An examination of the ingestion, bioaccumulation, and depuration of titanium dioxide nanoparticles by the blue mussel (Mytilus edulis) and the eastern oyster (Crassostrea virginica)

Marine Environmental Research 110 (2015) 45e52

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An examination of the ingestion, bioaccumulation, and depuration of titanium dioxide nanoparticles by the blue mussel (Mytilus edulis) and the eastern oyster (Crassostrea virginica) John J. Doyle*, J. Evan Ward, Robert Mason University of Connecticut, Department of Marine Sciences, 1080 Shennecossett Road, Groton, CT 06340, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 March 2015 Received in revised form 29 July 2015 Accepted 30 July 2015 Available online 31 July 2015

The production rates of titanium dioxide (TiO2) nanoparticles for consumer products far exceed the pace at which research can determine the effects of these particles in the natural environment. Sedentary organisms such as suspension-feeding bivalves are particularly vulnerable to anthropogenic contaminants, such as nanoparticles, that enter coastal environments. The purpose of this work was to examine the ingestion, bioaccumulation, and depuration rates of TiO2 nanoparticles by two species of suspensionfeeding bivalves, the blue mussel (Mytilus edulis) and the eastern oyster (Crassostrea virginica). Two representative TiO2 nanoparticles, UV-Titan M212 (Titan) and Aeroxide P25 (P25), were delivered to the animals either incorporated into marine snow or added directly to seawater at a concentration of 1.0 mg/ L for exposure periods of 2 and 6 h. After feeding, the animals were transferred to filtered-seawater and allowed to depurate. Feces and tissues were collected at 0, 12, 24, 72, and 120 h, post-exposure, and analyzed for concentrations of titanium by inductively coupled plasma-mass spectrometry. Results indicated that the capture and ingestion (i.e., transfer to the gut) of TiO2 nanoparticles by both mussels and oysters was not dependent on the presence of marine snow, and weight-standardized clearance rates of bivalves exposed to TiO2 nanoparticles were not significantly different than those of unexposed control animals. Both species ingested about half of the nanoparticles to which they were exposed, and >90% of the nanoparticles were egested in feces within 12 h, post-exposure. The findings of this study demonstrate that mussels and oysters can readily ingest both Titan and P25 nanoparticles regardless of the form in which they are encountered, but depurate these materials over a short period of time. Importantly, bioaccumulation of Titan and P25 nanoparticles does not occur in mussels and oysters following exposures of up to 6 h. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Nanoparticles Mussels Oysters Ingestion Bioaccumulation Depuration

1. Introduction Currently, the annual production of nanoscale titanium dioxide (TiO2) in the United States is estimated to be approximately 1.4 million metric tons with environmental loads expected to reach 2,000,000 to 6,000,000 tons over the next decade (Robichaud et al., 2009). The production and widespread use of nano-enabled products will lead to the release of nanoparticles (NPs) into the environment, either directly via firsthand use and spills, or indirectly via release from landfills, waste incineration plants, and

* Corresponding author. Gloucester Marine Genomics Institute, 6 Rowe Square, Gloucester, MA 01930, USA. E-mail addresses: [email protected] (J.J. Doyle), [email protected] (J.E. Ward), [email protected] (R. Mason). http://dx.doi.org/10.1016/j.marenvres.2015.07.020 0141-1136/© 2015 Elsevier Ltd. All rights reserved.

wastewater treatment facilities (Cheng et al., 2004; Moore, 2006). Attempts to calculate the current concentrations of TiO2 NPs in the environment found concentrations of TiO2 NPs in runoff water collected from painted facades as high as 3.5
108 particles/L (Kaegi et al., 2008). In addition, Mueller and Nowack (2008) predicted the concentration of TiO2 NPs in aquatic environments to be approximately 0.7e16 mg/L. Coastal ecosystems in close proximity to densely populated, industrialized regions are particularly vulnerable to the infiltration of anthropogenic materials such as NPs into coastal waters (Moore, 2006). These systems support a rich diversity of organisms including bivalve molluscs, which are benthic, suspension-feeders pervasive in coastal and estuarine waters. Bivalves play a critical role in nutrient cycling and material exchange between the surrounding water and sediments, and are capable of clearing

