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Accumulation and distribution of multiwalled carbon nanotubes in zebrafish (Danio rerio) Hanna Maja Maes, Felix Stibany, Sebastian Giefers, Benjamin Daniels, Björn Deutschmann, Werner Baumgartner, and Andreas Schaeffer Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es503006v • Publication Date (Web): 26 Sep 2014 Downloaded from http://pubs.acs.org on September 30, 2014
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Accumulation and distribution of multiwalled carbon nanotubes in zebrafish (Danio rerio)
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Hanna M. Maes*1, Felix Stibany1, Sebastian Giefers1, Benjamin Daniels1, Björn Deutschmann1, Werner Baumgartner2, and Andreas Schäffer1
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Institute for Environmental Research (Biology V), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany 2 Department for Cellular Neurobionics (Biology II), RWTH Aachen University, Lukasstrasse 1, 52056 Aachen, Germany *Corresponding author:
mail: [email protected] phone: +49-241-80-26698 fax: +49-241-80-22182
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Abstract
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No data on the bioaccumulation and distribution of multiwalled carbon nanotubes (MWCNT) in
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aquatic vertebrates is available till now. We quantified uptake and elimination of dispersed
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radiolabelled MWCNT (14C-MWCNT; 1 mg/L) by zebrafish (Danio rerio) over time. The influences of
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the feeding regime and presence of dissolved organic carbon (DOC) on accumulation of the
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nanomaterial were determined. The partitioning of radioactivity to different organs and tissues was
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measured in all experiments. A bioaccumulation factor of 16 L/kg fish wet weight was derived.
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MWCNT quickly associated with the fish and steady state was reached within one day. After transfer
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to clear medium, MWCNT were quickly released to the water phase, but on average 5 mg
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MWCNT/kg fish dry weight remained associated with the fish. The nanomaterial mainly accumulated
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in the gut of all fish. Feeding led to lower internal concentrations due to facilitated elimination via
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the digestive tract. In presence of DOC, a tenfold less was taken up by the fish after 48 h of exposure
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compared to without DOC. Quick adhesion to and detachment from superficial tissues were
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observed. Remarkably, little fractions of the internalized radioactivity were detected in the blood and
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muscle tissue of exposed fish. The part accumulated in these fish compartments remained constant
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during the elimination phase. Hence, biomagnification of MWCNT in the food chain is possible and
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should be subject of further research.
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Introduction
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Carbon nanotubes (CNT) are considered promising materials in nanotechnology1. Investment in
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nanotechnology research and development as well as nanoparticle production volumes are growing
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rapidly worldwide2, 3. Based on the assumption that this will lead to increasing CNT release in the
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environment, a lot of research has recently been dedicated to investigating the possible (eco)toxicity
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of this nanomaterial4. However, little information is available on the impact of CNT and other
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carbonaceous nanoparticles on aquatic vertebrates. Some studies found that these nanomaterials
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did not affect exposed organisms5, but it was also reported that single walled CNT (SWCNT) exerted
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respiratory toxicity to rainbow trout (Oncorhynchus mykiss)6, that SWCNT and fullerenol altered the
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antioxidant capacity of adult zebrafish (Danio rerio)7, that multiwalled CNT (MWCNT) and fullerene
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caused toxicity in zebrafish embryos8, 9, and that double walled CNT (DWCNT) physically blocked
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epithelial tissue of larvae of the African clawed frog (Xenopus laevis)10. As can be extracted from a
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recent review, there is a distinct lack on data on bioaccumulation of CNT11. This is consistent with the
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limited number of analytical methods available to differentiate carbon-based nanoparticles from
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organic matrices, and thus, to detect these materials and measure their concentration in biological
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samples12-15. Using mainly microscopic techniques, it was observed that CNTs clogged the filter
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apparatus and covered the carapace of daphnids16, precipitated on the gills of rainbow trout6, or
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filled the gastrointestinal tract of several aquatic invertebrates16-18 after exposure of the organisms
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via aqueous dispersions of CNT. From research published up to now, it seems that nanotubes do not
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pass the gut epithelium to accumulate in exposed aquatic organisms4, 15, 19. Benthic invertebrates and
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earthworms were shown to incorporate CNT from sediment, food, and soil matrices, but the
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nanomaterials did not appear to pass the gut epithelium either20-22. Since most described effects
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could not be linked to internal distribution of nanoparticles and/or their agglomerates, the
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underlying mechanisms of possible CNT toxicity remain subject of many current investigations. In a
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recent study, SWCNT were tracked in fish using near infrared fluorescence after single gavage
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We studied the accumulation and distribution of MWCNT for the first time in a vertebrate after
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waterborne exposure. Adult zebrafish were exposed to radiolabeled MWCNT (14C-MWCNT) for up to
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one month and uptake of radioactivity by the fish was quantified over time. Afterwards, exposed fish
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were transferred to clear water to examine elimination of MWCNT. The influences of the feeding
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regime and the presence of dissolved organic carbon (DOC) on MWCNT accumulation in zebrafish
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were additionally determined. In all experiments, fish were dissected to assess the fraction of
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MWCNT partitioned to different fish compartments.
