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Effect of triclosan and triclocarban biocides on biodegradation of estrogens in soils
Chemosphere 77 (2009) 1381–1386
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Effect of triclosan and triclocarban biocides on biodegradation of estrogens in soils Ali Shareef a,*, Sina Egerer b, Rai Kookana a a
Centre for Environmental Contaminant Research, Commonwealth Scientific and Industrial Research Organization (CSIRO), Land and Water, Private Mail Bag 2, Glen Osmond, South Australia 5064, Australia b Department of Soil Science and Soil Ecology, Geographical Institute, Ruhr-University Bochum, 150, 44780 Bochum, Germany1
a r t i c l e
i n f o
Article history: Received 2 June 2009 Received in revised form 10 September 2009 Accepted 10 September 2009 Available online 4 October 2009 Keywords: Antimicrobial agents Endocrine disrupting chemicals 17b-Estradiol 17a-Ethynylestradiol
a b s t r a c t We have investigated the effect of antimicrobials triclosan (TCS) and triclocarban (TCC) on biodegradation of 17b-Estradiol (E2) and 17a-Ethynylestradiol (EE2) in a sandy soil from South Australia. Two separate batch studies were conducted. In the first, the rates of loss of E2 and EE2 were determined at time intervals of 0, 3, 7, 14, 21, 28 and 56 d after initial spiking of soil with each estrogen at 1 mg kg1 and the antimicrobials at 10 and 100 mg kg1. Little loss of E2 and EE2 (<15%) under sterile conditions was noted compared to rapid loss in non-sterile soil (>60% in 24 h). There were no measurable effects on estrogen degradation by the two antimicrobials at spiked concentrations up to 100 mg kg1. The experiments were repeated to study degradation rates of the estrogens within the first 24 h (0, 3, 8, 24 h), 3 d and then weekly to 56 d. Again, E2 and EE2 degradation was not significantly affected by the presence of TCS up to 100 mg kg1 (p > 0.05). However TCS did significantly affect biodegradation of the estrogens when the soils were spiked with 1000 mg kg1 of TCS (p < 0.0005). In contrast, presence of TCC in soil showed no significant effect on biodegradation of the two compounds up to 1000 mg kg1 (p > 0.05). Considering environmental concentrations of the antimicrobials reported in the literature, it is highly unlikely these biocides would have any adverse impact on biodegradation of E2 or EE2 in soils. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction The release of organic contaminants of emerging concern such as endocrine disrupting chemicals, and pharmaceuticals and personal care products into the environment has received increasing public concern recently. Evidence in the scientific literature suggesting the detrimental impact of such chemicals on aquatic organisms and wildlife as well as the concerns about the potential impact on human health have mounted over recent years (Rubin and Niskar, 1999; Heberer, 2002; Allmyr et al., 2008). Municipal wastewater is one of the major sources of environmental pollution for numerous organic contaminants such as endocrine disrupting chemicals and pharmaceuticals and personal care products. For example, treated effluents can induce estrogenic activity in recipient waters up to several kilometers from the outfalls, with feminization of male fish indicated by the imposex condition or the production of the egg–yolk protein vitellogenin (Harries et al., 1997; Jobling et al., 1998; Metcalfe et al., 2001; Wibe et al., 2002). Steroid hormones are excreted by humans and animals in a water-soluble conjugated forms that are either estrogenically inactive or have a lower potency. However these glucuronide or sulfate * Corresponding author. Tel.: +61 8 8303 8474; fax: +61 8 8303 8565. E-mail address: [email protected] (A. Shareef). 1 Present address. 0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.09.018
conjugates are metabolized and released into the environment as fully active estrogenic hormones through treated sewage effluents (Tabak et al., 1981; Desbrow et al., 1998; Ternes et al., 1999; Hanselman et al., 2003). 17a-Ethynylestradiol (EE2) is of major concern because it is a relatively stable synthetic hormone which is commonly found in the formulation of oral contraceptives (Johnson and Sumpter, 2001). Although less stable, the natural hormone 17b-Estradiol (E2) and its metabolites also raise significant concern because E2 is the principal endogenous steroid hormone in vertebrates, stimulating the growth and development of the female sex organs. Triclosan (TCS) and triclocarban (TCC) are two common antimicrobial agents with very similar antifungal and antibacterial properties. They are used in numerous household and personal care products such as toothpaste, soaps and detergents, household sponges, plastic cutting boards, socks and undergarments (European Commission, 2005). TCS has been more widely used globally in a broader range of consumer products (0.3–1.0%) compared with its original use in hospitals and health care facilities (Schweizer, 2001). TCC is a high production volume chemical in the US with market ranges between 250 and 500 metric tones per year (US EPA, 2002). During common wastewater treatment the removal rates of estrogens as well as TCS and TCC from the aqueous-phase are relatively high due to their generally low aqueous solubilities and hydrophobic properties (i.e. log KOW P 3.9) (Stasinakis et al.,
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2007; Ying and Kookana, 2007; Ying et al., 2008). These two antimicrobials, which enter wastewater treatment plants at concentration levels in the order of low lg L1 (e.g. Ying and Kookana, 2007; Stasinakis et al., 2008a), show aqueous-phase removal efficiencies of up to 97% (Heidler et al., 2006; Stasinakis et al., 2007). Removal rates of E2, EE2 are also generally above 90% (Lee and Liu, 2002; Andersen et al., 2003). At the same time these compounds exhibit moderate to high sorption affinity for the solid phase and have a tendency to accumulate in sludges and sediments where they can persist (Ying et al., 2002; Heidler et al., 2006; Heidler and Halden, 2007). Heidler et al. (2006) demonstrated that no significant transformation of TCC takes place during the common sludge treatment of anaerobic digestion for 19 d. Approximately 76% of the TCC input of wastewater treatment plant was recovered in the dewatered, digested sludge after treatment resulting in an accumulation of TCC in the sludge at levels of 51 mg kg1 dry weight (Heidler et al., 2006). Ternes et al. (2002) reported that E2 and EE2 persisted in activated and digested sludge at levels up to 49 and 17 lg kg1, respectively. Treated sludges from wastewater treatment plants are often applied to agricultural land. Therefore these ‘biosolids’ may potentially introduce many endocrine disrupting chemicals, and pharmaceuticals and personal care products including the steroid estrogens as well as biocides such as TCS and TCC into soil environment. The fate, transport and distribution of these compounds may be influenced by many processes taking place in the soil/sediment environments including sorption and degradation. A growing body of research suggests that biodegradation can play a significant role in the fate of these organic compounds in soils. These include estrogens as well as TCS and TCC (Colucci et al., 2001; Colucci and Topp, 2001; Ying et al., 2007; Stumpe and Marschner, 2009) with little or no loss under anaerobic or sterile conditions (Andersen et al., 2003; Ying and Kookana, 2005; Heidler et al., 2006; Heidler and Halden, 2007). Due to the hydrophobic and persistent nature of these antimicrobial agents, it is likely that they can accumulate in the sediment phase in the aquatic environment. Although most wastewater treatment plants rely on activated sludge processes to treat wastewater, currently very little is known about the toxicity of these antimicrobial compounds specific to activated sludge microorganisms. In a recent investigation on toxicities of TCS, phenol and copper (II) to activated sludge microorganisms, Neumegen and co-workers (2005) reported that TCS had the highest relative toxicity based on EC50 values (TCS: 1.82 mg L1, phenol: 2.70 mg L1 and Cu(II): 18.3 mg L1). Stasinakis et al. (2008b) who investigated toxicity of TCS and the xenoestrogen 4-nonylphenol (NP) reported lower EC50 values of 0.22 mg L1 and 3.51 mg L1 for TCS and NP respectively, for a marine bacterium Vibrio fischeri. Based on these findings, the authors suggested that the presence of TCS can have a detrimental effect on the nitrification process in activated sludge systems in wastewater treatment plants. The impact of these biocides on soil microorganisms is largely unknown. Recently Waller and Kookana (2009) investigated the impact of TCS on some soil microbiological processes (i.e. substrate induced nitrification and respiration, and some selected enzyme activity assays) using two contrasting soils from South Australia. The addition of TCS at 50 mg kg1 affected substrate induced respiration in the clay soil but not in the sandy soil. However, at 5 mg kg1 TCS affected the nitrogen cycle in the sandy soil (a concentration that is within typical levels of TCS in Australian biosolids which can range from 0.09 to 16.79 mg kg1 (Ying and Kookana, 2007)). This may indicate that nitrifying bacteria were sensitive to low levels of TCS in soils. Considering that the co-occurrence of estrogens and the antimicrobials in wastewater treatment plant effluents, sediments and biosolids is of significant concern, the objective of the current study was to investigate (i) whether or not the biodegradation of E2 and EE2 may be significantly influenced by presence of the antimicrobial agent TCS or TCC in soil and if so (ii) at what concentra-
tions these effects may become significant. Answers to both of these questions are desirable for better understanding of likely environmental fate of estrogens in sediment and biosolids matrices.
