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Uptake of Contaminants of Emerging Concern by the Bivalves Anodonta californiensis and Corbicula fluminea Niveen S. Ismail, Claudia E. Müller, Rachel R. Morgan, and Richard G. Luthy* Department of Civil and Environmental Engineering and ReNUWIt Engineering Research Center, Stanford University, Stanford, California 94305, United States S Supporting Information *

ABSTRACT: Uptake of seven contaminants regularly detected in surface waters and spanning a range of hydrophobicities (log Dow −1 to 5) was studied for two species of freshwater bivalves, the native mussel Anodonta californiensis and the invasive clam Corbicula f luminea. Batch systems were utilized to determine compound partitioning, and flowthrough systems, comparable to environmental conditions in effluent dominated surface waters, were used to determine uptake and depuration kinetics. Uptake of compounds was independent of bivalve type. Log bioconcentration factor (BCF) values were correlated with log Dow for nonionized compounds with the highest BCF value obtained for triclocarban (TCC). TCC concentrations were reduced in the water column due to bivalve activity. Anionic compounds with low Dow values, i.e., clofibric acid and ibuprofen, were not removed from water, while the organic cation propranolol showed biouptake similar to that of TCC. Batch experiments supported compound uptake patterns observed in flow-through experiments. Contaminant removal from water was observed through accumulation in tissue or settling as excreted pseudofeces or feces. The outcomes of this study indicate the potential utility of bivalve augmentation to improve water quality by removing hydrophobic trace organic compounds found in natural systems.

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inants.13,14 However, the fate of trace organic contaminants in bivalves is not well studied. Limited information on contaminant fate in other aquatic organisms such as worms, fish, and snails indicates the potential for bioaccumulation and biotransformation.5,6,15,16 In the present study, we aim to gain further insight into the uptake of a variety of emerging contaminants having different physicochemical properties by use of two species of freshwater bivalves: the native mussel Anodonta californiensis and the invasive clam Corbicula fluminea. The compounds studied include pharmaceuticals, personal care products, herbicides, and flame retardants. The pharmaceuticals are ibuprofen (anti-inflammatory), propranolol (beta-blocker), and clofibric acid (the metabolite of a cholesterol-lowering drug). Clofibric acid is also an active ingredient in herbicides, and another herbicide studied is diuron. Triclocarban is an antimicrobial or biocide that is found in many personal care products including soaps and detergents. Tris(1,3-dichloro-2propyl)phosphate (TDCPP) and tris(2-chloroethyl) phosphate (TCEP) are commonly used flame retardants. All of these compounds are not fully removed by conventional wastewater treatment processes and are detected in surface waters.1,17,18

INTRODUCTION Unregulated trace organic contaminants are detected in aquatic systems due to release from a variety of sources including agricultural runoff, animal wastes, and municipal wastewater.1 These contaminants of emerging concern (CECs) exhibit a range of physicochemical properties, and many of these compounds are ubiquitous in the aquatic environment due to their polar and nonvolatile nature, continual environmental input, and relative stability.2,3 Limited information is currently available on the bioavailability and metabolic degradation pathways of many of these contaminants in the aquatic environment.4−7 CECs include pharmaceuticals, personal care products, herbicides, and flame retardants. The bioavailability of hydrophobic legacy contaminants, such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), polyaromatic hydrocarbons (PAHs), and metals, has been studied using benthic organisms such as bivalves that are exposed to contaminants in aqueous and particulate phases.8−10 With many of these hydrophobic compounds uptake is correlated with the fraction that is predominantly available via the dissolved phase.11,12 Bivalves filter large volumes of water and can remove particulate matter from the water column as well as from deposited sediment. Through filter-feeding and deposit-feeding processes, bivalves can accumulate contaminants that are dissolved in water or desorbed from particulate matter.11,12 Bivalves are used as biomonitors due to their ability to hyperaccumulate contam© 2014 American Chemical Society

