Low Bioavailability of Silver Nanoparticles Presents Trophic Toxicity to

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Low Bioavailability of Silver Nanoparticles Presents Trophic Toxicity to Marine Medaka (Oryzias melastigma) Jian Wang and Wen-Xiong Wang* Division of Life Science, State Key Laboratory of Marine Pollution, Hong Kong University of Science and Technology (HKUST), Clearwater Bay, Kowloon, Hong Kong, China S Supporting Information *

ABSTRACT: Concerns for the potential risks of silver nanoparticles (AgNPs) to aquatic organisms have increased. The present study investigated the trophic transfer of AgNPs from brine shrimp (Artemia salina) nauplii to marine medaka. We found that the aggregated AgNPs (20 and 80 nm) and well dispersed 80-nm AgNPs (stabilized by 20 μM Tween 20) could be readily accumulated by brine shrimp, while far less well-dispersed 20-nm AgNPs were accumulated. The assimilation efficiency (AE) of AgNPs in medaka fed AgNPs-contaminated brine shrimp was low (99% and Tween 20, PREMIUM, 0.05, one-way ANOVA).

body wt., twice a day) was conducted on the second day. Because nearly 90% of the accumulated Ag was excreted by medaka within 16 h and further depuration (36 h, data not shown) did not show significant difference (p > 0.05, one-way ANOVA) from that at 26 h, the AE was determined by the retained percentage of Ag in medaka at 26 h. Another experiment was conducted to quantify the AEs of AgNPs (20 nm) at different food Ag concentrations. Ag-contaminated food was prepared by exposing brine shrimp to radioactive 20-nm AgNPs (i.e., 250, 500, and 1000 μg/L) following the same procedures as described above. Chronic Dietary Exposure of AgNPs to Marine Medaka. A chronic (28 day) dietary exposure of AgNPs (20 nm) was conducted to investigate whether the long-term dietary exposure could cause any toxic effect in medaka. Brine shrimp were prepared similarly to the AE experiment by exposing them to 200 and 1000 μg/L AgNPs (20 nm) for 4 h. A control food (Ag-free brine shrimp) and food containing AgNO3 (by exposing brine shrimp to 1000 μg/L AgNO3) were prepared for comparison. The final total food Ag concentrations were determined by AAS and are listed in Table 1. For each treatment, a total of 30 adult medaka (2-month-old) were acclimated in 20 L of natural seawater (salinity 30 psu, pH 7.89 ± 0.07, temperature 25 ± 0.5 °C) with gentle aeration (DO > 6.5 mg/L) and fed with control food for 1 week at 5% body wt./d (twice a day). Feces were removed and half of the seawater was renewed every day. After acclimation, the Agspiked food was used instead of the control food. During the chronic exposure period, 5 fish from each treatment were removed every 7 days for total body weight and length measurement. The fish were immediately transferred and stored at −80 °C for later enzymatic activities assay. On day 28, 5 fish were depurated for 12 h and dissected (only intestine was separated) for Ag content assay. Fish growth gross parameters were calculated as below.

