND RAI S. KOOKANA CSIRO Land and Water, Adelaide Laboratory, PMB 2, Glen Osmond SA 5064, Australia

We studied degradation of five endocrine-disrupting chemicals (EDCs), namely bisphenol A (BPA), 17β-estradiol (E2), 17R-ethynylestradiol (EE2), 4-tert-octyl phenol (4-tOP), and 4-n-nonyl phenol (4-n-NP), in the marine environment using a marine sediment and seawater collected from the coastal area near Adelaide, South Australia. This laboratory study showed that all five EDCs were degraded in seawater within 56 days. However, a lag phase preceding their rapid degradation in seawater was observed for BPA, E2, and EE2. On the other hand, 4-t-OP and 4-nNP dissipated rapidly due to abiotic as well as biotic factors without any lag phase. Under the aerobic conditions, the five EDCs were found to degrade in the sediment, with E2 and 4-n-NP showing fastest degradation, with estimated halflives of 4.4 and 5.8 d, respectively, followed by BPA (t1/2 14.5 d), EE2 (t1/2 >20 d), and 4-t-OP (t1/2 >20 d). Under anaerobic conditions in the marine sediment, little or no degradation of the five EDCs was noted, except E2 which showed a slow continuous degradation rate during the 70-day study. Approximately 50% of E2 was degraded after 70 d anaerobic incubation. The study showed that the five EDCs studied here will degrade relatively rapidly under aerobic conditions in marine sediment and seawater but are likely to show much longer persistence under anoxic conditions.

Introduction Marine and coastal environments near big cities around the world have received oil spills, stormwater runoff, and in some cases sewage effluents (1). This leads to direct input of many different classes of pollutants, including endocrine-disrupting chemicals (EDCs), through the sewage effluents and industrial wastewater. Furthermore, antifouling agents such as TBT are released from the paint on ships and boats in many ports and marinas (1). These chemicals or chemical mixtures have already caused problems such as imposex of oysters and molluscs (2-4) and high levels of heavy metals (e.g., Hg, Pb, and Cd) and persistent organochlorines and nonylphenols in marine species (5-7). Consequently, there is an emerging interest in the fate and behavior of EDCs that may be present in such effluents in the environment and on their possible effects on natural ecosystems as well as on human health (8). Five representative EDCs were chosen in this study: natural estrogenic steroid 17β-estradiol (E2), synthetic steroid 17R-ethynylestradiol (EE2), industrial chemical bisphenol A (BPA), and surfactant degradation products 4-tert-octyl * Corresponding author phone: +61 08 83038474; fax: +61 08 83038565; e-mail: [email protected]. 1256

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phenol (4-t-OP) and 4-n-nonyl phenol (4-n-NP). These five chemicals have been found in sewage effluents at concentrations ranging from ng/L to µg/L (9-15, and some references therein). Various researchers have reviewed the fate of the five EDCs in the environment, including studies on BPA (16), NP and OP (14, 17-20), and E2 and EE2 (15). Some information on their distribution and circumstantial evidence of fate in seawater and marine sediment has been reported (21-25). Persistence of OP and NP in marine sediment was observed by Isobe et al. (23), Marcomini et al. (24), and Shang et al. (25). However, only limited experimental data are available in regard to the behavior and fate of the five compounds in the aquatic environment. Thus, this study was designed to investigate the degradation of the five EDCs in seawater under aerobic conditions and in marine sediment under aerobic and anaerobic conditions.

