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Waterborne and dietary accumulation of well-dispersible hematite nanoparticles by zebrafish at different life stages
Environmental Pollution 259 (2020) 113852
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Waterborne and dietary accumulation of well-dispersible hematite nanoparticles by zebrafish at different life stages* Bin Huang 1, Yu-Qing Cui 1, Wen-Bo Guo , Liuyan Yang , Ai-Jun Miao * State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu Province 210023, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 September 2019 Received in revised form 17 November 2019 Accepted 17 December 2019 Available online 20 December 2019
The widespread use of nanoparticles (NPs) has drawn considerable attention because of their potential toxicity and the environmental consequences thereof. However, the effects of the exposure route and life stage of an organism on the bioaccumulation and toxicity of NPs are largely unknown. In the present study, we investigated the accumulation kinetics (uptake, assimilation, and efflux) and tissue distribution of waterborne and dietary hematite NPs (HemNPs) during three life stages (embryo, larva, and adult) of the zebrafish Danio rerio. For all zebrafish life stages, the waterborne accumulation of well-dispersed HemNPs increased linearly with exposure time but decreased after reaching a maximum. The increase in HemNPs accumulation followed the order embryo > larva > adult. Compared with the waterborne route, the dietary accumulation of HemNPs in larval and adult zebrafish fluctuated, reaching a maximum after each food refreshment and then decreasing until the next food addition. Similar to waterborne exposure, adult fish accumulated less dietary HemNPs than did larvae. Nevertheless, dietary HemNPs mostly accumulated in the intestinal tract, with smaller amounts in the truncus, head, and gills, as compared with their waterborne counterparts. Moreover, in the gonad no dietary HemNPs were detected whereas accumulation via waterborne HemNPs was significant. Despite the low assimilation efficiency of dietary HemNPs, biodynamic modeling showed that the diet was the main source of particle accumulation in zebrafish. Thus, both the life stage and the exposure route should be considered in evaluations of the environmental risks of NPs. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Bioaccumulation Exposure route Hematite nanoparticles Life stage Zebrafish
1. Introduction Engineered nanoparticles (NPs) are defined as particles with a diameter between 1 and 100 nm in at least two dimensions (Klaine et al., 2008). Due to their extraordinary physicochemical characteristics, NPs have a very wide range of applications such that their annual production is approaching one million tons (Keller and Lazareva, 2013). However, an inevitable consequence of the use and production of NPs has been their release into aquatic environments, where their potential risks to aquatic ecosystems have become a highly relevant concern. Several recent studies have investigated the toxic effects of NPs in aquatic organisms at different trophic levels (Skjolding et al., 2016), but there have been
* This paper has been recommended for acceptance by Sarah Harmon. * Corresponding author. School of the Environment, Nanjing University, Mail box 24, Xianlin Road 163, Nanjing 210023, Jiangsu Province, China. E-mail address: [email protected] (A.-J. Miao). 1 These authors contributed equally to this paper.
https://doi.org/10.1016/j.envpol.2019.113852 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
few systematic investigations of the toxicity of NPs at the different life stages of these organisms. Moreover, although the bioaccumulation kinetics of NPs is one of the most important determinants of toxicity, it has yet to be fully examined, including the contribution of different routes (e.g., waterborne vs. dietary pathways) of bioaccumulation. Zebrafish (Danio rerio) is a model aquatic organism in toxicological studies (Bar-Ilan et al., 2013; Griffitt et al., 2007; Wu et al., 2018; Yang et al., 2013). Its cardiovascular, nervous, and digestive systems are similar to those of mammals (Lewis and Eisen, 2003) and its genome has a high degree of similarity with that of humans (Renier et al., 2007). In addition, the three growth stages (embryo, larva, and adult) of zebrafish allow toxicity to be tested as a function of developmental stage. Embryogenesis in zebrafish occurs rapidly and is thus suitable to screen the developmental toxicity of pollutants (Kimmel et al., 1995). The most sensitive stage in toxicity testing is the larval stage (Zhang et al., 2003). In addition, zebrafish embryos and larvae are transparent, which facilitates direct in vivo monitoring of the development of cells, tissues, and organs
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(Fishman, 1999). Toxicological studies of adult zebrafish have largely focused on chronic effects, mostly to fill the data gaps resulting from studies restricted to embryonic and larval stages (Chen et al., 2011). Aquatic organisms accumulate pollutants mainly via waterborne (i.e., from the water directly) and dietary (i.e., from food) routes (Wang et al., 2018). Pollutants that accumulate through these two routes differ in their distribution patterns and toxicity (Liang et al., 2007). Wang et al. (2018) found that the toxicity of dietary arsenate to Daphnia magna was less than that of its waterborne counterpart. Such toxicity difference was explained by the fact that dietary arsenate had a lower total accumulation, a higher distribution in the gut and non-sensitive subcellular fractions, and a higher rate of transformation to less toxic arsenic species. However, the few studies of the bioaccumulation of NPs have not allowed a consensus to be reached regarding the relative importance of waterborne vs. dietary routes of accumulation. Liu et al. (2019) reported that waterborne Fe2O3 NPs accounted for > 73.6% of the Fe2O3 NPs that accumulated in D. magna. By contrast, in the study of Zhu et al. (2010), dietary exposure was the major route of accumulation of TiO2 NPs in D. rerio, although the biomagnification factor was only in the range of 0.009e0.024. In the present study, we systematically examined the waterborne and dietary accumulation kinetics (uptake, assimilation, and efflux) of well-dispersible polyacrylic acid (PAA)-coated hematite nanoparticles (HemNPs) in zebrafish at different life stages. The distribution of the accumulated HemNPs in the organs of adult zebrafish was also studied. HemNPs were chosen because of their wide-ranging applications, including in catalysis, pigments, gas sensors, water treatment, and medical research. In addition, the low toxicity (no observed effect concentration > 100 mg Fe/L for zebrafish) and negligible dissolution of HemNPs minimize the potential interference from the adverse effects of NPs and from the metal ions (Huang et al., 2016). Therefore, they are especially suitable for bioaccumulation studies. To improve the detection limit and exclude the potential interference of background Fe, 55Felabeled HemNPs with a detection limit at picogram level were used throughout the whole study. The protozoan Tetrahymena thermophila and the zooplankton D. magna served as food for larval and adult zebrafish, respectively. Both organisms are universally distributed in freshwater ecosystems and their ability to ingest NPs has been documented (Huang et al., 2018; Tan et al., 2016). The overall objectives of our study were to explore the contributions of waterborne and dietary routes to the bioaccumulation of NPs and to determine whether NPs accumulation in aquatic organisms
½HemNPsemb ¼
through a filtration system and maintained at a temperature of 28 ± 1 C. The fish were exposed to a light/dark cycle of 12/12 h and fed daily with Zeigler fish food. Selected male and female parent zebrafish were additionally fed daily with fresh Artemia salina larvae to supplement their nutrition until mating and spawning. After spawning, the fertilized embryos were collected 0.5 h postfertilization (hpf), washed with modified Holtfreter’s medium (MH, pH ¼ 7.0, Table S1, Supporting Information), and maintained at 28 ± 1 C in MH medium that was refreshed daily. At 4 days postfertilization (dpf), the embryos had mostly hatched, but the yolk was not consumed completely until 6 dpf. All of the following experiments described below were carried out in triplicate at 28 ± 1 C in fish exposed to a 12:12 h light/dark cycle. 55 Fe-labeled HemNPs (primary particle size, 5e10 nm; specific activity, 13.6 mCi g1 Fe; Fe proportion, 29%) were synthesized by titration hydrolysis and then PAA-coated, as described in a previous study by our group (Huang et al., 2016). The hydrodynamic diameter and zeta potential of HemNPs in the experimental medium (i.e., MH medium) were determined using a dynamic light scattering (DLS) particle sizer (ZetaPALS, Brookhaven Instruments, NY, USA). The morphology of the labeled HemNPs in MH was visualized by transmission electron microscopy (TEM; JEM-200CX, JEOL, Tokyo, Japan). 2.2. Accumulation of HemNPs in embryos Triplicate samples of zebrafish embryos (0.5 hpf) were exposed to 55Fe-labeled HemNPs at a concentration of 30 mg Fe L1 and sampled at 0.5, 1.5, 3, 6, 12, 24, 36, 48, and 72 h. Such concentration of HemNPs was selected to ensure reliable determination of their bioaccumulation at different life stages. The exposure duration was chosen to explore the effects of hatching on HemNPs accumulation. At each time point, 10 embryos were collected from each replicate and rinsed three times with MH to remove HemNPs weakly adsorbed on their surfaces. The embryos were dried at 60 C until a constant weight was reached, digested in 0.2 mL of concentrated HNO3, and diluted with Milli-Q water (18.2 MU) to 4 mL, such that the final HNO3 concentration was 3.5% (w/v). The radioactivity of 55 Fe in the digest was quantified using liquid scintillation counting (LSC, Tri-Carb 2800 TR; PerkinElmer, USA) after the addition to the samples of 10 mL of scintillation cocktail (Oxysolve C-400; Zinsser Analytic, Frankfurt, Germany) (Huang et al., 2017). The amount of HemNPs that accumulated in the embryos ([HemNPs]emb, mg Fe g1 dry weight, dw) was calculated according to Eq. (1):
Radioactivity per dry weight embryo ½HemNPsmed Radioactivity per liter experimental medium
changes as a function of their life stage. The results of this study contribute to a better understanding of the bioaccumulation of NPs in aquatic ecosystems and facilitate assessments of the ecological risks posed by these particles to aquatic organisms. 2. Materials and methods 2.1. Organisms and HemNPs Adult zebrafish (Danio rerio, AB strain) were purchased from the local fish market. Before the start of the experiments, they were acclimated for at least 2 weeks in aerated tap water circulated
(1)
where [HemNPs]med (mg Fe L1) is the HemNPs concentration in the experimental medium, which was determined at the beginning and end of the experiment. The average dry weight of an embryo was 68.6 mg, as obtained by weighing 500 embryos of 24 hpf in total. To determine whether the NPs were able to pass through the chorion and accumulate inside the embryo, another 10 embryos were collected after a 24-h exposure to 30 mg Fe L1 of HemNPs and rinsed three times with fresh MH. The chorion of each embryo was then removed using forceps under a stereomicroscope. The 55 Fe radioactivity in the chorion and in the dechorionated embryos was calculated according to Eq. (1).