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Materials and Methods
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Preparation of 14C-MWCNT test suspensions for exposure of zebrafish
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One batch of
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synthesized (as described in the SI) and used to perform all experiments. 500 µg of
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agglomerates were weighed on a microbalance (0.0001 mg readability; Mettler Toledo UMX2,
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Mettler Toledo GmbH, Germany) and added to a 1000 mL test beaker, filled with 500 mL of test
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medium, which contained 294.0 mg/L CaCl2, 123.3 mg/L MgSO4, 63.0 mg/L NaHCO3, and 5.3 mg/L
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KCl. The beaker was placed in an ice bath and the MWCNT material was homogenized by means of
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ultrasonication with a microtip (Sonoplus HD 2070, Bandelin, Germany) set to a 0.2 seconds pulse at
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70 W – 0.8 seconds pause regime for 5 minutes. The procedure was repeated two times. This
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method resulted in the presence of small agglomerates and single nanotubes of 0.2 to 1.0 µm tube
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length (both referred to as MWCNT) in the medium, as was visualized by means of transmission
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electron microscopy (TEM; SI Figure S3). In case DOC was supplemented to the medium, a stock
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solution was prepared by mixing 2.5 g Aldrich humic acid (Sigma Aldrich Chemie GmbH, Germany)
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and 0.4 g NaOH (Carl Roth GmbH, Germany) in 1 L of distilled water. After stirring overnight, the
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suspension was centrifuged for 15 minutes at 350 g and then filtered through three filters of
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different pore sizes (1.2, 0.8, and 0.45 µm) to remove suspended particles. The pH was set to 7 with
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HCl before the total organic carbon (TOC) content was determined using a TOC-Analyzer (Shimadzu
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C-MWCNT with a specific radioactivity of 1.3 ± 0.1 MBq/mg (SI Table S5) was 14
C-MWCNT
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Europe GmbH, Duisburg, Germany). The volume to add to 500 mL medium to obtain 8 mg DOC/L was
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calculated and amounted to about 6 mL. In this case, the MWCNT agglomerates were sonicated in
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about 494 mL and 6 mL DOC stock solution was added afterwards. The 14C-MWCNT concentration (1
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mg/L) and homogeneity of the test dispersion were verified by measuring the amount of radioactivity
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in replicate samples directly after sonication. Four samples of 1 mL were mixed with 5 mL of
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scintillation cocktail (Insta-Gel PlusTM, Perkin Elmer, Germany) and subjected to liquid scintillation
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counting (LSC; LS 5000 TD, Beckmann Instruments GmbH, Germany; detection limit: 1 Bq
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corresponding to 0.8 ng MWCNT). Using a radioactive standard (SPEC-CHEC-14C, Perkin Elmer,
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Germany), it was verified that the presence of DOC did not influence LSC measurements in the
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applied amounts. After placing the test beakers in a heated water bath (25 ± 1°C), one adult female
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fish was added to the MWCNT suspension.
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Test setup
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Adult female zebrafish (Danio rerio held as described in the SI; 453 ± 103 mg wet weight) were
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individually exposed to 1 mg
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was shown that this rather high concentration was necessary to be able to detect radioactivity in fish
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compartments, but did not cause adverse effects on the growth and condition of zebrafish (data not
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shown). Females were selected for this bioaccumulation study as they are bigger than male fish of
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the same age. For comparability reasons, only one sex was tested. At least five fish were subjected to
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each treatment, i.e. in separate test beakers. Since preliminary experiments had shown that data of
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MWCNT accumulation involved large scatter, more replicates were provided for important sampling
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points. Moreover, data of different experiments involving the same treatment and exposure duration
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were pooled, when no significant differences in the results were observed. In the uptake experiment,
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uptake of MWCNT by the fish was quantified over time. Sampling of the water phase and the fish
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was performed after 3 h, 1 day, 2 days, 1 week, 2 weeks, 3 weeks, and 4 weeks, as described below.
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In the elimination experiment, fish were exposed to 14C-MWCNT for two weeks, and then rinsed and
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transferred to clear water to quantify elimination of MWCNT. Samples were taken after exposure,
14
C-MWCNT/L in 500 mL test medium. In preliminary experiments, it
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and after an elimination period of 4, 24, 48, and 168 h. In both tests, fish were fed every second day
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before the MWCNT suspension or clear medium was completely renewed (water renewal every 48
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h). They were supplied with 2% of their wet weight in Artemia nauplii that were consumed within ten
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minutes. In the feeding experiment, the influence of the feeding regime on MWCNT accumulation in
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the fish was determined. Fish were either not fed or fed once a day with 1% of their body weight in
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Artemia nauplii during a 48 h exposure period, or additionally fed every second day with 2% of their
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body weight in Artemia nauplii after complete water renewal during a 168 h exposure period. In the
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DOC experiment, it was examined whether DOC has an influence on accumulation of MWCNT in
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zebrafish. Fish were exposed to 1 mg MWCNT/L in the presence or absence of 8 mg DOC/L. The fish
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were not fed and the water was not renewed during this experiment. Sampling was performed after
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3, 24, and 48 h.
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Sampling
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At each sampling point, fish were caught with a steel grid and rinsed to remove MWCNT containing
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water from the skin. Their length (L) and weight (W) were measured to determine the condition
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factor (CF: W/L³) that was compared with the CF of five control fish held under the same conditions
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in clear water. Afterwards, they were anaesthetized in a saturated benzocaine (Sigma Aldrich,
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Germany) solution in tap water. Depending on the aim of the experiment, only the gut of the fish was
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removed, or they were fully dissected in order to study the distribution of CNT in zebrafish (see SI).
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Except for the blood, all tissues were separately placed on small pre-weighed pieces of aluminium
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foil, dried in an oven at 60 °C, and weighed on a microbalance. All samples were transferred to stable
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glass test tubes, covered with tissue solubilizer (BTS-450, Beckman Coulter, USA), and incubated for
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24 h at 37°C. Afterwards, a 35% hydrogen peroxide solution was added to bleach the samples. They
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were transferred to 20 mL scintillation vials using methanol to rinse the glass tubes. The vials were
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filled with scintillation cocktail, and subjected to LSC after storage overnight at 4°C in the dark. The
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amount of radioactivity in samples of unexposed fish, which were bleached and solubilized in the
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same way as those of exposed fish, was below the limit of detection. Adding a known amount of 14C6 ACS Paragon Plus Environment
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MWCNT to blank tissue samples, a recovery higher than 95% could be derived (data not shown). To
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make a balance of the total radioactivity present in the system (SI Figure S7), the MWCNT
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concentration in the medium was again measured. Thereto, the remaining water in the test beakers
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was sonicated as described above, in order to again homogenize the test dispersion, now containing
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settled agglomerates of MWCNT. The radioactivity in four 1 mL water samples per test beaker was
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measured by means of LSC.