into the fresh medium overnight allowing equilibrium with the medium before the experiment, while AgNPs were introduced into the medium immediately before the experiment. Because AgNPs rapidly aggregated in seawater, a parallel experiment was set up to investigate the bioaccumulation of stabilized AgNPs by adding Tween 20 (20 μM) to all treatments. The impact of Tween 20 (20 μM) on the bioaccumulation of AgNO3 was also studied. After incubation at 25 °C for 24 h (light/dark cycle = 14 h:10 h), the hydrodynamic size of AgNPs was measured and brine shrimp were collected. The animals were dried, weighed, and digested before the measurement of total Ag content using AAS. Bioconcetration factors (BCFs, L/g) of different Ag species in brine shrimp were calculated by dividing total body Ag concentration (μg/g) by the normalized (see below for normalization) water Ag concentration (μg/L). A short-term (4 h) uptake experiment was also conducted following the same procedures. Brine shrimp were sampled at 1, 2, 3, and 4 h for total Ag content analysis. The influx rate (ng/ g/h) was calculated as the slope of the linear regression between the newly accumulated Ag in brine shrimp and the exposure time. For all the uptake experiments, no mortality of brine shrimp was observed, and water samples (0.5 mL, before and after the exposure) were collected to measure the actual Ag concentration in the medium. Dissolution of AgNPs (at 1 mg/ L) in each treatment was also quantified at 0, 4, and 24 h, using 3-kDa centrifugal filter units (pore size around 1 nm, Millipore) following the same method as the previous study.27 In general, change of Ag concentration during the short-term uptake experiment was within 20%, while there was a sharp decrease of Ag concentration during the 24-h bioaccumulation experiment (especially in the groups treated with AgNPs). The average of the initial and final water Ag concentrations was used in the calculation. Assimilation Efficiency of AgNPs in Medaka. Assimilation efficiency of AgNPs in marine medaka (6-month-old adult, 0.325 ± 0.02 g, 27.0 ± 0.7 mm) was determined using our well established pulse-chase feeding methodology.28 The brine shrimp was first exposed to 1 mg/L of 110mAgNO3 or radioactive AgNPs (20 and 80 nm) in the presence and absence of Tween 20 (20 μM) for 4 h. After the radiolabeling, the brine shrimp was collected and fed to medaka in 200 mL of seawater. The feeding duration lasted for 15 min, which was enough for the fish to finish the given diet. The initial ingested total Ag was determined right after the pulse-chase feeding, and fish were allowed to depurate in the clean seawater. The radioactivity of the fish was assayed at different time intervals (i.e., 0, 3, 9, 15− 18, and 26 h). After each measurement, the seawater was renewed to reduce the potential uptake of radioactive Ag from water (e.g., 110mAg released from feces). Routine feeding (5%

Condition factor (b): b = log(Wwet /a)/log Lfish

where Wwet (mg) is the wet weight of the fish and Lfish (mm) is the length of fish total body length. For certain species, a was a constant and condition factor b indicates the fitness of the fish.29 The constant a and the reference condition factor b were obtained by weighing and measuring regularly kept healthy medaka of different ages (n = 31) and an exponential regression was then applied (Supporting Information (SI) Figure S1). The obtained constant a (i.e., 0.0265) was used for all the condition factor calculations and the reference condition factor b (i.e., 2.88) was used to evaluate the fitness of the fish. 8154

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Figure 1. Characterization of AgNPs. Transmission electronic microscopy (TEM) images of commercial 20-nm AgNPs (A, scale bar 20 nm), radiolabeled 20-nm AgNPs (B, scale bar 20 nm), commercial 80-nm AgNPs (C, scale bar 100 nm), and radiolabeled 80-nm AgNPs (D, scale bar 100 nm). The images for radiolabeled AgNPs were obtained by using stable Ag+ instead of 110mAg+ for reaction and a high resolution of single AgNPs is also shown in the small window on the top right corner. Energy-dispersive X-ray spectroscopy (EDX) image of randomly selected 80-nm AgNPs after radiolabeling (E). Size distribution of the above-mentioned four kinds of AgNPs (n = 200), “M” in the legend refers to radiolabeled nanoparticle (F).

free crude enzyme extraction (n = 3), indicating the negligible impacts of Ag during bioassay. Radioactivity Measurement and Statistics Analysis. Radioactivity of 110mAg was measured using a Wallac 1480 NaI (T1) gamma counter (Wallac, Turku, Finland). The counting time was 2 min to yield propagated counting errors 0.05, oneway ANOVA) in size was found after radiolabeling. The EDX results for randomly selected AgNPs also showed an unchanged