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particulate matter from significant volumes of water (Dame, 1993, 1996). Furthermore, bivalves are commonly regarded as living marine resources as they are a crucial component of the food web, and they provide a source of jobs and sustenance to people living in coastal regions. The abundance of bivalves in near-shore environments, their sedentary lifestyle, and predilection for suspensionfeeding could make these animals more susceptible to NPs
et al., 2008). entering coastal waters (Gagne In the marine environment, few particles exist in a solitary state. Depending on the time of year, greater than 70% of natural particles of disparate size and quality are incorporated into marine snow (Alldredge et al., 1993). Marine snow forms when organic (living and detrital) and inorganic particles in marine and freshwater environments begin to agglomerate (Alldredge and Silver, 1988; Kiørboe et al., 1990). Particle agglomeration is enhanced by transparent exopolymeric particles (TEP) that are formed from mucopolysaccharides released in abundance by bacteria, phytoplankton, and benthic suspension-feeders (Alldredge et al., 1993; Heinonen et al., 2007; Li et al., 2008; Simon et al., 2002). TEP coats the surfaces and increases the “stickiness” of organic and inorganic particles suspended in the water column (Alldredge and Silver, 1988; Jackson, 1990; Kiørboe et al., 1990; Passow, 2002; Simon et al., 2002). As particles are subjected to physical forces such as shear, Brownian motion, and differential settling, they collide, stick together, and eventually form marine snow (Jackson, 1990; Kiørboe et al., 1990; Simon et al., 2002). Therefore, NPs reaching coastal waters, or resuspended from the sediments during storm events, will likely be incorporated into marine snow, making them potentially more bioavailable to marine organisms. Particle size is a critical determining factor in the capture and ingestion of particulate matter by suspension-feeding bivalves (Ward and Shumway, 2004). For example, bivalves can capture particles greater than 5 mm with an efficiency of approximately 100%. Capture efficiency, however, decreases as particles become smaller (Møhlenberg and Riisgård, 1978; Riisgård, 1988). A few bivalve species, such as the ribbed mussel (Geukensia demissa), are able to capture 1-mm particles with approximately 50% efficiency, but for most species of bivalves the capture efficiency of particles smaller than 1 mm is less than 20% (Kach and Ward, 2008; Ward and Shumway, 2004). Conversely, particles less than 5 mm that agglomerate, or particles incorporated into marine snow, will have a greater particle size, and as a result, will be captured at a higher efficiency. For example, Ward and Kach (2009) observed extremely low capture efficiencies of freely-suspended 100-nm fluorescent polystyrene particles in the blue mussel (Mytilus edulis), and the eastern oyster (Crassostrea virginica), however, capture efficiency increased significantly when NPs were incorporated into marine snow. Additionally, agglomerated particles and particles incorporated into marine snow possess larger diameters than constituent particles resulting in enhanced settling rates (Stokes Law; Hill, 1998; Waite et al., 2000). Greater settling could lead to increased encounter and bioavailability of NPs to suspension-feeding and benthic organisms. Many studies allude to the ingestion of aggregated NPs by suspension-feeding bivalves, but do not include ecologically-relevant experiments addressing particle capture and
et al., 2008). Only the study by Montes ingestion efficiencies (Gagne et al. (2012) examines the ingestion and bioaccumulation of NPs (CeO2 and ZnO) in the Mediterranean mussel (Mytilus galloprovincialis). Hence, there is a paucity of data regarding the capture, ingestion, and depuration rates of NPs in suspension-feeding bivalves. The purpose of this work was to examine two aspects of bivalveNP interactions. First, we determined if the form of delivery affected ingestion and bioaccumulation of NPs by exposing bivalves

to NPs incorporated into marine snow, an ecologically-relevant natural hetero-aggregation, and to freely-suspended NPs in single-exposure experiments. Second, we determined if multiple exposures affected ingestion, bioaccumulation, and depuration of NPs. The two types of TiO2 NPs tested, UV-Titan M212 and Aeroxide P25, are widely used in a variety of commercial products, but they differ in their specific crystalline-phase composition and presence/ absence of a surface coating. The NPs were delivered to two species of bivalves, the blue mussel and the eastern oyster at a concentration of 1 mg/L. These two species are among the most abundant, commercially-relevant bivalves in near-shore estuarine ecosystems along the eastern seaboard and Gulf coasts of the United States, making them likely candidates for NP exposure and subsequent consumption by humans. A concentration of 1 mg/L was chosen for these experiments as the effects of TiO2 NPs on several species of bivalves have been demonstrated to occur following exposure to concentrations ranging from approximately 1e10 mg/L (Canesi et al., 2010a, 2010b; Ciacci et al., 2012). 2. Materials and methods 2.1. Collection and characterization of natural seawater All containers used in these experiments were acid washed (10% HCl) prior to use. Natural seawater was collected in 20-L carboys from the rocky intertidal zone at Avery Point in Groton, Connecticut. Before experiments were conducted, all seawater was passed through a 210-mm sieve to remove large material. The pH, salinity, chlorophyll, and total suspended solids (TSS) were measured to characterize the chemical parameters of the seawater. The pH was determined using a bench-top pH meter (Accumet AB15 Plus; Accumet Probe Model 13-620-223A), and the salinity (practical salinity units; PSU) was measured using a YSI salinometer (Model 30). To analyze the concentration of chlorophyll present, seawater was drawn up into a syringe and passed through a pre-ashed, preweighed GF/F filter. The process was repeated until there was a visible color on the filter. The filter was then treated with 1 mL of magnesium carbonate, air dried, folded in half, and stored in aluminum foil at 20  C until processed. The concentration of chlorophyll-a was determined using the acidification method outlined by the Environmental Protection Agency (Arar and Collins, 1997). Total suspended solids were calculated by vacuum-filtering 1 L of seawater through a pre-ashed, pre-weighed GF/C filter. The filters were dried to a constant mass in an oven at 70  C to determine the concentration of total suspended solids present. 2.2. Nanoparticles UV-Titan M212, a surface-coated TiO2 NP, was obtained from Sachtleben Pigments Oy (93% rutile TiO2, 6% Al2O3, and 1% glycerin; hereafter referred to as Titan). Aeroxide P25, a TiO2 NP commonly used in environmental studies (e.g. Couleau et al., 2012; Weir et al., 2012; Libralato et al., 2013), was obtained through the National Institute of Standards and Technology (NIST SRM 1898 see http:// www-s.nist.gov/srmors/view_cert.cfm?srm¼1898; 76% anatase TiO2, 24% rutile TiO2; hereafter referred to as P25). X-ray diffraction (XRD) analysis of the Titan NPs showed the characteristic rutile crystalline phase and a mean particle size of 86.0 nm ± 32.0 (Doyle et al., 2014). Analysis of the P25 NPs demonstrated both the rutile and anatase crystalline phases and a mean particle size of 28.0 nm ± 4.0. Both types of NPs were found to agglomerate rapidly when immersed in solutions of high ionic strength (Doyle et al., 2014). Stock suspensions for the Titan and P25 NPs were created by adding TiO2 powder to ultrapure Milli-Q water (MQ-water;