Received: Revised: Accepted: Published: 9211

March 7, 2014 June 21, 2014 July 13, 2014 July 13, 2014 dx.doi.org/10.1021/es5011576 | Environ. Sci. Technol. 2014, 48, 9211−9219

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at the Colorado School of Mines. All solutions were prepared using HPLC grade water or Milli-Q water from a Millipore system. Bivalve Collection and Preparation. The clam Corbicula f luminea was collected from the Guadalupe River in San Jose, CA. The length of C. f luminea used in experiments varied but the average length was 2.8 ± 0.7 cm. The average dry weight of C. f luminea tissue used in experiments was 0.11 ± 0.05 g. The mussel Anodonta californiensis was collected from either the San Andreas Reservoir in San Francisco, CA or the South Fork Eel River near Branscomb, CA. The length of A. californiensis used in experiments varied but the average length was 5.8 ± 0.6 cm. The average dry weight of A. californiensis tissue used in experiments was 0.39 ± 0.2 g. After cleaning of shells, bivalves were placed in aquaria containing synthetic freshwater in a 15 °C controlled temperature room and fed Nanochloropsis sp. (4−6 μm diameter, Florida Aquafarms, Dade City, FL) algae until ready for use. Prior to experimental use, the bivalves were depurated in individual beakers containing synthetic freshwater for 72 h, and their shells were scrubbed and rinsed. Mass Balance Experimental Setup. Mass balance experiments were performed using a batch approach in which individual bivalves were placed in beakers containing 100 mL of algal suspension (C. f luminea) or 250 mL of algal suspension (A. californiensis) for 24 h followed by 72 h of depuration in synthetic freshwater. Beakers were prepared with Nanochloropsis algae diluted with synthetic freshwater to approximately 106 cells/mL. The algal suspension was spiked with TCC, TDCPP, TCEP, propranolol, ibuprofen, and clofibric acid to achieve a 250 ng/L concentration 24 h prior to the start of the experiment. Diuron was spiked at 2500 ng/L. Six replicate beakers were prepared for each species of bivalve. Two control beakers without bivalves were prepared; one contained synthetic freshwater and unspiked algae, and one contained the spiked algal mixture. After 24 h, the media and feces or pseudofeces were swirled together and collected in 50-mL centrifuge tubes from beakers in which bivalves had fully cleared the water of visible algae. The media and fecal matter mixture was centrifuged at 1000 × g for 30 min to separate (pseudo)feces from media. Centrifugation was repeated three times to ensure proper separation. Feces and pseudofeces were not differentiated in these experiments; hence a combination of the two excretions was assumed and is referred to as (pseudo)feces. The media was decanted into 50-mL centrifuge tubes. Beakers were also rinsed with 10 mL of methanol to remove any analytes sorbed onto the walls. Bivalves were then placed in synthetic freshwater for depuration. The depuration water was refreshed every 24 h and collected for analysis along with any produced feces. After depuration the bivalve tissue was removed from the shell with a scalpel and placed in a centrifuge tube. Shells were rinsed with methanol to remove any sorbed analytes. All collected samples were stored at −20 °C until analysis. Flow-through System Setup and Conditions. Two identical 19-L Plexiglas flow-through tanks were designed with a distributed inlet, constant level outflow, and a circulation pump to achieve uniform mixing. For each flow-through experiment, one tank contained bivalves and another tank without bivalves served as the control. Nitrified secondary treated wastewater effluent was collected from Palo Alto Regional Quality Control Plant ahead of the dual media filters. The collected wastewater had already passed through the fixed film reactors and aeration basin resulting in removal of

