This study investigated the toxicity of double walled carbon nanotubes (DWCNTs) to three aquatic organisms, namely,
Carbon nanotubes (CNTs) are the most widely studied class of engineered nanomaterials. This is due to carbon’s unique hybridisation properties and the sensitivity to variations in the synthesis conditions allowing tailoring of these nanostructures for specific applications [
The synthesis and use of CNTs lead to their release into the environment, particularly the aquatic environment. This may be as a result of the use of CNTs and through wastewater containing nanowaste and runoff from landfills [
Aquatic toxicology has been identified as a useful tool for assessing environmental risks associated with CNTs [
The aquatic ecotoxicity of ENMs is greatly influenced by their speciation in aquatic environments [
This study reports the influence of ionic strength (based on Na and Ca) and humic acid on the acute toxicity of pristine and oxidised DWCNTs to three aquatic organisms representing three different trophic levels, that is, primary consumers (algae),
Double-walled carbon nanotubes were synthesised in our laboratories using the catalytic vapour deposition method as described by Flahaut and coworkers [
Surface morphology and DWCNT diameter were determined using high resolution transmission electron microscopy (HR TEM, JEOL Jem-2100) and scanning electron microscopy (SEM, JEOL JSM 7500F coupled with EDX). Elemental composition of DWCNTs was determined using energy dispersive X-ray spectroscopy (EDX) and surface functionality was determined using Raman spectroscopy (Perkin Elmer Raman Microscope, RamanMicro 200) and Fourier transform infrared spectroscopy (Perkin Elmer Spectrum 100 FT-IR-spectrometer). Hydrodynamic size and zeta potential in aqueous DWCNT suspensions were determined using dynamic light scattering (Malvern Zetasizer Nano series, NanoZS).
Moderately hard reconstituted waters with varying ionic Ca2+ strengths were prepared by varying the amount of calcium carbonate. Reconstituted waters with ionic strengths of 0.00449 M Ca2+, 0.0226 M Ca2+, and 0.0339 M Ca2+ were prepared. The salts used to prepare reconstituted water were kept constant when Ca2+ was varied. The salts were 0.2400 g of NaHCO3, 0.0200 g of KCL, and 0.1500 g of MgSO4 and these were added to 5 L of Milli-Q water. The DWCNT suspensions were prepared by adding 50 mg of DWCNTs to 100 mL of the prepared waters with varying ionic strengths. The suspensions were sonicated for 2 h and were shaken at 30 min intervals. The suspensions were then refrigerated at 4°C for 24 h.
A stock solution of humic acid was prepared by dissolving 50 mg of humic acid powder containing 20% ash (Fluka) in 100 mL of Milli-Q water. The humic acid powder contained ash estimated to be 20% of the mass of the powder. It was important to first eliminate the ash content from the solution and determine the exact concentration of humic acid in the stock solution. The solution was filtered with a 0.45
Double-walled carbon nanotubes stock suspensions were prepared by adding 5 mg of carbon nanotubes (pristine and oxidised) to 100 mL of Milli-Q water (pH 7). Sodium chloride was added to the DWCNT suspension to vary the ionic strength in suspensions from 0.00335, 0.00585, 0.00835, and 0.0139 M NaCl. The suspensions were then sonicated for 2 h and refrigerated at 4°C for 24 h (to stabilise the suspensions).
The hydrodynamic particle size was determined and zeta potential measurements were done for each of the different salinity and humic acid conditions using dynamic light scattering.
The algal toxicity tests were carried out using the Algaltoxkit sourced from MicroBioTests Inc. (Cat. # 030229). The test kit contained prepared nutrient stock solutions for the culturing medium, matrix dissolving medium, algal beads, and plastic long cells. In our tests, the long cells were replaced with glass Erlenmeyer flasks.
Algal culturing medium (2 L) was prepared by adding 20 mL of nutrient stock solution A and adding 2 mL (each) of nutrient stock solutions B, C, and D. The medium was aerated and set aside. The
The DWCNT stock solution was prepared by mixing 10 mg of dry DWCNT and 100 mL of culture medium in 100 mL Schott bottles. The mixture was then sonicated for 2 h in an ultrasonication bath. The suspensions were prepared 24 h before toxicity tests were carried out and were refrigerated at 4°C.
