Bioaccumulation, biodistribution，and depuration of 13C-labelled fullerenols in zebrafish through dietary exposure

Ecotoxicology and Environmental Safety 191 (2020) 110173

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Bioaccumulation, biodistribution，and depuration of fullerenols in zebrafish through dietary exposure

13

C-labelled

T

Qiuyue Shia,c, Han Zhanga, Chenglong Wangb, Hongyun Rena, Changzhou Yana, Xian Zhanga,∗, Xue-Ling Changb,∗∗ a

Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China Key Lab for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China c University of Chinese Academy of Sciences, Beijing, 100049, China b

ARTICLE INFO

ABSTRACT

Keywords: Fullerenols Dietary exposure Accumulation Depuration Tissues

In aquatic organisms, dietary exposure to nanomaterials is not only one of the important uptake pathways, but it is also one method to assess the transmission risk of the food chain. To address this concern, we quantitatively investigated the accumulation and depuration of fullerenols in the tissues of zebrafish after exposure to fullerenols-contaminated Daphnia magna. After exposure to 13C-labelled fullerenol solution at a concentration of 2.5 mg/L for 72 h, the steady state concentration of fullerenols in D. magna was 31.20 ± 1.59 mg/g dry weight. During the 28 d uptake period for zebrafish, fullerenols in the tissues increased in a tissue- and day-dependent manner, and the major target tissues of fullerenols were the intestines and liver, followed by the gill, muscle, and brain. The kinetic parameters of uptake and depuration were also quantitatively analyzed. After depuration for 15 d, a certain amount of residual fullerenols remained in the tissues, especially the brain, where approximately 64 d may be needed to achieve 90% of the cumulative concentration depuration. The calculated distributionbased trophic transfer factors (TTFd values) (from 0.26 to 0.49) indicated that the tissue biomagnification of fullerenols by zebrafish through dietary exposure may not occur. Transmission electron microscopy (TEM) confirmed the presence of fullerenols in D. magna and the tissues of zebrafish. Our research data are essential for thoroughly understanding of the fate of nanoparticles through the dietary exposure pathway and directing future tissue bioeffect studies regarding target tissues for further research.

1. Introduction Increased environmental release of nanomaterials is a likely outcome of the growth in manufacture and use of nanomaterials in commerce，which have raised concerns about their health risks and environmental impacts (Nel et al., 2013; Wang et al., 2012). Understanding the environmental fate, transformation, and potential for accumulation and trophic transfer is necessary for environmental risk assessment of nanomaterials (Nanna et al., 2014). Carbon nanomaterials as star materials in nanomaterials, have become one of the most widely used commercial nanomaterials in the world because of their unique physical and chemical properties (Dai et al., 2012; Jariwala et al., 2013). With the release of carbon nanomaterials into the aquatic environment, which will lead to bioaccumulation，biological effects, toxicity and hazards to human health through the trophic

transfer of the food chain. At present, there have been many studies published on the accumulation, biological effects and toxicity of carbon nanomaterials (such as fullerenes) in aquatic organisms (Sumi and Chitra, 2017; Tervonen et al., 2010; Yamawaki and Iwai, 2006). The carbon nanoparticles in the aquatic environment (such as fullerenes and carbon nanotubes) have been found, and the predicted concentration of fullerenes was up to 0.31 μg/L (Pérez et al., 2009). To understand the behaviour of carbon nanomaterials in organisms and aquatic environment, it is necessary to study the biological processes of nanomaterials. Studying the biological processes of nanoparticles in organisms is not only conducive to revealing the fate of nanoparticles, but also conducive to revealing their toxic mechanism. The biological processes of nanomaterials in organisms include absorption, bioaccumulation, biodistribution, metabolism, and excretion. Previous reports have suggested that exposure pathways of nanomaterials to organisms can affect

Corresponding author. Corresponding author. E-mail addresses: [email protected] (Q. Shi), [email protected] (H. Zhang), [email protected] (C. Wang), [email protected] (H. Ren), [email protected] (C. Yan), [email protected] (X. Zhang), [email protected] (X.-L. Chang). ∗

∗∗

https://doi.org/10.1016/j.ecoenv.2020.110173 Received 2 July 2019; Received in revised form 25 December 2019; Accepted 2 January 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

