Ocean acidification increases the accumulation of titanium dioxide nanoparticles (nTiO2) in edible bivalve mollusks and poses a potential threat to seafood safety

Large amounts of anthropogenic CO2 in the atmosphere are taken up by the ocean, which leads to ‘ocean acidification’ (OA). In addition, the increasing application of nanoparticles inevitably leads to their increased release into the aquatic environment. However, the impact of OA on the bioaccumulation of nanoparticles in marine organisms still remains unknown. This study investigated the effects of OA on the bioaccumulation of a model nanoparticle, titanium dioxide nanoparticles (nTiO2), in three edible bivalves. All species tested accumulated significantly greater amount of nTiO2 in pCO2-acidified seawater. Furthermore, the potential health threats of realistic nTiO2 quantities accumulated in bivalves under future OA scenarios were evaluated with a mouse assay, which revealed evident organ edema and alterations in hematologic indices and blood chemistry values under future OA scenario (pH at 7.4). Overall, this study suggests that OA would enhance the accumulation of nTiO2 in edible bivalves and may therefore increase the health risk for seafood consumers.

Effects of ocean acidification on the accumulation of nTiO 2 in various tissues of three edible bivalve species. The nTiO 2 contents accumulated in the gills, foot and mantles of blood clam (Tegillarca granosa), hard clam (Meretrix meretrix), and venus clam (Cyclina sinensis) after a 21-day 100 μg/L nTiO 2 exposure at pH 7.4 and 7.8 were about 1.34 and 1.16 times greater than those raised in the ambient pH of 8.1, respectively ( Fig. 1, p < 0.05). This finding suggests that ocean acidification increases the accumulation of nTiO 2 in the bivalve species (Table S1).
The impacts of ocean acidification on seafood safety: mouse assays. Upon feeding with 2.5 mg/kg nTiO 2 per day, an equivalent dose simulating the intake of nTiO 2 through consuming contaminated bivalves raised under pH 7.4, the numbers of white blood cell (WBC) and lymphocytes (Lym) in mice were significantly greater than those under ambient pH of 8.1 (Table 2). Similarly, the levels of alanine transaminase (ALT), alkaline phosphatase (ALP), and creatinine (Crea), as well as the ALT/aspartate transaminase (AST) ratio were significantly induced upon feeding with an equivalent amount of nTiO 2 accumulated in the bivalves raised under ocean acidification scenarios (Table 3). Evident liver edema indicated by sinusoidal expansion (arrows in Fig. 2a) and inflammatory cell infiltration (or hydropic degeneration) (circles in Fig. 2a) was detected in mice fed with 2.5 mg/kg nTiO 2 daily (Fig. 2a). In addition, inflammatory cell infiltration and slight swelling of renal tubular epithelial cells (circles in Fig. 2b) were also observed in the kidneys of these mice.  Table 1. Physicochemical properties of titanium dioxide nanoparticles (mean ± SE). Mean values that do not share the same superscript were significantly different at p < 0.05. * The particle hydrodynamic diameters and zeta potential were tested at a dose of 0.1 mg L −1 nTiO 2 . www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Seafood, including edible bivalves, contributes almost 20% of animal proteins for over three billion people in the world 39 . However, the presence of contaminants in seafood poses threats to consumers' health and therefore draws increasing attention from both the scientific community and regulatory authorities, such as the Food and Agriculture Organization (FAO) 39 , the Food and Drug Administration (FDA) 40 and the European Food Safety Authority (EFSA) 41 . The fast growth of the production and application of nanoparticles in a variety of products    Renal tubular epithelial cells were slightly swollen (circles) in the 2.5 mg/kg BW group. No obvious pathological changes were found in the heart, spleen and lung among the different treatment groups.
www.nature.com/scientificreports www.nature.com/scientificreports/ will inevitably add onto the release of nanoparticles into the environment, which can accumulate in marine organisms, including commercial seafood species, posing potential threats to human health 22,24,42 . Although some evidence suggests that seafood safety may worsen under future climate scenarios 7,43 , the plausible effects of future ocean acidification scenarios on the safety of seafood with respect to nanoparticle contaminations have been largely overlooked.