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Data interpretation
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In order to calculate the bioaccumulation factor (BAF), a one-compartment model was fitted to the
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uptake and elimination data (according to Connell, 199823):
= ∗
∗ 1 − ∗ + ∗ ∗
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where Cb and Cw are the concentrations of MWCNT detected in the fish and the water, t represents
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time, and k1 and k2 are the uptake and elimination rate constants. Since the amount of MWCNT in
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the water body did not change a lot due to uptake by the fish (SI Figure S7) and since the water was
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renewed every second day, Cw can be regarded as invariable. It amounted to 1 and 0 mg/L during the
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uptake and depuration phase, respectively. At steady state Cb/Cw equals k1/k2, giving the BAF.
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Differences between treatments were tested after checking the data for outliers by means of
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Dixon/Grubbs test. Then, the data were checked for normality and variance homogeneity with
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Shapiro-Wilk’s test and Levene’s test, respectively. Statistical differences were identified by means of
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pairwise comparison using Student’s t-test in case of homoscedasticity and by Welch’s t-test in case
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of heteroscedasticity. For the evaluation of statistically significant fluctuations over time, Dunnett’s t-
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test was applied when variances were homogeneous and Welch’s t-test with Bonferroni adjustment
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when variances were heterogeneous. A significance level (α) of 0.05 was chosen for all tests.
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Results
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There was no significant difference in the condition factor (CF) of exposed and control fish at all time
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points (SI Table S1). In the uptake experiment, MWCNT quickly associated with zebrafish over time, 7 ACS Paragon Plus Environment
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but the variability between replicates was extremely high (with coefficients of variation of 70 to
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150%). Steady state was already reached within 24 h (Fig. 1, A, Y1). Maximal concentrations were
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measured after two weeks of exposure, where on average 6% (25 µg) of the total MWCNT amount
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present in the water was taken up by the fish (Table 3). The mean MWCNT level (± standard
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deviation) in all complete fish of the 24 h up to 4 weeks treatments amounted to 73 (± 93) ng
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MWCNT/mg fish dry weight (= 16 ng MWCNT/mg fish wet weight) (Fig. 1, A, Y1), and since steady
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state was reached, a bioaccumulation factor of 73 on dry weight (16 on wet weight) could be
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calculated (BAF = Cb/Cw = (73 mg/kg)/(1 mg/L)). In the elimination experiment, MWCNT were quickly
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rejected to the water phase, but after 24 h, no further elimination of the material could be observed
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(Fig. 1, B), i.e. the fish MWCNT concentrations after 24, 48, and 168 h of presence in clear water were
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not significantly different, amounting to 5 ng MWCNT/mg fish dry weight on average (SI Table S4).
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The data were modelled over 48 h, the time between the beginning of the depuration phase and the
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first water renewal. For complete fish, an elimination rate constant (k2) of 0.14 h-1 was calculated
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(Fig. 1, B). With this value, an uptake rate constant (k1) of 9.64 L*kg-1*h-1 was derived. Dividing k1 by
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k2, a BAF of 70 is obtained, verifying the above calculated BAF using Cb and Cw at steady state.
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MWCNT mainly accumulated in the digestive tract of the fish (Table 1, 2, 3; SI Table S2, S3, S4), which
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is not necessarily presenting a bioavailable fraction. Hence, it was removed and measured separately
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from the total remaining fish body. The one-compartment model did not fit to the data of fish from
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which the gut was removed. In the uptake phase, steady state was directly reached, i.e. no
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differences in the fish MWCNT content were observed over time (Fig. 1, A, Y2). Similarly, elimination
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was quicker than predicted by the model (Fig. 1, C). After 4 h, most of the MWCNT were no longer
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associated with the gut excised fish and the internal MWCNT content remained constant up to the
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last sampling point of 168 h, amounting to 0.24 ng/mg fish dry weight on average.
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A
206 B
C
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Fig. 1 Concentration of multiwalled carbon nanotubes (MWCNT) in zebrafish on dry weight (dw) after exposure to 1 mg MWCNT/L via the water phase in function of time. The upper graph (A) shows uptake by the fish over time. Filled rounds represent the concentrations in complete fish (Y-axis on the left: Y1), non-filled rounds those in fish from which the gut was removed (Y-axis on the right: Y2). The graphs below (B, C) present elimination of MWCNT from two weeks exposed zebrafish (time = 0 h) that were transferred to clear water. Again filled and non-filled rounds represent complete and gut excised fish, respectively. A one compartment model is plotted to all data (R²: determination coefficient).
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Feeding of the fish led to significantly facilitated elimination of MWCNT via the digestive tract, as was
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shown in the feeding experiment (Fig. 2). Within an exposure period of 48 h without water renewal,
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unfed fish had an MWCNT body burden that was on average 45 times higher than fish that were daily
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supplied with uncontaminated Artemia nauplii (Fig. 2, A). After 168 h, fish that were fed once a day
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accumulated less MWCNT than fish that were fed once every second day and much less than fish
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that were not fed at all (Fig. 2, B). No significant differences in the MWCNT content of fish from
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which the gut was removed were observed (Fig. 2, D). The differences originated from the fraction in
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the gut (Fig. 2, C). In the DOC experiment, it was shown that the presence of DOC in the medium also
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resulted in the accumulation of lower amounts of MWCNT by the fish (Fig. 3). After 48 h, the total
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concentrations in fish exposed to MWCNT in presence and absence of DOC amounted to 9 and 91 9 ACS Paragon Plus Environment
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ng/mg dry weight, respectively (Fig. 3; SI Table S2 and Table S3). Compared to unfed fish exposed
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without DOC, MWCNT got quicker associated with the gills and to larger relative amounts when DOC
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was present in the medium (Table 1 vs. Table 2). The sum of MWCNT present in the skin, filet, and
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blood (= rest fish), and the gills was relatively larger with (11%) than without (2%) DOC after 48 h of
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exposure. Furthermore, relatively less was present in the gut with (89%) than without (96%) DOC
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(Table 1 vs. Table 2).