2. Experimental 2.1. Materials 17b-Estradiol (E2), CAS [50–28-2], and 17a-Ethynylestradiol (EE2), CAS [57–63-6], both with purities of 98% or higher, as well as triclosan (TCS), CAS [3380–34-5] and triclocarban (TCC), CAS [101–20-2], with a purity of 99%, were obtained from Sigma–Aldrich (Sydney, Australia). HPLC grade anhydrous solvents (purity >99%) including methanol, acetone and acetonitrile were supplied by Biolab (Sydney, Australia). Table 1 presents the selected physicochemical properties of E2, EE2, TCS and TCC. The soil used for this study was a surface sandy loam soil collected from a depth of 0–15 cm from agricultural land at Roseworthy Farm (34.533°S, 138.733°E) in South Australia. The same soil has been used by Waller and Kookana (2009) in the study referred to earlier, which indicated that TCS had some negative impact of some of the microbial processes in this soil. It was air dried and ground to 62 mm before use. The soil characteristic properties are given in Table 2.
2.2. Methods Two replicate studies were conducted to determine the effect of the antimicrobial agents on the degradation of the estrogens. The first study involved a long-term investigation where degradation of E2 and EE2 were evaluated at time intervals of 0, 1, 3, 7, 14, 21, 28 and 56 d after initial spiking of the estrogens (1 mg kg1) and the antimicrobials (10 and 100 mg kg1). Due to the rapid loss of both E2 and EE2 observed in the initial experiments it was decided to repeat the experiment where time intervals of 0, 1, 3, 8, 24 h were added to the original time sequence. Also because no effect on biodegradation of the estrogens was observed with TCS or TCC spiked concentrations up to 100 mg kg1, an additional set of samples were included in the repeat experiment where the spiked concentration of TCS and TCC was increased up to 1000 mg kg1. To prepare the samples, individual stock solutions of the two estrogens were prepared at 100 mg L1 in methanol from which 50 lL aliquots were added to 5 g of soil (pre-weighed and moisture content adjusted to 50% of WHC using sterile milliQ water) in 20 mL glass culture tubes to achieve a concentration of 1.0 mg kg1 of E2 or EE2. TCS and TCC were spiked at three different concentrations of 10, 100 and 1000 mg kg1 by transferring appropriate volumes of stock solutions of TCS or TCC prepared in acetone. After spiking with the chemicals, the contents of the tubes were vortex mixed, aerated and incubated under dark conditions in a temperature controlled room maintained at 20 ± 1 °C. Sterile control tubes were prepared by autoclaving at 120 °C under a chamber pressure of 300 kPa for 1 h for three consecutive days before spiking with an estrogen. The spiking of the control tubes were done inside a laminar flow cabinet followed by sealing the tubes with screw caps with Teflon liners to ensure sterile conditions are maintained. The concentration of E2 and EE2 were then monitored at intervals from 0, 3, 8, 24 h (short study only), 1, 3, 7 d and then weekly for 56 d. All tests were conducted in triplicates and parallel samples were prepared for monitoring microbial activity in the tubes. The microbial activity in soil was monitored using substrate induced respiration technique following Organization for Economic Cooperation and Development protocol (Waller and Kookana, 2009).
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A. Shareef et al. / Chemosphere 77 (2009) 1381–1386 Table 1 Physicochemical properties of the selected estrogens and antimicrobials.
a b c d
Compound
17b-Estradiol (E2)
17a-Ethynylestradiol (EE2)
Triclosan (TCS)
Triclocarban (TCC)
mol.wt (g mol1) Log KOW Solubility (mg L1)
272 3.94a 1.51c
296 4.15a 9.2 c
289 4.7b 1.97–4.6b
316 4.9b 0.11d
Yu et al. (2004). Halden and Paull (2005). Shareef et al. (2006a). European Commission (2005).
Table 2 Physicochemical properties of the soil from South Australia.
a b
Clay (%) 10
Silt (%) 4
Sand (%) 84
CaCO3 (%) <0.5
MWHCa (%) 28.9
Total carbon (%) 1.1
Organic carbon (%) 0.85
Total nitrogen (%) 0.11
NH4–N (mg kg1) 1.8
NO3–N (mg kg1) 2.1
ECb (dS m1) 0.09
pH (0.01 M CaCl2) 5.4
MWHC = Maximum Water Holding Capacity. EC = Electrical Conductivity.
2.3. Extraction and analyses At the selected incubation time intervals, whole soil sample and sterile control tubes (5 g) were extracted twice by ultrasonication for 10 min with 10 mL of methanol:acetone (1:1, v/v). After centrifugation for 30 min (800 g), the liquid phase was separated by decanting into graduated conical bottom culture tubes in which the samples were concentrated to dryness under a gentle stream of nitrogen, and reconstituted in 1.0 mL of methanol. The optimised extraction method was developed after testing recoveries using a number of common solvents or combination of solvent (e.g. methanol, acetone, acetonitrile, ethyl acetate). The extraction method was also evaluated for the experimental spiking concentration ranges. The extracts were analysed by reverse phase high performance liquid chromatography with UVdetection (Agilent chromatograph equipped with photodiode array detector). The optimum wavelength for analysis was 280 nm. A Phenomenex Fusion RP C18 column (150 mm long, 4.6 mm I.D., 4.6 lm particle size) (Phenomenex, Sydney, Australia) protected by a guard column with matching stationary phase was used. Optimum chromatographic separation with baseline resolution of peaks was achieved using a mobile phase gradient at a linear flow rate of 1.0 mL min1 using acetonitrile (A) and water with 0.1% acetic acid (B) with the following gradient program: 0–8 min 40–60% A, 8–18 min 60–85% A, then dropped to 40% A from 18 to 20 min and held for 2 min. Calibration standards were prepared by serial dilution of the original standards used for spiking with methanol. A six-point linear calibration curves (ranges of 0.25–6 mg L1 for estrogens and 50–500 mg L1 for TCS and TCC) with correlation coefficients >0.99 were used for the determination of analyte concentrations in the samples. Appropriate dilutions of samples were required for quantitative determination of TCS and TCC for samples with the two higher spiked concentrations. The total run time for analysis of each sample was 20 min. 2.4. Analytical method validation and data analyses For the purpose of this study, a reliable and robust analytical method was developed for simultaneous analysis of the estrogens E2, EE2 and TCS or TCC using HPLC–UV detection, following a simple ultrasonic extraction method. The retention times from base-
line resolved peaks for E2 (5.6 min), EE2 (6.5 min) and TCS and TCC (15.0 min) were used to identify the analytes. The method allowed simultaneous and sensitive determination of E2, EE2 and either TCS or TCC in a single analysis. Co-elution of TCS and TCC was not an issue in our analyses given that our samples contained only TCS or TCC, not both. Extraction recoveries were greater than 80% for all the analytes. The mean extraction recoveries of 92 ± 11% and 90 ± 10% for E2 and EE2 respectively were used in calculations of the residual concentrations. The measured concentrations of E2, EE2, TCS or TCC have been normalised for simplicity of data interpretation. This was done by dividing the real measured concentration of the analytes by the concentration of the time-zero samples, taken as the nominal concentration. There was no significant difference between nominal and measured concentration of the analytes. Time-zero samples were spiked last, and extracted immediately to avoid possible loss due to degradation. Statistical analysis of the data with univariate analysis of variance (ANOVA) with Tuckey procedure was performed using SPSS v.15.1 for Windows. 3. Results and discussion 3.1. Biodegradation of estrogens – effect of antimicrobials Fig. 1 presents the aerobic biodegradation behaviours of E2 (1a) and EE2 (1b) spiked at 1 mg kg1 soil over a period of 56 d (from the long-term study) in the presence and absence of TCS (10 and 100 mg kg1). The corresponding data from the abiotic control treatment for the estrogens are also included. E2 degraded rapidly with approximately 70% of the initial spike concentration dissipating within a day. EE2 dissipation was slower with approximately 60% of initial concentration lost in one day. There was virtually no change in the concentration of E2 and EE2 under sterile conditions up to 56 d indicating that abiotic loss under sterile conditions was negligible. The effect of the addition of TCS at concentrations of 10 and 100 mg kg1 soils on the degradation of E2 and EE2 are also shown in the Fig. 1a and b respectively. There was no significant difference between E2 and EE2 dissipation in the presence of either of TCS at both spiked level (10 or 100 mg kg1) (p > 0.05). Almost complete loss of E2 was observed by 14 d even in the presence of TCS at both concentrations. Fig. 1c shows rapid biodegradation of TCS at both 10 and 100 mg kg1 measured in the same samples. However
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Fig. 1. Biodegradation of 1 mg kg1 (a) 17b-Estradiol (E2) and (b) 17a-Ethynylestradiol (EE2) in the presence of triclosan (TCS) at spiked concentrations of 10 (h) and 100 (4) mg kg1, and in the absence of TCS (s). (c) Biodegradation of triclosan (TCS) at 10 (h) and 100 (4) mg kg1. Filled circles represent abiotic controls and the error bars represent standard deviations. Data presented as percent residual concentrations have been normalised by dividing measured concentration by initial spiking concentrations.