J.J. Doyle et al. / Marine Environmental Research 110 (2015) 45e52

MilliPore Synthesis A10 system) to achieve a concentration of 250 mg/L. Each stock suspension was placed on a stir plate and subjected to ultrasonication (Fisher Scientific FB-505) at 30% power (ca. 16.0 W; Taurozzi et al., 2012) for 30 min. The requisite volume (188 mL) from each of the stock suspensions was diluted in seawater to obtain a concentration of 50 mg/L. The 50 mg/L stock suspensions were ultrasonicated a second time at 30% for 30 min to help disperse the NPs and disrupt those agglomerations that initially form following immersion in seawater (Doyle et al., 2014). The two 50 mg/L stock suspensions were diluted in seawater (1:50) to obtain two working suspensions each at a final concentration of 1.0 mg/L. The working suspensions were used immediately to generate marine snow or in the feeding experiments (see below). Blanks were prepared in the same manner as described above, except that MQ-water was substituted for the NP stock suspensions. 2.3. Preparation of the animals Mussels, collected from local populations, and oysters, obtained from the Noank Shellfish Cooperative (Noank, CT, USA), were cleaned of all fouling organisms and sediment prior to the experiments. A Velcro® strip was adhered to one of the animals' shells using a two-part marine epoxy (Ward and Kach, 2009). Animals were held in seawater aquaria in an environmental chamber, and fed the microalga Tetraselmis sp. (Wikfors et al., 1996) for several days while acclimating to a temperature between 18 and 20  C. Approximately 24 h before the commencement of the feeding experiments, the bivalves were secured to craft sticks with Velcro® and transferred to a large holding tray filled with aerated seawater, fed Tetraselmis sp., and allowed to acclimate. 2.4. Single-exposure feeding experiments 2.4.1. Production of marine snow Marine snow was produced in the laboratory using the rollertable method as described previously (Shanks and Edmondson, 1989; Ward and Kach, 2009; Doyle et al., 2014). The working suspensions of Titan and P25 were placed on a stir plate, mixed, and then poured into 1-L Nalgene rolling bottles in quarter-liter aliquots. After dispensing each aliquot, working suspensions were stirred again to ensure thorough mixing. This process was repeated until the rolling bottles were full. Each bottle was shaken by hand, and an initial 15-mL water sample was removed and stored at 4  C. For each type of NP, six bottles were designated for the feeding experiments, three bottles were used to determine the amount of nanoparticle incorporation into marine snow, and one bottle served as a blank (no NPs added). These twenty bottles were placed on a roller table at 15 rpm for 72 h, which is a sufficient period of time to incorporate >50% of the TiO2 NPs suspended in the seawater (Doyle et al., 2014). After 72 h, the bottles were removed from the roller table and either immediately used in the feeding experiments, or allowed to stand for 2 h so that large marine aggregations and snow could settle to the bottom and be collected (ca. 60 mm; see Hill, 1998). Marine snow was transferred from each bottle to an individually labeled 15-mL Falcon tube and stored at 4  C. 2.4.2. Feeding experiments Experiments were conducted in an environmental chamber set at 20 C. Mussels and oysters were exposed to one of three treatments: 1) a marine-snow treatment that contained either Titan or P25 NPs incorporated into the marine snow, 2) a freely-suspended treatment that contained one of the two NPs spiked directly into bottles containing seawater just prior to the start of the feeding experiment (final concentration ¼ 1 mg/L), and 3) a blank