231 A
B
232 D
C
233 234 235 236 237 238 239
Fig. 2 Concentration of multiwalled carbon nanotubes (MWCNT) in zebrafish (gut) after exposure to 1 mg MWCNT/L via the water phase for 48 or 168 h, during which food was provided in different regimes (no food, one time per two days, and once per day) and the MWCNT dispersion was either renewed one time every 48 h or not at all. The values are expressed on dry weight (dw). The rounds, the asterisks, the vertical bars, and the error bars represent the data points for separately exposed fish, outliers, the mean, and the standard deviation on the mean of remaining replicates, respectively. Significant differences between treatments are indicated by different letters.
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Fig. 3 Concentration of multiwalled carbon nanotubes (MWCNT) in zebrafish after exposure to 1 mg CNT/L via the water phase in function of time and in absence (-) or presence (+) of dissolved organic carbon (DOC, 8 mg/L). The mean of five replicates is presented by the vertical bars to which the error bars represent the standard deviation, all expressed on fish dry weight (dw). Significant differences between DOC treatments at one time point are indicated by the asterisks.
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As was mentioned above, MWCNT mainly accumulated in the gut of all fish (Table 1, 2, 3; SI Table S2,
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S3, S4). Large relative amounts of radioactivity were also detected in gills, skin and muscle samples of
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briefly exposed fish (3 h). Afterwards, only low percentages of the total amount were associated to
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these fish compartments. No distribution to the liver, the gonads, and the brain was observed.
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However, low amounts of radioactivity were detected in blood of fish exposed for more than one
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week. The average total amount of radioactivity in blood samples of unfed fish (exposed for only one
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week) was higher than in fish fed every second day (exposed for two weeks), corresponding to 7.6
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and 2.5 ng 14C-MWCNT, respectively (Table 2 vs. Table 3). During the elimination phase, the absolute
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blood MWCNT content did not significantly change, leading to an increase of the relative amount of
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radioactivity in the blood current over time. On the other hand, the absolute amount of MWCNT
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associated with the rest of the fish, i.e. the skin and filet, drastically decreased (from ca. 100 ng to 20
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ng) within the first 4 h of depuration. Afterwards, the relative amount of CNT in the rest fish
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increased, but the absolute amount corresponded to about 20 to 30 ng 14C-MWCNT at all elimination
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sampling points (Table 3). The MWCNT concentration in the skin and filet indeed decreased within
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the first four hours of elimination and remained more or less constant thereafter (SI Table S4). It is
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suggested that quick detachment from the skin occurred, and that the remaining radioactivity
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tissue was demonstrated for the gills, to which no more MWCNT were attached after 168 h of
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depuration (Table 3). Similarly, quick adhesion to the fish surface was measured in the uptake phase:
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on average 16 and 26% of the total amount of MWCNT associated with the fish were detected in gill
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tissue and skin within 3 h (Table 2). It seems therefore that connection of MWCNT to the fish exterior
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is reversible. During the depuration period, the part accumulated in internal fish compartments
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remained constant (as shown for blood and assumed for the filet), and the main MWCNT fraction
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was purged from the gut and detached from the gills and skin (as shown for the gills and assumed for
270
the skin).
271 272 273 274 275 276 277
Table 1 Distribution of multiwalled carbon nanotubes (MWCNT) in different compartments of zebrafish, which were 14 exposed to 1 mg C-MWCNT/L via the water phase in the presence of 8 mg dissolved organic carbon/L. The fraction in each fish compartment is expressed as the percentage of the total amount of radioactivity detected in the fish. The sum represents the total amount, and is expressed as the corresponding amount of MWCNT in µg. The rest is the relative amount of radioactivity measured in the residual fish, from which gut and gills were removed. Values that are struck out represent outliers (M: mean; SD: standard deviation).
exposure time (h) 3
elimination time (h) 0
M SD 24
0
M SD 48
0
M SD
gut (%) 8.2 68.6 11.4 5.5 6.4 7.9 2.6 90.4 93.2 99.0 97.3 12.9 95.0 3.9 92.2 83.8 95.4 84.4 89.4 89.0 5.0
gills (%) 43.8 4.8 12.4 39.4 40.0 28.1 18.1 3.0 0.2 0.3 0.5 28.6 1.0 1.3 7.1 9.8 1.3 3.5 8.7 6.1 3.6
rest = fish-gut-gills (%) 48.0 26.6 76.2 55.1 53.5 51.9 17.7 6.7 6.6 0.7 2.2 58.5 4.0 3.1 0.8 6.4 3.3 12.1 1.9 4.9 4.5
sum (µg) 0.035 0.122 0.107 0.039 0.035 0.068 0.043 0.054 0.198 0.709 0.197 0.020 0.235 0.277 0.368 0.048 1.602 0.655 0.662 0.667 0.580
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Table 2 Distribution of multiwalled carbon nanotubes (MWCNT) in different compartments of unfed zebrafish, which 14 were exposed to 1 mg C-MWCNT/L via the water phase. No water renewal was performed. The fraction in each fish compartment is expressed as the percentage of the total amount of radioactivity detected in the fish. The sum represents the total amount, and is expressed as the corresponding amount of MWCNT in µg. Values that are struck out represent outliers (M: mean; SD: standard deviation; DL: detection limit).