where Cfish (μg/g, dry wt.) is the Ag concentration in fish after 28-day chronic exposure and Cbrine shrimp (μg/g, dry wt.) is the Ag concentration in prepared brine shrimp food. For enzymatic activity measurements, fish in each treatment were homogenized in Tris-HCl buffer (10 mM) and used for subsequent total protein, superoxide dismutase (SOD), catalase (CAT), and Na+/K+-ATPase activities assays (SI). To avoid artifacts, the Ag content in crude enzyme extractions from each Ag treatment at week 4 was determined and found to be below the detection limit of AAS (0.05 μg/L). The activities of SOD, CAT, and Na+/K+-ATPase were further assayed in the presence of AgNO3 (0.05 μg/L), using silver-free crude enzyme extraction. No significant difference (p > 0.05, one-way ANOVA) of enzymatic activities was detected from the silver8155

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chemical composition. Negligible change was also found in the hydrodynamic size of AgNPs, with mean sizes (before and after radiolabeling, respectively) of 27.6 ± 5.5 and 29.4 ± 3.3 nm for 20-nm AgNPs, and 93 ± 6.3 and 91 ± 2.3 nm for 80-nm AgNPs. However, a slight increase of zeta-potential of AgNPs (before and after radiolabeling) occurred for both 20-nm AgNPs (from −38.2 ± 4.2 to −32.1 ± 3.2 mV) and 80-nm AgNPs (from −32.1 ± 1.2 to −28.5 ± 2.5 mV). In the bioaccumulation experiment, aggregation of citrate-coated AgNPs (20 and 80 nm) proceeded immediately after mixing with seawater. Visually, there was a sharp color change from bright yellow to dark gray for 20-nm AgNPs. The mean hydrodynamic sizes were 1652.7 (polydispersity index 2.43) and 3212 nm (polydispersity index 1.42) for 20- and 80-nm AgNPs, respectively, and the mean zeta-potentials were −10.04 and −12.32 mV for 20- and 80-nm AgNPs, respectively, after 24-h exposure. However, addition of Tween 20 (20 μM) effectively stabilized AgNPs against aggregation during 24-h exposure. The mean hydrodynamic size of 80-nm AgNPs was 104.7 nm with extraordinary homogeneity (polydispersity index 0.031) after 24-h exposure. However, slight aggregation still proceeded for 20-nm AgNPs (hydrodynamic size 140.6 nm), which can also be inferred from the high polydispersity index (i.e., 0.781). Continuous dissolution of AgNPs proceeded during the 24-h waterborne AgNPs exposure. The most dissolution at 24 h was observed for 20-nm AgNPs (i.e., 15.2%), while dissolution was 6.6%, 5.3%, and 1.7%, respectively, for the 80-nm AgNPs, 20-nm AgNPs (Tween 20 stabilized), and 80-nm AgNPs (Tween 20 stabilized). Uptake Kinetics of AgNPs in Brine Shrimp. Brine shrimp exposed to 20-nm AgNPs, 80-nm AgNPs, and 80-nm AgNPs stabilized by Tween 20 were able to accumulate AgNPs after 24 h of exposure, as demonstrated by the AgNPs aggregates in the gut (SI Figure S3). In contrast, only a few displayed such aggregates in the 20-nm AgNPs treatment stabilized by Tween 20. Consistently, two distinguished patterns were observed in the total body burden of Ag (Figure 2A). The groups exposed to 20-nm AgNPs, 80-nm AgNPs, and 80-nm AgNPs stabilized by Tween 20 accumulated elevated Ag with the increase of exposure concentration, with the calculated BCFs ranging from 5 to 21 L/g. However, brine shrimps were only able to accumulate 90−900 μg/g Ag when they were exposed to 20-nm AgNPs stabilized by Tween 20, which was similar to that of AgNO3 with or without the presence of Tween 20. No significant difference (p > 0.05, two-way ANOVA) of body accumulation of AgNO3 was found between treated and untreated Tween 20, indicating a minimal effect of Tween 20 on AgNO3 bioaccumulation. The short-term uptake experiment verified this distinguished bioaccumulation pattern (Figure 2B), with a constant uptake rate of 0.58 μg/g/h (r2 = 0.97) for 20-nm AgNPs stabilized by Tween 20, which were close to 1.02 μg/g/h (r2 = 0.94) measured for AgNO3 (Tween 20 treated) or 1.16 μg/g/h (r2 = 0.95) measured for AgNO3 (without Tween 20). However, elevated uptake rates were observed for the other treatments, indicating an enhanced accumulation of AgNPs by brine shrimp. Assimilation Efficiency of AgNPs in Marine Medaka. After pulse feeding, fish were able to excrete nearly 90% of the accumulated Ag within 16 h, leading to a final AE less than 6% for all the treatments at 26 h (Figure 3A). Briefly, the impact of Tween 20 was minor on the assimilation of Ag from AgNO3contaminated brine shrimp (AgNO3-Artemia) by medaka. About 4% of the total ingested Ag was retained at 26 h in