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2.3. Accumulation of HemNPs in larvae 2.3.1. Waterborne accumulation experiment The experimental procedure for the waterborne uptake kinetics of HemNPs by 6-dpf larvae was similar to that described above for the embryo accumulation experiment. Briefly, 100 healthy larvae in each container were exposed to 30 mg Fe L1 of 55Fe-labeled HemNPs for 6, 12, 24, 36, 48, 60, 72, 84, and 96 h. At each time point, all 100 larvae from each of the three replicate containers were collected and washed three times with MH. The larvae were then euthanized with 0.02% (w/v) tricaine methanesulfonate (MS-222) and dried to a constant weight. The radioactivity (55Fe) in the digested larvae was measured by LSC and the result used to calculate larval HemNPs accumulation ([HemNPs]larva). To examine the concentration-dependent bioaccumulation of HemNPs, another 100 larvae in triplicate were exposed for 24 h to HemNPs at concentrations of 0.3, 1, 3, 10, and 30 mg Fe L1 after which [HemNPs]larva was determined as described above. The uptake rate of HemNPs at different [HemNPs]med was then calculated as [HemNPs]larva divided by the exposure time. 2.3.2. Dietary accumulation experiment Tetrahymena thermophila containing 55Fe-labeled HemNPs was prepared as described in the Supporting Information and then used at a concentration of 1 105 cells mL1 to feed 2400 healthy larvae equally dispensed into 48 containers (50 individuals in each container). The exposure medium and the food were refreshed daily during the 96-h experiment. Every 6 h, 50 larvae in triplicate were collected from three containers for the measurement of [HemNPs]larva. 2.3.3. Assimilation experiment The assimilation efficiency (AE) of HemNPs was determined via the pulse-feeding method (Wang and Fisher, 1999). Briefly, 100 healthy larvae in each of 21 containers were fed for 30 min with T. thermophila (1 105 cells mL1) containing 55Fe-labeled HemNPs (Supporting Information). The feeding time was selected based on the gut passage time (>1e1.5 h) of food in zebrafish larvae. Subsequently, the larvae were washed three times with MH and transferred into fresh medium with the same density of T. thermophila but without HemNPs. This depuration step lasted 24 h, during which time 100 individuals were collected from each of three containers after 0, 2, 4, 6, 8, 12, and 24 h and the HemNPs retained in the larvae were quantified. 2.4. Accumulation of HemNPs in adults 2.4.1. Waterborne accumulation experiment Adult fish (3e4 months, 2e3 cm) were acclimated in MH for at least 24 h before the uptake kinetics experiment. Sixty individuals in triplicate were exposed to HemNPs (30 mg Fe L1) in 600 mL of MH without food for 144 h. Every 24 h, 10 individuals from each replicate were collected for the determination of [HemNPs]adult. In addition to the uptake kinetics, the concentration-dependent accumulation of HemNPs was measured. For this purpose, 10 individuals in triplicate were exposed to 0.3, 1, 3, 10, and 30 mg Fe L1 of HemNPs for 24 h, after which [HemNPs]adult and the uptake rate were calculated. 2.4.2. Dietary accumulation experiment Daphnia magna containing 55Fe-labeled HemNPs was prepared as adult fish food as described in the Supporting Information. Seventy-two adult zebrafish dispensed in 72 containers (1 individual per container) were fed daily with 10 daphnids (~2.5% dry weight of each fish). Such ration of food was chosen to ensure that
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the feeding behavior was completed within 15 min in case of significant depuration of HemNPs from the daphnids. The exposure medium was also refreshed daily during the 144-h experiment. Every 24 h, four individuals in triplicate (12 in total) were collected for the determination of [HemNPs]adult. 2.4.3. Assimilation experiment This experiment was similar to the larval assimilation experiment except that each container had one individual (84 in total) that was fed with 10 HemNPs-containing daphnids. Afterward, the medium was refreshed without any addition of HemNPs and the concentration of HemNPs retained in the fish (four individuals in triplicate) was monitored after 0, 2, 4, 6, 8, 12, and 24 h of depuration. 2.4.4. Efflux experiment Sixty adult fish in triplicate were exposed to 30 mg Fe L1 of HemNPs for 72 h. They were then collected, washed three times to remove the HemNPs weakly adsorbed on their surfaces, and transferred into fresh MH without HemNPs. This depuration step lasted 36 h, during which 10 individuals were collected at 0, 2, 4, 6, 12, and 36 h and the concentration of retained HemNPs was measured. The depuration medium was refreshed every 12 h to minimize the re-assimilation of HemNPs released from the fish. 2.4.5. Distribution experiment According to the results of the waterborne and dietary accumulation experiments, 20 adult zebrafish were collected either after being exposed to 30 mg Fe L1 of HemNPs for 3 days or after being fed HemNPs-containing daphnids for the same amount of time. The intestinal tract, gills, head, gonad, other viscera (heart, liver, pancreas, gallbladder, kidney, and swim bladder), and truncus (i.e., skin, scales, muscles, bones, and other residues) were isolated and the respective concentration of HemNPs was quantified as described in the accumulation experiment. Only the distribution of HemNPs in adult fish was examined herein, as the accumulation of HemNPs in larvae was low and the size of each larva was too small to be dissected. 2.5. Modeling and statistical analysis The data on HemNPs uptake, assimilation, and efflux were analyzed by biodynamic modeling (Dang et al., 2009; Fan and Reinfelder, 2003; Zhang and Wang, 2006). Detailed information on the calculation of the uptake (ku) and efflux (ke) rate constants, the kinetic order b, the AE, the food bioconcentration factor (BCFf), food intake rate (IR), and trophic transfer factor (TTF) is provided in the Supporting Information. Significant differences were defined as a p-value < 0.05, based on a Student’s t-test or a one-way analysis of variance with post-hoc multiple comparisons (Tukey or Tamhane; SPSS 11.0 by SPSS, Chicago, USA). The analysis of variance took into consideration both the normality (Kolmogorov-Smirnov and Shapiro-Wilk tests) and the homogeneity of variance (Levene’s test) of the data. 3. Results 3.1. Physicochemical properties of HemNPs The TEM results showed that HemNPs were well dispersed in MH, with an average size of 10.1 nm, based on the measurement of at least 1000 particles randomly chosen from the TEM images (Fig. 1a). The size was also consistent with the DLS results, according to which the average hydrodynamic diameter was 33.2 ± 1.1 nm (Fig. 1b) and the zeta potential was 26 mV.
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Fig. 1. (a) Transmission electron microscopy images of hematite nanoparticles in modified Holtfreter’s medium. (b) The hydrodiameter distribution of the particles as obtained by dynamic light scattering.
3.2. HemNPs accumulation in embryos Exposure of the embryos to 30 mg Fe L1 of HemNPs resulted in an increase in [HemNPs]emb from 0 mg Fe g1 dw at the beginning to 1264.1 mg Fe g1 dw at 36 h (Fig. 2a). Thereafter [HemNPs]emb decreased steadily, to 621.4 mg Fe g1 dw at 48 h, at which time the embryos hatched, and further to 44.2 mg Fe g1 dw at the end. As for the HemNPs accumulated in the embryos, most of them (92.3%) were found in the chorion, with only 0.4% in the embryonic cells (Fig. 2b).