The present study showed that future ocean acidification scenarios will lead to an increased accumulation of nTiO 2 in marine edible bivalves, which may be due to a synergic effect of pCO 2 -driven ocean acidification on the uptake of nTiO 2 from the environment and the exclusion of nTiO 2 out of the body of bivalves. First, ocean acidification may induce nTiO 2 accumulation by changing the aggregate size of nTiO 2 in seawater. In aqueous solution, nanoparticles tend to aggregate forming large particles and the extent of aggregation is dependent on various factors including the surface charge of nanoparticle and the pH value of medium [29][30][31] . It has been shown that marine bivalves capture and ingest larger nanoparticle aggregates more efficiently than those in suspension 38,44 . For example, particles larger than 4 μm in diameter can be easily ingested by bivalve species through the process of filter feeding. However, the nanoparticle uptake efficiency of bivalves through this filtration process decreases asymptotically with the decreasing particle size of the nanoparticle 45 . According to previous studies, the pH at the point of zero charge (pH zpc ) of nanoparticles has important implications for their aggregation, as electrostatic repulsion between nanoparticles of similar potential decreases when the solution pH approaches the pH zpc 29,31,35 .
Our data showed that the zeta potential of nTiO 2 approach the critical pH zpc value of 0 in pCO 2 -acidified seawater, which subsequently reduced the electrostatic repulsion between nTiO 2 favoring the formation of larger aggregates. Therefore, enhanced nTiO 2 accumulation under future ocean acidification scenarios could be partially due to easier ingestion of bigger aggregates by marine bivalves.
Second, the increase of nTiO 2 accumulation in bivalves could also be attributed to an increase in nTiO 2 uptake through other pathways. Theoretically, all substances in the marine environment could diffuse directly into the body via damaged tissues 46 . As increased pCO 2 can bring about severe tissue damage to marine organisms 47,48 , ocean acidification may facilitate the entry of nanoparticles into marine organisms by causing tissue lesion. In addition, nanoparticles can also enter the extrapallial fluid between the soft tissues and shell of marine bivalves through incorporation into the nacreous layer 49 . As mantle and labial palps have been confirmed as the target organs for the internalization of nanoparticles in marine invertebrates 50 , nanoparticles deposited on the shell surfaces may be transported into the extrapallial fluid and subsequently accumulate in the body. Since ocean acidification can result in shell dissolution and nanoparticle deposition acceleration 5,51 , increases in shell permeability and nanoparticle deposition on the shell surface induced by pCO 2 acidification would facilitate the uptake of nTiO 2 through the shell-extrapallial fluid pathway.
Third, since some of the nanoparticles ingested will be excreted from the body 52 , a hampered nTiO 2 exclusion induced by ocean acidification may also account for the increased nTiO 2 accumulation. The exclusion of most exogenous substances is an energy-consuming process, and it has been shown that ocean acidification may constrain energy availability for toxicant metabolism in marine invertebrates 7,53 . Therefore, the exclusion of nanoparticle could be reduced to some extent in elevated pCO 2 conditions, leading to an increase in nanoparticle accumulation.