In addition to examining uptake of these compounds, we consider the application of bivalves as biofilters to improve water quality. In various ecosystems it has been estimated that dense beds of bivalves may filter the entire water column in a day.19,20 For example, zebra mussel beds in the Hudson River have been estimated to filter the entire water column in 1−3 days.19 Due to their ability to filter large volumes of water, bivalves have been considered for removal of particulate matter and reduction of eutrophication.21−23 Suggested applications of bivalves include reduction of nutrients from aquaculture wastewater, removal of particulate matter in rivers, and final clarification of treated wastewater.24−26 For example, implementation of oysters for nutrient management in shrimp aquaculture leads to a reduction of nitrogen levels in the effluent by 72% and phosphorus levels by 86%.27 These bivalves are managed through harvesting to ensure nutrients removed from this system do not re-enter via fecal matter or dead bivalves, and this process is being termed “nutrient bioextraction”.28,29 The deployment and management of bivalves has yet to be applied to the removal of trace organic contaminants. This is the first study to expose either the freshwater mussel A. californiensis or the invasive clam C. f luminea to a suite of emerging compounds in order to quantify kinetic uptake and depuration, as well as consider possible applications for improving water quality. Batch and flow-through systems were employed in the study. The batch system experiments were used to determine the fate of the studied contaminants via analysis of bivalve tissue, feces or pseudofeces, and water. The flow-through system experiments were utilized to determine kinetic parameters and contaminant removal from water. Secondary treated wastewater was utilized in the flow-through experiments to expose bivalves to environmentally relevant conditions. Because 71% of native U.S. freshwater mussels are at risk of extinction, quantifying water quality improvement beyond particulate matter reduction can contribute to initiatives to protect and restore native bivalve species such as A. californiensis.30−32 The wide distribution of the invasive clam C. f luminea allows for application of results to a large geographic region. Finding a way to maximize use of invasive species can be just as important as finding reasons to protect and restore native species.21,22

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EXPERIMENTAL SECTION Chemicals and Standards. All reagents were purchased from Fisher Scientific (Santa Clara, CA) at the highest available purity. The properties of the test chemicals are listed in Supplementary Table S1. Stock solutions of triclocarban (TCC), tris(1,3-dichloro-2-propyl)phosphate (TDCPP), tris(2-chloroethyl) phosphate (TCEP), diuron, propranolol, ibuprofen, and clofibric acid were prepared in HPLC grade methanol and stored at 4 °C. All chemicals for stock solutions were purchased from Sigma-Aldrich (St. Louis, MO) with the exception of TCC, which was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The internal standards propranolol-d7 (CDN isotopes, Pointe-Claire, Quebec), tris(2-chloroethyl)phosphate-d12 (Toronto Research Chemicals, Toronto, Ontario), triclocarban-13C6 (Santa Cruz Biotechnology, Santa Cruz, CA), ibuprofen-d3 (Sigma-Aldrich, St. Louis, MO), clofibric acid-d4 (CDN isotopes, Pointe-Claire, Quebec), and diuron-d6 (Crescent Chemical Company, Islandia, New York) were also prepared in HPLC grade methanol and stored at 4 °C. Triclocarban-d7 was provided by Professor C. Higgins 9212

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conditions with some modifications (Supporting Information). 33 Surrogate recoveries were evaluated for each compound using the labeled version of the parent compound with the exception of TDCPP. TCEP-d12 was used as the surrogate for TDCPP. Recoveries varied depending on sample type and compound, and ranged from 27% for TDCPP in particulate matter to 106% for clofibric acid in water. Particulate matter refers to algae, (pseudo)feces, and matter collected on filters. Details of the analytical conditions are listed in Supplementary Table S3, and details of compound recoveries and repeatability are in Supplementary Tables S4, S5, and S6. Blanks, which were run with each batch of samples extracted, did not exhibit any of the analytes above the quantification limit. Unexposed bivalves were also tested for background levels of the target compounds. The analyte levels were at or below the quantification limit in unexposed bivalves. Lipid content was measured as 3.5 ± 0.03% for A. californiensis and 4.3 ± 0.04% for C. f luminea using previously published methods.34 Statistical analysis of results was completed using SPSS (IBM v 22).