Figure 2. Bioaccumulation of AgNPs and AgNO3 by brine shrimp. Brine shrimp were exposed to a series of total Ag concentration (i.e., 0−1000 μg/L) for 24-h. Tween 20 was applied to keep the AgNPs (20 or 80 nm) well suspended in the seawater (A). Short-term uptake of AgNPs and AgNO3 by brine shrimp. brine shrimp were exposed to 1000 μg Ag/L and sampled at 1, 2, 3, and 4 h (B). Data are mean ± SD (n = 3).

treatments both with and without the addition of 20 μM Tween 20 with no statistical difference (p > 0.05, one-way ANOVA). For different particle sizes (20 nm, 80 nm, and aggregated), the overall AE of AgNPs-contaminated brine shrimp (AgNPs-Artemia) was as low as that of AgNO3-Artemia. For 80-nm AgNPs stabilized by Tween 20 contaminated food, only 0.4% Ag was retained at 26 h, while about 4% of Ag was retained for the other AgNPs-Artemia treatments. Similarly low AE was also observed in the subsequent AE experiment at different dietary AgNPs concentrations. Over 90% of the ingested AgNPs was depurated within the first 9 h, with the final AE values of 0.31−0.53% for the low, middle, and high Ag food loads (Figure 3B). No significant difference (p > 0.05, one-way ANOVA) of AE was found among these three treatments. Chronic Dietary Exposure of AgNPs to Marine Medaka. After chronic dietary exposure to different concentrations of AgNPs or AgNO3 for 28 days, medaka were able to accumulate Ag with the highest body burden of Ag (1.98 ± 0.53 μg/g, dry wt.) recorded for the high AgNPs-Artemia treatment (Table 1). Additionally, 5%, 10%, and 4% of Ag accumulation was in intestine for AgNO3 (i.e., 10.9 μg/g, dry wt.), low AgNPs (i.e., 28.2 μg/g, dry wt.), and high AgNPs (i.e., 8156

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Figure 3. Retentions of Ag in the medaka fed with AgNPs (20 and 80 nm) and AgNO3 contaminated brine shrimp, which were prepared at the same Ag exposure concentration (1 mg/L). Peer experiments using Tween 20 stabilized AgNPs (20 and 80 nm) and AgNO3 were also investigated (A). Retentions of Ag in the medaka fed with AgNPs (20 nm) contaminated brine shrimp, which were prepared at Ag exposure concentrations of 250, 500, and 1000 μg/L, labeled as low, middle, and high, respectively (B). Data are mean ± SD (n = 6).

Figure 4. Whole body Na+/K+-ATPase activity of medaka after chronic exposure; activity is expressed as the ability of Na+/K+-ATPase within 1 mg of protein to decompose 1 μmol ATP per hour (μmol Pi/ mg protein/h) (A). Whole body SOD activity of medaka after chronic exposure; SOD activity unit (U) is defined as inhibition 50% reduction of NBT and SOD activity was expressed as SOD activity unit per mg protein (U/mg protein) (B). Data are mean ± SD (n = 5).