Fig. 2. Biodegradation of 1 mg kg1 (a) 17b-Estradiol (E2) and (b) 17a-Ethynylestradiol (EE2) in the presence of triclocarban (TCC) at spiked concentrations of 10 (h) and 100 (D) mg kg1, and in the absence of TCC (s). (c) Biodegradation of triclocarban (TCC) at 10 (h) and 100 (D) mg kg1. Filled circles represent abiotic controls and the error bars represent standard deviations. Data presented as percent residual concentrations have been normalised by dividing measured concentration by initial spiking concentrations.
DT50 (time taken to dissipate 50% of initial concentration) was approximately 14 d. On the other hand, no significant degradation of TCS was observed under abiotic conditions. The fate data for TCS are similar to the findings from a recent study by Ying et al. (2007). The parallel study on substrate induced respiration, where samples were treated the same way as the biodegradation tests but without the antimicrobials, indicated microbial activity in the non-sterile soils (e.g. 14CO2 evolved per g soil per h gradually decreased from 7.5 mg kg1 h1 on d-0 to 5 mg kg1 h1 on d-56) under the conditions of this incubation study. Respiration rate (reflecting active microbial community) in the autoclaved samples was very low (<0.1 mg kg1 h1 14CO2 evolved for the period of study). Fig. 2 shows the effect of the second antimicrobial agent TCC on the biodegradation of E2 and EE2. Again about 80% of the initial E2 degraded in 1 d (Fig. 2a), whereas EE2 loss within the first day was approximately 60%. There was no significant difference between the results for biodegradation of E2 or EE2 with or without added TCC up to a concentration of 100 mg kg1 (p > 0.05). The degradation of TCC in the samples is displayed in Fig. 2c. There was no significant degradation of TCC at either concentration under both biotic and abiotic conditions which was consistent with previous studies (Ying et al., 2007). In the present study, DT50 for E2 was approximately 3 h while that for EE2 was 6 h. Although we observed very rapid loss of the two estrogens, data on the relative rates of degradation of the two estrogens are consistent with previous works investigating the degradation of estrogens in the environment. It has now been well documented in the scientific literature that E2 and EE2 undergo aerobic biodegradation with relatively short half-lives (<2 d). For example in a study examining the persistence of estrogens in soils, Colucci et al. (2001) reported that E2 was removed rapidly with a DT50 of
0.5 d at 30 °C under a range of different conditions and soil textures. The half-life of EE2 ranged between 7.7 d at 4 °C and 3.0 d at 30 °C (Colucci and Topp, 2001). Our results are consistent with other studies on the removal rates of estrogens in soils- even though the half-lives can vary due to different measurement methods and soil conditions (Ying and Kookana, 2005). Given the very rapid dissipation of the two estrogens and the lack of any significant impact of the antimicrobials up to100 mg kg1, we decided to investigate if there were any short-term impacts (within 24 h) of the two antimicrobials and if any adverse effect would be measurable at a higher concentration (1000 mg kg1) in soil. Since wastewaters are often continuously discharged into the aquatic environments, allowing little time for degradation of estrogens, we considered that even the short-term effects of antimicrobials may be important. 3.2. Effect of elevated concentrations of TCS and TCC The effect of increasing the spiked level of TCS on the biodegradation of 1 mg kg1 E2 and EE2 are shown in Fig. 3a and b respectively. Also it should be noted that data presented in these figures captured degradation time frame from 0 to 24 h taken at 0, 3, 8 and 24 h after treatment of the soil with the estrogens and the antimicrobials. The results from these repeat experiments confirmed the findings from the previous experiments demonstrating no impact of TCS up to 100 mg kg1 spiked concentration on degradation of both E2 and EE2. However, these results clearly show significant suppression of biodegradation of both E2 and EE2 when the soil was spiked with TCS concentration of a 1000 mg kg1 (Fig. 3a and b) (p < 0.0005). The addition of 1000 mg kg1 TCS resulted in 15–20% suppression of degradation of both E2 and EE2. The biodegradation of the estrogens in the presence of 1000 mg kg1 for the entire
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Fig. 4. Biodegradation of 1 mg kg1 (a) 17b-Estradiol (E2) and (b) 17a-Ethynylestradiol (EE2) in the presence of triclocarban (TCC) at spiked concentrations of 10 (h), 100 (4) and (.)1000 mg kg1, and (s) in the absence of TCC and the error bars represent standard deviations. Data presented as percent residual concentrations have been normalised by dividing measured concentration by initial spiking concentrations.
Fig. 3. Biodegradation of 1 mg kg1 (a) 17b-Estradiol (E2) and (b) 17a-Ethynylestradiol (EE2) for 0–24 h in the presence of triclosan (TCS) at spiked concentrations of 10 (h), 100 (D) and (.)1000 mg kg1, and (s) in the absence of TCS and the error bars represent standard deviations. (c) Biodegradation data for 1–56 days of E2 (.) and EE2 (d) in the presence of 1000 mg kg1. Open symbols (triangles and circles) display the biodegradation of 1000 mg kg1 TCS in E2 and EE2 respectively. Data presented as percent residual concentrations have been normalised by dividing measured concentration by initial spiking concentrations.
experiment is given in Fig. 3c. At 56 d, 25–30% of both E2 and EE2 still remained in the samples, while approximately 50% of TCS had depleted. The data for fate of the estrogens at 10 and 100 mg kg1 concentrations of TCS up to 56 d, has been described earlier (Fig. 1), where there was no significant impact on the degradation of E2 or EE2 in the presence of TCS at these concentrations. Fig. 4a and b display the results from the experiment repeated involving E2 and EE2 with three different concentrations of TCC ranging from 10 to 1000 mg kg1. While the general degradation behaviours of both estrogens were reproducible in these tests, there was no significant effect on the degradation of either E2 or EE2 even at 1000 mg kg1 concentration of TCC (p > 0.05). Also, given the slow biodegradation of TCC (>20% dissipation in 56 d) and the lack of any significant effect on the biodegradation of the estrogens (Fig. 2c), it is highly unlikely that TCC can have any effect of the fate of E2 or EE2, both of which have already been shown to undergo rapid biodegradation. Although environmental fate and ecotoxicological effects of TCS and TCC is largely unknown, the aerobic biodegradation has been proposed by some recent studies as one of the major pathways for their fate in the environment (Ying et al., 2007). A recent investigation by Waller and Kookana (2009) indicated that 5 mg kg1 TCS had some impact of microbial processes, especially the nitrogen cycle. The same study reported that in a clay soil, the TCS showed an effect on the microbial respiration, but only at a spiked concentration of 50 mg kg1. While the specialist bacteria such as those involved in the nitrogen cycle may be affected by TCS, the overall microbial activity is not. Moreover, the lack of any significant impact on the biodegradation behaviours of E2 and EE2 in
the presence of TCS or TCC up to a concentration of 100 mg kg1 may therefore suggest that the biodegradation of E2 and EE2 is primarily driven by specific microbial consortia which are not affected at levels as high as 100 mg kg1. Also, the fact that there was little loss of E2 or EE2 under the sterile conditions show that there were no other physicochemical processes (e.g. sorption, Shareef et al., 2006b) involved in the loss of the estrogens. A number of quality control measures were incorporated into this study to ensure the data presented here were free from any experimental artefacts. For example, the concentrations of TCS and TCC were measured along with the estrogens. There was no significant loss of either TCS or TCC even in the samples with lowest spiking concentration of 10 mg kg1 for up to 3 d, at which point most of the estrogen have dissipated. However TCS degraded down to 20–40% at the end of the experiment (56 d), while the 80–95% of TCC still persisted. These data corresponded well with a recent study by Ying and co-workers (2007) on the biological degradation of TCS and TCC in soil. The lack of significant microbial toxicity due to the presence of the antimicrobials was also confirmed from the measurement of microbial activity via substrate induced respiration in parallel sample. 3.3. Environmental significance In a recent Australian study investigating effect of TCS to microbial activity in soils, Waller and Kookana (2009) conducted a hazard assessment on terrestrial toxicology of TCS using a worst-case scenario based on soils amended with Australian biosolids containing high TCS concentration levels from literature (16.79 mg kg1) (Ying and Kookana, 2007). It was suggested that a dilution factor of 150 in the soil was likely to occur. Based on this, the predicted environmental concentrations (PEC) of TCS in soil are likely to be in the sub mg kg1 level. Data on TCS and TCC levels in sediments are scarce and it is difficult to predict PEC in sediments. However, Singer and co-workers (2002) reported that while TCS has a high photochemical removal rate in waters, it could accumulate in river sediments over time (53 lg kg1 in uppermost layer in lake sediments in this Swiss study). The same authors also attributed slow degradation rates of TCS in lake sediments to the high concentrations of TCS in 30 year old sediments.