ammonia. The collected wastewater was passed through a 325mesh sieve to remove larger particulates. Wastewater effluent was mixed with a small amount of Nanochloropsis algae stock solution (2% by volume) to ensure a sufficient food source for the bivalves. Spiking of the trace organic contaminants was identical to that listed in the batch experiment, but the final concentration varied due to the presence of these compounds in the wastewater prior to spiking and some sorption of compounds to the system surfaces. TCC in particular was impacted by sorption to system surfaces. The duration, hydraulic residence time, total tank volume, and sampling frequency are detailed in Table 1 for A. californiensis. Table 1. Parameters for Flow-through Experiment for A. californiensis no. of bivalves duration: exposure/depuration (days) water volume in tank (L) hydraulic residence time (days) water sample intervals/location bivalve sampling (day no.) no. of bivalves at each sample point (n)

17 14/7 9 3 daily/control + bivalve inlet/outlet + source tank exposure: 1, 2, 3, 6, 9, 14 depuration: 1, 2, 3, 5, 7 exposure: 2 depuration: 1

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RESULTS AND DISCUSSION Contaminant Partitioning and Fate. A mass balance approach from the batch experiments showed the fate of contaminants after 24 h of exposure and 72 h of depuration and allowed comparison of uptake between the two species (Figure 1). Uptake patterns of compounds for C. f luminea and A.

Conditions for the flow-through experiments were determined on the basis of measured algal clearance rates. The average algal clearance rates from batch experiments were determined as 80 mL/h (330 mL/h gdw) for an individual C. f luminea and 200 mL/h (590 mL/h gdw) for an individual A. californiensis. Experimental conditions for the C. f luminea flowthrough system are available in Supplementary Table S2. The two bivalves removed at each of the sampling days listed in Table 1 were immediately sacrificed. Bivalve tissue was removed in the same manner described in the batch mass balance experiments. Water samples were filtered through 0.7μm glass microfiber filters. The filters and filtrate were saved in centrifuge tubes. After exposure, depuration of the remaining 5 bivalves was completed in a batch mode as described for the batch mass balance experiment. A single bivalve was sampled and sacrificed at each depuration sampling day. All samples were stored at −20 °C until analysis. Sample Preparation. Detailed information on sample preparation is available in Supporting Information. Briefly, all samples were spiked with a labeled surrogate standard mixture prior to extraction and cleanup. Bivalve tissue, feces, filters, and algae were homogenized or ground with a mortar and pestle, freeze-dried, and then extracted with methanol three times. Based on method development results, a triple methanol extraction provided sufficient compound recovery; a fourth and fifth extraction did not improve compound recovery. Algae and (pseudo)feces extracts were cleaned with a liquid−liquid extraction with hexane to remove chlorophyll and lipids if necessary. All extracts were cleaned with solid phase extraction (hydrophilic−lipophilic balance (HLB)) following a slight modification of a standard protocol by Vanderford et al. (Supporting Information).33 Water samples were extracted with HLB cartridges. Sample Analysis. Concentrations of trace organic contaminants were analyzed using liquid chromatography−tandem mass spectrometry (LC−MS/MS) using previously described

Figure 1. Average mass distribution of contaminants after a 24 h batch exposure followed by 72 h of depuration for A. californiensis and C. f luminea (n = 6).

californiensis were not species-specific (p > 0.05, Mann− Whitney U test). Removal of the contaminant from the water column was achieved through retention in bivalve tissue or sedimentation via (pseudo)feces production. Sorption of contaminants to shells did not contribute to the mass balance. Control beakers contained the spiked algal mixture without bivalves and were measured at the start and end of the experiment to test for abiotic factors that could result in 9213

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Figure 2. Uptake and depuration kinetics of four analytes in A. californiensis. The solid and dotted lines represent first-order kinetics for uptake and depuration, respectively, and rs values show the fit between experimental and modeled data (U = uptake and D = depuration). Fit lines were omitted for compounds with rs (spearman’s rho) values less than 0.7. Reported concentration is based on tissue dry weight. Note differences in y-axis scale depending on the compound.