A total of six exposure concentrations arranged in a geometric series with a separation factor of 2 were prepared, with the highest DWCNT concentration being 100 mg/L. The diluted DWCNT suspensions (25 mL in each flask) were placed in Erlenmeyer flasks. A total of 10 000 algal cells (volume of stock solution added was calculated using the cell density in stock solution) were added to each Erlenmeyer flask. Each DWCNT concentration was prepared in duplicate. Controls consisting of duplicate negative (i.e., no DWCNTs added) and positive (reference toxicant CdCl2, 100 mg/L) were included in each bioassay. The algae were incubated for a period of 96 h in an incubator under a 16 : 8 h, light : dark regime.
Two samples (0.5 mL) from each Erlenmeyer flask were collected at 24 h intervals. Algal cell densities in samples were determined by cell counting using a haemocytometer chamber under a light microscope. Calculations of growth inhibition were done according to the OECD guidelines [
Algae cells were counted using a haemocytometer chamber with at least two counts for each replicate. Manual cell counting was required because the determination of cell density by UV/Vis absorption was not possible due to the large variations in the background spectrum of the test suspensions containing CNT. The lack of quicker, less laborious, and validated methods to quantify green algae in the presence of CNT and particulate matter in general has made manual cell counting the only option.
Test organisms,
Fish were sourced from Kirsten Aquaculture, situated at Modimolle, South Africa. The age of the fish used for exposure experiments ranged from 10 d to 21 d. The fish were exposed for 96 h to different DWCNT concentrations (500 mg/L, 250 mg/L, 125 mg/L, 62.5 mg/L, 31.25 mg/L, and 15.6 mg/L) using the static nonrenewal test. The tests were conducted in 600 mL low form glass beakers using moderately hard reconstituted water and five fish were placed in each beaker. The exposure tests were done in triplicate, with four negative controls (no DWCNTs added) and four positive controls (reference toxicant, 1000 mg/L K2Cr2O7). The experiments and controls were prepared in accordance with the OECD Guidelines 203 (2002) (pH 6 to 8.5, dissolved oxygen >80%, conductivity <10
As an estimate of relative lethal toxicity, EC50/LC50 values were based on the cumulative mortality observed at the end of a desired exposure time. EC50/LC50 values for each test species together with their respective 95% confidence intervals (CI) were calculated using the most appropriate statistical method for the specific toxicity data. The USEPA Probit Analysis Program used for calculating LC/EC values (version 1.5) was initially used to calculate the EC50/LC50 values [
The SEM micrograph of pristine DWCNTs showed a mixture of catalyst residues and some protruding fibre-like structures (Figure
(a) SEM micrograph of pristine DWCNTs showing catalyst residues. (b) SEM micrograph of oxidised DWCNTs showing nanotubes of different sizes and some catalyst residues.
The surface morphology of DWCNTs was analysed using TEM. Figure
TEM micrographs of (a) pristine and (b) oxidised DWCNTs. The pristine DWCNTs have excess catalyst on their surfaces. The oxidised DWCNTs have clean surfaces with scattered dark spots which are catalyst residues. (c) A graph showing the diameter size distribution for the synthesized DWCNTs.
Oxidation of DWCNTs was confirmed by FT-IR spectroscopy. The peak at around 1 721 nm is characteristic of the C=O group. Some O-H groups were identified at the 3 492 nm peak and C-O groups were identified at around 1 207 nm for the oxidised DWCNTs but were insignificant in the case of pristine DWCNTs (Figure
FT-IR of both pristine and oxidised DWCNTs illustrating the major functional groups present in oxidised DWCNTs which are not found in pristine DWCNTs.
The quality of DWCNTs was determined using Raman spectroscopy. The D-band peak which was observed at approximately 1300 cm−1, for both pristine and oxidised DWCNTs, is related to the presence of defects on the walls of CNTs (Figure
Raman spectra of pristine and oxidised DWCNTs showing different intensities of the D- and G-bands in both pristine and oxidised DWCNTs.
Pristine DWCNTs contained a large amount of magnesium and molybdenum which originated from the magnesium oxide supported Mo/Co catalyst used for the synthesis of DWCNTs. Some oxygen was also present in pristine DWCNTS (Figure
EDX spectra of (a) pristine and (b) oxidised DWCNTs showing their elemental composition.
After oxidation, DWCNTs still contained some of the molybdenum. Magnesium oxide in the catalyst is used as a support for the molybdenum and acid treatment removed most of the magnesium oxide leaving the molybdenum (Figure
The oxygen fundamentally found to be present in DWCNTs is a result of oxidation in the presence of nitric acid. Carbon, as expected, was also detected.