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their biological processes (Gupta et al., 2017; Hou et al., 2013). In aquatic organisms, there are two exposure pathways: the aqueous and dietary pathway. Current studies of other nanomaterials have suggested that the biodistribution of nanoparticles in fish occur more easily by the dietary exposure pathway than through the aqueous exposure pathway (Federici et al., 2007; Ramsden et al., 2009). Therefore, the potential exposure risk to fish tissues through dietary exposure is greater than that of aqueous exposure. Dietary exposure facilitates nanoparticle accumulation in internal tissues, which enhances the risk of tissue bioeffect or toxicity. Thus, it is important to study the distribution of nanomaterials in organisms through the dietary exposure pathway to assess the biological effects and ecological risk. At present, research on the distribution of carbon nanomaterials is just at the level of distribution or/and accumulation through different administration routes. For instance, the distribution of water-miscible fullerene after intravenous administration to rats showed that it was distributed rapidly to various tissues in a time-dependent manner, and most of the material was retained in the body after one week (Yamago et al., 1995). Similarly, C60–OH translocated to a much wider range of tissues after intraperitoneal injection, including the stomach, kidneys, lung, intestine, blood, muscle and bone, where it showed high accumulation levels (Wang et al., 2016). Despite the studies of carbon nanoparticle distribution that have been conducted in vertebrates (Wang et al., 2016; Yamago et al., 1995), there is a key knowledge gap: the lack of in-depth studies on the biological processes of carbon nanomaterials in organisms such as biodistribution, uptake and depuration kinetics via the dietary exposure pathway. This information is important to improve our understanding of the environmental fate of carbon nanoparticles and to direct future biological effect or toxicity studies regarding the target tissues. To address a gap in knowledge in carbon nanomaterials biodistribution and bioaccumulation in organisms through dietary exposure pathway, this study examined the biological processes involved in the uptake, distribution, depuration and trophic transfer of one class of CNBs, fullerenols, to zebrafish exposed through dietary pathway. Fullerenols were selected as the target nanomaterials because they had more potential applications in the field of life science as a result of their extensive range of biological activities (Gao et al., 2011; Injac et al., 2009; Jiao et al., 2010). Moreover, in our previous study, it was demonstrated that there was high bioaccumulation of 13C-labelled fullerenols in the bodies of D. magna after exposure in artificial freshwater, which may pose a potential exposure risk to their predators, such as fish (Du et al., 2016). Stable isotope (13C) labelling of the carbon skeleton is a well-established approach for the quantification of carbon nanomaterials in biological samples by analyzing the 13C/12C ratio with isotope ratio mass spectrometry (IRMS) (Chen et al., 2017; Wang et al., 2018). The results of this study will provide quantitative information to fully understand the ecological risks and underlying biological effects of carbon nanomaterials.

maintained in artificial freshwater aerated for 3 d at 20 ± 1 °C with a 16:8 h light/dark cycle. D. magna were fed once a day with a green algae culture of Scenedesmus obliquus. The 7-day-old D. magna were used for the uptake experiments. Adult zebrafish (length 2.90 ± 0.4 cm; weight 0.178 ± 0.012 g (mean ± SD) were obtained from the State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Science. Zebrafish (80 individuals) were acclimated to the laboratory conditions as described by Du et al. (2012). Feeding ceased 3 d before exposure. 2.3. Preparation and analysis of fullerenol solutions A stock solution of fullerenols was made by adding 10 mg of 13Clabelled fullerenols to 1000 mL of artificial freshwater (KCl, 1.2 mg/L; CaCl2·2H2O, 58.6 mg/L; NaHCO3, 13.0 mg/L; and MgSO4·2H2O, 24.5 mg/L) (Petersen et al., 2009). Then, the stock solution of 13C-labelled fullerenols was diluted with artificial freshwater to a final concentration of 2.5 mg/L, which was the exposure concentration chosen for the uptake experiment. The exposure concentration was selected based on the mortality and accumulation amount of the preliminary experiment. After sonication in an ice bath using an ultrasonic cell pulveriser (VCX800, Sonics) for 10 min, the size of the fullerenol agglomerates remained stable during the period of the exposure experiment. The morphology of fullerenols in the stock solution was characterized by transmission electron microscopy (TEM, Tecnai F30, Philips-FEI, Netherlands) with 120 kv accelerating voltage. Particle size distributions of the fullerenols in the exposure solutions were measured by a Zetasizer Nano ZS system and dynamic light scattering (DLS) (ZEN 3600, Malvern, U.K.). The fullerenols remained stable in artificial freshwater because no precipitate was observed after 72 h. 2.4. Bioaccumulation of

13

C-labelled fullerenols in Daphnia magna

The aim of our bioaccumulation experiment was to confirm the time to steady state (proper exposure time) and dietary 13C-labelled fullerenols content (initial nominal dietary fullerenols) for sequential analysis. The time to steady state was used in the preparation of the diet, including 13C-labelled fullerenols, for zebrafish consumption. We exposed 7-day-old D. magna to a13C-labelled fullerenol solution of 2.5 mg/ L. Specifically, D. magna were transferred to clean artificial freshwater for 2 h to allow for intestine purging and acclimated after 1 d without feeding. A total of 100 organisms were added to each glass beaker containing 1 L of artificial freshwater with 2.5 mg of 13C-labelled fullerenols. No feeding occurred during the uptake period. Five D. magna from each glass beaker were sampled after 2, 5, 8, 12, 16, 24, 36, 48 and 72 h of exposure. The water quality itself did not change throughout water quality parameter measurements (such as pH 7.64, dissolved oxygen 7.23 mg/L, total nitrogen 21.45 mg/L and total phosphorus 0.02 mg/L). The sampled D. magna were placed in clean water and pipetted vigorously to remove fullerenol nanoparticles attached to their carapaces. Following this, D. magna were freeze-dried in a freeze dryer (Boyikang, Beijing, China) and weighed (Mettler Toledo microbalance) into tin capsules for carbon isotope analysis to determine the 13C-labelled fullerenols concentration within them. After 36 h of exposure, another five D. magna from each beaker were sampled and observed using a transmission electron microscope (TEM) (H-7650, Hitachi, Japan). The time at which steady state was reached was determined to be 24 h.