Fig. 2. (a) Changes in the concentration of HemNPs in embryos ([HemNPs]emb) during a 72-h exposure to 30 mg Fe L1 of HemNPs. (b) The distribution of HemNPs in the chorion (CH) and in dechorionated embryos (DE) after a 24-h exposure to 30 mg Fe L1 of HemNPs. The data are the mean ± standard deviation (n ¼ 3).
phases to 151.6 mg Fe g1 dw during the third 24-h phase and further to 223.5 mg Fe g1 dw over the last 24 h. In the assimilation experiment, a substantial amount of HemNPs (45.6 mg Fe g1 dw) accumulated in the larvae after their pulse feeding. Transfer of the HemNPs-containing larvae to fresh medium without HemNPs resulted in a rapid decrease in [HemNPs]larva, to 1.5 mg Fe g1 dw, after 12 h of depuration. Thereafter, the value leveled off. The AE (proportion of HemNPs retained in the larvae at the end of depuration) thus obtained was 4.1% (Fig. 3d). 3.4. HemNPs accumulation in adult fish
3.3. HemNPs accumulation in larvae In the waterborne accumulation experiment, [HemNPs]larva increased linearly, from 0 mg Fe g1 dw at the beginning to 66.7 mg Fe g1 dw at 84 h, and then slightly decreased until the end of the experiment, to 49.3 mg Fe g1 dw (Fig. 3a). HemNPs accumulation was concentration-dependent, indicated by the approximately 8fold increase in [HemNPs]larva as [HemNPs]med increased from 0.3 to 30 mg Fe L1. Accordingly, on an exponential scale, the uptake rate increased linearly with increasing [HemNPs]med (Fig. 3b); the uptake rate constant ku was 5.00 L kg1 d1. Dietary accumulation was more complicated than waterborne accumulation with respect to the time-related change of [HemNPs]larva. [HemNPs]larva reached a maximum between each food refreshment and the maximum value increased with increasing exposure time (Fig. 3c). Thus, [HemNPs]larva increased from 0 mg Fe g1 dw at the beginning to 112.6 mg Fe g1 dw at 18 h and then decreased to 55.8 mg Fe g1 dw until the next food refreshment at 24 h. The maximum value of [HemNPs]larva increased from 82.8 to 112.6 mg Fe g1 dw for the first two 24-h
Similar to the waterborne accumulation of HemNPs in larvae, [HemNPs]adult increased linearly with exposure time during the first 72 h of the 144-h exposure of the fish to 30 mg Fe L1 of HemNPs. This was followed by a steady decrease from the maximum (10.4 mg Fe g1 dw) to 3.4 mg Fe g1 dw at the end of the experiment (Fig. 4a). The accumulation of HemNPs increased as [HemNPs]med increased, resulting in a linear correlation on an exponential scale between the uptake rate of HemNPs and [HemNPs]med (Fig. 4b). The uptake rate constant ku thus obtained was 0.66 L kg1 d1. Similar to larval accumulation, an irregular correlation between [HemNPs]adult and exposure time was determined in the dietary accumulation experiment. Namely, [HemNPs]adult increased quickly, to 34.0e48.2 mg Fe g1 dw, after the daily feeding of adult fish with HemNPs-containing daphnids and then steadily decreased, to 0.27e0.56 mg Fe g1 dw, until the next feeding (Fig. 4c). An assimilation experiment was then performed to measure the proportion of dietary HemNPs subsequently utilized by the adult zebrafish. As shown in Fig. 4d, [HemNPs]adult decreased
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Fig. 3. (a) Changes in the concentration of HemNPs in larvae ([HemNPs]larva) during a 96-h exposure of the larvae to 30 mg Fe L1 of HemNPs. (b) The uptake rates of HemNPs in larvae at different HemNPs concentrations in the experimental medium ([HemNPs]med of 0.3, 1, 3, 10, and 30 mg Fe L1). (c) Changes in [HemNPs]larva in larvae fed daily with HemNPs-containing Tetrahymena thermophila during a 96-h period. (d) The proportion of HemNPs retained in the larvae after depuration for 0, 2, 4, 6, 8, 12, and 24 h in the assimilation experiment. The dashed line in (b) is the linear regression between the HemNPs uptake rate and [HemNPs]med on an exponential scale. The data are the mean ± standard deviation (n ¼ 3).
quickly within the first 4 h of the 24-h depuration period but then remained relatively constant. Accordingly, 2.9% of the initially accumulated HemNPs was retained at the end of the assimilation experiment. Given this low accumulation of HemNPs from the dietary route, only the efflux of the particles accumulated through the waterborne source was examined. After a 3-day exposure to 30 mg Fe L1 of HemNPs, [HemNPs]adult was approximately 10.4 mg Fe g1 dw but it decreased in two phases when the fish were transferred to depuration medium (Fig. 4e). The efflux rate constants (ke) thus obtained for the fast and slow phases were 0.056 (ke1) and 0.012 h1 (ke2), respectively. An analysis of the tissue distribution of the HemNPs showed that they had mainly accumulated in the intestinal tract (78.7 and 94.2%), followed by the truncus (6.8% and 1.7%), head (6.8% and 0.9%), and gills (3.2% and 0.4%), regardless of the accumulation route (Fig. 4f). However, a comparison of the two routes showed the accumulation of significantly (p < 0.05, t-test) more waterborne than dietary HemNPs in the gills, truncus, and gonad. In fact, in the gonad, while the waterborne route accounted for 1.7% of the accumulated HemNPs there was no accumulation in this tissue via the dietary route. Although the difference was not significant (p > 0.05, t-test), a lower concentration of waterborne than of dietary HemNPs in the intestinal tract was observed.
4. Discussion 4.1. Waterborne accumulation In the present study, HemNPs mainly accumulated in the zebrafish chorion (>92%), with only 0.4% or less detected inside the embryos, indicating that the chorion served as a barrier to HemNPs entry into the embryos (Kashiwada, 2006). This barrier effect can be explained by the unique structure of the chorion, whose three protein layers have a total thickness of 1.5e2.5 mm. The inner and middle layers contain regularly arranged tapered pores (diameter, 0.5e0.7 mm) whose narrow ends face outwards. By contrast, the outer layer is powdery, which blocks the pores and thus presumably prevented HemNPs from entering the perivitelline space (Rawson et al., 2000). In addition, the internalization of HemNPs may have been inhibited by their complexation with the chorion (Huang et al., 2015). Auffan et al. (2014) found that AgNPs adsorbed onto the surface of the chorion via their complexation with the thiol group of chorionic proteins. The adsorption of HemNPs onto the chorion may have resulted in the reconstitution of its outer layer via the secretion by the embryo of additional substances, resulting in the steady absorption of the NPs (Fig. 2a) (Chang and Huang, 2002; Zhao et al., 2016). When the chorions were shed from the embryos, the larvae thus obtained were able to orally ingest the particles, providing a new pathway for their accumulation from the water. In addition, because the skin and branchial membrane of the larvae were not
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Fig. 4. (a) Changes in the concentration of HemNPs in adult fish ([HemNPs]adult) exposed for 144 h to 30 mg Fe L1 of HemNPs. (b) The uptake rates of HemNPs in adult fish at [HemNPs]med of 0.3, 1, 3, 10, and 30 mg Fe L1. (c) Changes in [HemNPs]adult in adult fish fed daily with HemNPs-containing Daphnia magna during a 144-h period. (d) The proportion of HemNPs retained in adult fish after depuration for 0, 2, 4, 6, 8, 12, and 24 h in the assimilation experiment. (e) The proportion of HemNPs retained in adult fish after depuration for 0, 2, 4, 6, 12, and 36 h in the efflux experiment. (f) The distribution of HemNPs in the gills, gonad, intestinal tract (IT), other viscera (OV), truncus, and head of adult zebrafish exposed to 30 mg Fe L1 of HemNPs or fed for 3 days with HemNPs-containing D. magna. The dashed line in (b) is the linear regression between the HemNPs uptake rate and [HemNPs]med on an exponential scale. The dashed lines in (e) are the bilinear regression between the proportion of HemNPs retained in the fish (exponential scale) and the depuration time in the efflux experiment. The data are the mean ± standard deviation (n ¼ 3).