Materials and Methods Chemicals. Hormone steroids including 17β-estradiol (E2) and 17R-ethynylestradiol (EE2) were obtained from Aldrich (Australia); and 4-tert-octyl phenol (4-t-OP) was obtained from Chem Service (Australia). Bisphenol A (BPA) and 4-nnonyl phenol (4-n-NP) were purchased from Fluka (Riedelde Hae¨n, Australia). The physiochemical properties of these chemicals are given in Table 1. HPLC grade methanol and acetonitrile were obtained from BDH (England). Stock solutions (100 mg/L) of each standard, as well as mixtures, were prepared in methanol. Marine Sediment and Seawater. The marine sediment and seawater used in this study were collected from the coastal area around Adelaide, South Australia. The sediment was taken from St. Kilda beach and seawater was collected from a jetty nearby. Marine sediment and seawater were stored in a refrigerator at 4 °C before use. The water content of the wet sediment was about 15%. The physiochemical properties of the marine sediment and seawater were analyzed and are presented in Table 2. Aerobic Degradation in Seawater. The degradation potential of the five EDCs (BPA, E2, EE2, 4-t-OP, and 4-nNP) was studied at a concentration of 5 µg/L each in seawater. Two 2-L samples of fresh seawater were collected in two 2.5-L Winchester bottles from a jetty in an Adelaide beach, South Australia. One of the bottles was sterilized by autoclaving 3× within 3 d and used as a sterile control. Both bottles (sterilized and unsterilized water) were spiked by adding 10 µL of the stock solution with a concentration of 100 mg/L for each chemical. The bottles, covered with cotton wool bungs, were incubated at 20 ( 3 °C and aerated by bubbling air through them at a rate of approximately 1 L/h. All the bottles were shaken before sampling. The concentrations of the five compounds were monitored on days 0, 1, 2, 4, 7, 13, 21, 28, 35, 42, 49, and 56. A 100-mL aliquot was sampled each time from the two bottles and each sample was analyzed by online solid-phase extraction HPLC. Following the above experiment, the dissipation of 4-tOP and 4-n-NP in seawater was further investigated without air bubbling. Seawater samples of 800 mL each were measured into four 1-L Schott bottles, and two of the bottles were sterilized. Then all the bottles were spiked with the two compounds at the required concentration (5 µg/L). The lid of each bottle was tightened after spiking to minimize any loss due to volatilization. Other conditions used were the same as above. Degradation in Marine Sediment. Biodegradation of the five EDCs in marine sediment was undertaken under aerobic and anaerobic conditions. In the experiments, 5 g of marine 10.1021/es0262232 CCC: $25.00

 2003 American Chemical Society Published on Web 02/13/2003

TABLE 1. Physiochemical Properties of the Five EDCs chemical name bisphenol A (BPA) 17β-estradiol (E2) 17R-ethynylestradiol (EE2) 4-t-octylphenol (4-t-OP) 4-n-nonylphenol (4-n-NP)

molecular water solubility weight (mg/L at 20 °C) log Kowa 228.0 272.4 296.4 206.0 220.0

120b 13c 4.8c 12.6d 1.57f

3.32b 3.94c 4.15c 4.12e 5.76f

a Octanol-water partition coefficient. b Howard (26). c Lai et al. (27). Ahel and Giger (28). e Ahel and Giger (29). f Calculated based on EPI Suite from U.S. EPA. d

sediment and 5 mL of seawater from Adelaide coast, South Australia were used to make slurry during all experiments. The concentration applied for each chemical was 1 µg/g in the sediment by adding into each tube 50 µL of the mixture stock solution with a concentration of 100 mg/L for each compound. After spiking, the bottles or tubes were mixed with a vortex-mixer. All five compounds were present in each microcosm and interaction effects on degradation rates were assumed to be negligible. The incubation temperature used in the studies was 20 °C. The concentrations of the five compounds were monitored on days 0, 1, 3, and 7, and then weekly until 70 d. All experiments were performed in duplicate, and duplicate sterile controls were monitored at the same times. Aerobic Study. Sediment and water were weighed into 100-mL Schott bottles. One-half of the bottles were sterilized by autoclaving 3× within 3 d, and used as controls. Chemicals were spiked at the selected concentration (1 µg/g). All bottles were incubated in a temperature-controlled chamber. Those bottles were shaken for one minute at each sampling time. The small volume of the slurry media in the large bottles plus sufficient headspace ensured aerobic conditions. Two bottles were sacrificed from every treatment at each sampling time. Anaerobic Study. Sediment and water were weighed into Hungate anaerobic culture tubes (16 × 125 mm). Half of the tubes were sterilized by autoclaving 3× within 3 d and used as controls. These Hungate tubes were placed into an anaerobic incubation chamber filled with nitrogen gas. The lids of these tubes were loosened to facilitate gas exchange. Resazurin was added at a concentration of 0.0002% into two tubes as a redox indicator. The reducing conditions in the tubes were seen to have been reached when resazurin turned from red to colorless. It took nearly a month in the anaerobic incubation chamber until the tubes containing the redox indicator resazurin turned colorless. Chemicals were spiked into each tube at the selected concentration (1 µg/g) and the lids of all tubes were tightened after spiking. All operations were performed inside the anaerobic incubation chamber. Then all the tubes were incubated in the same chamber as in the aerobic study. Two tubes were sacrificed from every treatment at each sampling time. Extraction and Analysis. Each of the sediment-seawater slurry samples was extracted twice with 20 mL of ethyl acetate by shaking 2 h each time. The extracts were dried under a gentle nitrogen stream and redissolved in methanol. The recoveries for BPA, E2, EE2, 4-t-OP, and 4-n-NP were 95(0.8%, 95(3.4%, 95(3.7%, 108(8.3%, and 88(4.6%, respectively. Water samples were directly analyzed by online solid-phase extraction and HPLC (30). PRP-1 cartridges from Varian were used in on-line solid-phase extraction of water samples using a Prospekt on-line preconcentration unit. The cartridge was first conditioned each time with methanol followed by water at a flow rate of 5 mL/min. Then 50 mL of water sample was passed through the cartridge at the same flow rate. The chemicals adsorbed on the cartridge were