2. Materials and methods 2.1. Materials 13

C-labelled fullerenols were obtained from the Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Science. The methods for the synthesis of 13C-labelled fullerenols have been described by Wang et al. (2014). Additionally, in order to remove impurity (such as toluene) in the synthesis of fullerenols, vacuum evaporation at 0.009 MPa and multiple washing were done.

2.5. Diets for zebrafish with/without

13

C-labelled fullerenols

Based on the trophic relationship between zebrafish (predator) and D. magna (prey), the D. magna accumulated 13C-labelled fullerenols were prepared as diets for zebrafish consumption. According to the steady state time (24 h) of uptake confirmed above, 7-day-old D. magna were exposed to 2.5 mg/L13C-labelled fullerenols for 24 h, then filtered and rinsed five times with clean water to be used for the zebrafish diets.

2.2. Experimental animal D. magna were obtained from the State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. In detail, D. magna were 2

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D. magna with/without 13C-labelled fullerenols were used in the following dietary exposure experiment.

were then calculated according to the w13c values of the 13C-fullerenols, D. magna, and zebrafish tissues using eq (1) and expressed as milligrams per gram of dry weight (mg/g).

2.6. Dietary acclimation of zebrafish to D. magna Eighty zebrafish were placed in aquarium (50 L) containing aerated artificial freshwater at 25 °C with a photoperiod of 14:10 h light/dark based on the Code for Zebrafish Culture, China Zebrafish Resource Centre. Zebrafish were acclimated to feed on D. magna once a day for 2 weeks. The average feed weight for each fish per day was 0.01 g wet weight D. magna (approximately 17 D. magna), which was within the feed rate of 3%–5%. Fish faeces in the aquarium were removed every day, and 80% of the volume was renewed once every 2 d. The aquarium was cleaned once a week. 2.7. Dietary exposure of zebrafish to

w13c (control)

w13c (fullerenols )

(1)

2.10. Data analysis Statistical analysis was conducted using the statistical software package SPSS, version 13.0 (SPSS, Chicago, IL). All data are expressed as the mean values of three individual observations together with the associated standard deviation (mean ± SD). Only when data were acceptable was statistical analysis performed by one-way analysis of variance (ANOVA) to evaluate significant differences between tissues. Two-way ANOVA was performed to evaluate the significant effect of time on the accumulation of fullerenols. ANOVA followed by Tukey's post-hoc test was performed to examine differences in the body burden of fullerenols bioaccumulation in D. magna at 24–72 h. The criterion for statistical significance was p < 0.05. Kinetic analysis was performed on the basis of fullerenols concentration in each tissue after dietary exposure using the pseudo-firstorder model, which is the classical kinetics model. The uptake rate constant (ku ) and depuration rate constant (ke ) were calculated from the first-order model with the following two equations (Yamada et al., 1994).

13

C-labelled fullerenols

Experimental culture conditions for zebrafish were the same as those under the acclimated conditions. We divided the eighty zebrafish acclimated to feed on D. magna into two groups: 40 zebrafish were fed D. magna with 13C-labelled fullerenols, while 40 zebrafish were fed D. magna without 13C-labelled fullerenols. Each group was set up in triplicate. In detail, each group of exposed zebrafish was evenly allocated to three glass experimental tanks. The remaining four zebrafish in each group were used as reserve. The average feed weight for each zebrafish per day was 0.01 g wet weight of D. magna (approximately 17 D. magna). Zebrafish were fed D. magna without fullerenols during the depuration period. No residual food remained during the experiment. Three zebrafish were selected randomly as samples from each group after feeding for 24 h on days 2, 7, 14, 21, 28, 31, 33, 37 and 43. Several tissue samples, such as the liver, intestine, gill, brain and muscle, were collected and dissected from the zebrafish. Following this, all tissue samples were freeze-dried in a freeze dryer (Boyikang, Beijing, China) and weighed (using a Mettler Toledo microbalance) into tin capsules for carbon isotope analysis to determine the 13C -labelled fullerenols concentrations within them.

Ct1 =

CF ku (1 ke

Ct 2 = Ct 3 e

e

ke t

)

ke t

(2) (3)

where Ct1 is the 13C-fullerenols concentration in the tissues by dietary uptake at the time of the uptake experiment; Ct3 is the 13C-fullerenols concentration in the tissues at the beginning of the depuration experiment; Ct2 is the 13C-fullerenols concentration in the tissues at time of the depuration experiment; t is the period of the uptake or depuration experiment; and CF is the 13C-fullerenols concentration in the feed. The half-live (t1/2 ) and fitted plateau value (Cf , equilibrium concentration) were calculated with the following two equation.

2.8. Localization of fullerenols in zebrafish To further study the fate of the fullerenols in the zebrafish body, TEM was used to determine the location and pathway of particle uptake across the gastrointestinal tract. Representative portions of the liver, intestine, gill, brain, and muscle of experimental and normal (control) zebrafish were fixed in 2.5% glutaraldehyde for 2 h at 4 °C. Samples were then rinsed with 0.1 M PBS 2 times for 10 min each time. The cleared samples were again fixed in 1% osmic acid for 1 h. Then, the samples were rinsed with 0.1 M PBS twice for 10 min each time, and dehydrated through a graded ethanol series and acetone. Samples were then embedded in Epon 812 substitute. Ultrathin sections of approximately 50–70 nm were cut with a glass knife on a Leica EM UC7 ultramicrotome and transferred to 200-mesh copper EM finder grids. Sections were stained with only uranyl acetate. Finally, sections were viewed on a Hitachi H-7650 TEM operating at 80 kV accelerating voltage. 2.9. Quantification of

w13c (sample )

wfullerenols =

t1/2 =

Cf =

log e 0.5 ke

CF ku ke

(4) (5)

The distribution-based trophic transfer factor (TTFd, unitless) was calculated to evaluate the trophic transfer in the specific tissues by eq (6).