fully developed, the weak barrier effects of these structures may have allowed the direct entry of the HemNPs (Chetty et al., 2016). Kwok et al. (2012) found that in juvenile Japanese medaka (Oryzias latipes) exposed to AgNPs, the particles were mainly concentrated in the gills and intestine. Those authors speculated that the gills were the initial site of AgNPs absorption. However, in our study, regardless of the new uptake routes that emerged at the larval stage, the amount of bioaccumulated HemNPs was much less in the
larvae than in the embryos, due to the loss of the chorion and thus of its high accumulation capacity (Bohme et al., 2015). Unlike in the larvae, in the adult fish the gills and epidermis were mature, which would have hindered the accumulation of HemNPs. This explains why only 4.0% and 6.8% of the total accumulated HemNPs were found in the gills and truncus, respectively. Similarly, Zhao et al. (2011) reported that the amount of CuO NPs that had accumulated in the skin and scale of carp (Cyprinus carpio)
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accounted for only 1.6% of total NPs accumulation. Overall, the intestinal tract was the dominant site of HemNPs accumulation in adult fish (Zhang et al., 2015). However, a substantial fraction of the HemNPs was subsequently depurated in two distinct phases, indicative of the distribution of the particles in different organs (Liu et al., 2019). Fan et al. (2016) also reported the two-phase eliminations of TiO2 NPs by D. magna: rapidly during the first 6 h and then gradually during the following 18 h of a 24-h efflux period. The authors proposed that the fast efflux corresponded to particles concentrated in the gut (Khan et al., 2015) and the slow efflux to particles adsorbed on non-gut tissues, microvilli, or the peritrophic membrane (Isaacson et al., 2017). The decreasing accumulation of HemNPs from the embryonic to the larval and then to the adult stage may result from a decreased specific surface area with age or a progressive increase in the zebrafish’s ability to regulate the bioaccumulation of NPs. A 24-h exposure of the embryos, larvae, and adults to 30 mg Fe L1 of HemNPs resulted in a HemNPs content of 1140.7, 21.9, and 4.6 mg Fe g1 dw, respectively. Growth-dependent accumulation was further evidenced by the values of the uptake rate constant ku and the kinetic coefficient b of the larval and adult fish. The coefficient b indicates the saturation degree of HemNPs uptake: the smaller the value of b, the more saturated the accumulation (Zhang and Wang, 2006). In the present study, the ku (b) of larval and adult fish was 5.00 L kg1 d1 (0.42) and 0.66 L kg1 d1 (0.50), respectively. Thus, the uptake of HemNPs by adult fish was slower and saturation degree was also lower, compared with the uptake by the larvae. However, despite the decrease in HemNPs accumulation after it reached a maximum in all three zebrafish life stages, the underlying mechanisms differed between embryos and larvae/adults. Namely, the decrease in the HemNPs content in embryos was due to the loss of the chorion at the end of the uptake experiment, while in larval and adult zebrafish the efflux of the HemNPs accounted for the loss. 4.2. Dietary accumulation HemNPs accumulation through the dietary route in the larval stage was characterized by a periodic fluctuation together with a time-related increase in the maximum value of each 24-h cycle. This can be explained by the low rate of assimilation (i.e., quick depuration of foods from the gut) and the growth-dependent increase in predation (Fig. 3c). The latter resulted in an increase of HemNPs accumulation after each food refreshment. Thus, as predation ability increased with the increasing age of the zebrafish, the maximum HemNPs accumulation during each food refreshment cycle increased as well. Reitan et al. (1998) also found that the increase in the specific growth rate of turbot (Scophthalmus maximus) larvae was accompanied by an increase in the larval drinking rate, from 14 nL larva1 h1 at day 2 post-hatching to 120 nL larva1 h1 at day 11 post-hatching. However, the low AE and quick efflux of the HemNPs in the larvae resulted in a significant decline in particle accumulation after the maximum during each food refreshment cycle. Low AE values (<6%) were also reported by Wang and Wang (2014) in their study of the uptake of AgNPs of different sizes by marine medaka (Oryzias melastigma). The variation in HemNPs taken up by adult zebrafish via the dietary route was similar to that by larvae. The only difference was the unchanged maximum during each feeding cycle, as there was no significant growth of the adult fish during the experimental period. The dietary accumulation of HemNPs by adult fish was less than that by the larvae, which can be attributed to the lower AE of the adults (2.9% vs. 4.1%). This difference demonstrated the better ability of adult fish to regulate particle accumulation. Nevertheless, the possibility of a lower HemNPs content in the food of the adult fish than in the larval food cannot be excluded. The low AEs of
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HemNPs in both larvae and adults also indicated a low transport efficiency of HemNPs from the food to either life stage. This conclusion was supported in the adult fish by the very low TTF (4.4 104 ≪ 1), which indicated that there was no biomagnification of HemNPs under the conditions of the present study. Most of the studies reported in the literature have similarly shown a low transport efficiency of NPs along the food chain of aquatic organisms and thus a very low risk of biomagnification. Zhu et al. (2010) studied the trophic transfer of TiO2 NPs from daphnia to zebrafish and determined a TTF of only 0.009e0.024. A low transport efficiency of only 0.01e0.04 for dietary AgNPs was also reported by Wang and Wang, 2014, but the authors observed that the particles still inhibited the enzyme activity of medaka and reduced the growth of the fish. Together, these results demonstrate that in assessments of the ecological risk of NPs dietary accumulation routes cannot be ignored.
4.3. Waterborne vs. dietary accumulation A number of studies have examined the distribution of either waterborne or dietary NPs in fish and reported results similar to ours. Nevertheless, the two routes have only rarely been compared directly. The present study examined both waterborne and dietary uptake and showed that, although HemNPs accumulated via both routes were mainly concentrated in the zebrafish intestine, dietary uptake resulted in higher intestinal concentrations than direct uptake from the water phase. In the latter, however, HemNPs were more easily transported to other organs of the fish, including the gonad, which may result in biological effects distinct from those associated with dietary ingestion. Wu et al. (2018) reported that the aqueous-phase accumulation of TiO2 NPs in the zebrafish gonad affected not only the zebrafish itself but also its offspring. To further investigate the differences in the contributions of waterborne and dietary routes to HemNPs accumulation in zebrafish, the fraction of particles accumulated through food ingestion (ff) was calculated (Supporting Information). Fixing the value of BCFf (i.e., 10, 100, and 1000 L kg1) allowed ff at different [HemNPs]med to be calculated. As shown in Fig. 5, at a low [HemNPs]med and a high BCF, the dietary route accounted for most of the accumulation of HemNPs. This may in fact be the case in aquatic environments in nature, given their generally low NP concentrations and BCF values typically >10 L kg1. Our simulation further demonstrates the need to consider dietary accumulation in evaluations of the ecological risks of NPs.
Fig. 5. The contribution of dietary HemNPs to the total accumulation of HemNPs in adult zebrafish (ff) at different ambient concentrations of HemNPs ([HemNPs]med) under a BCFf of 10, 100, and 1000 L kg1, as predicted by biodynamic modeling.
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5. Conclusion The present study compared the accumulation kinetics of waterborne and dietary HemNPs in zebrafish at three different life stages. Although waterborne HemNPs accumulated to a greater extent in embryos than in either larvae or adults, most of the particles were shed together with the chorion during hatching and thus posed little risk to the fish. The decreasing concentration of waterborne and dietary HemNPs from the larval to the adult stage may result from a decreased specific surface area with age or the better ability of mature fish to regulate the bioaccumulation of HemNPs. In contrast to the uptake kinetics of waterborne particles, which showed a linear correlation between HemNPs accumulation and initial exposure time, HemNPs accumulation through the dietary route fluctuated, indicative of a low AE and a high predation rate during each food refreshment cycle. Despite the low AE of dietary HemNPs, the contribution of this route to HemNPs accumulation in zebrafish was much greater than that of the waterborne route, according to biodynamic modeling. Overall, both waterborne and dietary routes as well as differences in the NPs accumulation ability of organisms at different life stages should be considered in evaluations of the environmental risks of HemNPs. Declaration of competing interest There are no conflicts of interest to declare. CRediT authorship contribution statement Bin Huang: Writing - original draft, Investigation, Formal analysis. Yu-Qing Cui: Methodology, Investigation, Formal analysis. Wen-Bo Guo: Investigation. Liuyan Yang: Writing - review & editing, Funding acquisition. Ai-Jun Miao: Conceptualization, Methodology, Supervision, Writing - review & editing. Acknowledgements We thank three reviewers for their constructive suggestions on this paper. This work was supported by the National Natural Science Foundation of China (21822605, 21677068, and 41807479), Chinese Public Science and Technology Research Funds for Ocean Projects (201505034), the China Postdoctoral Science Foundation (2017M622781), and the State Key Laboratory of Pollution Control and Resource Reuse Foundation (PCRRF18020). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113852. References Auffan, M., Matson, C.W., Rose, J., Arnold, M., Proux, O., Fayard, B., Liu, W., Chaurand, P., Wiesner, M.R., Bottero, J.Y., Di Giulio, R.T., 2014. Salinity-dependent silver nanoparticle uptake and transformation by Atlantic killifish (Fundulus heteroclitus) embryos. Nanotoxicology 8, 167e176. Bar-Ilan, O., Chuang, C.C., Schwahn, D.J., Yang, S., Joshi, S., Pedersen, J.A., Hamers, R.J., Peterson, R.E., Heideman, W., 2013. TiO2 Nanoparticle exposure and illumination during zebrafish development: mortality at parts per billion concentrations. Environ. Sci. Technol. 47, 4726e4733. Bohme, S., Stark, H.J., Reemtsma, T., Kuhnel, D., 2015. Effect propagation after silver nanoparticle exposure in zebrafish (Danio rerio) embryos: a correlation to internal concentration and distribution patterns. Environ. Sci. -Nano 2, 603e614. Chang, Y.S., Huang, F.L., 2002. Fibroin-like substance is a major component of the outer layer of fertilization envelope via which carp egg adheres to the substratum. Mol. Reprod. Dev. 62, 397e406. Chen, J.Y., Dong, X., Xin, Y.Y., Zhao, M.R., 2011. Effects of titanium dioxide nanoparticles on growth and some histological parameters of zebrafish (Danio rerio) after a long-term exposure. Aquat. Toxicol. 101, 493e499.