exposure time (h) 3
elimination time (h) 0
M SD 24
0
M SD 48
0
M SD 168
0
M SD
gut (%) 1.2 6.3 2.7 6.8 29.3 4.3 2.7 31.0 99.0 92.3 99.5 75.0 91.3 11.3 99.1 91.2 94.9 97.5 98.8 96.3 3.3 99.7 99.8 99.9 98.6 99.9 99.6 0.6
gills (%) 7.9 32.9 24.6 8.0 6.0 15.9 12.1 35.0 0.4 2.5 0.2 24.2 12.4 16.2 0.5 0.0 4.2 1.8 0.4 1.4 1.7 0.11 0.03 0.02 0.36 0.03 0.03 0.00
blood (%)
Article
Accumulation and distribution of multiwalled carbon nanotubes in zebrafish (Danio rerio) Hanna Maja Maes, Felix Stibany, Sebastian Giefers, Benjamin Daniels, Björn Deutschmann, Werner Baumgartner, and Andreas Schaeffer Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es503006v • Publication Date (Web): 26 Sep 2014 Downloaded from http://pubs.acs.org on September 30, 2014
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Accumulation and distribution of multiwalled carbon nanotubes in zebrafish (Danio rerio)
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Hanna M. Maes*1, Felix Stibany1, Sebastian Giefers1, Benjamin Daniels1, Björn Deutschmann1, Werner Baumgartner2, and Andreas Schäffer1
6 7 8 9
1
10 11 12
Institute for Environmental Research (Biology V), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany 2 Department for Cellular Neurobionics (Biology II), RWTH Aachen University, Lukasstrasse 1, 52056 Aachen, Germany *Corresponding author:
mail: [email protected] phone: +49-241-80-26698 fax: +49-241-80-22182
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Abstract
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No data on the bioaccumulation and distribution of multiwalled carbon nanotubes (MWCNT) in
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aquatic vertebrates is available till now. We quantified uptake and elimination of dispersed
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radiolabelled MWCNT (14C-MWCNT; 1 mg/L) by zebrafish (Danio rerio) over time. The influences of
40
the feeding regime and presence of dissolved organic carbon (DOC) on accumulation of the
41
nanomaterial were determined. The partitioning of radioactivity to different organs and tissues was
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measured in all experiments. A bioaccumulation factor of 16 L/kg fish wet weight was derived.
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MWCNT quickly associated with the fish and steady state was reached within one day. After transfer
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to clear medium, MWCNT were quickly released to the water phase, but on average 5 mg
45
MWCNT/kg fish dry weight remained associated with the fish. The nanomaterial mainly accumulated
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in the gut of all fish. Feeding led to lower internal concentrations due to facilitated elimination via
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the digestive tract. In presence of DOC, a tenfold less was taken up by the fish after 48 h of exposure
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compared to without DOC. Quick adhesion to and detachment from superficial tissues were
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observed. Remarkably, little fractions of the internalized radioactivity were detected in the blood and
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muscle tissue of exposed fish. The part accumulated in these fish compartments remained constant
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during the elimination phase. Hence, biomagnification of MWCNT in the food chain is possible and
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should be subject of further research.
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Introduction
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Carbon nanotubes (CNT) are considered promising materials in nanotechnology1. Investment in
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nanotechnology research and development as well as nanoparticle production volumes are growing
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rapidly worldwide2, 3. Based on the assumption that this will lead to increasing CNT release in the
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environment, a lot of research has recently been dedicated to investigating the possible (eco)toxicity
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of this nanomaterial4. However, little information is available on the impact of CNT and other
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carbonaceous nanoparticles on aquatic vertebrates. Some studies found that these nanomaterials
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did not affect exposed organisms5, but it was also reported that single walled CNT (SWCNT) exerted
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respiratory toxicity to rainbow trout (Oncorhynchus mykiss)6, that SWCNT and fullerenol altered the
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antioxidant capacity of adult zebrafish (Danio rerio)7, that multiwalled CNT (MWCNT) and fullerene
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caused toxicity in zebrafish embryos8, 9, and that double walled CNT (DWCNT) physically blocked
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epithelial tissue of larvae of the African clawed frog (Xenopus laevis)10. As can be extracted from a
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recent review, there is a distinct lack on data on bioaccumulation of CNT11. This is consistent with the
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limited number of analytical methods available to differentiate carbon-based nanoparticles from
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organic matrices, and thus, to detect these materials and measure their concentration in biological
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samples12-15. Using mainly microscopic techniques, it was observed that CNTs clogged the filter
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apparatus and covered the carapace of daphnids16, precipitated on the gills of rainbow trout6, or
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filled the gastrointestinal tract of several aquatic invertebrates16-18 after exposure of the organisms
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via aqueous dispersions of CNT. From research published up to now, it seems that nanotubes do not
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pass the gut epithelium to accumulate in exposed aquatic organisms4, 15, 19. Benthic invertebrates and
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earthworms were shown to incorporate CNT from sediment, food, and soil matrices, but the
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nanomaterials did not appear to pass the gut epithelium either20-22. Since most described effects
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could not be linked to internal distribution of nanoparticles and/or their agglomerates, the
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underlying mechanisms of possible CNT toxicity remain subject of many current investigations. In a
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recent study, SWCNT were tracked in fish using near infrared fluorescence after single gavage
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We studied the accumulation and distribution of MWCNT for the first time in a vertebrate after
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waterborne exposure. Adult zebrafish were exposed to radiolabeled MWCNT (14C-MWCNT) for up to
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one month and uptake of radioactivity by the fish was quantified over time. Afterwards, exposed fish
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were transferred to clear water to examine elimination of MWCNT. The influences of the feeding
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regime and the presence of dissolved organic carbon (DOC) on MWCNT accumulation in zebrafish
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were additionally determined. In all experiments, fish were dissected to assess the fraction of
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MWCNT partitioned to different fish compartments.