Previous investigations on the impacts of nanoparticles on food safety, including nTiO 2 , were generally conducted with excessive doses 36 . In addition, although it has been suggested that nanoparticles can accumulate in marine organisms such as gastropods 54 , amphipods 20 , bivalves 42 and fish 24 , the health consequences of oral exposure to nanoparticle-contaminated seafood at environmentally realistic concentrations remains unknown. Our data showed that a 30-day oral exposure to nTiO 2 at the equivalent doses of seafood consumption under future ocean acidification scenarios can threaten health in terms of organ lesion and alterations in hematologic indices and blood chemistry values. For instance, increased WBC and lymphocyte counts indicated an inflammatory reaction upon feeding with nTiO 2 at the dose equivalent to oral intake via consuming contaminated seafood under future ocean acidification scenarios 55 . In addition, evident injuries were detected in liver since the organ is the main target organ for nanoparticles via oral exposure [56][57][58][59] . Similar kidney and hepatic injuries in mice after gastrointestinal nTiO 2 exposure has been observed previously, and it was suggested that these injuries are associated with alterations in inflammatory cytokine expression and reduction in detoxification of nTiO 2 60 . According to our results obtained, ocean acidification would induce the bioaccumulation of nTiO 2 in marine edible bivalves and cause liver and kidney injuries in mice at the equivalent dose of seafood consumption, indicating an increased health risk by consuming seafood under future ocean acidification scenarios. We speculate that this health risk could be underestimated, given the relatively short accumulation duration (30 days) examined in the present study. In addition, both ocean acidification and nanoparticles have been reported to facilitate the accumulation of other toxic pollutants through complex interactions 9,61 , further increasing seafood safety risk. Though it is generally accepted that the concentrations of nanoparticles in the environment will grow steadily and reach as high as mg/L level in water system 33 , little is known about its environmental concentrations. Future studies should consider long-term effects of nanoparticles and pH in the context of future ocean acidification as well as populations in different geographic locations.

Methods
This study was carried out in three steps namely collection of bivalves from Yueqing Bay  62 . Prior to experiments, bivalves were acclimatized in sand-filtered seawater (pH 8.10 ± 0.03, salinity 21.3 ± 0.6‰) at an ambient water temperature 25.3 ± 0.5 °C with constant aeration and a natural light/dark cycle for at least 7 days. Bivalves were fed daily with microalgae (Tetraselmis chuii) at the satiation feed rate during the acclimation, and selected bivalves were cultured overnight in filtered seawater without feeding before the exposure experiment. pCo 2 -driven seawater acidification. Sand-filtered seawater from the clam-sampling site, Yueqing Bay, was used throughout the experiment. To simulate the present and near-future projections of the Intergovernmental Panel on Climate Change (IPCC), one ambient present pH (pH at 8.1) and two lower pH levels (pH 7.8 and 7.4, representing ocean acidification scenarios in 2100 and 2300, respectively) were used in the present study 2,3 . Seawater at different pH values was achieved and maintained by continuous aeration with CO 2 -air mixture with corresponding predicted pCO 2 , which was obtained by mixing CO 2 -free air and pure CO 2 gas at known flow rates using flow controllers (Fig. S3) 7,9 . Throughout the experiment, pH calibrated with standard US National Bureau of Standards buffers (pH NBS ), salinity, temperature, total alkalinity (TA) and carbonate system parameters including dissolved inorganic carbon (DIC), aragonite saturation state (Ωara) and calcite saturation state (Ωcal) were monitored daily. Seawater pH was measured with a Sartorius PB-10 pH meter (Sartorius, Germany) with an accuracy of ±0.01. Salinity was measured using a conductivity meter (Multi 3410 WTW, Germany) with an accuracy of ±0.5% measurements. Total alkalinity measurements were performed by potentiometric titration with an automatic titrator system (SMTitrino 702, Metrohm). Throughout the experiment, seawater pH NBS was measured and adjusted by pH/ORP controllers (PC-2110, House, China; pH fluctuations were controlled within 0.01 units) to maintain the desired pH. The carbonate system parameters were calculated from the measured pH, salinity, temperature and TA values using the open-source program CO2SYS, as described previously 10 . The pH fluctuations were within 0.01 units over the 21 days exposure in all tanks. Seawater carbonate chemistry parameters in each tank measured and calculated daily for all treatments during the exposure were summarized in Table 4.

Characterization of nanoparticles.