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While the present study has demonstrated that TCS may impact biodegradation of the estrogens in soils when spiked at 1000 mg kg1, considering the expected concentrations of the antimicrobial in the environment in the low mg kg1 levels (Ying and Kookana, 2007; Stasinakis et al., 2008a), it is unlikely that TCS would have any adverse impact on biodegradation of estrogens in soils and sediments. By implications this study provides a reassurance that in situations where hormones and biocides co-occur, their accumulation in sediment or soils would not be facilitated by TCC or TCS. However, it is not clear if these findings would be applicable to other organic contaminants, requiring specialist microorganism in their biodegradation. Further investigations on the overall impact of the antimicrobial agents on the functional microbial communities may provide useful information for extending the findings of this study to other soils and environmental conditions. 4. Conclusions The study shows that the biodegradation of E2 or EE2 (spiked at 1 mg kg1), was virtually unaffected by TCS up to 100 mg kg1. However we can confirm that at higher concentration, TCS (e.g. TCS spike concentration of 1000 mg kg1 was tested in this study) can have a significant influence on short-term degradation of both E2 and EE2. There was no measurable effect of TCC at even at 1000 mg kg1 on biodegradation of E2 or EE2 may indicate the lack of any significant impact on microbial activity, especially on those microorganisms that may be involved in the degradation of the selected estrogens. On the basis of the lack of any significant adverse effects, even at very high spiked concentrations, of the two biocides on readily biodegradable hormones, we infer that at the environmental concentrations of the two antimicrobials reported in literature, it is highly unlikely that the two compounds would have any adverse impact on biodegradation of E2 or EE2 in soils or sediments. Acknowledgements The authors thank Catherine Fiebiger, Natasha Waller, Marianne Woods and Simon Toze from CSIRO, and Kate Langdon from University of Adelaide. References Allmyr, M., Harden, F., Toms, L.M.L., Mueller, J.F., McLachlan, M.S., Adolfsson-Erici, M., Sandborgh-Englund, G., 2008. The influence of age and gender on triclosan concentrations in Australian human blood serum. Sci. Total Environ. 393, 162– 167. Andersen, H., Siegrist, H., Halling-Sorensen, B., Ternes, T.A., 2003. Fate of estrogens in a municipal sewage treatment plant. Environ. Sci. Technol. 37, 4021–4026. Colucci, M.S., Topp, E., 2001. Persistence of estrogenic hormones in agricultural soils: II. 17a-Ethynylestradiol. J. Environ. Qual. 30, 2077–2080. Colucci, M.S., Bork, H., Topp, E., 2001. Persistence of estrogenic hormones in agricultural soils: I. 17b-Estradiol and estrone. J. Environ. Qual. 30, 2070–2076. Desbrow, C., Routledge, E.J., Brighty, G.C., Sumpter, J.P., Waldock, M., 1998. Identification of estrogenic chemicals in STW effluent. 1. Chemical fractionation and in vitro biological screening. Environ. Sci. Technol 32, 1549– 1558. European Commission, 2005. Opinion on triclocarban for other uses than as a Preservative. EC Scientific Committee on Consumer Products Report No. SCCP/ 0851/04. pp. 1–45. Halden, R.U., Paull, D.H., 2005. Co-occurrence of triclocarban and triclosan in US. water resources. Environ. Sci. Technol 39, 1420–1426. Hanselman, T.A., Graetz, D.A., Wilkie, A.C., 2003. Manure-borne estrogens as potential environmental contaminants: a review. Environ. Sci. Technol. 37, 5471–5478. Harries, J.E., Sheahan, D.A., Jobling, S., Matthiessen, P., Neall, P., Sumpter, J.P., Tylor, T., Zaman, N., 1997. Estrogenic activity in five United Kingdom rivers detected
by measurement of vitellogenesis in caged male trout. Environ. Toxicol. Chem. 16, 534–542. Heberer, T., 2002. Occurrence, fate, and removal of pharmaceutical residues in the aquatic environment: a review of recent research data. Toxicol. Lett. 131, 5–17. Heidler, J., Halden, R.U., 2007. Mass balance assessment of triclosan removal during conventional sewage treatment. Chemosphere 66, 362–369. Heidler, J., Sapkota, A., Halden, R.U., 2006. Partitioning, persistence, and accumulation in digested sludge of the topical antiseptic triclocarban during wastewater treatment. Environ. Sci. Technol. 40, 3634–3639. Jobling, S., Nolan, M., Tyler, C.R., Brighty, G., Sumpter, J.P., 1998. Widespread sexual differences in wild fish. Environ. Sci. Technol. 32, 2498–2506. Johnson, A.C., Sumpter, J.P., 2001. Removal of endocrine-disrupting chemicals in activated sludge treatment works. Environ. Sci. Technol. 35, 4697–4703. Lee, H.B., Liu, D., 2002. Degradation of 17b-Estradiol and its metabolites by sewage bacteria. Water Air Soil Pollut. 134, 351–366. Metcalfe, C.D., Mackenzie, T.L., Kiparissis, Y., 2001. Estrogenic potency of chemicals detected in sewage treatment plant effluents as determined by in vivo assays with Japanese Medaka (Oryzia Latipes). Environ. Toxicol. Chem. 20, 297–308. Neumegen, R.A., Fernandez-Alba, A.R., Chisti, Y., 2005. Toxicities of triclosan, phenol, and copper sulfate in activated sludge. Environ. Toxicol. 20, 160–164. Rubin, C.H., Niskar, A.S., 1999. Endocrine disruptors: an emerging environmental health problem. J. Med. Assoc. Georgia., 27–30. Schweizer, H.P., 2001. Triclosan: a widely used biocide and its link to antibiotics. FEMS Microbiol. Lett. 202, 1–7. Shareef, A., Angove, M.J., Wells, J.D., Johnson, B.B., 2006a. Aqueous solubilities of estrone, 17b-Estradiol, 17a-Ethynylestradiol, and bisphenol A. J. Chem. Eng. Data 51, 879–881. Shareef, A., Angove, M.J., Wells, J.D., Johnson, B.B., 2006b. Sorption of bisphenol A, 17a-Ethynylestradiol and estrone to mineral surfaces. J. Colloid Interface Sci. 297, 62–69. Singer, H., Mueller, S., Tixier, C., Pillonell, L., 2002. Triclosan: occurrence and fate of a widely used biocide in the aquatic environment: field measurements in wastewater treatment plants, surface waters, and lake sediments. Environ. Sci. Technol. 36, 4998–5004. Stasinakis, A.S., Petalas, A.V., Mamais, D., Thomaidis, N.S., Gatidou, G., Lekkas, T.D., 2007. Investigation of triclosan fate and toxicity in continuous-flow activated sludge systems. Chemosphere 68, 375–381. Stasinakis, A.S., Gatidou, G., Mamais, D., Thomaidis, N.S., Lekkas, T.D., 2008a. Occurrence and fate of endocrine disrupters in Greek sewage treatment plants. Water Res. 42, 1796–1804. Stasinakis, A.S., Mamais, D., Thomaidis, N.S., Danika, E., Gatidou, G., Lekkas, T.D., 2008b. Inhibitory effect of triclosan and nonylphenol on respiration rates and ammonia removal in activated sludge systems. Ecotoxicol. Environ. Saf. 70, 199–206. Stumpe, B., Marschner, B., 2009. Factors controlling the biodegradation of 17bEstradiol, estrone and 17a-Ethinylestradiol in different natural soils. Chemosphere 74, 556–562. Tabak, H.H., Bloomhuff, R.N., Bunch, R.L., 1981. Steroid hormones as water pollutants II. Studies on the persistence and stability of natural urinary and synthetic ovulation-inhibiting hormones in untreated and treated wastewaters. Dev. Ind. Microbiol. 22, 497–519. Ternes, T.A., Stumpf, M., Mueller, J., Haberer, K., Wilken, R.D., Servos, M., 1999. Behavior and occurrence of estrogens in municipal sewage treatment plants – I. Investigations in Germany, Canada and Brazil. Sci. Total Environ. 