47

treatment that contained seawater or marine snow with no TiO2 NPs added to account for background concentrations of titanium. Bottles containing these three treatments were arranged on multiposition stir plates. In addition, one settling control containing Titan or P25 NPs, but no animal, was added to the stir plates. The settling control was used to determine the amount of particulates that settled out of suspension in the feeding experiment described below (Ward et al., 1992). During the experiments, each bottle was supplied with gentle aeration and mixed with a stir bar for 10 s every 5 min to ensure that marine snow and particulate matter remained in suspension (Kach and Ward, 2008; Ward and Kach, 2009). Prior to the animals being placed into the bottles, a 20-mL water sample was removed from each bottle for particle analysis (particles >2 mm). One bivalve was then placed into the center of each bottle (except settling controls) and its craft stick secured to the rim by means of a wooden clip (Ward and Kach, 2009). Animals were allowed to feed for 2 h with time commencing after they showed signs of feeding (i.e., shells open, mantles extended). After 2 h, animals were removed from the 1-L bottles, individually labeled, and stored at 20  C until analyzed. Feces produced by the animals were collected and placed in individually labeled 15-mL Falcon tubes and also stored at 20  C. A 20-mL sample was removed from each bottle for determination of final particle concentrations (particles >2 mm). 2.5. Multiple-exposure feeding experiments Based on the results of the single-exposure feeding experiments (see Section 4.2.1), only the freely-suspended condition (no marine snow) was used in the multiple-exposure experiments, and only oysters were exposed. The experimental set-up was the same as described above, with NP stock suspensions (Titan and P25) being prepared and added directly into appropriate bottles filled with seawater at a final concentration of 1 mg/L. Three bottles containing the experimental treatment were prepared for each time interval for a total of fifteen bottles. Additionally, two blank treatments containing bivalves but no NPs, and one settling control containing NPs, but no oyster, were also prepared. Blank treatments were prepared to account for background concentrations of titanium that naturally occur in the organisms' tissues, whereas the settling controls were used to determine how much of the NPs remained in suspension. This experiment was repeated a total of four times to obtain a sufficient number of replicates for statistical analysis. Bottles were arranged on stir plates and each was supplied with gentle aeration and a stir bar to ensure mixing. Prior to the oysters being placed into the bottles, initial water samples were collected for determination of particle concentrations (particles >2 mm). One oyster was then placed in the center of the bottle (except settlingcontrols) and its craft stick secured to the rim by means of a wooden clip (Ward and Kach, 2009). Animals were allowed to feed for 2 h with time commencing after they showed signs of feeding (i.e., shells open, mantles extended). After 2 h of feeding, all feces were removed and final water samples collected from each bottle and placed in an appropriately labeled container. Each oyster was rapidly transferred into a new 1-L Nalgene bottle containing the same treatment to which it was previously exposed. The entire feeding process was repeated (2-h of feeding), oysters were transferred a second time, and a third cycle was completed for a total of six hours of exposure. Immediately after exposure (6 h), six oysters were sacrificed and stored at 20  C. These animals were designated as 0-h (time, postexposure). The remaining oysters were transferred to aerated, 1-L beakers containing 0.22-mm filtered seawater (no NPs). A mixed phytoplankton diet (Reed Mariculture) was added to the water to

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give a final cell concentration of 10,000 cells/mL. Groups of oysters were allowed to depurate for 12, 24, 72, and 120 h, post-exposure, with replicate animals being sacrificed when each time interval was reached. Sacrificed animals were labeled and stored at 20  C until analyzed. Once a day, feces were collected from each beaker, the water was replaced with fresh 0.22-mm filtered seawater, and additional shellfish food was added (final concentration of 10,000 cells/mL). 2.6. Calculation of particle clearance rates Feeding activity (i.e., clearance rates) was calculated for each bivalve to determine which animals were filtering water and actually being exposed to the NPs. This is a critical consideration when using animals that can isolate themselves from the external environment, and ensured that only animals that were feeding were included in the analysis of NP capture. An electronic particle counter (Coulter multisizer) was used to analyze the number of particles greater than 2 mm in size that were present in the initial and final water samples. These particles included natural seston present in the seawater and large agglomerations of NPs. Clearance rates for each animal were calculated using the following formula (Coughlan, 1969):

CR ¼ ðM=tÞ½lnðC0 =Ct Þ  ½lnðC0 blank=Ct blankÞ; where CR ¼ clearance rate (ml/min); M ¼ volume of seawater in each container (ml); t ¼ time over which the experiment was conducted (min); C0 ¼ concentration of particles in the container at the start; Ct ¼ concentration in the container at the end; C0blank ¼ concentration of particles in the settling control container at the start; and Ctblank ¼ concentration in the control container at the end. Clearance rates were standardized to a 0.5 g dry-tissue-mass animal using the following allometric equation:

Weight  standardized CRS ¼ ðWS =WO Þb $CRO ; where CRS (L/h/0.5 g) is the clearance rate for a standard bivalve of dry tissue mass WS (0.5 g); CRO (L/h) is the observed clearance rate for a bivalve of dry tissue mass WO (g); and b is the exponent relating feeding to body mass (Bayne and Newell, 1983). 2.7. Analysis of water, marine snow, tissue, and fecal samples The concentration of titanium in all samples was determined by means of an ELAN DRC II inductively coupled plasma-mass spectrometer (ICP-MS; Perkin Elmer). The ICP-MS was tuned to detect the titanium-47 isotope in the tissue and feces samples to avoid interference from the high levels of the titanium-48 isotope found in seawater. The analytical error of the ICP-MS was calculated as 0.910 ± 0.06 mg/L (mean ± standard deviation of six solutions containing TiO2 at a concentration of 1 mg/L). The limits of detection of the ICP-MS were calculated as 37.5 mg/L. Samples were prepared for analysis as described below. Just prior to analysis, all samples were ultrasonicated at 60% power (ca. 50 W) for 1 min. Marine snow samples were centrifuged for two minutes at 1500
g (2730 rpm), and the seawater was removed with a Pasteur pipette. Samples were stored at 20  C overnight, and then lyophilized for 24 h to remove any remaining moisture. Marine snow was digested in 2 mL of 18 M optima grade sulfuric acid and 16 M optima grade nitric acid (3:7 v/v ratio) for 24 h (Lawrence et al., 1999), and then diluted to 1% in MQ-water. Samples were stored at 4  C for one year and analyzed along with the initial water samples (see below). For analysis, samples were bath sonicated for 5 min, and then a 3.5 mL aliquot was removed from each and

diluted with ultrapure MQ-water (ca. 1:14 dilution). Sample dilutions were then ultrasonicated at 60% power for 1 min, and analyzed using ICP-MS. Initial water samples were allowed to settle for a period of one year, and then centrifuged at 1500
g for 10 min. The seawater supernatant was removed with a pipette because salts interfere with elemental analysis, and 14 mL of MQ-water were added to each Falcon tube. The samples were vortexed and then bath sonicated for 5 min. The samples were transferred to individually labeled 50-mL Falcon tubes. The 15-mL Falcon tubes that originally contained each sample were rinsed two times with 14 mL of MQwater and bath sonicated for 5 min after each addition. Each wash was then added to the appropriate 50-mL Falcon tube and the volume was adjusted to 49.5 mL with MQ-water. Finally, 0.5 mL of 18 M H2SO4 and 16 M HNO3 in a 3:7 ratio (volume/volume) was added to acidify each sample for analysis. The visceral mass, mantle, and gills of bivalves were removed by dissection and placed in 20-mL scintillation vials. Tissues were stored at 20  C overnight and then lyophilized for 48 h to remove any remaining moisture. A dry mass was obtained and the organs digested in 2 mL of 18 M H2SO4 and 16 M HNO3 in a 3:7 ratio (volume/volume) for 24 h. Following digestion, the samples were agitated on a vortex and the digest was diluted to a 1% solution using MQ-water. Feces collected at each time interval were centrifuged at 3220
g (5860 rpm) for 5 min, and the supernatant removed. The feces were washed once with 5 mL of MQ-water and the procedure repeated. Washed feces were lyophilized, massed, digested in acid, and prepared as described above for tissue samples. Preliminary experiments indicated that >99% of all TiO2 was present in the gills, visceral mass, and feces, so only these samples were analyzed. Tissues and feces were analyzed using ICP-MS for titanium to examine the concentration of TiO2 present. Concentrations of TiO2 were normalized to the dry mass of the tissue and fecal material. Mean values for the tissues and feces of the blank animals (not exposed to TiO2) were subtracted from those of exposed animals to account for any natural titanium that may have been present in the animals' tissues. Finally, feces-control assays for each particle type were set-up in the same manner as the freely-suspended feeding experiments. Briefly, numerous fecal ribbons were collected from animals that were not exposed to NPs and placed in bottles containing Titan or P25 NPs (1 mg/L; no animals). The bottles were aerated and stirred in the same manner as those used in the exposure experiments, and the feces were incubated for two hours. After incubation, the feces were collected and analyzed as described above. The purpose of these control assays was to examine whether the Titan and P25 NPs suspended in seawater adhered to the surface of the feces during the feeding experiments, and whether the small amount of water that was concomitantly sampled during collection of the feces would produce elevated titanium signatures during ICP-MS analysis. 2.8. Statistical analysis A two-way analysis of variance (ANOVA) test was used to examine the effects of NP type (Titan versus P25) and the form of delivery (incorporated in marine snow versus freely suspended) on the total amount of TiO2 ingested (concentration in visceral mass, gills, feces combined) by each species of bivalve. For the multipleexposure experiments, a one-way ANOVA was used to compare the total amount of each NP type ingested (concentration in visceral mass, gills, pooled feces) by oysters over the 6-hr experimental period. One-way ANOVAs also were used to compare the mass of the Titan and P25 NPs egested in oyster feces at each sampling time. A one-way ANOVA was used to compare the mean weight-