In this study, nTiO 2 were purchased from Shanghai Klamar Reagent Co. Ltd, China, and the size and shape of the nTiO 2 particles were determined using transmission electron microscopy (TEM, JEM-1230, JEOL, Tokyo, Japan). Crystal structure of the particles was identified using X-ray powder diffractometry (XRD, Rigaku D/MAX 2550/PC, Tokyo, Japan), and surface area was measured through Brunauer-Emmett-Teller (BET) adsorption measurements (Micromeritic TriStarII 3020, Micrometritics Instrument Corp., Norcross, GA). The majority of nTiO 2 used in this study was anatase crystals with irregular shapes and a surface area of 60.65 m 2 /g ( Fig. S4 and Table 1). To minimize weighing errors and ensure concentration accuracy, a stock solution of 1 g/L nTiO 2 was prepared daily by dispersing the nTiO 2 in ultrapure water followed by sonication for 15 mins 57,63,64 . Test solutions of nTiO 2 were prepared immediately prior to use by diluting the stock solution with 0.1-μm membrane-filtered seawater (pH 8.10, salinity 21.3‰). Particle hydrodynamic diameter and zeta potential of nTiO 2 in seawater at different pH values (ambient pH 8.1, pH 7.8 and 7.4) were tested with the Zetasizer Nano ZS90 (Malvern Instruments Ltd, Malvern, UK).

Bioaccumulation experiment.
Since nTiO 2 is mostly introduced into the environment via sewage discharge and it remains about 100 μg/L nTiO 2 in the waste water effluents after treatment 33,36 , seawater in polluted areas could be contaminated at equivalent magnitudes, especially when the steady increase of environmental nTiO 2 is taken into account. Therefore, 100 μg/L was chosen to simulate the environmental concentration of nTiO 2 in the polluted areas in this study. After one week of acclimation, bivalves (90 individuals for each species) were randomly assigned into plastic tanks, with a total seawater volume of 30 L containing approximately 100 μg/L nTiO 2 and maintained under the desired pH levels. In total, 27 experimental tanks and 270 bivalves (10 individuals per tank × 3 replicate tanks × 3 pH levels × 3 species) were used in the present study. Bivalves were fed with T. chuii twice a day during the experiment and the seawater was changed daily with corresponding pre-acidified seawater containing the desired concentration of nTiO 2 . No mortality was observed throughout the experimental period.  www.nature.com/scientificreports www.nature.com/scientificreports/ Following that of Johnston 65 , Hooper 66 , and Gaiser 67 , in the present study an exposure time of 21 days was adopted to avoid the effect of stress syndrome. After 21-day exposure to 100 μg/L nTiO 2 at different pH levels, five live individuals of each species were randomly taken from each tank and purged in sand-filtered seawater overnight. After rinsing with ultrapure water, the bivalve individuals were dissected on ice and the gill, mantle and foot muscle of each individual were peeled off and stored separately at −20 °C for TiO 2 residue analysis. TiO 2 concentrations in the various tissues were calculated and expressed in mg/kg dry weight for each individual. Similarly, the entire soft body of bivalves was peeled off to determine TiO 2 concentration (expressed in mg/kg wet weight), which was later used to calculate the equivalent oral exposure dose of nTiO 2 used in the mice toxicology assays.

Content analysis of nTiO 2 .
To obtain working concentration of nTiO 2 in the seawater for each experimental group (pH at 8.1, 7.8, and 7.4, respectively), 1 mL of seawater samples (three replicates for each experimental group) were taken after seawater renewal. In addition, approximately 0.1-0.3 g of each tissue or the entire soft body were used to determine the amount of accumulated nTiO 2 . The contents of TiO 2 in both tissue and seawater samples were determined following that of the National Standard of China (GB5009.246-2016) 62 . In brief, the water and tissue samples were digested in ultrapure nitric acid overnight. After adding 0.5 mL H 2 O 2 , mixtures were heated with an electric heating plate until samples were completely digested, and the remaining nitric acid was removed until colorless and clear solutions were achieved. The solutions were diluted to 3 mL with 2% nitric acid and used for titanium concentration measurement using inductively coupled plasma atomic emission spectrometry (ICP-MS, PE NexION 300X, USA). Indium (20 ng/ml) was taken as the internal standard and the detection limit of titanium was 0.074 ng/ml. The background and working concentrations of nTiO 2 in seawater during the 21-day experiment are listed in Table 5.