Chetty, S.S., Praneetha, S., Basu, S., Sachidanandan, C., Murugan, A.V., 2016. Sustainable, rapid synthesis of bright-luminescent CuInS2-ZnS alloyed nanocrystals: multistage nano-xenotoxicity assessment and intravital fluorescence bioimaging in zebrafish-embryos. Sci. Rep. 6, 26078. Dang, F., Zhong, H., Wang, W.X., 2009. Copper uptake kinetics and regulation in a marine fish after waterborne copper acclimation. Aquat. Toxicol. 94, 238e244. Fan, C.W., Reinfelder, J.R., 2003. Phenanthrene accumulation kinetics in marine diatoms. Environ. Sci. Technol. 37, 3405e3412. Fan, W.H., Liu, L.L., Peng, R.S., Wang, W.X., 2016. High bioconcentration of titanium dioxide nanoparticles in Daphnia magna determined by kinetic approach. Sci. Total Environ. 569, 1224e1231. Fishman, M.C., 1999. Zebrafish genetics: the enigma of arrival. Proc. Natl. Acad. Sci. U.S.A. 96, 10554e10556. Griffitt, R.J., Weil, R., Hyndman, K.A., Denslow, N.D., Powers, K., Taylor, D., Barber, D.S., 2007. 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Isaacson, C.W., Sigg, L., Ammann, A.A., Stadnicka-Michalak, J., Schirmer, K., 2017. Interactions of TiO2 nanoparticles and the freshwater nematode Plectus aquatilis: particle properties, kinetic parameters and bioconcentration factors. Environ. Sci. -Nano 4, 712e719. Kashiwada, S., 2006. Distribution of nanoparticles in the see-through medaka (Oryzias latipes). Environ. Health Perspect. 114, 1697e1702. Keller, A.A., Lazareva, A., 2013. Predicted releases of engineered nanomaterials: from global to regional to local. Environ. Sci. Technol. Lett. 1, 65e70. Khan, F.R., Paul, K.B., Dybowska, A.D., Valsami-Jones, E., Lead, J.R., Stone, V., Fernandes, T.F., 2015. Accumulation dynamics and acute toxicity of silver nanoparticles to Daphnia magna and Lumbriculus variegatus: implications for metal modeling approaches. Environ. Sci. Technol. 49, 4389e4397. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic-development of the zebrafish. Dev. 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Algal foods reduce the uptake of hematite nanoparticles by downregulating water filtration in Daphnia magna. Environ. Sci. Technol. 53, 7803e7811. Rawson, D.M., Zhang, T., Kalicharan, D., Jongebloed, W.L., 2000. Field emission scanning electron microscopy and transmission electron microscopy studies of the chorion, plasma membrane and syncytial layers of the gastrula-stage embryo of the zebrafish Brachydanio rerio: a consideration of the structural and functional relationships with respect to cryoprotectant penetration. Aquacult. Res. 31, 325e336. Reitan, K.I., Natvik, C.M., Vadstein, O., 1998. Drinking rate, uptake of bacteria and microalgae in turbot larvae. J. Fish Biol. 53, 1145e1154. Renier, C., Faraco, J.H., Bourgin, P., Motley, T., Bonaventure, P., Rosa, F., Mignot, E., 2007. Genomic and functional conservation of sedative-hypnotic targets in the zebrafish. Pharmacogenetics Genom. 17, 237e253. 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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Waterborne and dietary accumulation of well-dispersible hematite nanoparticles by zebrafish at different life stages* Bin Huang 1, Yu-Qing Cui 1, Wen-Bo Guo , Liuyan Yang , Ai-Jun Miao * State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu Province 210023, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 September 2019 Received in revised form 17 November 2019 Accepted 17 December 2019 Available online 20 December 2019
The widespread use of nanoparticles (NPs) has drawn considerable attention because of their potential toxicity and the environmental consequences thereof. However, the effects of the exposure route and life stage of an organism on the bioaccumulation and toxicity of NPs are largely unknown. In the present study, we investigated the accumulation kinetics (uptake, assimilation, and efflux) and tissue distribution of waterborne and dietary hematite NPs (HemNPs) during three life stages (embryo, larva, and adult) of the zebrafish Danio rerio. For all zebrafish life stages, the waterborne accumulation of well-dispersed HemNPs increased linearly with exposure time but decreased after reaching a maximum. The increase in HemNPs accumulation followed the order embryo > larva > adult. Compared with the waterborne route, the dietary accumulation of HemNPs in larval and adult zebrafish fluctuated, reaching a maximum after each food refreshment and then decreasing until the next food addition. Similar to waterborne exposure, adult fish accumulated less dietary HemNPs than did larvae. Nevertheless, dietary HemNPs mostly accumulated in the intestinal tract, with smaller amounts in the truncus, head, and gills, as compared with their waterborne counterparts. Moreover, in the gonad no dietary HemNPs were detected whereas accumulation via waterborne HemNPs was significant. Despite the low assimilation efficiency of dietary HemNPs, biodynamic modeling showed that the diet was the main source of particle accumulation in zebrafish. Thus, both the life stage and the exposure route should be considered in evaluations of the environmental risks of NPs. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Bioaccumulation Exposure route Hematite nanoparticles Life stage Zebrafish
1. Introduction Engineered nanoparticles (NPs) are defined as particles with a diameter between 1 and 100 nm in at least two dimensions (Klaine et al., 2008). Due to their extraordinary physicochemical characteristics, NPs have a very wide range of applications such that their annual production is approaching one million tons (Keller and Lazareva, 2013). However, an inevitable consequence of the use and production of NPs has been their release into aquatic environments, where their potential risks to aquatic ecosystems have become a highly relevant concern. Several recent studies have investigated the toxic effects of NPs in aquatic organisms at different trophic levels (Skjolding et al., 2016), but there have been
* This paper has been recommended for acceptance by Sarah Harmon. * Corresponding author. School of the Environment, Nanjing University, Mail box 24, Xianlin Road 163, Nanjing 210023, Jiangsu Province, China. E-mail address: [email protected] (A.-J. Miao). 1 These authors contributed equally to this paper.
https://doi.org/10.1016/j.envpol.2019.113852 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
few systematic investigations of the toxicity of NPs at the different life stages of these organisms. Moreover, although the bioaccumulation kinetics of NPs is one of the most important determinants of toxicity, it has yet to be fully examined, including the contribution of different routes (e.g., waterborne vs. dietary pathways) of bioaccumulation. Zebrafish (Danio rerio) is a model aquatic organism in toxicological studies (Bar-Ilan et al., 2013; Griffitt et al., 2007; Wu et al., 2018; Yang et al., 2013). Its cardiovascular, nervous, and digestive systems are similar to those of mammals (Lewis and Eisen, 2003) and its genome has a high degree of similarity with that of humans (Renier et al., 2007). In addition, the three growth stages (embryo, larva, and adult) of zebrafish allow toxicity to be tested as a function of developmental stage. Embryogenesis in zebrafish occurs rapidly and is thus suitable to screen the developmental toxicity of pollutants (Kimmel et al., 1995). The most sensitive stage in toxicity testing is the larval stage (Zhang et al., 2003). In addition, zebrafish embryos and larvae are transparent, which facilitates direct in vivo monitoring of the development of cells, tissues, and organs
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(Fishman, 1999). Toxicological studies of adult zebrafish have largely focused on chronic effects, mostly to fill the data gaps resulting from studies restricted to embryonic and larval stages (Chen et al., 2011). Aquatic organisms accumulate pollutants mainly via waterborne (i.e., from the water directly) and dietary (i.e., from food) routes (Wang et al., 2018). Pollutants that accumulate through these two routes differ in their distribution patterns and toxicity (Liang et al., 2007). Wang et al. (2018) found that the toxicity of dietary arsenate to Daphnia magna was less than that of its waterborne counterpart. Such toxicity difference was explained by the fact that dietary arsenate had a lower total accumulation, a higher distribution in the gut and non-sensitive subcellular fractions, and a higher rate of transformation to less toxic arsenic species. However, the few studies of the bioaccumulation of NPs have not allowed a consensus to be reached regarding the relative importance of waterborne vs. dietary routes of accumulation. Liu et al. (2019) reported that waterborne Fe2O3 NPs accounted for > 73.6% of the Fe2O3 NPs that accumulated in D. magna. By contrast, in the study of Zhu et al. (2010), dietary exposure was the major route of accumulation of TiO2 NPs in D. rerio, although the biomagnification factor was only in the range of 0.009e0.024. In the present study, we systematically examined the waterborne and dietary accumulation kinetics (uptake, assimilation, and efflux) of well-dispersible polyacrylic acid (PAA)-coated hematite nanoparticles (HemNPs) in zebrafish at different life stages. The distribution of the accumulated HemNPs in the organs of adult zebrafish was also studied. HemNPs were chosen because of their wide-ranging applications, including in catalysis, pigments, gas sensors, water treatment, and medical research. In addition, the low toxicity (no observed effect concentration > 100 mg Fe/L for zebrafish) and negligible dissolution of HemNPs minimize the potential interference from the adverse effects of NPs and from the metal ions (Huang et al., 2016). Therefore, they are especially suitable for bioaccumulation studies. To improve the detection limit and exclude the potential interference of background Fe, 55Felabeled HemNPs with a detection limit at picogram level were used throughout the whole study. The protozoan Tetrahymena thermophila and the zooplankton D. magna served as food for larval and adult zebrafish, respectively. Both organisms are universally distributed in freshwater ecosystems and their ability to ingest NPs has been documented (Huang et al., 2018; Tan et al., 2016). The overall objectives of our study were to explore the contributions of waterborne and dietary routes to the bioaccumulation of NPs and to determine whether NPs accumulation in aquatic organisms
½HemNPsemb ¼
through a filtration system and maintained at a temperature of 28 ± 1 C. The fish were exposed to a light/dark cycle of 12/12 h and fed daily with Zeigler fish food. Selected male and female parent zebrafish were additionally fed daily with fresh Artemia salina larvae to supplement their nutrition until mating and spawning. After spawning, the fertilized embryos were collected 0.5 h postfertilization (hpf), washed with modified Holtfreter’s medium (MH, pH ¼ 7.0, Table S1, Supporting Information), and maintained at 28 ± 1 C in MH medium that was refreshed daily. At 4 days postfertilization (dpf), the embryos had mostly hatched, but the yolk was not consumed completely until 6 dpf. All of the following experiments described below were carried out in triplicate at 28 ± 1 C in fish exposed to a 12:12 h light/dark cycle. 55 Fe-labeled HemNPs (primary particle size, 5e10 nm; specific activity, 13.6 mCi g1 Fe; Fe proportion, 29%) were synthesized by titration hydrolysis and then PAA-coated, as described in a previous study by our group (Huang et al., 2016). The hydrodynamic diameter and zeta potential of HemNPs in the experimental medium (i.e., MH medium) were determined using a dynamic light scattering (DLS) particle sizer (ZetaPALS, Brookhaven Instruments, NY, USA). The morphology of the labeled HemNPs in MH was visualized by transmission electron microscopy (TEM; JEM-200CX, JEOL, Tokyo, Japan). 2.2. Accumulation of HemNPs in embryos Triplicate samples of zebrafish embryos (0.5 hpf) were exposed to 55Fe-labeled HemNPs at a concentration of 30 mg Fe L1 and sampled at 0.5, 1.5, 3, 6, 12, 24, 36, 48, and 72 h. Such concentration of HemNPs was selected to ensure reliable determination of their bioaccumulation at different life stages. The exposure duration was chosen to explore the effects of hatching on HemNPs accumulation. At each time point, 10 embryos were collected from each replicate and rinsed three times with MH to remove HemNPs weakly adsorbed on their surfaces. The embryos were dried at 60 C until a constant weight was reached, digested in 0.2 mL of concentrated HNO3, and diluted with Milli-Q water (18.2 MU) to 4 mL, such that the final HNO3 concentration was 3.5% (w/v). The radioactivity of 55 Fe in the digest was quantified using liquid scintillation counting (LSC, Tri-Carb 2800 TR; PerkinElmer, USA) after the addition to the samples of 10 mL of scintillation cocktail (Oxysolve C-400; Zinsser Analytic, Frankfurt, Germany) (Huang et al., 2017). The amount of HemNPs that accumulated in the embryos ([HemNPs]emb, mg Fe g1 dry weight, dw) was calculated according to Eq. (1):
Radioactivity per dry weight embryo ½HemNPsmed Radioactivity per liter experimental medium
changes as a function of their life stage. The results of this study contribute to a better understanding of the bioaccumulation of NPs in aquatic ecosystems and facilitate assessments of the ecological risks posed by these particles to aquatic organisms. 2. Materials and methods 2.1. Organisms and HemNPs Adult zebrafish (Danio rerio, AB strain) were purchased from the local fish market. Before the start of the experiments, they were acclimated for at least 2 weeks in aerated tap water circulated
(1)
where [HemNPs]med (mg Fe L1) is the HemNPs concentration in the experimental medium, which was determined at the beginning and end of the experiment. The average dry weight of an embryo was 68.6 mg, as obtained by weighing 500 embryos of 24 hpf in total. To determine whether the NPs were able to pass through the chorion and accumulate inside the embryo, another 10 embryos were collected after a 24-h exposure to 30 mg Fe L1 of HemNPs and rinsed three times with fresh MH. The chorion of each embryo was then removed using forceps under a stereomicroscope. The 55 Fe radioactivity in the chorion and in the dechorionated embryos was calculated according to Eq. (1).