finally eluted with methanol and analyzed by HPLC with fluorescence detection. A Varian high-performance liquid chromatographic system was used to analyze the five EDCs in this study. The system consisted of an autosampler (model 9100), a Prospekt on-line preconcentration unit (model 9200), a solvent delivery system (model 9012 pump), and a fluorescence detector (model 9070). The instrument was equipped with a reversed phase column (Adsorbosphere C18, 5 µ, 250 × 4 mm) from Alltech. For compounds of interest, the fluorescence detector settings were 230 nm excitation and 290 nm emission. The mobile phase for gradient elution were Milli-Q water- and acetonitrile (ACN)-delivered at a constant flow rate of 1 mL/ min. The gradient program of the mobile phase was as follows: 30% ACN and 70% water at 0 min, 40% ACN and 60% water at 5 min, 60% ACN and 40% water at 10 min, 80% ACN and 20% water at 20 min, isocratic purge until 30 min, and increasing to 100% ACN and 0% water at 35 min. The total run time for an HPLC analysis was 35 min. The injection volume of standards and samples was 50 µL for injection using the autosampler and 50 mL of water for the on-line sample preparator. The extracts were quantified by calibration curves of the external standards. The detection limit of the on-line solid-phase extraction HPLC was 10 ng/L for E2, EE2, and BPA, and 30 ng/L for 4-t-OP and 4-n-NP; whereas the detection limit of the HPLC method with normal autosampler injection was 10 µg/L for E2, EE2, and BPA and 30 µg/L for 4-t-OP and 4-n-NP.

Results and Discussion Aerobic Degradation in Seawater. All the five EDCs in seawater were degraded almost completely (>90%) within 56 d after treatment (DAT) (Figure 1). However, BPA, E2, and EE2 underwent an acclimation stage followed by rapid degradation. Within 35 d after treatment, little degradation of BPA occurred, but there was rapid degradation in the following week. BPA concentration dropped from 4.15 µg/L at 35 DAT to 1.34 µg/L at 42 DAT, and then to 0.4 µg/L at 56 DAT. E2 and EE2 had similar degradation behavior although E2 degraded slightly faster than EE2 in seawater. After 28 d following treatment, the concentrations of E2 and EE2 were nearly 4 µg/L in seawater. Then E2 and EE2 concentrations decreased to only 0.14 µg/L and 0.38 µg/L, respectively, at 42 DAT. In contrast, 4-t-OP and 4-n-NP showed degradation behavior different from that of the other EDCs in seawater. A significant loss was observed in the sterile seawater in the first 2 d for 4-n-NP and 13 d for 4-t-OP. After this initial loss, their concentrations in the sterile seawater remained stable with little change. A comparison of sterile and nonsterile treatments showed that following the initial abiotic loss, biodegradation was mainly responsible for the further loss in nonsterile seawater. Within a week, 4-n-NP concentration decreased from 5 µg/L to only 0.06 µg/L in seawater. In contrast, 4-t-OP in seawater underwent much slower biodegradation and took 42 DAT to reach a concentration of 0.03 µg/L. Aerobic degradation of the five EDCs in seawater demonstrates that they could be degraded by aerobes in seawater once the microorganisms in the water became acclimated to the chemicals. Greater than 90% degradation of BPA within 4 d was observed by Dorn et al. (31) in laboratory experiments using three river waters from near plastics manufacturing facilities. But Stone and Watkinson (32) reported that aerobic biodegradation of BPA was less than 1% within 28 d in OECD testing. The bacterial assemblages in the river waters near plastics factories may have developed the capability to degrade BPA in water, whereas in media without previous exposure to BPA, degradation rates are much slower. Fast biodegradation of E2 in water samples from English rivers VOL. 37, NO. 7, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Physiochemical Properties of a Marine Sediment and Seawatera pH