and TDCPP as these compounds did not show a high level of sorption to particulate matter (Supplementary Figure S1). For the compounds examined in this experiment we hypothesize that uptake occurred via particle ingestion and tissue sorption as a result of the filter feeding action of bivalves. The mode of uptake was dependent on compound hydrophobicity, with the exception of propranolol, which is a weak base and exists as an organic cation at pH less than the pKa of 9.5.36 When considering these bivalves in the context of restoration or for treatment purposes, it is important to understand the ultimate fate of the compound removed from the water column. While bivalves retain removed TCC and propranolol in their tissue with some excretion of the compounds back in the water column after depuration, TDCPP and diuron removal exhibit a different uptake pattern. These compounds are not retained in the bivalve tissue but are excreted in (pseudo)feces during the initial exposure period with additional excretion during depuration. Previous studies showed that bivalves have the ability to shed accumulated contaminants to varying degrees when depurated in clean water. For example depuration of Mytilus sp. mussels exposed to PCBs and PAHs took over 2 months to reach background levels of native unexposed mussels, whereas complete depuration of diazepam to background levels in those mussels took 3 days.6,11,37,38 Retention of compounds in tissue versus excretion via (pseudo)feces will impact management strategies regarding the potential use of bivalves for treatment purposes. These batch experiments showed the partitioning and ultimate fate of the spiked compounds on an individual bivalve basis. Since mass balance totals were less than 100% for most compounds, the possibility exists for biotransformation of some of these compounds, which could help close the mass balance. It has been observed that metabolites of several of the studied compounds have been found in aquatic and benthic organisms. Studies indicate that transformation of TCC occurs in

changes in analyte concentration (Supplementary Figure S1). Abiotic factors did not play a role in reduction of contaminants in the water column based on results from control beakers for TCC, propranolol, TDCPP, and ibuprofen. Of the recoverable TCC, which is the most hydrophobic of the compounds and does not ionize in water, 80% was removed from the water column with a roughly equal amount distributed between bivalve tissue and (pseudo)feces. In contrast, ibuprofen showed virtually no removal by the bivalves with the compound remaining in the water column. The results for the other compounds fell between these two limits of 0−80% removal as shown in Figure 1. The mean values and standard deviations are listed in Supplementary Table S7. The exposure route of these compounds varied on the basis of their sorption to the algal food source, with the proportion of the spiked compound sorbed on algae versus that dissolved in water shown in Supplementary Figure S1. Removal via particulate matter uptake was evident for TCC, the most hydrophobic of the suite of compounds tested. We also observed TCC uptake by bivalves when the compound was spiked directly into synthetic freshwater devoid of particulate matter (Supplementary Figure S2). The uptake of TCC from synthetic freshwater indicates a mechanism of removal through sorption onto tissue in addition to removal via ingestion of particulate matter with sorbed contaminant. Our data are consistent with previous studies demonstrating that bivalves can remove hydrophobic, particulate-sorbed compounds as well as hydrophobic dissolved compounds. Uptake via ingestion of particulate-sorbed compounds has been widely reported for PCBs and PBDEs.9−11,35 C. f luminea has also been shown to bioaccumulate PCBs from the aqueous phase as well.12 Direct uptake by a marine mussel of two moderately hydrophobic pharmaceuticals, tetrazepam (log Kow= 3.20) and diazepam (log Kow = 2.82), from the aqueous phase has been observed.6 Uptake directly from water may have occurred for propranolol 9214

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available for TCC in fish, worms, and snails.5,15,39 The uptake and depuration rates can vary among different organisms with lower uptake observed in fish versus in worms and snails. This lower observed bioaccumulation in fish may be attributed to different modes of feeding and the ability of the organism to metabolize the analyte. The value of the half-life (t1/2) is derived from depuration data and is used to express the elimination rate of accumulated compounds. For example in fish larvae, TCC elimination t1/2 was 1 h,39 whereas t1/2 for bivalves in this study was 2.3 days. Schebb et al. showed that fish metabolism of TCC through enzymatic activity was the predominant factor resulting in the fast elimination of TCC in 1 h. The ability of bivalves to biotransform TCC via metabolic processes is not known, but the batch experiments indicate that bivalves may metabolize TCC since recovery of the compound was approximately 70% (Figure 1). Metabolism of TCC may account for some of the unrecovered TCC in the mass balance. Bioconcentration Factors (BCF). The bioconcentration factors were calculated from experimental concentrations in mussel and water as well as from the model uptake and depuration rates (k1 and k2).