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2.3. Accumulation of HemNPs in larvae 2.3.1. Waterborne accumulation experiment The experimental procedure for the waterborne uptake kinetics of HemNPs by 6-dpf larvae was similar to that described above for the embryo accumulation experiment. Briefly, 100 healthy larvae in each container were exposed to 30 mg Fe L1 of 55Fe-labeled HemNPs for 6, 12, 24, 36, 48, 60, 72, 84, and 96 h. At each time point, all 100 larvae from each of the three replicate containers were collected and washed three times with MH. The larvae were then euthanized with 0.02% (w/v) tricaine methanesulfonate (MS-222) and dried to a constant weight. The radioactivity (55Fe) in the digested larvae was measured by LSC and the result used to calculate larval HemNPs accumulation ([HemNPs]larva). To examine the concentration-dependent bioaccumulation of HemNPs, another 100 larvae in triplicate were exposed for 24 h to HemNPs at concentrations of 0.3, 1, 3, 10, and 30 mg Fe L1 after which [HemNPs]larva was determined as described above. The uptake rate of HemNPs at different [HemNPs]med was then calculated as [HemNPs]larva divided by the exposure time. 2.3.2. Dietary accumulation experiment Tetrahymena thermophila containing 55Fe-labeled HemNPs was prepared as described in the Supporting Information and then used at a concentration of 1 105 cells mL1 to feed 2400 healthy larvae equally dispensed into 48 containers (50 individuals in each container). The exposure medium and the food were refreshed daily during the 96-h experiment. Every 6 h, 50 larvae in triplicate were collected from three containers for the measurement of [HemNPs]larva. 2.3.3. Assimilation experiment The assimilation efficiency (AE) of HemNPs was determined via the pulse-feeding method (Wang and Fisher, 1999). Briefly, 100 healthy larvae in each of 21 containers were fed for 30 min with T. thermophila (1 105 cells mL1) containing 55Fe-labeled HemNPs (Supporting Information). The feeding time was selected based on the gut passage time (>1e1.5 h) of food in zebrafish larvae. Subsequently, the larvae were washed three times with MH and transferred into fresh medium with the same density of T. thermophila but without HemNPs. This depuration step lasted 24 h, during which time 100 individuals were collected from each of three containers after 0, 2, 4, 6, 8, 12, and 24 h and the HemNPs retained in the larvae were quantified. 2.4. Accumulation of HemNPs in adults 2.4.1. Waterborne accumulation experiment Adult fish (3e4 months, 2e3 cm) were acclimated in MH for at least 24 h before the uptake kinetics experiment. Sixty individuals in triplicate were exposed to HemNPs (30 mg Fe L1) in 600 mL of MH without food for 144 h. Every 24 h, 10 individuals from each replicate were collected for the determination of [HemNPs]adult. In addition to the uptake kinetics, the concentration-dependent accumulation of HemNPs was measured. For this purpose, 10 individuals in triplicate were exposed to 0.3, 1, 3, 10, and 30 mg Fe L1 of HemNPs for 24 h, after which [HemNPs]adult and the uptake rate were calculated. 2.4.2. Dietary accumulation experiment Daphnia magna containing 55Fe-labeled HemNPs was prepared as adult fish food as described in the Supporting Information. Seventy-two adult zebrafish dispensed in 72 containers (1 individual per container) were fed daily with 10 daphnids (~2.5% dry weight of each fish). Such ration of food was chosen to ensure that
3
the feeding behavior was completed within 15 min in case of significant depuration of HemNPs from the daphnids. The exposure medium was also refreshed daily during the 144-h experiment. Every 24 h, four individuals in triplicate (12 in total) were collected for the determination of [HemNPs]adult. 2.4.3. Assimilation experiment This experiment was similar to the larval assimilation experiment except that each container had one individual (84 in total) that was fed with 10 HemNPs-containing daphnids. Afterward, the medium was refreshed without any addition of HemNPs and the concentration of HemNPs retained in the fish (four individuals in triplicate) was monitored after 0, 2, 4, 6, 8, 12, and 24 h of depuration. 2.4.4. Efflux experiment Sixty adult fish in triplicate were exposed to 30 mg Fe L1 of HemNPs for 72 h. They were then collected, washed three times to remove the HemNPs weakly adsorbed on their surfaces, and transferred into fresh MH without HemNPs. This depuration step lasted 36 h, during which 10 individuals were collected at 0, 2, 4, 6, 12, and 36 h and the concentration of retained HemNPs was measured. The depuration medium was refreshed every 12 h to minimize the re-assimilation of HemNPs released from the fish. 2.4.5. Distribution experiment According to the results of the waterborne and dietary accumulation experiments, 20 adult zebrafish were collected either after being exposed to 30 mg Fe L1 of HemNPs for 3 days or after being fed HemNPs-containing daphnids for the same amount of time. The intestinal tract, gills, head, gonad, other viscera (heart, liver, pancreas, gallbladder, kidney, and swim bladder), and truncus (i.e., skin, scales, muscles, bones, and other residues) were isolated and the respective concentration of HemNPs was quantified as described in the accumulation experiment. Only the distribution of HemNPs in adult fish was examined herein, as the accumulation of HemNPs in larvae was low and the size of each larva was too small to be dissected. 2.5. Modeling and statistical analysis The data on HemNPs uptake, assimilation, and efflux were analyzed by biodynamic modeling (Dang et al., 2009; Fan and Reinfelder, 2003; Zhang and Wang, 2006). Detailed information on the calculation of the uptake (ku) and efflux (ke) rate constants, the kinetic order b, the AE, the food bioconcentration factor (BCFf), food intake rate (IR), and trophic transfer factor (TTF) is provided in the Supporting Information. Significant differences were defined as a p-value < 0.05, based on a Student’s t-test or a one-way analysis of variance with post-hoc multiple comparisons (Tukey or Tamhane; SPSS 11.0 by SPSS, Chicago, USA). The analysis of variance took into consideration both the normality (Kolmogorov-Smirnov and Shapiro-Wilk tests) and the homogeneity of variance (Levene’s test) of the data. 3. Results 3.1. Physicochemical properties of HemNPs The TEM results showed that HemNPs were well dispersed in MH, with an average size of 10.1 nm, based on the measurement of at least 1000 particles randomly chosen from the TEM images (Fig. 1a). The size was also consistent with the DLS results, according to which the average hydrodynamic diameter was 33.2 ± 1.1 nm (Fig. 1b) and the zeta potential was 26 mV.
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Fig. 1. (a) Transmission electron microscopy images of hematite nanoparticles in modified Holtfreter’s medium. (b) The hydrodiameter distribution of the particles as obtained by dynamic light scattering.