NH4-N (mg/L)

NO3-N (mg/L)

8.4

1.7

0.2

pH

TC (%)

OC (%)

9.5

8.8

0.1

a

Seawater PO4-P (mg/L) SO4) (mg/L) 0.1

3100

Marine Sediment CEC(NH4) cmol(+)/kg CO3 as CaCO3 (%) 2.4

72

TC (mg/L)

IC (mg/L)

OC (mg/L)

29

28

1

clay (%)

silt (%)

sand (%)

3

0.8

22.1

TC ) total carbon, IC ) inorganic carbon, OC ) organic carbon, and CEC ) cation exchange capacity.

FIGURE 2. Dissipation of 4-t-OP and 4-n-NP in seawater without air bubbling during incubation.

FIGURE 1. Aerobic degradation of the five EDCs in seawater with air bubbling during incubation. Control samples were sterilized and used as a comparison in the study. with half-lives of 0.2 to 9 d has been reported by Jurgens et al. (33). Compared to E2, EE2 was much more resistant to biodegradation in the water from English rivers (33). The rapid loss of 4-t-OP and 4-n-NP in seawater shortly following treatment was caused by abiotic processes such as volatilization, because there were parallel losses of these two chemicals in the sterile and nonsterile seawater. 4-t-OP and 4-n-NP are chemically stable compounds at 20 °C, but they have high vapor pressures, e.g. 0.0023 mmHg for nonylphenols (34). Occurrence of NP and OP in air have been reported with their concentrations up to 70 ng/m3 for NP in the coastal atmosphere of the New York-New Jersey Bight (35), and up to 1.0 ng/m3 for OP and 56.3 ng/m3 for NP at a coastal site (Sandy Hook) of the Lower Hudson River Estuary (36). Initial rapid loss of 4-t-OP and 4-n-NP in the seawater may be caused by volatilization due to shaking and airing in the present 1258

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experiment. Sorption on glassware may also account for some of the loss because these two compounds are highly hydrophobic and can adsorb onto solid phase (Table 1). Therefore, their rapid loss in the seawater is believed to be caused mainly by initial volatilization due to air bubbling and sorption on glassware and later biodegradation. This is also supported by our further laboratory experiments on the two compounds, which showed much smaller loss of 4-t-OP and 4-n-NP in sterile seawater water without shaking or air bubbling (Figure 2). Some loss observed in sterile seawater in this case may have been caused by their adsorption on glassware. The half-lives of 4-t-OP and 4-n-NP in nonsterile seawater without air-bubbling were 60 and 5 d, respectively. 4-t-OP and 4-n-NP have similarities in their structures: phenol with an alkyl chain. The only difference is that 4-t-OP has a highly branched alkyl chain, which makes 4-t-OP more resistant to microbial degradation than 4-n-NP. Aerobic Degradation in Marine Sediment. Within 70 d of incubation, all five endocrine-disrupting chemicals (BPA, E2, EE2, 4-t-OP, and 4-n-NP) in the marine sediment were completely degraded under aerobic conditions (Figure 3). Indeed, E2 and 4-n-NP degraded very quickly with half-lives of 4.4 and 5.8 d, respectively, based on the first-order reaction kinetics. The concentration of E2 in the sediment decreased from 1 µg/g at the beginning to 0.54 µg/g at 1 DAT and to 0.04 µg/g at 21 DAT. The concentration of 4-n-NP also decreased to 0.58 µg/g at 1 DAT and to 0.05 µg/g at 21 DAT. This demonstrates ready degradation o f these two chemicals under aerobic conditions. BPA in the sediment was degraded under aerobic conditions with a half-life of 14.5 d. Its concentration decreased from 0.87 µg/g at 14 DAT to 0.015 µg/g at 28 DAT. EE2 and 4-t-OP were degraded much more slowly with half-lives of >20 d. EE2 concentration in the sediment decreased from 1 µg/g at 0 DAT to 0.69 µg/g at 21 DAT, to 0.31 µg/g at 28 DAT, and to 0.06 µg/g at 42 DAT. For 4-t-OP, there was an acclimation period with a concentration of 0.84 µg/g at 21 DAT, followed by quick degradation with its concentration decreasing to only 0.09 µg/g within a week. Anaerobic Degradation in Marine Sediment. Little or no degradation of the five EDCs, except E2, was observed under anaerobic conditions (Figure 4). E2 in the marine sediment