TTFd = Cd1/ Cd2

(6)

where TTFd is the trophic transfer factor on day n (n = 28); Cd1 is the fullerenols concentration in the specific tissue (mg/g dry weight) and Cd2 is the fullerenols concentration in the feed (mg/g dry weight). 3. Results

13

C-labelled fullerenols

3.1. Characterization of fullerenols

The quantification of 13C-labelled fullerenols was conducted according to the method described by Chang et al. (2014) with slight modifications. Carbon isotope ratios (13C/12C) in D. magna and zebrafish were determined by isotope-ratio mass spectrometry (IRMS, Delta V Advantage, Thermo Fisher Scientific, Bremen, Germany). Moreover, the C elemental contents were measured according to the peak areas obtained from mass spectrometry. Labelled urea was analyzed as a check standard for the accuracy and precision of the isotope ratios after every 12 samples. According to the carbon elemental content, the 13 C/12C ratio was converted to 13C concentration and expressed as w13c . The concentrations of 13C-fullerenols in D. magna, and zebrafish tissues

As shown in Fig. 1A- and Fig. 1B, the average particle size and zeta potential of fullerenols at a concentration of 2.5 mg/L were 264.0 ± 40.2 nm (n = 4) and −38.5 ± 2.9 mV (n = 4) as determined through dynamic light scattering (DLS) and a Nano ZS Zeta (Malvern Instruments Ltd.), respectively. The value of the zeta potential (−38.5 ± 2.9 mV) suggested that the fullerenol agglomerates were stable in the solution based on the relationship between zeta potential and system stability (Ghadimi et al., 2011). The morphology of 13Cfullerenol agglomerates characterized by TEM is shown in Fig. 1C. The diameters of 13C-fullerenol agglomerates suspended in artificial 3

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Fig. 1. Morphology and size distribution of 13C-labelled fullerenols in artificial freshwater: (A–B) size distribution and zeta potential of fullerenols in artificial freshwater (2.5 mg/L) (n = 4); (C) TEM image.

freshwater were approximately 65–250 nm. More details about the precise structure of the fullerenols can be found in our previous report (Wang et al., 2014). 3.2. Bioaccumulation of

13

C-labelled fullerenols in D. magna

D. magna has a high possibility to accumulate nanoparticles in vivo (Petersen et al., 2009), which may be attributable to their unique uptake behaviour of filtering nanoparticles. Then, nanoparticles that have accumulated in D. magna could transfer to a higher consumer through the dietary exposure pathway, which could become a major potential nanoparticle exposure pathway for a higher trophic level of aquatic organisms. No mortality was observed in any D. magna group throughout the whole experiment. D. magna took up the fullerenols from the fullerenol solution at a concentration of 2.5 mg/L. The exposure period lasted for 72 h, during which the concentrations of 13Cfullerenols in D. magna were measured by IRMS at different sampling times. As shown in Fig. 2, fullerenol concentrations in D. magna increased rapidly during the first 12 h and then continued to increase until reaching a steady state from 12 to 24 h. Steady state was reached at approximately 24 h, due to no significant variation through contrast of the cumulative amount (p > 0.05) at the three sample times (i.e., 24, 36, 48, and 72 h) using one-way analysis of variance (ANOVA). After the end of the exposure period, the exposed D. magna were collected for the following experiment. The maximum body burden of fullerenols in D. magna was calculated to be 31.20 ± 1.59 mg/g dry weight after reaching the steady state. Similarly, in our previous study, fullerenol concentrations in D. magna exposed to 1.0 mg/L fullerenols accumulated up to 13 mg/g dry weight at the steady state (Du et al., 2016). Both these results possessed the same order of magnitude in accumulative amount of fullerenols, which suggested the stability of the accumulative capacity in D. magna for fullerenols.

Fig. 2. The uptake of fullerenols by D. magna exposed to fullerenol solution with initial concentration of 2.5 mg/L. ANOVA followed by Tukey's post-hoc test was performed to examine differences in body burden of fullerenols bioaccumulation in D. magna at 24–72 h (a indicate values not significantly greater at 24–72 h, p > 0.05). The date are presented as the mean ± standard deviation (SD) (n = 3).