225, 81–90. Ternes, T.A., Andersen, H., Gilberg, D., Bonerz, M., 2002. Determination of estrogens in sludge and sediments by liquid extraction and GC/MS/MS. Anal. Chem. 74, 3498–3504. US Environment Protection Agency, 2002. High Production Volume (Hpv) Chemical Challenge Program Data Availability and Screening Level Assessment for Triclocarban. US EPA Triclocarban Consortium, Report No. 201-141886A. pp. 1–44. Waller, N.J., Kookana, R.S., 2009. Effect of triclosan on microbiological activity in Australian soils. Environ. Toxicol. Chem. 28, 65–70. Wibe, A.E., Rosenqvist, G., Jenssen, B.M., 2002. Disruption of male reproductive behavior in threespine stickleback Gasterosteus aculeatus exposed to 17bEstradiol. Environ. Res. 90, 136–141. Ying, G.-G., Kookana, R.S., 2005. Sorption and degradation of estrogen-likeendocrine disrupting chemicals in soil. Environ. Toxicol. Chem. 24, 2640–2645. Ying, G.-G., Kookana, R.S., 2007. Triclosan in wastewaters and biosolids from Australian wastewater treatment plants. Environ. Int. 33, 199–205. Ying, G.-.G., Kookana, R.S., Ru, Y.-J., 2002. Occurrence and fate of hormone steroids in the environment. Environ. Int. 28, 545–551. Ying, G.-G., Yu, X.Y., Kookana, R.S., 2007. Biological degradation of triclocarban and triclosan in a soil under aerobic and anaerobic conditions and comparison with environmental fate modelling. Environ. Pollut. 150, 300–305. Ying, G.-G., Kookana, R.S., Kumar, A., 2008. Fate of estrogens and xenoestrogens in four sewage treatment plants with different technologies. Environ. Toxicol. Chem. 27, 87–94. Yu, Z., Xiao, B., Huang, W., Peng, P., 2004. Sorption of steroid estrogens to soils and sediments. Environ. Toxicol. Chem. 23, 531–539.
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Effect of triclosan and triclocarban biocides on biodegradation of estrogens in soils Ali Shareef a,*, Sina Egerer b, Rai Kookana a a
Centre for Environmental Contaminant Research, Commonwealth Scientific and Industrial Research Organization (CSIRO), Land and Water, Private Mail Bag 2, Glen Osmond, South Australia 5064, Australia b Department of Soil Science and Soil Ecology, Geographical Institute, Ruhr-University Bochum, 150, 44780 Bochum, Germany1
a r t i c l e
i n f o
Article history: Received 2 June 2009 Received in revised form 10 September 2009 Accepted 10 September 2009 Available online 4 October 2009 Keywords: Antimicrobial agents Endocrine disrupting chemicals 17b-Estradiol 17a-Ethynylestradiol
a b s t r a c t We have investigated the effect of antimicrobials triclosan (TCS) and triclocarban (TCC) on biodegradation of 17b-Estradiol (E2) and 17a-Ethynylestradiol (EE2) in a sandy soil from South Australia. Two separate batch studies were conducted. In the first, the rates of loss of E2 and EE2 were determined at time intervals of 0, 3, 7, 14, 21, 28 and 56 d after initial spiking of soil with each estrogen at 1 mg kg1 and the antimicrobials at 10 and 100 mg kg1. Little loss of E2 and EE2 (<15%) under sterile conditions was noted compared to rapid loss in non-sterile soil (>60% in 24 h). There were no measurable effects on estrogen degradation by the two antimicrobials at spiked concentrations up to 100 mg kg1. The experiments were repeated to study degradation rates of the estrogens within the first 24 h (0, 3, 8, 24 h), 3 d and then weekly to 56 d. Again, E2 and EE2 degradation was not significantly affected by the presence of TCS up to 100 mg kg1 (p > 0.05). However TCS did significantly affect biodegradation of the estrogens when the soils were spiked with 1000 mg kg1 of TCS (p < 0.0005). In contrast, presence of TCC in soil showed no significant effect on biodegradation of the two compounds up to 1000 mg kg1 (p > 0.05). Considering environmental concentrations of the antimicrobials reported in the literature, it is highly unlikely these biocides would have any adverse impact on biodegradation of E2 or EE2 in soils. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction The release of organic contaminants of emerging concern such as endocrine disrupting chemicals, and pharmaceuticals and personal care products into the environment has received increasing public concern recently. Evidence in the scientific literature suggesting the detrimental impact of such chemicals on aquatic organisms and wildlife as well as the concerns about the potential impact on human health have mounted over recent years (Rubin and Niskar, 1999; Heberer, 2002; Allmyr et al., 2008). Municipal wastewater is one of the major sources of environmental pollution for numerous organic contaminants such as endocrine disrupting chemicals and pharmaceuticals and personal care products. For example, treated effluents can induce estrogenic activity in recipient waters up to several kilometers from the outfalls, with feminization of male fish indicated by the imposex condition or the production of the egg–yolk protein vitellogenin (Harries et al., 1997; Jobling et al., 1998; Metcalfe et al., 2001; Wibe et al., 2002). Steroid hormones are excreted by humans and animals in a water-soluble conjugated forms that are either estrogenically inactive or have a lower potency. However these glucuronide or sulfate * Corresponding author. Tel.: +61 8 8303 8474; fax: +61 8 8303 8565. E-mail address: [email protected] (A. Shareef). 1 Present address. 0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.09.018
conjugates are metabolized and released into the environment as fully active estrogenic hormones through treated sewage effluents (Tabak et al., 1981; Desbrow et al., 1998; Ternes et al., 1999; Hanselman et al., 2003). 17a-Ethynylestradiol (EE2) is of major concern because it is a relatively stable synthetic hormone which is commonly found in the formulation of oral contraceptives (Johnson and Sumpter, 2001). Although less stable, the natural hormone 17b-Estradiol (E2) and its metabolites also raise significant concern because E2 is the principal endogenous steroid hormone in vertebrates, stimulating the growth and development of the female sex organs. Triclosan (TCS) and triclocarban (TCC) are two common antimicrobial agents with very similar antifungal and antibacterial properties. They are used in numerous household and personal care products such as toothpaste, soaps and detergents, household sponges, plastic cutting boards, socks and undergarments (European Commission, 2005). TCS has been more widely used globally in a broader range of consumer products (0.3–1.0%) compared with its original use in hospitals and health care facilities (Schweizer, 2001). TCC is a high production volume chemical in the US with market ranges between 250 and 500 metric tones per year (US EPA, 2002). During common wastewater treatment the removal rates of estrogens as well as TCS and TCC from the aqueous-phase are relatively high due to their generally low aqueous solubilities and hydrophobic properties (i.e. log KOW P 3.9) (Stasinakis et al.,