87 88
Materials and Methods
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Preparation of 14C-MWCNT test suspensions for exposure of zebrafish
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One batch of
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synthesized (as described in the SI) and used to perform all experiments. 500 µg of
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agglomerates were weighed on a microbalance (0.0001 mg readability; Mettler Toledo UMX2,
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Mettler Toledo GmbH, Germany) and added to a 1000 mL test beaker, filled with 500 mL of test
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medium, which contained 294.0 mg/L CaCl2, 123.3 mg/L MgSO4, 63.0 mg/L NaHCO3, and 5.3 mg/L
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KCl. The beaker was placed in an ice bath and the MWCNT material was homogenized by means of
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ultrasonication with a microtip (Sonoplus HD 2070, Bandelin, Germany) set to a 0.2 seconds pulse at
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70 W – 0.8 seconds pause regime for 5 minutes. The procedure was repeated two times. This
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method resulted in the presence of small agglomerates and single nanotubes of 0.2 to 1.0 µm tube
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length (both referred to as MWCNT) in the medium, as was visualized by means of transmission
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electron microscopy (TEM; SI Figure S3). In case DOC was supplemented to the medium, a stock
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solution was prepared by mixing 2.5 g Aldrich humic acid (Sigma Aldrich Chemie GmbH, Germany)
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and 0.4 g NaOH (Carl Roth GmbH, Germany) in 1 L of distilled water. After stirring overnight, the
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suspension was centrifuged for 15 minutes at 350 g and then filtered through three filters of
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different pore sizes (1.2, 0.8, and 0.45 µm) to remove suspended particles. The pH was set to 7 with
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HCl before the total organic carbon (TOC) content was determined using a TOC-Analyzer (Shimadzu
14
C-MWCNT with a specific radioactivity of 1.3 ± 0.1 MBq/mg (SI Table S5) was 14
C-MWCNT
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Europe GmbH, Duisburg, Germany). The volume to add to 500 mL medium to obtain 8 mg DOC/L was
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calculated and amounted to about 6 mL. In this case, the MWCNT agglomerates were sonicated in
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about 494 mL and 6 mL DOC stock solution was added afterwards. The 14C-MWCNT concentration (1
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mg/L) and homogeneity of the test dispersion were verified by measuring the amount of radioactivity
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in replicate samples directly after sonication. Four samples of 1 mL were mixed with 5 mL of
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scintillation cocktail (Insta-Gel PlusTM, Perkin Elmer, Germany) and subjected to liquid scintillation
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counting (LSC; LS 5000 TD, Beckmann Instruments GmbH, Germany; detection limit: 1 Bq
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corresponding to 0.8 ng MWCNT). Using a radioactive standard (SPEC-CHEC-14C, Perkin Elmer,
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Germany), it was verified that the presence of DOC did not influence LSC measurements in the
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applied amounts. After placing the test beakers in a heated water bath (25 ± 1°C), one adult female
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fish was added to the MWCNT suspension.
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Test setup
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Adult female zebrafish (Danio rerio held as described in the SI; 453 ± 103 mg wet weight) were
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individually exposed to 1 mg
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was shown that this rather high concentration was necessary to be able to detect radioactivity in fish
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compartments, but did not cause adverse effects on the growth and condition of zebrafish (data not
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shown). Females were selected for this bioaccumulation study as they are bigger than male fish of
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the same age. For comparability reasons, only one sex was tested. At least five fish were subjected to
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each treatment, i.e. in separate test beakers. Since preliminary experiments had shown that data of
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MWCNT accumulation involved large scatter, more replicates were provided for important sampling
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points. Moreover, data of different experiments involving the same treatment and exposure duration
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were pooled, when no significant differences in the results were observed. In the uptake experiment,
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uptake of MWCNT by the fish was quantified over time. Sampling of the water phase and the fish
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was performed after 3 h, 1 day, 2 days, 1 week, 2 weeks, 3 weeks, and 4 weeks, as described below.
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In the elimination experiment, fish were exposed to 14C-MWCNT for two weeks, and then rinsed and
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transferred to clear water to quantify elimination of MWCNT. Samples were taken after exposure,
14
C-MWCNT/L in 500 mL test medium. In preliminary experiments, it
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and after an elimination period of 4, 24, 48, and 168 h. In both tests, fish were fed every second day
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before the MWCNT suspension or clear medium was completely renewed (water renewal every 48
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h). They were supplied with 2% of their wet weight in Artemia nauplii that were consumed within ten
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minutes. In the feeding experiment, the influence of the feeding regime on MWCNT accumulation in
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the fish was determined. Fish were either not fed or fed once a day with 1% of their body weight in
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Artemia nauplii during a 48 h exposure period, or additionally fed every second day with 2% of their
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body weight in Artemia nauplii after complete water renewal during a 168 h exposure period. In the
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DOC experiment, it was examined whether DOC has an influence on accumulation of MWCNT in
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zebrafish. Fish were exposed to 1 mg MWCNT/L in the presence or absence of 8 mg DOC/L. The fish
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were not fed and the water was not renewed during this experiment. Sampling was performed after
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3, 24, and 48 h.
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Sampling
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At each sampling point, fish were caught with a steel grid and rinsed to remove MWCNT containing
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water from the skin. Their length (L) and weight (W) were measured to determine the condition
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factor (CF: W/L³) that was compared with the CF of five control fish held under the same conditions
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in clear water. Afterwards, they were anaesthetized in a saturated benzocaine (Sigma Aldrich,
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Germany) solution in tap water. Depending on the aim of the experiment, only the gut of the fish was
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removed, or they were fully dissected in order to study the distribution of CNT in zebrafish (see SI).
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Except for the blood, all tissues were separately placed on small pre-weighed pieces of aluminium
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foil, dried in an oven at 60 °C, and weighed on a microbalance. All samples were transferred to stable
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glass test tubes, covered with tissue solubilizer (BTS-450, Beckman Coulter, USA), and incubated for
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24 h at 37°C. Afterwards, a 35% hydrogen peroxide solution was added to bleach the samples. They
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were transferred to 20 mL scintillation vials using methanol to rinse the glass tubes. The vials were
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filled with scintillation cocktail, and subjected to LSC after storage overnight at 4°C in the dark. The
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amount of radioactivity in samples of unexposed fish, which were bleached and solubilized in the
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same way as those of exposed fish, was below the limit of detection. Adding a known amount of 14C6 ACS Paragon Plus Environment
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MWCNT to blank tissue samples, a recovery higher than 95% could be derived (data not shown). To
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make a balance of the total radioactivity present in the system (SI Figure S7), the MWCNT
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concentration in the medium was again measured. Thereto, the remaining water in the test beakers
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was sonicated as described above, in order to again homogenize the test dispersion, now containing
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settled agglomerates of MWCNT. The radioactivity in four 1 mL water samples per test beaker was
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measured by means of LSC.