oligochaete worms, but the mechanisms of the transformation are not well studied. Transformation may be due to microbiota in the gut or enzyme induction.15 Biotransformation of TCC in larval fish is reported to occur from enzymatic activity and follows metabolism pathways similar to those in mammals.39 Metabolites of ibuprofen and propranolol have also been identified in fish with biotransformation due to enzymatic activity in livers and gills.16,40,41 Although metabolism of TDCPP and TCEP has not been studied in aquatic organisms, metabolism of these compounds has been suggested as a result of studies in humans; however, transformation results in humans cannot always be used to predict results in aquatic species.41−44 Limited data exist examining the metabolic ability of bivalves to biotransform organic contaminants, but results indicate that enzymes in digestive glands may be the primary forces of transformation.45 Uptake and Depuration Kinetics. Uptake and depuration kinetics were determined from flow-through system experiments for both species of bivalves. The 28-day exposure experiment completed with C. f luminea showed that steady state was achieved prior to 14 days (Supplementary Figure S3). A 14-day exposure duration was then chosen for A. californiensis based on results from the C. f luminea experiment. First-order kinetics were assumed to determine uptake (k1, day−1) and depuration (k2, day−1) rate constants.6 An average tank water concentration (ng/g) was used in the calculation. Cmussel represents the concentration of analytes in tissue expressed as ng/gdw: Cmussel =

k1 Cwater(1 − e−k 2t ) k2

BCF =

C k1 = mussel k2 Cwater

(2)

A significant difference did not exist for the two methods used to calculate BCF (p > 0.05). BCF values were similar for the two species of bivalves. A strong significant linear correlation exists between log BCF and hydrophobicity for compounds with a log Dow >1 (Figure 3). Log Dow, also

(1)

The experimental and fitted results of uptake and depuration of TCC, propranolol, ibuprofen, and TCEP are shown in Figure 2. With the exception of propranolol, uptake by the bivalves reached steady state for all compounds. Comparison of experimental data with the first-order kinetic model showed a good fit for TCC, propranolol, and TCEP. Uptake followed first-order kinetics for these compounds. Depuration experimental and model data were highly correlated but not significant for TCC and TCEP. While TCEP uptake by bivalves did follow first-order kinetics based on statistical analysis (Figure 2), the uptake concentrations in the tissue were an order of magnitude lower than that observed for TCC and propranolol. The low values for TCEP indicate that very little of the analyte accumulated in the tissue, which corresponds with the results from the mass balance batch experiment (Figure 1). Highest uptake was observed for TCC and propranolol, which corresponds to the accumulation in tissue observed in the mass balance batch experiments. Uptake was not observed for ibuprofen. Uptake and depuration results for diuron, TDCPP, and clofibric acid are presented in Supplementary Figure S4, but first-order kinetic processes did not fit these compounds. TDCPP concentration decreased in bivalve tissue over time, which could possibly be attributed to excretion of the compound via (pseudo)feces as observed in the mass balance experiments or metabolism of the compound. In the mass balance experiment, TDCPP was not retained in the bivalve tissue but was removed from the water column predominantly through the sedimentation of excreted (pseudo)feces. Published data do not exist for uptake and depuration kinetics of these compounds in bivalves, but these values are

Figure 3. Linear relationship between log BCF and log Dow (apparent Kow at the system pH) for compounds with log Dow > 1. Ionized compounds with a log Dow < 1 did not follow a linear relationship with BCF.

referred to as apparent log Kow at the system pH, was utilized to account for ionization of the analytes based on pH 6.8 of the nitrified secondary treated wastewater effluent. The BCF may be estimated on the basis of compound hydrophobicity for the bivalves for un-ionized compounds (log Dow >1) using the following expression: log BCF = 0.65 log Dow + 0.37 9215