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2007; Ying and Kookana, 2007; Ying et al., 2008). These two antimicrobials, which enter wastewater treatment plants at concentration levels in the order of low lg L1 (e.g. Ying and Kookana, 2007; Stasinakis et al., 2008a), show aqueous-phase removal efficiencies of up to 97% (Heidler et al., 2006; Stasinakis et al., 2007). Removal rates of E2, EE2 are also generally above 90% (Lee and Liu, 2002; Andersen et al., 2003). At the same time these compounds exhibit moderate to high sorption affinity for the solid phase and have a tendency to accumulate in sludges and sediments where they can persist (Ying et al., 2002; Heidler et al., 2006; Heidler and Halden, 2007). Heidler et al. (2006) demonstrated that no significant transformation of TCC takes place during the common sludge treatment of anaerobic digestion for 19 d. Approximately 76% of the TCC input of wastewater treatment plant was recovered in the dewatered, digested sludge after treatment resulting in an accumulation of TCC in the sludge at levels of 51 mg kg1 dry weight (Heidler et al., 2006). Ternes et al. (2002) reported that E2 and EE2 persisted in activated and digested sludge at levels up to 49 and 17 lg kg1, respectively. Treated sludges from wastewater treatment plants are often applied to agricultural land. Therefore these ‘biosolids’ may potentially introduce many endocrine disrupting chemicals, and pharmaceuticals and personal care products including the steroid estrogens as well as biocides such as TCS and TCC into soil environment. The fate, transport and distribution of these compounds may be influenced by many processes taking place in the soil/sediment environments including sorption and degradation. A growing body of research suggests that biodegradation can play a significant role in the fate of these organic compounds in soils. These include estrogens as well as TCS and TCC (Colucci et al., 2001; Colucci and Topp, 2001; Ying et al., 2007; Stumpe and Marschner, 2009) with little or no loss under anaerobic or sterile conditions (Andersen et al., 2003; Ying and Kookana, 2005; Heidler et al., 2006; Heidler and Halden, 2007). Due to the hydrophobic and persistent nature of these antimicrobial agents, it is likely that they can accumulate in the sediment phase in the aquatic environment. Although most wastewater treatment plants rely on activated sludge processes to treat wastewater, currently very little is known about the toxicity of these antimicrobial compounds specific to activated sludge microorganisms. In a recent investigation on toxicities of TCS, phenol and copper (II) to activated sludge microorganisms, Neumegen and co-workers (2005) reported that TCS had the highest relative toxicity based on EC50 values (TCS: 1.82 mg L1, phenol: 2.70 mg L1 and Cu(II): 18.3 mg L1). Stasinakis et al. (2008b) who investigated toxicity of TCS and the xenoestrogen 4-nonylphenol (NP) reported lower EC50 values of 0.22 mg L1 and 3.51 mg L1 for TCS and NP respectively, for a marine bacterium Vibrio fischeri. Based on these findings, the authors suggested that the presence of TCS can have a detrimental effect on the nitrification process in activated sludge systems in wastewater treatment plants. The impact of these biocides on soil microorganisms is largely unknown. Recently Waller and Kookana (2009) investigated the impact of TCS on some soil microbiological processes (i.e. substrate induced nitrification and respiration, and some selected enzyme activity assays) using two contrasting soils from South Australia. The addition of TCS at 50 mg kg1 affected substrate induced respiration in the clay soil but not in the sandy soil. However, at 5 mg kg1 TCS affected the nitrogen cycle in the sandy soil (a concentration that is within typical levels of TCS in Australian biosolids which can range from 0.09 to 16.79 mg kg1 (Ying and Kookana, 2007)). This may indicate that nitrifying bacteria were sensitive to low levels of TCS in soils. Considering that the co-occurrence of estrogens and the antimicrobials in wastewater treatment plant effluents, sediments and biosolids is of significant concern, the objective of the current study was to investigate (i) whether or not the biodegradation of E2 and EE2 may be significantly influenced by presence of the antimicrobial agent TCS or TCC in soil and if so (ii) at what concentra-
tions these effects may become significant. Answers to both of these questions are desirable for better understanding of likely environmental fate of estrogens in sediment and biosolids matrices.
2. Experimental 2.1. Materials 17b-Estradiol (E2), CAS [50–28-2], and 17a-Ethynylestradiol (EE2), CAS [57–63-6], both with purities of 98% or higher, as well as triclosan (TCS), CAS [3380–34-5] and triclocarban (TCC), CAS [101–20-2], with a purity of 99%, were obtained from Sigma–Aldrich (Sydney, Australia). HPLC grade anhydrous solvents (purity >99%) including methanol, acetone and acetonitrile were supplied by Biolab (Sydney, Australia). Table 1 presents the selected physicochemical properties of E2, EE2, TCS and TCC. The soil used for this study was a surface sandy loam soil collected from a depth of 0–15 cm from agricultural land at Roseworthy Farm (34.533°S, 138.733°E) in South Australia. The same soil has been used by Waller and Kookana (2009) in the study referred to earlier, which indicated that TCS had some negative impact of some of the microbial processes in this soil. It was air dried and ground to 62 mm before use. The soil characteristic properties are given in Table 2.
2.2. Methods Two replicate studies were conducted to determine the effect of the antimicrobial agents on the degradation of the estrogens. The first study involved a long-term investigation where degradation of E2 and EE2 were evaluated at time intervals of 0, 1, 3, 7, 14, 21, 28 and 56 d after initial spiking of the estrogens (1 mg kg1) and the antimicrobials (10 and 100 mg kg1). Due to the rapid loss of both E2 and EE2 observed in the initial experiments it was decided to repeat the experiment where time intervals of 0, 1, 3, 8, 24 h were added to the original time sequence. Also because no effect on biodegradation of the estrogens was observed with TCS or TCC spiked concentrations up to 100 mg kg1, an additional set of samples were included in the repeat experiment where the spiked concentration of TCS and TCC was increased up to 1000 mg kg1. To prepare the samples, individual stock solutions of the two estrogens were prepared at 100 mg L1 in methanol from which 50 lL aliquots were added to 5 g of soil (pre-weighed and moisture content adjusted to 50% of WHC using sterile milliQ water) in 20 mL glass culture tubes to achieve a concentration of 1.0 mg kg1 of E2 or EE2. TCS and TCC were spiked at three different concentrations of 10, 100 and 1000 mg kg1 by transferring appropriate volumes of stock solutions of TCS or TCC prepared in acetone. After spiking with the chemicals, the contents of the tubes were vortex mixed, aerated and incubated under dark conditions in a temperature controlled room maintained at 20 ± 1 °C. Sterile control tubes were prepared by autoclaving at 120 °C under a chamber pressure of 300 kPa for 1 h for three consecutive days before spiking with an estrogen. The spiking of the control tubes were done inside a laminar flow cabinet followed by sealing the tubes with screw caps with Teflon liners to ensure sterile conditions are maintained. The concentration of E2 and EE2 were then monitored at intervals from 0, 3, 8, 24 h (short study only), 1, 3, 7 d and then weekly for 56 d. All tests were conducted in triplicates and parallel samples were prepared for monitoring microbial activity in the tubes. The microbial activity in soil was monitored using substrate induced respiration technique following Organization for Economic Cooperation and Development protocol (Waller and Kookana, 2009).
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a b c d
Compound
17b-Estradiol (E2)
17a-Ethynylestradiol (EE2)
Triclosan (TCS)
Triclocarban (TCC)
mol.wt (g mol1) Log KOW Solubility (mg L1)
272 3.94a 1.51c
296 4.15a 9.2 c
289 4.7b 1.97–4.6b
316 4.9b 0.11d
Yu et al. (2004). Halden and Paull (2005). Shareef et al. (2006a). European Commission (2005).
Table 2 Physicochemical properties of the soil from South Australia.
a b
Clay (%) 10
Silt (%) 4
Sand (%) 84
CaCO3 (%) <0.5
MWHCa (%) 28.9
Total carbon (%) 1.1
Organic carbon (%) 0.85
Total nitrogen (%) 0.11
NH4–N (mg kg1) 1.8
NO3–N (mg kg1) 2.1
ECb (dS m1) 0.09
pH (0.01 M CaCl2) 5.4
MWHC = Maximum Water Holding Capacity. EC = Electrical Conductivity.