Mice toxicology assays. Healthy Kunming (KM) male mice (8 weeks old) were purchased from the Experimental Animals Center of Zhejiang University and housed in plastic laboratory animal cages in room conditions (20 ± 2 °C, 60 ± 10% relative humidity, under a 12 h light/dark cycle) for a week. During the acclimation, a commercial pellet diet and deionized water were available ad libitum. After 7 days of acclimation, 15 adult mice were equally divided into 3 treatment groups (n = 5): the pH 8.1 exposed group, the pH 7.8 exposed group, and the pH 7.4 exposed group. All experiments were approved by the Animal Care Committee of Zhejiang University and all methods were performed in accordance with the Guidelines for the Care and Use of Animals for Research and Teaching at Zhejiang University.
The obtained mean values of the amount of nTiO 2 accumulated in M. meretrix, the lowest of the three bivalve species, were used to calculate oral exposure doses. According to previous dietary surveys and guidelines 68,69 , 150 g/person, equivalent to approximately 3 g/body weight (BW)/day, was used as the amount of daily intake of seafood. Based on the nTiO 2 concentrations detected in bivalve M. meretrix in the present study (5.05, 6.77, and 8.30 mg/kg at pH 8.1, 7.8 and 7.4, respectively), the daily intake of dietary nTiO 2 through consuming nTiO 2 contaminated seafood raised under pH 8.1, 7.8, and 7.4 was estimated to be 0.015, 0.020, and 0.025 mg/kg, respectively. Taking the interspecies extrapolation into consideration 70 , a 100-fold dose of the human exposure was used for mice assays (1.5, 2.0, and 2.5 mg/kg BW, respectively). After 15-min sonication in ultrapure water, nTiO 2 suspensions were given to mice by gavage once a day for 30 consecutive days. During the 30-day experiment, body weight was recorded every 5 days and any symptom or mortality was observed and recorded daily. After 30 days of nTiO 2 exposure, all mice were weighed and sacrificed after anesthetization. Blood samples were collected from the femoral artery in the groin area. Serum was obtained by centrifuging blood at 3000 rpm for 15 min. Tissues and organs such as heart, kidney, spleen, lung and liver were excised and weighed. Tissue samples for histopathologic examination were fixed in 10% neutral buffered formalin.
After weighing, the coefficients of various tissues to the body weight were calculated as the ratio of tissues (wet weight, mg) to body weight (g). Blood component parameters were determined with an auto hematology analyzer (BC-2800Vet, Shenzhen, China). Liver function was evaluated based on the serum levels of ALT, AST and ALP. Nephrotoxicity was determined by blood urea nitrogen (BUN) and Crea, which were determined using an automated biochemical analyzer (Hitachi 7170 A, Tokyo, Japan). All histopathological observations were performed according to standard laboratory procedures. Tissues were embedded in paraffin, sliced into 5-μm thicknesses and placed onto glass slides. After hematoxylin-eosin (HE) staining, the slides were examined and images were taken using an optical microscope (Nikon Eclipse Ci-L, Tokyo, Japan). The identity and analysis of the pathology slides were blind to the pathologist.
Statistical analyses. Differences in hydrodynamic diameter and zeta potential of nTiO 2 in seawater at different pH levels (pH 8.1, 7.8, and 7.4) were compared by one-way analyses of variance (One-way ANOVAs) followed by post-hoc Tukey tests using OriginPro 9.0.
The nTiO 2 concentrations accumulated in individuals were assessed using a linear mixed effects model with treatment pH as a fixed factor and the treatment tank as a random factor. In total, nine linear mixed effects models were performed for each tissue and species investigated (3 species × 3 tissues) using 'R' statistical package lme4 (R Development Core Team, 2012).  www.nature.com/scientificreports www.nature.com/scientificreports/ Differences in hematologic indices and blood chemistry values of the mice after oral exposure to different nTiO 2 doses were evaluated using one-way ANOVAs followed by post-hoc Tukey tests using OriginPro 9.0. Percentage data (e.g. percentages of monocytes, granulocytes and lymphocytes) were arcsine-square root transformed prior to analysis to meet the assumption of a normal distribution 71 .
For all analyses, Levene's test and Shapiro-Wilk's test were performed using OriginPro 9.0 to verify homogeneity of variance and normality, respectively. All data were presented as mean ± standard error (SE) and a p-value at p < 0.05 was taken as statistically significant.