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Figure 4. Mass flow comparison of mussel uptake, water outlet, and water inlet for four analytes. The shaded areas correspond to the modeled mussel sink mass flow (dark gray) and measured outlet (light gray). The line graphs represent the measured inlet and the combined outlet and mussel flow. Reported inlet and outlet values are based on an average value over 2 days, and the number of mussels in the system decreased over time from 17 to 6 due to periodic sampling.

our studies, the lack of correlation between BCF and apparent Dow for bivalves was particularly pronounced for propranolol (Figure 3). Weak bases, such as propranolol, may have a higher BCF than expected due to a charge attraction of the positively charged cation to the negatively charged cell membrane.48 This charge attraction may contribute to propranolol accumulating in the bivalve tissue via membrane sorption processes that are independent of lipid-like partitioning. Removal of Contaminants from Water. The uptake of TCC and propranolol was appreciable in the flow-through system, resulting in a reduction in concentration in the tank outlet in comparison to the tank inlet (Figure 4). However, as displayed in Figure 4, the absolute mass of TCC and propranolol retained in mussel tissue in the flow-through system decreased over time due to fewer mussels remaining in the tank because of sampling according to the schedule in Table 1 and the approach to steady state during the latter half of the experiment. Figure 4 also shows that during the first 3 days of the experiment the tank was in “startup” phase and may not have achieved steady conditions. For propranolol, the main removal mechanism leading to lower measured outlet values can be attributed to mussel uptake activity. For TCC, processes other than mussel uptake were contributing to lower outlet values as demonstrated by the sum of the outlet and mussel mass flows being less than the measured inlet mass flow value (Figure 4). Also a reduction between the inlet and outlet mass flow was observed in the control tank for TCC (Supplementary Figure S5). These factors may include abiotic processes such as sorption, which was also observed in the control tank. As previously mentioned, the metabolism of TCC as demonstrated in worms and fish, may also contribute to the difference between inlet mass flow and combined mussel and outlet mass

Highest BCF values were obtained for TCC, which corresponds to the most hydrophobic of the compounds studied. In order to test the ability of eq 3 to predict BCF based on the compound hydrophobicity, additional un-ionized compounds with greater hydrophobicities (log Dow > 4) should be tested. Since the relationship between BCF and Dow or Kow is based primarily on partitioning of the contaminant from the aqueous or dissolved phase into organism lipid, this relationship may not apply to the more complex system used in these experiments.11,46 The contaminants in our system both sorbed on particulate matter and remained in the aqueous phase, which may have impacted the calculation of the BCFs. BCF results for TCC and ibuprofen are consistent with published studies that determined BCF values for other aquatic organisms. The ibuprofen log BCF values estimated for fathead minnow and catfish ranged from 0.08 for muscle to 1.4 for plasma samples.7 This range corresponds to the log BCF value of 1.6 calculated for bivalves in this study. The BCF values for TCC for both species of bivalves were calculated at 3.9, which is within the same range as reported values of 2.9−3.4, with fish being at the lower end and snails at the higher end of the range.5,39 As might be expected, a linear regression relation with hydrophobicity does not apply for ionized compounds with log Dow < 1, and our results show no apparent relationship between BCF concentration and hydrophobicity for these compounds. Previous exposure studies with fish have shown that linear regression correlations between log Kow and log BCF hold best within the moderate-to-high log Kow range of 1−6.46,47 At low log Kow values, e.g., < 1, the bioconcentration of a compound is not solely based on accumulation in lipids and other components and processes likely influence accumulation.47 In 9216

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the high filtration efficiency and use of bivalves for improvement of water quality on a large scale, leading to the possible utility of bivalves for improvement of water quality in engineered systems or as part of ecological rehabilitation. Factors to consider in application of bivalves for water quality improvement are maintenance of an optimal population density of bivalves and management of (pseudo)feces production and exposed bivalves.21,27 Also, examining the potential of trophic transfer and biomagnification of compounds removed by the bivalves will help inform decisions on bivalve management strategies. The management approach chosen will vary on the basis of use of an invasive species already existing in a system or a reintroduction of a native species in decline. Quantifying bivalve uptake of trace organics found in surface waters can help with restoration efforts of native freshwater mussels or provide incentive to harness the ability of invasive species that already dominate waterways.