2.3. Extraction and analyses At the selected incubation time intervals, whole soil sample and sterile control tubes (5 g) were extracted twice by ultrasonication for 10 min with 10 mL of methanol:acetone (1:1, v/v). After centrifugation for 30 min (800 g), the liquid phase was separated by decanting into graduated conical bottom culture tubes in which the samples were concentrated to dryness under a gentle stream of nitrogen, and reconstituted in 1.0 mL of methanol. The optimised extraction method was developed after testing recoveries using a number of common solvents or combination of solvent (e.g. methanol, acetone, acetonitrile, ethyl acetate). The extraction method was also evaluated for the experimental spiking concentration ranges. The extracts were analysed by reverse phase high performance liquid chromatography with UVdetection (Agilent chromatograph equipped with photodiode array detector). The optimum wavelength for analysis was 280 nm. A Phenomenex Fusion RP C18 column (150 mm long, 4.6 mm I.D., 4.6 lm particle size) (Phenomenex, Sydney, Australia) protected by a guard column with matching stationary phase was used. Optimum chromatographic separation with baseline resolution of peaks was achieved using a mobile phase gradient at a linear flow rate of 1.0 mL min1 using acetonitrile (A) and water with 0.1% acetic acid (B) with the following gradient program: 0–8 min 40–60% A, 8–18 min 60–85% A, then dropped to 40% A from 18 to 20 min and held for 2 min. Calibration standards were prepared by serial dilution of the original standards used for spiking with methanol. A six-point linear calibration curves (ranges of 0.25–6 mg L1 for estrogens and 50–500 mg L1 for TCS and TCC) with correlation coefficients >0.99 were used for the determination of analyte concentrations in the samples. Appropriate dilutions of samples were required for quantitative determination of TCS and TCC for samples with the two higher spiked concentrations. The total run time for analysis of each sample was 20 min. 2.4. Analytical method validation and data analyses For the purpose of this study, a reliable and robust analytical method was developed for simultaneous analysis of the estrogens E2, EE2 and TCS or TCC using HPLC–UV detection, following a simple ultrasonic extraction method. The retention times from base-
line resolved peaks for E2 (5.6 min), EE2 (6.5 min) and TCS and TCC (15.0 min) were used to identify the analytes. The method allowed simultaneous and sensitive determination of E2, EE2 and either TCS or TCC in a single analysis. Co-elution of TCS and TCC was not an issue in our analyses given that our samples contained only TCS or TCC, not both. Extraction recoveries were greater than 80% for all the analytes. The mean extraction recoveries of 92 ± 11% and 90 ± 10% for E2 and EE2 respectively were used in calculations of the residual concentrations. The measured concentrations of E2, EE2, TCS or TCC have been normalised for simplicity of data interpretation. This was done by dividing the real measured concentration of the analytes by the concentration of the time-zero samples, taken as the nominal concentration. There was no significant difference between nominal and measured concentration of the analytes. Time-zero samples were spiked last, and extracted immediately to avoid possible loss due to degradation. Statistical analysis of the data with univariate analysis of variance (ANOVA) with Tuckey procedure was performed using SPSS v.15.1 for Windows. 3. Results and discussion 3.1. Biodegradation of estrogens – effect of antimicrobials Fig. 1 presents the aerobic biodegradation behaviours of E2 (1a) and EE2 (1b) spiked at 1 mg kg1 soil over a period of 56 d (from the long-term study) in the presence and absence of TCS (10 and 100 mg kg1). The corresponding data from the abiotic control treatment for the estrogens are also included. E2 degraded rapidly with approximately 70% of the initial spike concentration dissipating within a day. EE2 dissipation was slower with approximately 60% of initial concentration lost in one day. There was virtually no change in the concentration of E2 and EE2 under sterile conditions up to 56 d indicating that abiotic loss under sterile conditions was negligible. The effect of the addition of TCS at concentrations of 10 and 100 mg kg1 soils on the degradation of E2 and EE2 are also shown in the Fig. 1a and b respectively. There was no significant difference between E2 and EE2 dissipation in the presence of either of TCS at both spiked level (10 or 100 mg kg1) (p > 0.05). Almost complete loss of E2 was observed by 14 d even in the presence of TCS at both concentrations. Fig. 1c shows rapid biodegradation of TCS at both 10 and 100 mg kg1 measured in the same samples. However
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Fig. 1. Biodegradation of 1 mg kg1 (a) 17b-Estradiol (E2) and (b) 17a-Ethynylestradiol (EE2) in the presence of triclosan (TCS) at spiked concentrations of 10 (h) and 100 (4) mg kg1, and in the absence of TCS (s). (c) Biodegradation of triclosan (TCS) at 10 (h) and 100 (4) mg kg1. Filled circles represent abiotic controls and the error bars represent standard deviations. Data presented as percent residual concentrations have been normalised by dividing measured concentration by initial spiking concentrations.
Fig. 2. Biodegradation of 1 mg kg1 (a) 17b-Estradiol (E2) and (b) 17a-Ethynylestradiol (EE2) in the presence of triclocarban (TCC) at spiked concentrations of 10 (h) and 100 (D) mg kg1, and in the absence of TCC (s). (c) Biodegradation of triclocarban (TCC) at 10 (h) and 100 (D) mg kg1. Filled circles represent abiotic controls and the error bars represent standard deviations. Data presented as percent residual concentrations have been normalised by dividing measured concentration by initial spiking concentrations.
DT50 (time taken to dissipate 50% of initial concentration) was approximately 14 d. On the other hand, no significant degradation of TCS was observed under abiotic conditions. The fate data for TCS are similar to the findings from a recent study by Ying et al. (2007). The parallel study on substrate induced respiration, where samples were treated the same way as the biodegradation tests but without the antimicrobials, indicated microbial activity in the non-sterile soils (e.g. 14CO2 evolved per g soil per h gradually decreased from 7.5 mg kg1 h1 on d-0 to 5 mg kg1 h1 on d-56) under the conditions of this incubation study. Respiration rate (reflecting active microbial community) in the autoclaved samples was very low (<0.1 mg kg1 h1 14CO2 evolved for the period of study). Fig. 2 shows the effect of the second antimicrobial agent TCC on the biodegradation of E2 and EE2. Again about 80% of the initial E2 degraded in 1 d (Fig. 2a), whereas EE2 loss within the first day was approximately 60%. There was no significant difference between the results for biodegradation of E2 or EE2 with or without added TCC up to a concentration of 100 mg kg1 (p > 0.05). The degradation of TCC in the samples is displayed in Fig. 2c. There was no significant degradation of TCC at either concentration under both biotic and abiotic conditions which was consistent with previous studies (Ying et al., 2007). In the present study, DT50 for E2 was approximately 3 h while that for EE2 was 6 h. Although we observed very rapid loss of the two estrogens, data on the relative rates of degradation of the two estrogens are consistent with previous works investigating the degradation of estrogens in the environment. It has now been well documented in the scientific literature that E2 and EE2 undergo aerobic biodegradation with relatively short half-lives (<2 d). For example in a study examining the persistence of estrogens in soils, Colucci et al. (2001) reported that E2 was removed rapidly with a DT50 of
0.5 d at 30 °C under a range of different conditions and soil textures. The half-life of EE2 ranged between 7.7 d at 4 °C and 3.0 d at 30 °C (Colucci and Topp, 2001). Our results are consistent with other studies on the removal rates of estrogens in soils- even though the half-lives can vary due to different measurement methods and soil conditions (Ying and Kookana, 2005). Given the very rapid dissipation of the two estrogens and the lack of any significant impact of the antimicrobials up to100 mg kg1, we decided to investigate if there were any short-term impacts (within 24 h) of the two antimicrobials and if any adverse effect would be measurable at a higher concentration (1000 mg kg1) in soil. Since wastewaters are often continuously discharged into the aquatic environments, allowing little time for degradation of estrogens, we considered that even the short-term effects of antimicrobials may be important. 3.2. Effect of elevated concentrations of TCS and TCC The effect of increasing the spiked level of TCS on the biodegradation of 1 mg kg1 E2 and EE2 are shown in Fig. 3a and b respectively. Also it should be noted that data presented in these figures captured degradation time frame from 0 to 24 h taken at 0, 3, 8 and 24 h after treatment of the soil with the estrogens and the antimicrobials. The results from these repeat experiments confirmed the findings from the previous experiments demonstrating no impact of TCS up to 100 mg kg1 spiked concentration on degradation of both E2 and EE2. However, these results clearly show significant suppression of biodegradation of both E2 and EE2 when the soil was spiked with TCS concentration of a 1000 mg kg1 (Fig. 3a and b) (p < 0.0005). The addition of 1000 mg kg1 TCS resulted in 15–20% suppression of degradation of both E2 and EE2. The biodegradation of the estrogens in the presence of 1000 mg kg1 for the entire
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Fig. 4. Biodegradation of 1 mg kg1 (a) 17b-Estradiol (E2) and (b) 17a-Ethynylestradiol (EE2) in the presence of triclocarban (TCC) at spiked concentrations of 10 (h), 100 (4) and (.)1000 mg kg1, and (s) in the absence of TCC and the error bars represent standard deviations. Data presented as percent residual concentrations have been normalised by dividing measured concentration by initial spiking concentrations.