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Data interpretation
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In order to calculate the bioaccumulation factor (BAF), a one-compartment model was fitted to the
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uptake and elimination data (according to Connell, 199823):
= ∗
∗ 1 − ∗ + ∗ ∗
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where Cb and Cw are the concentrations of MWCNT detected in the fish and the water, t represents
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time, and k1 and k2 are the uptake and elimination rate constants. Since the amount of MWCNT in
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the water body did not change a lot due to uptake by the fish (SI Figure S7) and since the water was
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renewed every second day, Cw can be regarded as invariable. It amounted to 1 and 0 mg/L during the
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uptake and depuration phase, respectively. At steady state Cb/Cw equals k1/k2, giving the BAF.
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Differences between treatments were tested after checking the data for outliers by means of
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Dixon/Grubbs test. Then, the data were checked for normality and variance homogeneity with
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Shapiro-Wilk’s test and Levene’s test, respectively. Statistical differences were identified by means of
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pairwise comparison using Student’s t-test in case of homoscedasticity and by Welch’s t-test in case
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of heteroscedasticity. For the evaluation of statistically significant fluctuations over time, Dunnett’s t-
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test was applied when variances were homogeneous and Welch’s t-test with Bonferroni adjustment
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when variances were heterogeneous. A significance level (α) of 0.05 was chosen for all tests.
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Results
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There was no significant difference in the condition factor (CF) of exposed and control fish at all time
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points (SI Table S1). In the uptake experiment, MWCNT quickly associated with zebrafish over time, 7 ACS Paragon Plus Environment
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but the variability between replicates was extremely high (with coefficients of variation of 70 to
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150%). Steady state was already reached within 24 h (Fig. 1, A, Y1). Maximal concentrations were
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measured after two weeks of exposure, where on average 6% (25 µg) of the total MWCNT amount
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present in the water was taken up by the fish (Table 3). The mean MWCNT level (± standard
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deviation) in all complete fish of the 24 h up to 4 weeks treatments amounted to 73 (± 93) ng
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MWCNT/mg fish dry weight (= 16 ng MWCNT/mg fish wet weight) (Fig. 1, A, Y1), and since steady
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state was reached, a bioaccumulation factor of 73 on dry weight (16 on wet weight) could be
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calculated (BAF = Cb/Cw = (73 mg/kg)/(1 mg/L)). In the elimination experiment, MWCNT were quickly
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rejected to the water phase, but after 24 h, no further elimination of the material could be observed
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(Fig. 1, B), i.e. the fish MWCNT concentrations after 24, 48, and 168 h of presence in clear water were
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not significantly different, amounting to 5 ng MWCNT/mg fish dry weight on average (SI Table S4).
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The data were modelled over 48 h, the time between the beginning of the depuration phase and the
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first water renewal. For complete fish, an elimination rate constant (k2) of 0.14 h-1 was calculated
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(Fig. 1, B). With this value, an uptake rate constant (k1) of 9.64 L*kg-1*h-1 was derived. Dividing k1 by
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k2, a BAF of 70 is obtained, verifying the above calculated BAF using Cb and Cw at steady state.
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MWCNT mainly accumulated in the digestive tract of the fish (Table 1, 2, 3; SI Table S2, S3, S4), which
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is not necessarily presenting a bioavailable fraction. Hence, it was removed and measured separately
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from the total remaining fish body. The one-compartment model did not fit to the data of fish from
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which the gut was removed. In the uptake phase, steady state was directly reached, i.e. no
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differences in the fish MWCNT content were observed over time (Fig. 1, A, Y2). Similarly, elimination
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was quicker than predicted by the model (Fig. 1, C). After 4 h, most of the MWCNT were no longer
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associated with the gut excised fish and the internal MWCNT content remained constant up to the
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last sampling point of 168 h, amounting to 0.24 ng/mg fish dry weight on average.
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A
206 B
C
207 208 209 210 211 212 213
Fig. 1 Concentration of multiwalled carbon nanotubes (MWCNT) in zebrafish on dry weight (dw) after exposure to 1 mg MWCNT/L via the water phase in function of time. The upper graph (A) shows uptake by the fish over time. Filled rounds represent the concentrations in complete fish (Y-axis on the left: Y1), non-filled rounds those in fish from which the gut was removed (Y-axis on the right: Y2). The graphs below (B, C) present elimination of MWCNT from two weeks exposed zebrafish (time = 0 h) that were transferred to clear water. Again filled and non-filled rounds represent complete and gut excised fish, respectively. A one compartment model is plotted to all data (R²: determination coefficient).
214 215
Feeding of the fish led to significantly facilitated elimination of MWCNT via the digestive tract, as was
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shown in the feeding experiment (Fig. 2). Within an exposure period of 48 h without water renewal,
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unfed fish had an MWCNT body burden that was on average 45 times higher than fish that were daily
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supplied with uncontaminated Artemia nauplii (Fig. 2, A). After 168 h, fish that were fed once a day
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accumulated less MWCNT than fish that were fed once every second day and much less than fish
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that were not fed at all (Fig. 2, B). No significant differences in the MWCNT content of fish from
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which the gut was removed were observed (Fig. 2, D). The differences originated from the fraction in
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the gut (Fig. 2, C). In the DOC experiment, it was shown that the presence of DOC in the medium also
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resulted in the accumulation of lower amounts of MWCNT by the fish (Fig. 3). After 48 h, the total
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concentrations in fish exposed to MWCNT in presence and absence of DOC amounted to 9 and 91 9 ACS Paragon Plus Environment
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ng/mg dry weight, respectively (Fig. 3; SI Table S2 and Table S3). Compared to unfed fish exposed
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without DOC, MWCNT got quicker associated with the gills and to larger relative amounts when DOC
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was present in the medium (Table 1 vs. Table 2). The sum of MWCNT present in the skin, filet, and
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blood (= rest fish), and the gills was relatively larger with (11%) than without (2%) DOC after 48 h of
229
exposure. Furthermore, relatively less was present in the gut with (89%) than without (96%) DOC
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(Table 1 vs. Table 2).