3.3. Distribution, accumulation and depuration of in zebrafish through dietary exposure pathway

13

C-labelled fullerenols

To study the biological processes of fullerenols in zebrafish, we quantitatively studied the biodistribution, accumulation and depuration of fullerenols in zebrafish, which will contribute to the further 4

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Fig. 3. Tissue-specific accumulation and depuration of fullerenols in zebrafish by the dietary exposure pathway. One-way ANOVA revealed highly significant differences between the tissues at exposure 28 d or depuration 15 d (*p < 0.05). Two-way ANOVA revealed that time had significant effect on the accumulation of fullerenols (p < 0.05). Results presented are the average of the triplicate measurements, and the error bars show the standard error of each measurement.

depuration amount at different time points, tissue kinetics simulation curves were generated. As shown in Fig. 4, the uptake and depuration of fullerenols followed first-order kinetics, which indicated that fullerenols in zebrafish followed a continuous, dynamic and simultaneous accumulation process of absorption and depuration. During the uptake period, although the accumulation of fullerenols in different tissues was time-dependent, the cumulative trend of fullerenols was slightly different. As shown in Fig. 4A, the cumulative trend of fullerenols in the intestine increased gradually with time and tended to be stable after 28 d of exposure. Compared with the uptake of fullerenols in the intestine, the cumulative trend of fullerenols in the liver, muscle and brain continued to rise over time at 28 d after exposure (Fig. 4B–C, E), which suggested that reaching a stable state in these tissues would require a longer time, especially in the brain. Unlike other tissue accumulation trends, the accumulation in the gill reached a steady state 28 d after exposure (Fig. 4D). During the depuration period, the depuration trend of fullerenols in all tissues decreased with time after the beginning of depuration. In particular, the depuration trend of fullerenols in the gill significantly decreased and they tended to be completely purified by the end of the depuration period. In contrast, the depuration trend of fullerenols in the brain was slower, which indicated that the elimination of fullerenols in the brain needed a longer time. The kinetic parameters of absorption and depuration in different tissues may be obtained using simulation analysis. The uptake rate constants of fullerenols in the liver, intestine, gill, brain, and muscle were 0.05, 0.06, 0.05, 0.01 and 0.02 d−1, respectively. In addition, the depuration rate constants of fullerenols in the liver, intestine, gill, brain, and muscle were 0.10, 0.14, 0.20, 0.04 and 0.08 d−1, respectively. Because fullerenols concentrations in most tissues did not reach the steady state, the equilibrium concentrations of fullerenols in liver, intestine, muscle, brain and gill were forecasted by the fitted plateau value, which were 15.25, 14.63, 9.37, 8.73, and 8.08 mg/g, respectively (Table 1). According to the kinetic parameters, the higher accumulations of fullerenols in the intestine and liver were likely due to the uptake rate of fullerenols (uptake rate constants of 0.06 and 0.05 d−1, respectively). Similarly, the lowest cumulative amount of fullerenols in the brain was related to the slowest uptake rate of fullerenols (uptake rate constant of 0.01 d−1). As mentioned above, more than 75% of the cumulative fullerenols in the intestine, liver, and gill were purified after the end of the purification period, which may be related to the faster purification rates of these tissues (0.14, 0.10, and 0.20 d−1, respectively). In contrast, most of the fullerenols that reached the muscle and brain were not depurated after the end of the depuration experiment, probably because of the slower deputation rates (0.08 and 0.04 d−1, respectively) of these tissues. In the brain, it takes up to 60 d to purify 90% of the accumulated concentration. Furthermore, the kinetic parameters of the tissues could fully explain the time of residency of fullerenols in these tissues. For example, the intestine, liver, and gill had the highest rate of fullerenols uptake (uptake rate constants of 0.06, 0.05, and 0.05 d−1, respectively) but were also the tissues with the

intensive study of the possible biological effect mechanisms of absorption and metabolism. No mortality was observed in any group of zebrafish during the whole experiment. The experiment consisted of a 28 d uptake period and a 15 d depuration period. During the uptake period, zebrafish assimilated fullerenols by feeding on D. magna (containing 31.20 ± 1.59 mg/g dry weight fullerenols). After exposure, fullerenols were detected in the liver, intestine, gill, muscle, and brain of zebrafish, which indicated that fullerenols could transfer into zebrafish and reach the tissues through a dietary exposure pathway. During the 28 d uptake period, fullerenols accumulation in these same tissues occurred in a time-dependent manner (Fig. 3A). As shown in Fig. 3A, the fullerenols concentration in the tissues increased over time, and the maximum accumulation was observed on days 21 or 28. Moreover, the accumulation of fullerenols in different tissues was also distinct, showing a tissues-dependent manner. Especially, One-way ANOVA revealed highly significant differences between the tissues at exposure 28 days (p < 0.05). After 28 d of exposure, the fullerenols concentration decreased in the following order: intestine > liver > gill > muscle > brain. Obviously, the intestine and liver showed very high potential to accumulate fullerenols compared to the other tissues. The distribution and accumulation of fullerenols in zebrafish might be related to the administration method, the nature of the material itself, and the function of the tissues. To study the depuration levels of the different tissues, clearance pathways or the elimination mechanism in organisms, it is vital to assess the ecological risk of the nanomaterials. In this study, zebrafish exposed after the end of the exposure period were transferred to freshwater for 15 d depuration. As shown in Fig. 3B, fullerenol concentrations in zebrafish tissues decreased gradually over time in a timeand tissue-dependent manner. This is related to the excellent water solubility and bioavailability of fullerenols, which enhance the ability of these particles to cross biological barriers in zebrafish and consequently reduce the accumulation and retention of particles in these tissues. More than 60% of the cumulative amount in the liver was purified on the third day after depuration, suggesting that the liver had strong self-repair and protection abilities. One-way ANOVA revealed highly significant differences between the tissues at depuration 15 days (p < 0.05). Accumulated fullerenols in the intestine, liver, gill, muscle, and brain were not fully depurated within 15 d, with the amount of fullerenols remaining in each tissue of 10%, 24%, 25%, 46%, and 52% (compared to the fullerenol concentrations in the corresponding tissue at time zero after transfer to clean water), respectively. Obviously, the intestine has the strongest capacity to eliminate fullerenols. In contrast, the elimination capacity for fullerenols was the lowest in the brain. 3.4. Kinetics of fullerenols accumulation and depuration in different tissues To further explore the biological processes of fullerenols in zebrafish in depth, the tissue kinetics were analyzed on the basis of tissue accumulation and depuration concentration. Based on the tissue uptake and 5