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standardized clearance rate of mussels and oysters exposed to each type of TiO2 NP and the control suspension. Following ANOVA analyses, a Tukey's HSD post-hoc test was applied to examine differences between levels of the independent variables. Prior to statistical analyses, data were assessed for homoscedasticity and normality using an equality-of-variance test and a Kurtosis test, respectively. Data sets that did not meet the underlying assumptions were transformed by means of a natural-log transformation. In all tests, an alpha level of 0.05 was used. 3. Theory Knowledge of the ingestion and depuration rates as well as the extent of bioaccumulation of TiO2 NPs in suspension-feeding bivalves is critical to understanding the actual in vivo exposure in these animals to NPs in their environment and to assess NPassociated risk. By understanding how NPs are encountered by marine animals in the environment, we can begin to assess the cytotoxic and genotoxic effects of these materials. In vivo exposure studies such as this will provide realistic data essential to maintaining healthy populations of commercially and ecologically important shellfish, and ensuring the safety of seafood for human consumption. 4. Results 4.1. Characterization of seawater The physicochemical characteristics of the seawater used in the single- and multiple-exposure experiments were well within the optimal range for mussels and oysters inhabiting Long Island Sound (Table 1). The concentration of chlorophyll-a (a proxy for phytoplankton abundance) and suspended solids was sufficient to stimulate suspension-feeding in experimental bivalves, and provided ample particles for the determination of clearance rates (Ward and Shumway, 2004). 4.2. Feeding experiments 4.2.1. Single-exposures Visible marine snow aggregations formed over the 72-h rolling period. A comparison of the concentrations of titania in initial water (1e2 mg/L) and marine snow samples indicated that the mean percent incorporation of the Titan NPs was approximately 69% (±16.4% standard deviation), whereas the mean percent incorporation of P25 NPs was about 97% (±5.1% standard deviation). These incorporation values are similar to results obtained for the same NP types in prior experiments (see Doyle et al., 2014). After two hours of exposure, the highest fraction of titanium was associated with the visceral mass and feces of both mussels and oysters, regardless of treatment (Table 2). No background concentration of titanium was detected in the tissues of the blank animals (not exposed to TiO2 NPs). Neither particle type (Titan versus P25) nor mode of delivery (marine snow versus freely-suspended) affected the total amount of TiO2 NPs ingested by mussels and oysters. Additionally, no significant interaction effects were found between

49

the two independent variables (two-way ANOVA; p > 0.05; Fig. 1). No significant differences were found in weight-standardized clearance rates of mussels and oysters exposed to Titan or P25 NPs compared to control (unexposed) animals (one-way ANOVA; p > 0.05; Table 3). 4.2.2. Multiple-exposures The majority of both Titan and P25 NPs ingested by oysters during the six-hour exposure period were egested in the feces by the end of this period (Fig. 2; Table 2). That is, the amount of NPs remaining in the gill and visceral mass at the end of the six-hour exposure period was a small fraction of the amount originally ingested by the oysters. Hereafter, we use the term “taken up” to mean TiO2 particles that were ingested or potentially moved across the gill epithelium by phagocytes moving through the hemolymph. Taken up should not be confused with “uptake”, which is typically used to mean bioaccumulation of a material in an organism's tissues. The mass of Titan and P25 NPs egested during the six-hour exposure was significantly greater than the mass egested at the 12, 24, 72, and 120-h time intervals (one-way ANOVA; p < 0.05; Fig. 2). No significant differences were found in the masses of NPs egested during the 12, 24, 72, and 120-h time periods (one-way ANOVA; p > 0.05). By the end of the depuration period (120 h), 100% of all the Titan and P25 NPs taken up by the oysters had been egested (Fig. 2A, B). As was the case in the single-exposure experiments, no background concentration of titanium was detected in the tissues of the blank animals (not exposed to TiO2 NPs). Further, there was no significant difference in the mean weightstandardized clearance rate of oysters exposed to Titan and P25 NPs when compared to control (unexposed) animals (one-way ANOVA; p > 0.05; Table 3). The mean mass of Titan and P25 NPs associated with the feces collected in the feces-control assays was 0.010 mg and 0.001 mg, respectively (n ¼ 2). These masses represent less than 8% and 1% of the amount of Titan and P25 NPs, respectively, measured in the feces of bivalves exposed to the NPs. Therefore during the feeding experiments, the quantity of NPs that adhered to the surface of the feces and in the small amount of water concomitantly collected with the feces had a negligible impact on our conclusions. 5. Discussion During the single-exposure feeding experiments (2 h), mussels and oysters ingested the same amount of Titan and P25 NPs regardless of the form of delivery (incorporated into marine snow or freely-suspended). Only a small fraction of titanium was found in the gill tissue. These results are particularly relevant for an understanding of how bivalves might encounter NPs in the environment. Given the size of marine snow aggregations (10se100s of mm), constituent material would be captured by the gill of bivalves with an efficiency of about 100% (Ward and Shumway, 2004), much higher than that of particles <1 mm in size. Although >69% of both NP types were incorporated into marine snow, no differences in ingestion of freely-suspended NPs and NPs incorporated in marine snow were found. Findings of the current study differ from those of Ward and Kach (2009) who demonstrated that marine snow