flow.15,39 The mass flow for ibuprofen shows a difference in outlet and inlet values during days 2−6, which cannot be explained by mussel uptake. The control tank did not show a reduction in ibuprofen outlet values (Supplementary Figure S5). The observed decrease in ibuprofen concentration at the experimental outlet, without any apparent justification via mussel uptake, suggests in situ biotransformation of the compound that we were unable to characterize. Biotransformation of ibuprofen has been observed in fish studies.16,41 With fish, ibuprofen may be readily metabolized and biotransformed to polar metabolites that could be easily eliminated rather than accumulated.40 Metabolism of ibuprofen needs to be studied in bivalves to help explain the results observed in the flow-through system. TCEP mass flow shows that uptake by mussels did not contribute to reduction in outlet concentration, which corresponds to the low levels of accumulation in tissue (Figure 2). The behavior of mass flow rates for TDCPP, diuron, and clofibric acid are similar to TCEP or ibuprofen. In the mass balance batch experiments we observed additional uptake from water via sedimentation of (pseudo)feces. The flow-through system was designed as a well-mixed system, and hence excreted (pseudo)feces may have been resuspended in the solution and removal via sedimentation was not accounted for in our mass flow. Environmental Significance. We have demonstrated the fate of various trace organic contaminants spanning a range of hydrophobicities using two bivalve species. We have developed a relationship to predict BCF from compound Dow (apparent Kow) based on the uptake and depuration kinetics for compounds with log Dow >1. This relationship can help predict the bioaccumulation potential for other emerging contaminants, but additional compounds should be tested to confirm the validity of the relationship. We have also demonstrated the potential for bivalves to reduce water concentration of TCC via accumulation in tissue, and bivalves may also remove other hydrophobic compounds that do not ionize in environmental water. Additional studies are needed to examine the potential biotransformation and the long-term fate of the studied compounds. Biotransformation due to metabolic activity may influence the apparent bioaccumulation of the parent compound, and understanding biotransformation rates can help refine prediction of BCF values from Dow. Since bivalves are an important food source of the aquatic food web, the generated kinetic data can also be utilized in research designed to understand trophic transfer and biomagnification of these compounds in an aquatic system. The use of environmentally relevant concentrations and treated wastewater, which is analogous to effluent-dominated surface waters, provide the first indication of the potential efficacy of bivalves for removal of contaminants of emerging concern to improve water quality. Additional studies are needed to understand the concentration dependence of uptake of trace organics and the correlation with bivalve density. Elliott et al. showed a logarithmic relationship between chlorophyll a removal with mussel density using large-scale flume studies, but this relationship has not been examined for trace organic uptake.22 Previous efforts to model reduction of eutrophication through use of bivalves in a lake system can provide insight but cannot be applied directly to trace organic uptake since algal clearance rates have not been correlated to trace organic contaminant uptake rates.49 Results from those algal-based studies confirm

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ASSOCIATED CONTENT

S Supporting Information *

Additional information on method development, experimental design, and figures and tables referenced in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 650-721-2615. Fax: 650-725-9720. E-mail: luthy@ stanford.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation (NSF) Engineering Research Center for Reinventing the Nation’s Water Infrastructure (ReNUWIt) EEC-1028968, a Ford Foundation Predoctoral Fellowship, and a NSF Graduate Research Fellowship. We thank Dr. Jeanette Howard from the Nature Conservancy and San Francisco Public Utilities Commission for help with collecting bivalves, Palo Alto Regional Water Quality Control Plant for providing effluent, Dr. David Sedlak for reviewing the manuscript, Justin Jasper for testing samples for metabolites of propranolol, and Dr. Chris Higgins for supplying TCC-d7.

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REFERENCES

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