181 μg/g, dry wt.) dietary treatments, respectively, yielding a very high intestine Ag concentration. The calculated corresponding TTFs were 0.044, 0.009, and 0.011, respectively. No significant (p > 0.05, one-way ANOVA) difference of body weight was found among different treatments (Table 1), and the calculated condition factors (indicator of fitness) were all around 3.1 (larger than the reference value 2.88). These data suggested a sufficient food supply during exposure, which ruled out the possibility of variation of food supply during preparation. A slight decrease (but not significant) of mRGL (indicator for the development of digestion tract) of medaka was also observed when fed Ag-contaminated brine shrimp. However, feeding high AgNPs-Artemia did lead to a significant (p < 0.05, one-way ANOVA) reduction of total body length (94.6% of control) and WC (94.7% of control) of the fish. Compared with the control treatment, inhibition of whole body Na+/K+-ATPase activity was observed in both low and high AgNPs-Artemia treatments, and significant (p < 0.05, twoway ANOVA) reduction occurred in the high AgNPs-Artemia treatment over 28 days of chronic exposure (Figure 4A). Specifically, the inhibition of whole body Na+/K+-ATPase activity occurred from the first week and recovery was observed in the third week for AgNPs-Artemia treatments. However, medaka continuously exposed to high AgNPs-Artemia eventually led to a sharp decline in whole body Na+/K+-ATPase

activity on week 4. Besides, feeding medaka with AgNO3Artemia did not generate a significant different (p > 0.05, twoway ANOVA) profile from the control, whereas significant differences (p < 0.05, two-way ANOVA) from both low and high AgNPs-Artemia treatments were detected. Also, no significant difference (p > 0.05, two-way ANOVA) of the whole body SOD activity was detected in AgNO3-Artemia treatment from the control (Figure 4B), while suppression of this enzyme was observed within the first 2 weeks in AgNPsArtemia treatments. Remarkably, feeding medaka with high AgNPs-Artemia led to a significant reduction (p < 0.05, twoway ANOVA) of the whole body SOD activity from control. The whole body CAT activity displayed a large variability for all treatments (SI Figure S2) and no significant difference (p > 0.05, two-way ANOVA) was detected among all treatments.

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DISCUSSION Uptake of AgNPs in Brine Shrimp. Brine shrimp is a suspension feeder and captures suspended particles in the aquatic environment, but the efficiency of particle capture depends on the particle size.30 In this study, we found that the brine shrimp can well-accumulate AgNPs as small as 105 nm, not to mention the aggregated AgNPs (i.e., >1000 nm). Because dissolution of AgNPs was unavoidable in the high ionic 8157