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Fig. 4. Kinetic curves of accumulation and depuration of fullerenols in the tissue-specific of zebrafish by dietary exposure pathway. The date are presented as the mean ± SD (n = 3).

fastest elimination (depuration rate constants of 0.14, 0.10, and 0.20 d−1, respectively) and thus had short half-lives (5.08, 6.74, and 3.53 d−1, respectively) and short times of residency in these tissues (Table 1). Compared with other nanomaterials, such as AgNPs (Jang et al., 2014), the uptake and depuration rates of fullerenols in our study were faster (except for in the brain), which may be related to the properties of the nanomaterials, the exposure pathways, and different organisms. The hydrophilicity and good biocompatibility (Zuckerman and Kao, 2009) of fullerenols and the method of dietary exposure led to nanoparticles easily penetrating the cell membrane and distributing into tissues.

TTFd > 1 may lead to a potential higher health risk to that specific tissue. Thus, all the fitted TTFd values were less than 1, indicating that tissue biomagnification of fullerenols was unlikely to occur in this study. This could also be confirmed by the kinetic parameters mentioned above because the relative depurate rate was faster than that of uptake rate, which not result in a large accumulation or detention amount in the various tissues and finally not cause tissue biomagnification. These results indicated that the tissue trophic transfer potential of fullerenols was related to the uptake and metabolism mechanism in different tissues. Similarly, tissue biomagnification of graphene was not observed in zebrafish (Dong et al., 2018). Although tissue biomagnification of fullerenols did not occur, exposure for long periods of time may exert a negative impact on aquatic organisms.

3.5. Distribution-based trophic transfer factor The trophic transfer potential of fullerenols from D. magna to zebrafish tissues was evaluated using the distribution-based trophic transfer factor (TTFd). TTFd, is the ratio of tissue concentration in a high-trophic-level organism to the concentration in a low-trophic-level organism at steady state and is used to evaluate the specific tissue biomagnification through the transfer of the food chain (Dong et al., 2018). Since the steady state was not reached in zebrafish tissues, the fitted TTFd could be calculated through the value of the fitted plateau. The values of the fitted TTFd were 0.49, 0.47, 0.26, 0.28 and 0.30 in the liver, intestine, gill, brain and muscle, respectively (Table 1). TTFd represents the ability of nanoparticles from low-trophic-level organisms to transfer to the specific tissues of high-trophic-level organisms. A

3.6. Localization of fullerenols in D. magna and zebrafish For the identification and localization of fullerenols in organisms and tissues, TEM technique was used in this study. The application of TEM was more conducive to the study of the present and absorption pathways of the nanomaterials. The TEM images of fullerenols in different tissues could exactly reflect the presence and localization of the nanoparticles in the zebrafish body. In Fig. 5, these black particles indicated by the red arrows were speculated to be fullerenol nanoparticles by observing the tissue itself and comparing with the TEM images of the control tissue samples. In detail, these black particles did not apparently fuse with the surrounding tissues, which proved that they were

Table 1 Bioaccumulation and depuration parameters of fullerenols in zebrafish through dietary exposure pathwaya.

Liver Intestine Gill Brain Muscle a

ku (d−1)

k e (d−1)

R2

Fitted plateau (mg/g)

Half-life (t1/2 )(d)

90% concentration time of depuration (d)

TTFd

0.05 0.06 0.05 0.01 0.02

0.10 0.14 0.20 0.04 0.08

0.86 0.78 0.74 0.85 0.58

15.25 14.63 8.08 8.73 9.37

6.74 5.08 3.53 18.98 9.21

22.39 16.88 11.72 63.05 30.59

0.49 0.47 0.26 0.28 0.30

The values of ku and k e were derived from the following equation: Ct1 =

following equation t1/2 =

log e0.5 ke

and C f =

CF ku , ke

respectively.

CF ku (1 ke

6

e

k et ).