Characteristics such as size distribution, surface chemistry, surface charge, and dispersion in aqueous phase have been identified as essential in studying the environmental fate of DWCNTs. Such data are essential because the interactions of environmental and biological systems are dependent on them [
Surface morphology and the presence of impurities in pristine DWCNT were determined by TEM and SEM. Pristine DWCNTs had impurities on their surface while oxidised DWCNTs had very few impurities. Elemental analysis using EDX indicated that pristine DWCNTs contained residual magnesium and molybdenum content originating from the catalyst used during synthesis of DWCNTs. Flahaut and colleagues reported a similar finding of metal residue on DWCNTs and attributed these residues to poor dispersion of the metal catalyst. Some amount of oxygen was also present in as-prepared DWCNTS. The oxygen content originated from a MoO
Oxidation of DWCNTs was confirmed using FT-IR spectroscopy where vibrational peaks of C=O, C-O, and O-H bonds were detected in oxidised DWCNTs. From the Raman spectra of pristine and oxidised DWCNTs the
Ionic strength (IS) and natural organic matter were found to influence the surface charge on DWCNT surfaces resulting in reduced or increased stability of DWCNTs in water suspensions.
Increasing ionic strength caused an increased aggregation of both pristine and oxidised DWCNTs. The salts used in suspensions to vary ionic strength, CaCO3 and NaCl, had different effects on the colloidal stability of pristine and oxidised DWCNTs.
The addition of sodium chloride in the DWCNTs suspensions resulted in increased aggregation in both pristine and oxidised DWCNTs as the IS was increased (Figure
Hydrodynamic size and zeta potentials of DWCNTs at different IS (Na+) levels (error bars are the ranges of hydrodynamic size and zeta potentials).
A decrease in zeta potential for both pristine and oxidised DWCNTs was observed (Figure
The increase in hydrodynamic size in particles is as a result of higher aggregation caused by the increased salinity in water. The presence of electrolytes in water affects the stabilisation of colloids in the aqueous phase thus lowering the concentration of smaller particles in the water column [
Increased IS in water promotes aggregation in both pristine and oxidised DWCNTs as confirmed by the increased hydrodynamic particle size with increased hardness (Figure
Hydrodynamic size and zeta potentials of DWCNTs at different IS (Ca2+) levels.
It was found that zeta potential data correlated with the increased aggregation observed with increased IS. Pristine DWCNTs had a slightly lower average zeta potential at the different IS levels (Figure
Oxidised DWCNTs had a slight increase in zeta potential between IS of 0 M and 0.00449 M. Thereafter a consistent decrease in zeta potential was observed with increased cation content (Figure
Both DWCNT types had increased hydrodynamic sizes as the IS of water was increased. However, there was no noticeable increase in hydrodynamic size of both pristine and oxidised DWCNTs from the IS of 0.0226 to 0.0396 M. The zeta potential of oxidised DWCNTs increased slightly in IS of 0.00449 M and decreased in the higher IS values (Figure
There was a higher amount of smaller particles (DWCNT aggregates) suspended in water containing humic acid compared to the blanks. The zeta potentials of DWCNTs confirm that there was an increase in the repulsion energies of DWCNT particles.
Addition of humic acid to DWCNT suspensions resulted in more negative zeta potential values. It was shown that pristine DWCNTs had a consistent reduction in zeta potential up to around 2 mg/L of humic acid (Figure
Hydrodynamic size and zeta potentials of DWCNTs at different humic acid levels.