3.2. HemNPs accumulation in embryos Exposure of the embryos to 30 mg Fe L1 of HemNPs resulted in an increase in [HemNPs]emb from 0 mg Fe g1 dw at the beginning to 1264.1 mg Fe g1 dw at 36 h (Fig. 2a). Thereafter [HemNPs]emb decreased steadily, to 621.4 mg Fe g1 dw at 48 h, at which time the embryos hatched, and further to 44.2 mg Fe g1 dw at the end. As for the HemNPs accumulated in the embryos, most of them (92.3%) were found in the chorion, with only 0.4% in the embryonic cells (Fig. 2b).
Fig. 2. (a) Changes in the concentration of HemNPs in embryos ([HemNPs]emb) during a 72-h exposure to 30 mg Fe L1 of HemNPs. (b) The distribution of HemNPs in the chorion (CH) and in dechorionated embryos (DE) after a 24-h exposure to 30 mg Fe L1 of HemNPs. The data are the mean ± standard deviation (n ¼ 3).
phases to 151.6 mg Fe g1 dw during the third 24-h phase and further to 223.5 mg Fe g1 dw over the last 24 h. In the assimilation experiment, a substantial amount of HemNPs (45.6 mg Fe g1 dw) accumulated in the larvae after their pulse feeding. Transfer of the HemNPs-containing larvae to fresh medium without HemNPs resulted in a rapid decrease in [HemNPs]larva, to 1.5 mg Fe g1 dw, after 12 h of depuration. Thereafter, the value leveled off. The AE (proportion of HemNPs retained in the larvae at the end of depuration) thus obtained was 4.1% (Fig. 3d). 3.4. HemNPs accumulation in adult fish
3.3. HemNPs accumulation in larvae In the waterborne accumulation experiment, [HemNPs]larva increased linearly, from 0 mg Fe g1 dw at the beginning to 66.7 mg Fe g1 dw at 84 h, and then slightly decreased until the end of the experiment, to 49.3 mg Fe g1 dw (Fig. 3a). HemNPs accumulation was concentration-dependent, indicated by the approximately 8fold increase in [HemNPs]larva as [HemNPs]med increased from 0.3 to 30 mg Fe L1. Accordingly, on an exponential scale, the uptake rate increased linearly with increasing [HemNPs]med (Fig. 3b); the uptake rate constant ku was 5.00 L kg1 d1. Dietary accumulation was more complicated than waterborne accumulation with respect to the time-related change of [HemNPs]larva. [HemNPs]larva reached a maximum between each food refreshment and the maximum value increased with increasing exposure time (Fig. 3c). Thus, [HemNPs]larva increased from 0 mg Fe g1 dw at the beginning to 112.6 mg Fe g1 dw at 18 h and then decreased to 55.8 mg Fe g1 dw until the next food refreshment at 24 h. The maximum value of [HemNPs]larva increased from 82.8 to 112.6 mg Fe g1 dw for the first two 24-h
Similar to the waterborne accumulation of HemNPs in larvae, [HemNPs]adult increased linearly with exposure time during the first 72 h of the 144-h exposure of the fish to 30 mg Fe L1 of HemNPs. This was followed by a steady decrease from the maximum (10.4 mg Fe g1 dw) to 3.4 mg Fe g1 dw at the end of the experiment (Fig. 4a). The accumulation of HemNPs increased as [HemNPs]med increased, resulting in a linear correlation on an exponential scale between the uptake rate of HemNPs and [HemNPs]med (Fig. 4b). The uptake rate constant ku thus obtained was 0.66 L kg1 d1. Similar to larval accumulation, an irregular correlation between [HemNPs]adult and exposure time was determined in the dietary accumulation experiment. Namely, [HemNPs]adult increased quickly, to 34.0e48.2 mg Fe g1 dw, after the daily feeding of adult fish with HemNPs-containing daphnids and then steadily decreased, to 0.27e0.56 mg Fe g1 dw, until the next feeding (Fig. 4c). An assimilation experiment was then performed to measure the proportion of dietary HemNPs subsequently utilized by the adult zebrafish. As shown in Fig. 4d, [HemNPs]adult decreased
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Fig. 3. (a) Changes in the concentration of HemNPs in larvae ([HemNPs]larva) during a 96-h exposure of the larvae to 30 mg Fe L1 of HemNPs. (b) The uptake rates of HemNPs in larvae at different HemNPs concentrations in the experimental medium ([HemNPs]med of 0.3, 1, 3, 10, and 30 mg Fe L1). (c) Changes in [HemNPs]larva in larvae fed daily with HemNPs-containing Tetrahymena thermophila during a 96-h period. (d) The proportion of HemNPs retained in the larvae after depuration for 0, 2, 4, 6, 8, 12, and 24 h in the assimilation experiment. The dashed line in (b) is the linear regression between the HemNPs uptake rate and [HemNPs]med on an exponential scale. The data are the mean ± standard deviation (n ¼ 3).
quickly within the first 4 h of the 24-h depuration period but then remained relatively constant. Accordingly, 2.9% of the initially accumulated HemNPs was retained at the end of the assimilation experiment. Given this low accumulation of HemNPs from the dietary route, only the efflux of the particles accumulated through the waterborne source was examined. After a 3-day exposure to 30 mg Fe L1 of HemNPs, [HemNPs]adult was approximately 10.4 mg Fe g1 dw but it decreased in two phases when the fish were transferred to depuration medium (Fig. 4e). The efflux rate constants (ke) thus obtained for the fast and slow phases were 0.056 (ke1) and 0.012 h1 (ke2), respectively. An analysis of the tissue distribution of the HemNPs showed that they had mainly accumulated in the intestinal tract (78.7 and 94.2%), followed by the truncus (6.8% and 1.7%), head (6.8% and 0.9%), and gills (3.2% and 0.4%), regardless of the accumulation route (Fig. 4f). However, a comparison of the two routes showed the accumulation of significantly (p < 0.05, t-test) more waterborne than dietary HemNPs in the gills, truncus, and gonad. In fact, in the gonad, while the waterborne route accounted for 1.7% of the accumulated HemNPs there was no accumulation in this tissue via the dietary route. Although the difference was not significant (p > 0.05, t-test), a lower concentration of waterborne than of dietary HemNPs in the intestinal tract was observed.
4. Discussion 4.1. Waterborne accumulation In the present study, HemNPs mainly accumulated in the zebrafish chorion (>92%), with only 0.4% or less detected inside the embryos, indicating that the chorion served as a barrier to HemNPs entry into the embryos (Kashiwada, 2006). This barrier effect can be explained by the unique structure of the chorion, whose three protein layers have a total thickness of 1.5e2.5 mm. The inner and middle layers contain regularly arranged tapered pores (diameter, 0.5e0.7 mm) whose narrow ends face outwards. By contrast, the outer layer is powdery, which blocks the pores and thus presumably prevented HemNPs from entering the perivitelline space (Rawson et al., 2000). In addition, the internalization of HemNPs may have been inhibited by their complexation with the chorion (Huang et al., 2015). Auffan et al. (2014) found that AgNPs adsorbed onto the surface of the chorion via their complexation with the thiol group of chorionic proteins. The adsorption of HemNPs onto the chorion may have resulted in the reconstitution of its outer layer via the secretion by the embryo of additional substances, resulting in the steady absorption of the NPs (Fig. 2a) (Chang and Huang, 2002; Zhao et al., 2016). When the chorions were shed from the embryos, the larvae thus obtained were able to orally ingest the particles, providing a new pathway for their accumulation from the water. In addition, because the skin and branchial membrane of the larvae were not
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Fig. 4. (a) Changes in the concentration of HemNPs in adult fish ([HemNPs]adult) exposed for 144 h to 30 mg Fe L1 of HemNPs. (b) The uptake rates of HemNPs in adult fish at [HemNPs]med of 0.3, 1, 3, 10, and 30 mg Fe L1. (c) Changes in [HemNPs]adult in adult fish fed daily with HemNPs-containing Daphnia magna during a 144-h period. (d) The proportion of HemNPs retained in adult fish after depuration for 0, 2, 4, 6, 8, 12, and 24 h in the assimilation experiment. (e) The proportion of HemNPs retained in adult fish after depuration for 0, 2, 4, 6, 12, and 36 h in the efflux experiment. (f) The distribution of HemNPs in the gills, gonad, intestinal tract (IT), other viscera (OV), truncus, and head of adult zebrafish exposed to 30 mg Fe L1 of HemNPs or fed for 3 days with HemNPs-containing D. magna. The dashed line in (b) is the linear regression between the HemNPs uptake rate and [HemNPs]med on an exponential scale. The dashed lines in (e) are the bilinear regression between the proportion of HemNPs retained in the fish (exponential scale) and the depuration time in the efflux experiment. The data are the mean ± standard deviation (n ¼ 3).