The values of half-live and fitted plateau were derived from the

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Fig. 5. Distribution of 13C-labelled fullerenols in zebrafish: (A–E) TEM images of different tissues of the zebrafish fed fullerenols-contaminated D. magna, (F–J) TEM images of different tissues of the zebrafish fed control prey (unexposed D. magna). A and F, liver; B and G, intestine; C and H, brain; D and I, muscle; E and J, gill.

nanoparticles (Jang et al., 2014; Jung et al., 2014; Yamago et al., 1995). It is well known that the liver serves the major function of detoxification, including the metabolism of xenobiotics and the formation and excretion of bile (Kashiwada, 2006). Thus, the accumulation of fullerenols in the liver probably passed through the liver-bile-intestine metabolism system. In addition, studies have found that an increased amount of accumulated nanoparticles in the liver is due to the presence of opsonins specific to macrophages located in the liver, which exhibit stronger binding to nanoparticles (Moghimi and Patel, 1988). In this respect, the enhanced uptake of fullerenols in the liver is largely attributed to the macrophages residing in this tissue. The fullerenol concentrations in the gill were significantly lower than those in the intestine and liver during the uptake period. This result differed from the reported cumulative differences of nanomaterials in the intestine and gill of fish through aqueous phase exposure (Kashiwada, 2006), suggesting that dietary administration of fullerenols avoided the directly exposure of the gill to fullerenols during respiration. Thus, the distribution of fullerenols into the gill was likely achieved through the circulatory system. Because of the concentration of fullerenols in the freshwater with zebrafish was not detected, which avoided the possibility of re-exposure through the aqueous phase and fullerenols binding to zebrafish surface. The main reason was that fish faeces in the aquarium were removed every day, and 80% of the volume was renewed once every two days during dietary exposure. In addition, the D. magna as food were all eaten by zebrafish in a very short time, which would avoid the risk that fullerenols might leak from dead D. magna to the freshwater with zebrafish over time. 13C abundance detected in the water with zebrafish was the same as that in water without zebrafish. A small amount of fullerenols were also found in the muscles, suggesting once again that fullerenols entered the circulatory system. Fullerenol concentrations were detected in the brain during the uptake period, suggesting that fullerenols penetrated the blood-brain barrier to reach the brain. From the accumulation level, the amount of fullerenols in the brain was significantly lower than in the other tissues, which showed that only low amounts of fullerenols could penetrate the blood-brain barrier. This phenomenon has also been found in other studies (Kashiwada, 2006). For instance, a study on the distribution of nanoparticles in medaka found that 39.4 nm particles penetrated the bloodbrain barrier after 7 d’ of exposure (Kashiwada, 2006). Moreover, some studies of gold nanoparticles unequivocally confirmed the possibility that gold particles with a diameter of approximately 10 nm or less can penetrate the blood-brain barrier (Hillyer and Albrecht, 1998, 2001). In

foreign matter. In addition, these black particles were not found in the TEM images of the control tissue samples. Thus, these black particles, which only appeared in the exposed tissues, were likely to be fullerenol nanoparticles entering the tissues. The TEM image of D. magna indicated that the ingested fullerenols resided in the gut of D. magna and the particle sizes were approximately 80–350 nm (Fig. S1). The particle sizes of fullerenol agglomerates in different tissues were approximately 30–100 nm by TEM. This was the first intuitive evidence that carbon nanoparticles could enter the tissue of these organisms. 4. Discussion 4.1. The potential translocation pathways of fullerenols accumulation, biodistribution and depuration in zebrafish The highest increase in the fullerenols concentration was observed in the intestine compared with the other tissues during the 2 d of exposure, indicating that it is the initial site of uptake. Moreover, the intestine in this study showed the highest accumulation amount until the end of the exposure period, suggesting that ingestion was a major route of fullerenols uptake in zebrafish. This was related to the exposure pathway because the gastrointestinal tract was the initial target tissue following dietary exposure. The absorption of nanoparticles can pass through the membranes of the intestine, enter the circulation and contribute to the distribution of nanoparticles in fish, as reported in a previous study (Kashiwada, 2006), which suggested that the accumulation and distribution of fullerenols in zebrafish could also occur through this translocation pathway. Furthermore, fullerenols with good water solubility and biocompatibility (the ability of living tissues to react with inactive materials) easily penetrate the cell membrane (Chaudhuri et al., 2009), which adequately explains how fullerenols can penetrate the membranes of the intestine and enter the circulation. Similarly, an acute toxicology pathological study of nanoparticles to adult tilapia also showed that the major translocation pathway to enter the circulation of nanoparticles in fish may be via the gill-blood route and/or the intestine-blood route (Srinonate et al., 2015), because the gill and gastrointestinal organs are principal routes that are directly exposed to and take up the toxicants from ambient water into the fish body. Higher concentrations of fullerenols, only slightly below those of the intestine, were found in the liver, indicating an important role for this tissue in processing fullerenols during exposure. Other studies have also found that the liver is a predominant site for the accumulation of 7