Fig. 3. Biodegradation of 1 mg kg1 (a) 17b-Estradiol (E2) and (b) 17a-Ethynylestradiol (EE2) for 0–24 h in the presence of triclosan (TCS) at spiked concentrations of 10 (h), 100 (D) and (.)1000 mg kg1, and (s) in the absence of TCS and the error bars represent standard deviations. (c) Biodegradation data for 1–56 days of E2 (.) and EE2 (d) in the presence of 1000 mg kg1. Open symbols (triangles and circles) display the biodegradation of 1000 mg kg1 TCS in E2 and EE2 respectively. Data presented as percent residual concentrations have been normalised by dividing measured concentration by initial spiking concentrations.
experiment is given in Fig. 3c. At 56 d, 25–30% of both E2 and EE2 still remained in the samples, while approximately 50% of TCS had depleted. The data for fate of the estrogens at 10 and 100 mg kg1 concentrations of TCS up to 56 d, has been described earlier (Fig. 1), where there was no significant impact on the degradation of E2 or EE2 in the presence of TCS at these concentrations. Fig. 4a and b display the results from the experiment repeated involving E2 and EE2 with three different concentrations of TCC ranging from 10 to 1000 mg kg1. While the general degradation behaviours of both estrogens were reproducible in these tests, there was no significant effect on the degradation of either E2 or EE2 even at 1000 mg kg1 concentration of TCC (p > 0.05). Also, given the slow biodegradation of TCC (>20% dissipation in 56 d) and the lack of any significant effect on the biodegradation of the estrogens (Fig. 2c), it is highly unlikely that TCC can have any effect of the fate of E2 or EE2, both of which have already been shown to undergo rapid biodegradation. Although environmental fate and ecotoxicological effects of TCS and TCC is largely unknown, the aerobic biodegradation has been proposed by some recent studies as one of the major pathways for their fate in the environment (Ying et al., 2007). A recent investigation by Waller and Kookana (2009) indicated that 5 mg kg1 TCS had some impact of microbial processes, especially the nitrogen cycle. The same study reported that in a clay soil, the TCS showed an effect on the microbial respiration, but only at a spiked concentration of 50 mg kg1. While the specialist bacteria such as those involved in the nitrogen cycle may be affected by TCS, the overall microbial activity is not. Moreover, the lack of any significant impact on the biodegradation behaviours of E2 and EE2 in
the presence of TCS or TCC up to a concentration of 100 mg kg1 may therefore suggest that the biodegradation of E2 and EE2 is primarily driven by specific microbial consortia which are not affected at levels as high as 100 mg kg1. Also, the fact that there was little loss of E2 or EE2 under the sterile conditions show that there were no other physicochemical processes (e.g. sorption, Shareef et al., 2006b) involved in the loss of the estrogens. A number of quality control measures were incorporated into this study to ensure the data presented here were free from any experimental artefacts. For example, the concentrations of TCS and TCC were measured along with the estrogens. There was no significant loss of either TCS or TCC even in the samples with lowest spiking concentration of 10 mg kg1 for up to 3 d, at which point most of the estrogen have dissipated. However TCS degraded down to 20–40% at the end of the experiment (56 d), while the 80–95% of TCC still persisted. These data corresponded well with a recent study by Ying and co-workers (2007) on the biological degradation of TCS and TCC in soil. The lack of significant microbial toxicity due to the presence of the antimicrobials was also confirmed from the measurement of microbial activity via substrate induced respiration in parallel sample. 3.3. Environmental significance In a recent Australian study investigating effect of TCS to microbial activity in soils, Waller and Kookana (2009) conducted a hazard assessment on terrestrial toxicology of TCS using a worst-case scenario based on soils amended with Australian biosolids containing high TCS concentration levels from literature (16.79 mg kg1) (Ying and Kookana, 2007). It was suggested that a dilution factor of 150 in the soil was likely to occur. Based on this, the predicted environmental concentrations (PEC) of TCS in soil are likely to be in the sub mg kg1 level. Data on TCS and TCC levels in sediments are scarce and it is difficult to predict PEC in sediments. However, Singer and co-workers (2002) reported that while TCS has a high photochemical removal rate in waters, it could accumulate in river sediments over time (53 lg kg1 in uppermost layer in lake sediments in this Swiss study). The same authors also attributed slow degradation rates of TCS in lake sediments to the high concentrations of TCS in 30 year old sediments.
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While the present study has demonstrated that TCS may impact biodegradation of the estrogens in soils when spiked at 1000 mg kg1, considering the expected concentrations of the antimicrobial in the environment in the low mg kg1 levels (Ying and Kookana, 2007; Stasinakis et al., 2008a), it is unlikely that TCS would have any adverse impact on biodegradation of estrogens in soils and sediments. By implications this study provides a reassurance that in situations where hormones and biocides co-occur, their accumulation in sediment or soils would not be facilitated by TCC or TCS. However, it is not clear if these findings would be applicable to other organic contaminants, requiring specialist microorganism in their biodegradation. Further investigations on the overall impact of the antimicrobial agents on the functional microbial communities may provide useful information for extending the findings of this study to other soils and environmental conditions. 4. Conclusions The study shows that the biodegradation of E2 or EE2 (spiked at 1 mg kg1), was virtually unaffected by TCS up to 100 mg kg1. However we can confirm that at higher concentration, TCS (e.g. TCS spike concentration of 1000 mg kg1 was tested in this study) can have a significant influence on short-term degradation of both E2 and EE2. There was no measurable effect of TCC at even at 1000 mg kg1 on biodegradation of E2 or EE2 may indicate the lack of any significant impact on microbial activity, especially on those microorganisms that may be involved in the degradation of the selected estrogens. On the basis of the lack of any significant adverse effects, even at very high spiked concentrations, of the two biocides on readily biodegradable hormones, we infer that at the environmental concentrations of the two antimicrobials reported in literature, it is highly unlikely that the two compounds would have any adverse impact on biodegradation of E2 or EE2 in soils or sediments. Acknowledgements The authors thank Catherine Fiebiger, Natasha Waller, Marianne Woods and Simon Toze from CSIRO, and Kate Langdon from University of Adelaide. References Allmyr, M., Harden, F., Toms, L.M.L., Mueller, J.F., McLachlan, M.S., Adolfsson-Erici, M., Sandborgh-Englund, G., 2008. The influence of age and gender on triclosan concentrations in Australian human blood serum. Sci. Total Environ. 393, 162– 167. Andersen, H., Siegrist, H., Halling-Sorensen, B., Ternes, T.A., 2003. Fate of estrogens in a municipal sewage treatment plant. Environ. Sci. Technol. 37, 4021–4026. Colucci, M.S., Topp, E., 2001. Persistence of estrogenic hormones in agricultural soils: II. 17a-Ethynylestradiol. J. Environ. Qual. 30, 2077–2080. Colucci, M.S., Bork, H., Topp, E., 2001. Persistence of estrogenic hormones in agricultural soils: I. 17b-Estradiol and estrone. J. Environ. Qual. 30, 2070–2076. Desbrow, C., Routledge, E.J., Brighty, G.C., Sumpter, J.P., Waldock, M., 1998. Identification of estrogenic chemicals in STW effluent. 1. Chemical fractionation and in vitro biological screening. Environ. Sci. Technol 32, 1549– 1558. European Commission, 2005. Opinion on triclocarban for other uses than as a Preservative. EC Scientific Committee on Consumer Products Report No. SCCP/ 0851/04. pp. 1–45. Halden, R.U., Paull, D.H., 2005. Co-occurrence of triclocarban and triclosan in US. water resources. Environ. Sci. Technol 39, 1420–1426. Hanselman, T.A., Graetz, D.A., Wilkie, A.C., 2003. Manure-borne estrogens as potential environmental contaminants: a review. Environ. Sci. Technol. 37, 5471–5478. Harries, J.E., Sheahan, D.A., Jobling, S., Matthiessen, P., Neall, P., Sumpter, J.P., Tylor, T., Zaman, N., 1997. Estrogenic activity in five United Kingdom rivers detected
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