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environment, various degrees of dissolution were also quantified in this study. By combining with the uptake rate constants of different Ag species (i.e., dissolved Ag and AgNPs), we can estimate the contribution of dissolved Ag to the total accumulated AgNPs in brine shrimp. Using the average uptake rate constants calculated from the 4-h shortterm uptake study and the concentration of dissolved Ag at 4 h, maximum contribution was observed in the Tween 20 stabilized 20 AgNPs (i.e., 4.1%), and for the other three treatments such contribution would be 0.96), while very poor correlation was found for the AgNO3 treatments (r2 < 0.56) in this study. A similar phenomenon was also found in the accumulation of several manufactured nanoparticles (e.g., nano-TiO2, nano-CeO2, and nano-Au) by daphnids or fish,33 which indicated that elevation of nanomaterial concentrations in aquatic environments may increase the body burden of the accumulated NMs in aquatic organisms. Therefore, the extraordinary ability of these suspension feeders to accumulate nanoparticles (especially aggregated) may generate a high trophic transfer potential. Dietary Assimilation of AgNPs in Medaka. Although brine shrimp could accumulate a large quantity of AgNPs, how much of these accumulated AgNPs could be assimilated by the predator was largely unknown. Generally, the AE of dietary Ag was considered to be very low in teleost fish.23,34 In this study, only 4.4% dietary AgNO3 was retained in the teleost fish. Similar results were also found in freshwater brown trout (Salmo trutta, 10%, silver cyanide contaminated food),35 saltwater American plaice (Hippoglossoides platessoides, 4− 16%, 110mAgNO3 spiked food),36 and saltwater European plaice (Pleuronectes platessa, about 5%, 110mAgNO3 contaminated worm).37 Assimilation of metals in fish intestine first involves the diffusion of metals into an unstirred diffusion layer, followed by subsequent internalization.38,39 An in vitro experiment using intestine of rainbow trout (Oncorhynchus mykiss) showed that the mucus-bound Ag was the major component and the trans-flux of Ag through the intestine barrier was saturable, exhibiting a Michaelis−Menten kinetics with a KM of 180 nM.40 Although Ag+ was presumed to mimic Na+ or Cu+ and may take advantage of their pathways (e.g., Na+ channels, P-type Cu-ATPases, and Cu transporter Ctr1) for transportation, limited proportion of Ag+ was found in high ionic saline environment (complexation with bulk anions) as well as in the intestine (binding with biomolecules).34 Additionally, the activities of Na+/K+-ATPases are broadly similar in gill and intestinal tissue of marine teleosts,41 and inhibition of Na+/K+-ATPases was observed after chronic waterborne exposure of tidepool sculpins (Oligocottus maculosus) to AgNO3,42 which may also reduce the uptake Ag+

through the intestine barrier. All this evidence may partly explain the low AE in marine teleost fish. For dietary AgNPs, internalization of both AgNPs aggregates and dissolved Ag during food digestion may possibly occur simultaneously. Endocytosis should be the major uptake route for the direct internalization of AgNPs due to their large sizes.43 However, our study showed that the overall AEs of AgNPs was very low and no size effect was observed. Besides, feeding medaka with different food AgNPs (20 nm) concentration has no impact on AE. Furthermore, even after 28-day dietary exposure, medaka could only accumulate a small amount of Ag (daily AE of 1.6% and 0.31−0.47%, respectively, for dietary AgNO3 and AgNPs treatments), indicating a rather poor internalization ability of AgNPs. Similar low dietary transfer efficiency (TTF < 0.024) was also found in zebrafish fed daphnids previously exposed to nano-TiO2 after 14 days dietary exposure.44 Besides, medaka had a lower AE of Ag from dietary AgNPs than from dietary AgNO3, similar to earlier finding by Blickley et al.45 on mummichog (Fundulus heteroclitus) with CdSe/ZnS quantum dots or CdCl2 food. However, the forms of the assimilated metals are still unknown (particle, ion, or both). The newly developed single particle inductively coupled plasma mass spectrometry (spICPMS) coupled with alkaline digestion (using tetramethylammonium hydroxide) may provide precise information on the particles (size and concentration) within biological tissues.46 Chronic Dietary Toxicity of AgNPs to Marine Medaka. Although both the quantified AE and TTF of AgNPs by medaka were rather low, whether such a small amount of Ag could cause any toxic effects was unknown. Generally, toxicity of dietary exposure of ionic Ag to teleost fish was considered to be much lower than the waterborne exposure at the same silver mass base.34 Galvez and Wood47 showed that 58-day dietary exposure of rainbow trout to 3−3000 Ag2S mg/kg (wet wt.) spiked food caused only a significant decrease of feeding rate, whereas no effect on growth and survival was observed. Consistently, no significant difference of gross growth parameters (body weight, body length, condition factor, WC, and mRGL), SOD, and Na+/K+-ATPase activity between the control and the AgNO3 dietary treatment was found in this study. One possible reason for such nonobservable toxicity was due to the lack of toxic Ag species. Complexation with anions, such as Cl−, took place immediately after the introduction of AgNO3 into the seawater48 and further biotransformation of these Ag complexes may occur during uptake in brine shrimp (such as binding with sulfhydryl biomolecules).34 All these Ag species were considered far less toxic than Ag+.23 Additionally, the low dosage of AgNO3 (10.88 μg/g, dry wt.) used in this study may also account for this nonobserved toxic effect and more than 96% of ingested Ag was excreted after dietary Ag exposure. Different from AgNO3 dietary exposure, Ag appeared as AgNPs aggregates within AgNPs-Artemia (SI Figure S3), which may pose a very high regional Ag body burden in fish intestine. In this study higher concentration of Ag was retained in the intestine in AgNPs-Artemia treatments than that in AgNO3Artemia treatment (Table 1). The high Ag in the gut may produce severe lesions that compromise the structure integrity of the intestine.49 As an indicator of intestine development, a slight decline of mRGL was observed in all dietary Ag exposed fish with the most declines in high AgNPs-Artemia treatment. More severe impairment of the digestion tract (erosion of intestinal epithelium) was observed by Federici et al.50 after 8158