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this study, the particle size of the fullerenols in the brain was determined by TEM to be 38–59 nm (Fig. 5D). Thus, whether nanoparticles can finally reach the brain through the blood-brain barrier may be largely related to the particle size of the nanoparticles. In addition, research of largemouth bass exposure to fullerenes proposed that exposure to the brain could occur via olfactory neurons (Oberdörster, 2004). Therefore, more potential translocation pathways of nanoparticle accumulation and biodistribution in fish bodies need further study. According to the results of fullerenols depuration, the rapid excretion of fullerenols accumulated in the intestine may be due to the metabolism of zebrafish (such as the excretion of faeces). In addition, another study showed that fullerenols can be degraded by gut flora under cultivation in vitro (Li et al., 2018), which could be a potential route of elimination for fullerenols from the intestine of zebrafish. Previous studies have shown that both the rapid elimination of particles from the blood and their long-term retention in the organism are associated with the functions of the hepatobiliary system (Khlebtsov and Dykman, 2011). Furthermore, the uptake of nanomaterials by Kupffer cells is a major mechanism of particle accumulation and clearance in vivo (Khlebtsov and Dykman, 2011). Based on the above results, it can be inferred that zebrafish could eliminate fullerenols through its own metabolic mechanism, the liver-bile-intestine system, and the circulatory system. There is an alternative clearance mechanism that has been found in human immune cells, which is that gold nanoparticles can be eliminated from peripheral blood via an extracellular network formed by neutrophil granulocytes (Bartneck et al., 2010). Obviously, the elimination mechanism of nanoparticles in organisms is very complex. In this study, we preliminarily explored the potential elimination mechanism or depuration pathway of nanoparticles in zebrafish. More thorough studies on the elimination mechanism of nanoparticles in vivo need to be carried out at the levels of proteins, metabolites or molecules in the future.

carbon nanomaterials. 5. Conclusions In summary, we have quantitatively investigated the biological processes of fullerenols (including the uptake, bioaccumulation, biodistribution, depuration, and uptake-depuration kinetics) in zebrafish through a dietary exposure pathway. Our results provide the first direct evidence that fullerenols could be transferred from D. magna to zebrafish and further enter into tissues because the concentration of fullerenols was detected by IRMS in different zebrafish tissues after feeding with fullerenol-contaminated D. magna. During the 28 d uptake period, the cumulative amount of fullerenols in the tissues followed the order intestine > liver > gill > muscle > brain. Notably, the intestine and liver were the target tissues for fullerenols accumulation. Moreover, kinetic analysis of the absorption and depuration of fullerenols in different tissues further thoroughly explained the biological processes of fullerenols in zebrafish. The depuration rates of fullerenols in different tissues followed the following order: gill > intestine > liver > muscle > brain. All of the accumulation and depuration results suggested that the mechanisms or pathways of absorption and depuration of fullerenols in different tissues were distinct. The uptake and depuration results and the TTFd values fully explained that fullerenols would not be seriously retained in tissues, which confirmed that there was no risk of tissue biomagnification in zebrafish. Our research data will provide an important basis and support for understanding the safety, environmental and human health impacts of nanotechnologybased products. Notes The authors declare no competing financial interest. Author contributions section

4.2. The assessment of the ecological risk of carbon nanomaterials based on fullerenols

QYS participated in all exposures experiments, collected samples for IRMS analysis, date acquisition, statistical analysis, and writing manuscript. XZ and XLC conceived, designed and managed the study. XZ and QYS participated in the revision of the manuscript. HZ and QYS participated in the detection of all samples. CLW prepared and analysis 13 C-labelled fullerenols. HYR performed the transmission electron microscope slides of tissues. CZY helped to polish the language of the manuscript.

In a previous study, the exposure of fullerenols caused dose-dependent cell damage; that is, a high dose of fullerenols caused cytotoxic injury or cell death in vascular endothelial cells (ECs), and low-dose fullerenols (10 μg/mL for 8 d) inhibited cell attachment and delayed EC growth (Yamawaki and Iwai, 2006). These results suggested that fullerenols could enter the body of the organism and be retained for a considerable amount of time in various organs or cells, possibly caused a functional decline in the organs. Moreover, the toxicity or biological effects linked to the internal distribution of nanoparticles are essential to understand the underlying mechanism and ecological risk. The cumulative concentration of fullerenols in the tissues relative to the concentration in the aqueous solution will have a greater adverse effect on its organization. Thus, the internal exposure concentration of fullerenols in tissues could directly result in potential biological effects or toxicity and reflect the extent of potential damage to the target organs. In this study, the results of the tissue-specific accumulation of fullerenols in zebrafish indicated that the bioeffects of fullerenols were mainly aimed at the intestine, liver and gill, but the translocation of fullerenols to other tissues could pose a relatively weak threat risk elsewhere. A previous study found that carbon-based nanoparticles (CBNs) could induce marked lipid alterations in the brain, gonads and gastrointestinal tissue samples of zebrafish and cause elevations in global genomic methylation (Gorrochategui et al., 2017). Other studies have also found low toxicity from carbon nanoparticles in fish (Henry et al., 2007, 2011; Zhu et al., 2006). All of these results indicate that carbon nanomaterials could pose a certain degree of risk to tissues and organs in organisms, which contributes to the assessment of the ecological risk for the release of carbon nanomaterials into aquatic environments and guide the safe use of commercial products based on

Declaration of interest statement The authors declare no competing financial interest. Acknowledgments This work was supported by the National Natural Science Foundation (11475194). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2020.110173. References Bartneck, M., Keul, H.A., Zwadlo-Klarwasser, G., Groll, J., 2010. Phagocytosis independent extracellular nanoparticle clearance by human immune cells. Nano Lett. 10, 59–63. Chang, X.L., Ruan, L.F., Yang, S.T., Sun, B.Y., Guo, C.B., Zhou, L.J., Dong, J.Q., Yuan, H., Xing, G.M., Zhao, Y.L., Yang, M., 2014. Quantification of carbon nanomaterials in vivo: direct stable isotope labeling on the skeleton of fullerene C60. Environ. Sci.: Nano 1, 64–70.

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