Table 1 Parameters of the seawater used in the feeding experiments. Natural seawater was collected in the rocky intertidal at Avery Point in Groton, CT, USA. Data are means ± standard deviation; n ¼ 6 (single-exposure), n ¼ 15 (multiple-exposure); ranges given in parentheses. Parameter

Single-exposure

pH Salinity (ppt) Chlorophyll (mg/L) Total Suspended Solids (mg/L)

7.53 28.9 2.22 17.1

± ± ± ±

0.04 0.19 0.86 4.73

(7.47e7.56) (28.7e29.1) (1.33e3.35) (11.4e25.6)

Multiple-exposure 8.11 29.3 0.63 13.8

± ± ± ±

0.17 0.12 0.49 6.53

(7.97e8.45) (29.1e29.5) (0.05e1.59) (6.8e26.8)

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Table 2 Mass of TiO2 measured in the tissues and feces of bivalves in different treatments after exposure to NPs. For multiple-exposure experiments, values for the feces represent the mass of TiO2 egested during the experiments by oysters sacrificed at the end of the exposure period, or the mass of TiO2 egested over the depuration period (post-exposure) by oysters sacrificed after 12, 24, 72 and 120 h. Total values represent the sum of TiO2 measured in tissues, feces collected during the exposure period, and feces collected over the depuration period (multiple-exposure experiments only).

Gill (mg)

Visceral Mass (mg)

Feces (mg)

Total (mg)

0.01 ± 0.01 0.01 ± 0.01

0.17 ± 0.05 0.17 ± 0.06

0.18 ± 0.05 0.18 ± 0.06

0.01 ± 0.00 0.01 ± 0.00

0.12 ± 0.02 0.21 ± 0.04

0.13 ± 0.02 0.22 ± 0.04

0.01 ± 0.00 0.03 ± 0.02

0.11 ± 0.03 0.12 ± 0.03

0.12 ± 0.03 0.15 ± 0.04

0.10 ± 0.03 0.17 ± 0.03

0.10 ± 0.03 0.18 ± 0.03

0.01 ± 0.01 0.02 ± 0.01

0.26 ± 0.09 0.38 ± 0.12

0.27 ± 0.09 0.40 ± 0.12

0.05 ± 0.03 0.02 ± 0.01

0.45 ± 0.14 0.56 ± 0.13

0.02 ± 0.01 0.04 ± 0.02

0.49 ± 0.13 0.42 ± 0.06

0.03 ± 0.02 0.03 ± 0.01

0.37 ± 0.07 0.35 ± 0.06

significantly enhances ingestion of 100-nm polystyrene particles by the mussel, M. edulis, and oyster, C. virginica. The different outcomes of the two studies could be a result of the formation of homoagglomerates. Several studies have shown that TiO2 NPs will agglomerate when placed in high ionic solutions such as seawater €a € et al., 2011), even after sonication (Porter et al., 2008). As (Sillanpa demonstrated in Doyle et al. (2014), agglomerates ranging in size from 0.5 to 5 um form over a period of minutes to hours. Additionally, in the current experiment, as in the natural environment, production of TEP (Heinonen et al., 2007) by bivalves would further

Fig. 1. Total amount of Titan and P25 NPs ingested by mussels and oysters during the single-exposure experiments. “Snow” indicates that the NPs were delivered to the bivalves incorporated into marine snow, whereas “free” signifies that the NPs were delivered to the animals as freely-suspended particles (no marine snow). No significant differences were found between the amount of NPs ingested by bivalves in the marine snow and freely-suspended treatments. Data are means ± standard error (n ¼ 5e7).

enhance agglomeration resulting in formation of TiO2 particles that could be captured at an efficiency similar to that of the marine snow (Li et al. 2008). The different physicochemical surface properties of polystyrene and the shorter feeding time (45 min) used by Ward and Kach (2009) might have resulted in the formation of fewer agglomerations that could be efficiently captured, and a difference in the amount of 100-nm polystyrene particles ingested between the two forms of delivery (incorporated into marine snow versus freely-suspended). In the multiple-exposure experiments (6 h), measurable concentrations of Titan and P25 NPs were found in the gills and visceral masses of oysters only immediately following exposure; tissues sampled at 12 through 120 h showed no detectable concentrations of TiO2 NPs. The feces data support this finding, with the bulk of TiO2 NPs egested after 12 h and only trace amounts present thereafter. These results demonstrate that TiO2 NPs are removed quickly from the gill after capture, and depurated rapidly from the visceral mass after ingestion (<12 h). Past work has shown that bivalves transport particles from the gill on the order of minutes (Milke and Ward, 2003). Once in the stomach, heavier, denser particles are moved to the intestine by the ciliary selection tracts, and have shorter gut residence times compared to lighter, less dense particles (Reid, 1965; Brillant and MacDonald, 2000). These findings suggest that TiO2 NPs do not bioaccumulate in the tissues of oysters after six hours of exposure to a concentration of 1 mg/L. Mass-balance calculations indicated that the amount of Titan and P25 NPs taken up (ingested or in the gill) by the bivalves, as a fraction of the amount delivered (1 mg/L), was approximately 10%e 25% for the single-exposure, and 30%e60% for multiple-exposure experiments. Comparison of the standardized clearance rates of bivalves exposed to Titan and P25 NPs, however, indicated that they were filtering water at a rate similar to that of bivalves not exposed to NPs. Additionally, there was no significant relationship between the amount of NPs taken up by the bivalves and their clearance