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ACKNOWLEDGMENTS We thank the anonymous reviewers for their comments on this work, and the State Key Laboratory of Marine Pollution for providing the marine medaka. This study was supported by a General Research Fund from the Hong Kong Research Grants Council (663011) to W.-X.W.

exposing rainbow trout to 0.5 mg/L nano-TiO2 for 14 days. Therefore, future examination on the microstructure of intestinal walls is needed to provide more information on this impairment. Such damage to intestine may affect the absorption of nutrients and further impair the growth of fish. Besides, the ingestion of AgNPs-Artemia brought new Ag specie (Ag0) into the digestion tract. The dissolution and oxidation of this zerovalent Ag to Ag+ during digestion can directly impair the activity of Na+/K+-ATPase and carbonic anhydrase,34 which may partly explain the observed inhibition of whole body Na+/ K+-ATPase activity in AgNPs-Artemia treatments during the first 2 weeks. However, acclimation of medaka occurred on the third week, and both Na+/K+-ATPase and SOD activity increased to the control level, which was also observed in some waterborne Ag exposure experiments.42,51 Although medaka were able to acclimate AgNPs-Artemia, feeding continuously with high AgNPs-Artemia did cause a significant (p < 0.05, two-way ANOVA) inhibition of Na+/K+-ATPase activity over the chronic exposure. On the other hand, longterm exposure of Ag+ can impair the ion and water transportation across the intestinal epithelia in fish,52 which may also partly explain the observed significant reduction of WC (water loss was regarded as the main loss during drying) in the high AgNPs-Artemia treatment after chronic exposure. However, to verify such assumption-based Ag+ toxicity, future studies on the quantification of AgNPs dissolution as well as Ag+ speciation within the biological compartments are strongly needed. Additionally, as a stomachless fish, pH within the intestine of medaka was alkaline,53 which was unfavorable for the dissolution AgNPs.24,54 More severe dissolution may achieve in those stomach-equipped fish (e.g., salmon and catfish), where the pH within the stomach can reach 2,55 rendering a high potential of toxic effects on fish. To sum up, high BCF (> 5 L/g) of AgNPs (20 and 80 nm) and well dispersed 80-nm AgNPs (stabilized by 20 μM Tween 20) were observed in brine shrimp, while far less well-dispersed 20-nm AgNPs was accumulated (BCF < 2 L/g, similar to that of silver nitrate). The AE of Ag (AgNO3, 20- and 80-nm AgNPs) from contaminated brine shrimp in the medaka was low (< 6%) and indepenent of food Ag (20-nm AgNPs) concentration. However, even this small amount of assimilated Ag (from AgNPs contaminated food) generated toxic effects (e.g., inhibition of Na+/K+-ATPase and SOD activity, reduction of total body length, and WC) on medaka during the 28-day chronic dietary exposure. This study highlighted the potential of AgNPs-contaminated food by generating toxicity to marine fish. Further study of the behavior of AgNPs (e.g., dissolution, redispersion, and translocation) within the digestion tract is needed to facilitate our understanding of this toxic effect.

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