FIGURE 3. Aerobic degradation of the five EDCs in marine sediment. was degraded slowly with a half-life of 67 d. Approximately 50% of E2 still remained in the sediment after the 70-d experiment. Sulfate (SO2-) was the dominant electron acceptor (Table 2), confirmed by the formation of dark sulfide mineral on the surface of sediment. Therefore, sulfatereducing biodegradation is probably responsible for the loss of E2. The other chemicals (BPA, EE2, 4-t-OP, and 4-n-NP) remained unchanged 70 d after treatment. The present experiments demonstrated that the five EDCs (BPA, E2, EE2, 4-t-OP, and 4-n-NP) degraded in the marine sediment under aerobic conditions, but they were found persistent under anaerobic conditions, except E2 which showed some slow degradation. Biodegradation of EDCs has been reported to play a major role in the removal of these EDCs from aquatic environments (14-20). BPA has a OH substituent on both aromatic rings, and this is a possible site for biodegradation. But the two rings are joined by a quaternary carbon, which may inhibit its degradation by microbes. A long acclimation period may be needed for microbes to attack the chemical, as found in the seawater experiment. However, no acclimation was needed for its aerobic degradation in the marine sediment. This is probably due to a more active and diverse microbial community and richer nutrients in the sediment. Ike et al. (37) investigated biodegradation of BPA in the aquatic environments using 3 activated sludge and 44 river water microcosms. Only 6 river water microcosms could completely mineralize BPA, and the others showed accumulation of common metabolites, which persisted for more than one month. So far, most reported experiments (31, 32, 37-39) were done under aerobic conditions, so that little information

FIGURE 4. Anaerobic degradation of the five EDCs in marine sediment. is available in the literature about the degradation of BPA under anaerobic conditions. No loss of BPA in estuarine sediments was observed within 162 days in conditions promoting either methanogenesis, sulfate-reduction, iron(III)-reduction, or nitrate-reduction (40). The present study also shows that BPA will accumulate in anoxic marine sediment. Rapid biodegradation of E2 has been reported in aerobic experiments with activated sludge and biosolids (41, 42), and river sediments (33). However, EE2 was found more persistent under these conditions (33, 42). The current results support those observations and suggest that there was a very slow rate of degradation of E2 under anaerobic conditions. E2 has two OH groups on its structure, which is amenable to microbial attack. EE2 has a chemical structure similar to that of E2, but it also contains two quaternary carbon atoms which are directly adjacent to each other. Therefore, EE2 is more resistant to microbial degradation. Laboratory and field experiments all demonstrated that alkylphenols readily degraded under aerobic conditions but were persistent under anaerobic conditions (14 and references therein). The present study also shows that 4-n-NP is more amenable to biodegradation than 4-t-OP in the sediment under aerobic conditions possibly due to the straight side chain in 4-n-NP. Consistent with our studies, NP and OP have been reported to be persistent in the anoxic marine sediments collected from the Canadian coast (25), the Venice lagoon (24), and Tokyo Bay (23). The present study clearly demonstrated that aerobic conditions are favorable for the five EDCs to degrade in the VOL. 37, NO. 7, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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marine environment. Owing to the hydrophobic nature of these chemicals (Table 1), they are expected to adsorb onto the suspended and bed sediments. From the study it can be concluded that the five EDCs, except E2, are likely to be persistent and may accumulate in anoxic marine sediments. Further investigations are needed on these aspects.

Acknowledgments We thank J. Brooker and H. H. Peng (University of Adelaide) for their generous help in the biodegradation experiment. We are grateful to P. Dillon and A. Juhasz (CSIRO Land and Water) for the useful discussion. We also appreciate the valuable comments made by the three reviewers.

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Received for review October 8, 2002. Revised manuscript received January 12, 2003. Accepted January 20, 2003. ES0262232