J.J. Doyle et al. / Marine Environmental Research 110 (2015) 45e52

51

Table 3 Clearance rate data for bivalves used in the single- and multiple-exposure experiments. For both mussels and oysters, no significant differences were found between the clearance rates of bivalves exposed to Titan and P25 NPs, and those not exposed to NPs (control). Data are means ± standard error; n ¼ 3e13 (single-exposure), n ¼ 9e31 (multiple-exposure). Experiment

Single-exposure Mussel Oyster Multiple-exposure Oyster

Treatment Control (mL/min/0.5 g)

Titan (mL/min/0.5 g)

P25 (mL/min/0.5 g)

9.91 ± 3.93 9.24 ± 0.84

19.2 ± 2.08 10.4 ± 1.44

16.7 ± 1.67 11.3 ± 1.76

13.6 ± 1.84

12.1 ± 0.89

9.26 ± 0.96

rates (linear regression, p > 0.05; data not shown). These results suggest that although mussels and oysters cleared NPs from the water, most of the captured particles were rejected in pseudofeces. Bivalves have the ability to discriminate between particles, and can selectively reject inorganic and refractory material based on physicochemical surface properties (Ward and Shumway, 2004; PalesEspinosa et al., 2010; Rosa et al., 2013). Therefore, it is not surprising that both Titan and P25 agglomerations were rejected in pseudofeces. Pseudofeces tend to stay suspended in seawater, and collecting it would have potentially disturbed the animals causing interruptions in feeding. For that reason, pseudofeces were not collected in this study. Previous studies have shown that several different types of NPs, including TiO2, can be captured and ingested by bivalves (Koehler et al., 2008; Tedesco et al., 2010; Hanna et al., 2013; Canesi et al., 2014), however, the efficiency and rate of this capture has not been determined. Bivalves and other filter-feeders can isolate

themselves from the external environment by sealing shells or other structures. During these periods, the animals are not feeding and are not exposed to added materials. Additionally, size and physiological status affect the rate at which individuals pump water across their feeding structures, requiring that standardized clearance rates be determined in order to compare ingestion and uptake between replicate animals. We suggest that future studies on exposure and effects of NPs on filter-feeding organisms should consider the following: 1) agglomeration potential in the aqueous media in which the experiments are being conducted to estimate the effective size of the NP agglomerates to which the animals are exposed; 2) the efficiency and rate at which the NP agglomerates are captured; and 3) the rates at which the nanomaterial is rejected in pseudofeces and egested in feces. All of these processes are important considerations when determining the actual amount of material ingested and potentially accumulated in the animals, and will ultimately determine tissue exposure. 6. Conclusions Results of this study demonstrate that mussels and oysters can capture and ingest both Titan and P25 NPs at rates which are unaffected by the form of delivery (marine snow versus freelysuspended). For both types of TiO2 NPs, the amount ingested was similar and ranged from 10% to 60% of the quantity to which the bivalves were exposed, depending on duration of exposure. Nearly 100% of the ingested NPs were depurated over 12 h, post-exposure. There was no evidence of bioaccumulation of the two types of NPs in the gill or the visceral mass. Our findings are particularly important to managers who monitor shellfish beds, and who are concerned with the accumulation of anthropogenic materials by commercially important bivalves. Our studies suggest that for these two bivalve species, short-term exposure to TiO2 NPs is not a cause for concern, however, the potential for bioaccumulation still exists following low, chronic exposures (see Barmo et al., 2013). Acknowledgements We would like to thank Bridget Holohan, Prentiss Balcom, and Meghan Danley for their assistance with various aspects of this study. We also thank James Markow (Noank Shellfish Cooperative) and Gregg Rivara (Cornell Cooperative Extension) for providing us with oysters, and David Holbrook (National Institute of Standards and Technology) for supplying us with the P25 NPs. This research was supported by grants from Connecticut Sea Grant (R/P-1, NA10OAR4170095) and the National Science Foundation's, Directorate for Engineering (CBET-1336358) to J. Evan Ward and Robert Mason. We appreciate this support.

Fig. 2. The mass and percentage of (A) Titan NPs, and (B) P25 NPs egested in the feces of oysters over time in the multiple-exposure feeding experiments. “E” represents the end of the 6-h exposure period. Symbols designated by different letters indicate a significant difference in the mass of NPs egested in the feces. Data are means ± standard error (n ¼ 6e7).

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