fully developed, the weak barrier effects of these structures may have allowed the direct entry of the HemNPs (Chetty et al., 2016). Kwok et al. (2012) found that in juvenile Japanese medaka (Oryzias latipes) exposed to AgNPs, the particles were mainly concentrated in the gills and intestine. Those authors speculated that the gills were the initial site of AgNPs absorption. However, in our study, regardless of the new uptake routes that emerged at the larval stage, the amount of bioaccumulated HemNPs was much less in the
larvae than in the embryos, due to the loss of the chorion and thus of its high accumulation capacity (Bohme et al., 2015). Unlike in the larvae, in the adult fish the gills and epidermis were mature, which would have hindered the accumulation of HemNPs. This explains why only 4.0% and 6.8% of the total accumulated HemNPs were found in the gills and truncus, respectively. Similarly, Zhao et al. (2011) reported that the amount of CuO NPs that had accumulated in the skin and scale of carp (Cyprinus carpio)
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accounted for only 1.6% of total NPs accumulation. Overall, the intestinal tract was the dominant site of HemNPs accumulation in adult fish (Zhang et al., 2015). However, a substantial fraction of the HemNPs was subsequently depurated in two distinct phases, indicative of the distribution of the particles in different organs (Liu et al., 2019). Fan et al. (2016) also reported the two-phase eliminations of TiO2 NPs by D. magna: rapidly during the first 6 h and then gradually during the following 18 h of a 24-h efflux period. The authors proposed that the fast efflux corresponded to particles concentrated in the gut (Khan et al., 2015) and the slow efflux to particles adsorbed on non-gut tissues, microvilli, or the peritrophic membrane (Isaacson et al., 2017). The decreasing accumulation of HemNPs from the embryonic to the larval and then to the adult stage may result from a decreased specific surface area with age or a progressive increase in the zebrafish’s ability to regulate the bioaccumulation of NPs. A 24-h exposure of the embryos, larvae, and adults to 30 mg Fe L1 of HemNPs resulted in a HemNPs content of 1140.7, 21.9, and 4.6 mg Fe g1 dw, respectively. Growth-dependent accumulation was further evidenced by the values of the uptake rate constant ku and the kinetic coefficient b of the larval and adult fish. The coefficient b indicates the saturation degree of HemNPs uptake: the smaller the value of b, the more saturated the accumulation (Zhang and Wang, 2006). In the present study, the ku (b) of larval and adult fish was 5.00 L kg1 d1 (0.42) and 0.66 L kg1 d1 (0.50), respectively. Thus, the uptake of HemNPs by adult fish was slower and saturation degree was also lower, compared with the uptake by the larvae. However, despite the decrease in HemNPs accumulation after it reached a maximum in all three zebrafish life stages, the underlying mechanisms differed between embryos and larvae/adults. Namely, the decrease in the HemNPs content in embryos was due to the loss of the chorion at the end of the uptake experiment, while in larval and adult zebrafish the efflux of the HemNPs accounted for the loss. 4.2. Dietary accumulation HemNPs accumulation through the dietary route in the larval stage was characterized by a periodic fluctuation together with a time-related increase in the maximum value of each 24-h cycle. This can be explained by the low rate of assimilation (i.e., quick depuration of foods from the gut) and the growth-dependent increase in predation (Fig. 3c). The latter resulted in an increase of HemNPs accumulation after each food refreshment. Thus, as predation ability increased with the increasing age of the zebrafish, the maximum HemNPs accumulation during each food refreshment cycle increased as well. Reitan et al. (1998) also found that the increase in the specific growth rate of turbot (Scophthalmus maximus) larvae was accompanied by an increase in the larval drinking rate, from 14 nL larva1 h1 at day 2 post-hatching to 120 nL larva1 h1 at day 11 post-hatching. However, the low AE and quick efflux of the HemNPs in the larvae resulted in a significant decline in particle accumulation after the maximum during each food refreshment cycle. Low AE values (<6%) were also reported by Wang and Wang (2014) in their study of the uptake of AgNPs of different sizes by marine medaka (Oryzias melastigma). The variation in HemNPs taken up by adult zebrafish via the dietary route was similar to that by larvae. The only difference was the unchanged maximum during each feeding cycle, as there was no significant growth of the adult fish during the experimental period. The dietary accumulation of HemNPs by adult fish was less than that by the larvae, which can be attributed to the lower AE of the adults (2.9% vs. 4.1%). This difference demonstrated the better ability of adult fish to regulate particle accumulation. Nevertheless, the possibility of a lower HemNPs content in the food of the adult fish than in the larval food cannot be excluded. The low AEs of
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HemNPs in both larvae and adults also indicated a low transport efficiency of HemNPs from the food to either life stage. This conclusion was supported in the adult fish by the very low TTF (4.4 104 ≪ 1), which indicated that there was no biomagnification of HemNPs under the conditions of the present study. Most of the studies reported in the literature have similarly shown a low transport efficiency of NPs along the food chain of aquatic organisms and thus a very low risk of biomagnification. Zhu et al. (2010) studied the trophic transfer of TiO2 NPs from daphnia to zebrafish and determined a TTF of only 0.009e0.024. A low transport efficiency of only 0.01e0.04 for dietary AgNPs was also reported by Wang and Wang, 2014, but the authors observed that the particles still inhibited the enzyme activity of medaka and reduced the growth of the fish. Together, these results demonstrate that in assessments of the ecological risk of NPs dietary accumulation routes cannot be ignored.
4.3. Waterborne vs. dietary accumulation A number of studies have examined the distribution of either waterborne or dietary NPs in fish and reported results similar to ours. Nevertheless, the two routes have only rarely been compared directly. The present study examined both waterborne and dietary uptake and showed that, although HemNPs accumulated via both routes were mainly concentrated in the zebrafish intestine, dietary uptake resulted in higher intestinal concentrations than direct uptake from the water phase. In the latter, however, HemNPs were more easily transported to other organs of the fish, including the gonad, which may result in biological effects distinct from those associated with dietary ingestion. Wu et al. (2018) reported that the aqueous-phase accumulation of TiO2 NPs in the zebrafish gonad affected not only the zebrafish itself but also its offspring. To further investigate the differences in the contributions of waterborne and dietary routes to HemNPs accumulation in zebrafish, the fraction of particles accumulated through food ingestion (ff) was calculated (Supporting Information). Fixing the value of BCFf (i.e., 10, 100, and 1000 L kg1) allowed ff at different [HemNPs]med to be calculated. As shown in Fig. 5, at a low [HemNPs]med and a high BCF, the dietary route accounted for most of the accumulation of HemNPs. This may in fact be the case in aquatic environments in nature, given their generally low NP concentrations and BCF values typically >10 L kg1. Our simulation further demonstrates the need to consider dietary accumulation in evaluations of the ecological risks of NPs.
Fig. 5. The contribution of dietary HemNPs to the total accumulation of HemNPs in adult zebrafish (ff) at different ambient concentrations of HemNPs ([HemNPs]med) under a BCFf of 10, 100, and 1000 L kg1, as predicted by biodynamic modeling.
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5. Conclusion The present study compared the accumulation kinetics of waterborne and dietary HemNPs in zebrafish at three different life stages. Although waterborne HemNPs accumulated to a greater extent in embryos than in either larvae or adults, most of the particles were shed together with the chorion during hatching and thus posed little risk to the fish. The decreasing concentration of waterborne and dietary HemNPs from the larval to the adult stage may result from a decreased specific surface area with age or the better ability of mature fish to regulate the bioaccumulation of HemNPs. In contrast to the uptake kinetics of waterborne particles, which showed a linear correlation between HemNPs accumulation and initial exposure time, HemNPs accumulation through the dietary route fluctuated, indicative of a low AE and a high predation rate during each food refreshment cycle. Despite the low AE of dietary HemNPs, the contribution of this route to HemNPs accumulation in zebrafish was much greater than that of the waterborne route, according to biodynamic modeling. Overall, both waterborne and dietary routes as well as differences in the NPs accumulation ability of organisms at different life stages should be considered in evaluations of the environmental risks of HemNPs. Declaration of competing interest There are no conflicts of interest to declare. CRediT authorship contribution statement Bin Huang: Writing - original draft, Investigation, Formal analysis. Yu-Qing Cui: Methodology, Investigation, Formal analysis. Wen-Bo Guo: Investigation. Liuyan Yang: Writing - review & editing, Funding acquisition. Ai-Jun Miao: Conceptualization, Methodology, Supervision, Writing - review & editing. Acknowledgements We thank three reviewers for their constructive suggestions on this paper. This work was supported by the National Natural Science Foundation of China (21822605, 21677068, and 41807479), Chinese Public Science and Technology Research Funds for Ocean Projects (201505034), the China Postdoctoral Science Foundation (2017M622781), and the State Key Laboratory of Pollution Control and Resource Reuse Foundation (PCRRF18020). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113852. References Auffan, M., Matson, C.W., Rose, J., Arnold, M., Proux, O., Fayard, B., Liu, W., Chaurand, P., Wiesner, M.R., Bottero, J.Y., Di Giulio, R.T., 2014. Salinity-dependent silver nanoparticle uptake and transformation by Atlantic killifish (Fundulus heteroclitus) embryos. Nanotoxicology 8, 167e176. Bar-Ilan, O., Chuang, C.C., Schwahn, D.J., Yang, S., Joshi, S., Pedersen, J.A., Hamers, R.J., Peterson, R.E., Heideman, W., 2013. TiO2 Nanoparticle exposure and illumination during zebrafish development: mortality at parts per billion concentrations. Environ. Sci. Technol. 47, 4726e4733. Bohme, S., Stark, H.J., Reemtsma, T., Kuhnel, D., 2015. Effect propagation after silver nanoparticle exposure in zebrafish (Danio rerio) embryos: a correlation to internal concentration and distribution patterns. Environ. Sci. -Nano 2, 603e614. Chang, Y.S., Huang, F.L., 2002. Fibroin-like substance is a major component of the outer layer of fertilization envelope via which carp egg adheres to the substratum. Mol. Reprod. Dev. 62, 397e406. Chen, J.Y., Dong, X., Xin, Y.Y., Zhao, M.R., 2011. Effects of titanium dioxide nanoparticles on growth and some histological parameters of zebrafish (Danio rerio) after a long-term exposure. Aquat. Toxicol. 101, 493e499.
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