231 A
B
232 D
C
233 234 235 236 237 238 239
Fig. 2 Concentration of multiwalled carbon nanotubes (MWCNT) in zebrafish (gut) after exposure to 1 mg MWCNT/L via the water phase for 48 or 168 h, during which food was provided in different regimes (no food, one time per two days, and once per day) and the MWCNT dispersion was either renewed one time every 48 h or not at all. The values are expressed on dry weight (dw). The rounds, the asterisks, the vertical bars, and the error bars represent the data points for separately exposed fish, outliers, the mean, and the standard deviation on the mean of remaining replicates, respectively. Significant differences between treatments are indicated by different letters.
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Fig. 3 Concentration of multiwalled carbon nanotubes (MWCNT) in zebrafish after exposure to 1 mg CNT/L via the water phase in function of time and in absence (-) or presence (+) of dissolved organic carbon (DOC, 8 mg/L). The mean of five replicates is presented by the vertical bars to which the error bars represent the standard deviation, all expressed on fish dry weight (dw). Significant differences between DOC treatments at one time point are indicated by the asterisks.
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As was mentioned above, MWCNT mainly accumulated in the gut of all fish (Table 1, 2, 3; SI Table S2,
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S3, S4). Large relative amounts of radioactivity were also detected in gills, skin and muscle samples of
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briefly exposed fish (3 h). Afterwards, only low percentages of the total amount were associated to
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these fish compartments. No distribution to the liver, the gonads, and the brain was observed.
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However, low amounts of radioactivity were detected in blood of fish exposed for more than one
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week. The average total amount of radioactivity in blood samples of unfed fish (exposed for only one
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week) was higher than in fish fed every second day (exposed for two weeks), corresponding to 7.6
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and 2.5 ng 14C-MWCNT, respectively (Table 2 vs. Table 3). During the elimination phase, the absolute
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blood MWCNT content did not significantly change, leading to an increase of the relative amount of
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radioactivity in the blood current over time. On the other hand, the absolute amount of MWCNT
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associated with the rest of the fish, i.e. the skin and filet, drastically decreased (from ca. 100 ng to 20
257
ng) within the first 4 h of depuration. Afterwards, the relative amount of CNT in the rest fish
258
increased, but the absolute amount corresponded to about 20 to 30 ng 14C-MWCNT at all elimination
259
sampling points (Table 3). The MWCNT concentration in the skin and filet indeed decreased within
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the first four hours of elimination and remained more or less constant thereafter (SI Table S4). It is
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suggested that quick detachment from the skin occurred, and that the remaining radioactivity
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tissue was demonstrated for the gills, to which no more MWCNT were attached after 168 h of
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depuration (Table 3). Similarly, quick adhesion to the fish surface was measured in the uptake phase:
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on average 16 and 26% of the total amount of MWCNT associated with the fish were detected in gill
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tissue and skin within 3 h (Table 2). It seems therefore that connection of MWCNT to the fish exterior
267
is reversible. During the depuration period, the part accumulated in internal fish compartments
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remained constant (as shown for blood and assumed for the filet), and the main MWCNT fraction
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was purged from the gut and detached from the gills and skin (as shown for the gills and assumed for
270
the skin).
271 272 273 274 275 276 277
Table 1 Distribution of multiwalled carbon nanotubes (MWCNT) in different compartments of zebrafish, which were 14 exposed to 1 mg C-MWCNT/L via the water phase in the presence of 8 mg dissolved organic carbon/L. The fraction in each fish compartment is expressed as the percentage of the total amount of radioactivity detected in the fish. The sum represents the total amount, and is expressed as the corresponding amount of MWCNT in µg. The rest is the relative amount of radioactivity measured in the residual fish, from which gut and gills were removed. Values that are struck out represent outliers (M: mean; SD: standard deviation).
exposure time (h) 3
elimination time (h) 0
M SD 24
0
M SD 48
0
M SD
gut (%) 8.2 68.6 11.4 5.5 6.4 7.9 2.6 90.4 93.2 99.0 97.3 12.9 95.0 3.9 92.2 83.8 95.4 84.4 89.4 89.0 5.0
gills (%) 43.8 4.8 12.4 39.4 40.0 28.1 18.1 3.0 0.2 0.3 0.5 28.6 1.0 1.3 7.1 9.8 1.3 3.5 8.7 6.1 3.6
rest = fish-gut-gills (%) 48.0 26.6 76.2 55.1 53.5 51.9 17.7 6.7 6.6 0.7 2.2 58.5 4.0 3.1 0.8 6.4 3.3 12.1 1.9 4.9 4.5
sum (µg) 0.035 0.122 0.107 0.039 0.035 0.068 0.043 0.054 0.198 0.709 0.197 0.020 0.235 0.277 0.368 0.048 1.602 0.655 0.662 0.667 0.580
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Table 2 Distribution of multiwalled carbon nanotubes (MWCNT) in different compartments of unfed zebrafish, which 14 were exposed to 1 mg C-MWCNT/L via the water phase. No water renewal was performed. The fraction in each fish compartment is expressed as the percentage of the total amount of radioactivity detected in the fish. The sum represents the total amount, and is expressed as the corresponding amount of MWCNT in µg. Values that are struck out represent outliers (M: mean; SD: standard deviation; DL: detection limit).
exposure time (h) 3
elimination time (h) 0
M SD 24
0
M SD 48
0
M SD 168
0
M SD
gut (%) 1.2 6.3 2.7 6.8 29.3 4.3 2.7 31.0 99.0 92.3 99.5 75.0 91.3 11.3 99.1 91.2 94.9 97.5 98.8 96.3 3.3 99.7 99.8 99.9 98.6 99.9 99.6 0.6
gills (%) 7.9 32.9 24.6 8.0 6.0 15.9 12.1 35.0 0.4 2.5 0.2 24.2 12.4 16.2 0.5 0.0 4.2 1.8 0.4 1.4 1.7 0.11 0.03 0.02 0.36 0.03 0.03 0.00
blood (%)