Humic substances are a fraction of natural organic matter and are ubiquitous in the aqueous environments. Humic acids are rich in aromatic moieties which, in theory, can interact with electron-rich surfaces such as graphene surfaces. Humic substances are known to decrease sedimentation rates of carbonaceous nanomaterials in aqueous suspensions [
The general trend in the exposure experiments done for this study is that there is an increase in growth inhibition with an increase in DWCNT concentration. Figure
The growth inhibition caused by pristine DWCNTs was higher than that of oxidised DWCNTs. The EC50 of pristine DWCNTs was found to be 17.95 mg/L while oxidised DWCNTs had an EC50 of 10.93 mg/L. Varied environmental parameters such as humic acid content and ionic strength (using both monovalent and divalent cations) resulted in the reduction of toxicity of both pristine and oxidised DWCNTs to
Growth inhibition (EC50s and 95% confidence intervals) for
Exposure | Physicochemical characteristics | EC50 (mg/L) | Confidence limits (mg/L) |
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Pristine | 0.00335 (control reconstituted water) M Na | 17.95a | 9.8–26.72 |
0.00585 M Na | 29.47a | 22.95–37.65 | |
0.00835 M Na | 30.89a | 18.06–40.4 | |
0.0139 M Na | 52.31b | 33.96–76.1 | |
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Oxidised | 0.00335 (control reconstituted water) M Na | 10.93a | 6.71–15.59 |
0.00585 M Na | 27.22a | 19.56–38.9 | |
0.00835 M Na | 16.11a | 11.59–22.42 | |
0.0139 M Na | 49.31b | 27.24–58.89 | |
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Pristine | 0.00449 M Ca | 23.26a | 17.04–28.00 |
0.0226 M Ca | 25.57a | 16.07–34.93 | |
0.0339 M Ca | 31.52a | 21.56–50.92 | |
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Oxidised | 0.00449 M Ca | 22.86a | 15.59–42.42 |
0.0226 M Ca | 22.91a | 12.95–40.89 | |
0.03395 M Ca | 24.61a | 13.37–34.44 | |
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Pristine | 0 (control reconstituted water) M Na | 17.95b | 9.80–26.72 |
0.9 mg/L HA | 22.75a | 14.39–31.70 | |
1.81 mg/L HA | 26.56a | 10.95–34.61 | |
3.64 mg/L HA | 31.65a | 10.87–42.19 | |
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Oxidised | 0 (control reconstituted water) M Na | 10.93a | 6.71–15.59 |
0.9 mg/L HA | 13.59a | 8.71–21.19 | |
1.81 mg/L HA | 18.01a | 16.71–24.93 | |
3.64 mg/L HA | 20.56a | 6.58–34.15 |
The algal cells were found to agglomerate with DWCNTs where a large amount of cells are entrapped in the DWCNT agglomerates. Figure
A light microscope picture of agglomeration of
Carbon nanotubes, SWCNT, DWCNTs, and MWCNT, have been reported to have an effect on the growth rates of various algal species [
Humic acid reduced agglomeration of DWCNTs (Figure
Moreover, the amounts of shading presented by smaller agglomerates may have been lower than with larger agglomerates. This may be due to the fact that humic acid reduced the hydrodynamic size of DWCNT agglomerates in the exposure medium and increased the specific surface area for the interaction of algal cells with the DWCNTs. Shading effect has been reported to be the major contributor in the toxicity of CNTs to algae [
To vary the ionic strength, divalent and monovalent cations, respectively, were added to the exposure bioassays. These cations influence the rate of agglomeration of nanoparticles such as DWCNTs since Ca2+ and Na+ cause rapid agglomeration of CNTs. From Figures
Double-walled carbon nanotubes have been reported to have different toxicities to a marine algal species,
The acute toxicity of CNTs reported in the literature varies by orders of magnitude, especially for MWCNTs. The main reason for such variation is a result of differences in methods used to conduct toxicity tests and methods of evaluating cell densities in the algal toxicity test [
Oxidised DWCNTs were generally more toxic than pristine DWCNTs in all experiments. Wei and coauthors suggest that the toxicity is mediated through initial interaction between algal cells and CNTs [
The acute toxicities of both pristine and oxidised DWCNTs to
Lethal concentrations (LC50s and 95% confidence intervals) for
Exposure | Physicochemical characteristics | EC50 (mg/L) | Confidence limits (mg/L) |
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Pristine | 0.00335 (control reconstituted water) M Na | 2.801a | 1.58–3.95 |
0.00585 M Na | 2.710a | 1.21–7.58 | |
0.00835 M Na | 2.835a | 0.82–5.66 | |
0.0139 M Na | 3.163a | 2.10–6.02 | |
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Oxidised | 0.00335 (control reconstituted water) M Na | 4.480a | 2.09–7.49 |
0.00585 M Na | 3.320a | 1.88–6.04 | |
0.00835 M Na | 3.048a | 1.71–7.05 | |
0.0139 M Na | 3.541a | 1.52–6.35 | |
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Pristine | 0.00449 M Ca | 3.048a | 1.51–6.43 |
0.0226 M Ca | 2.81a | 1.58–3.95 | |
0.03395 M Ca | 3.327a | 1.72–6.44 | |
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Oxidised | 0.00449 M Ca | 3.77a | 1.01–5.49 |
0.0226 M Ca | 4.48a | 2.09–7.49 | |
0.03395 M Ca | 3.57a | 1.81–7.58 | |
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Pristine | 0 (control reconstituted water) M Na | 2.80a | 1.58–3.95 |
0.9 mg/L HA | 1.91a | 1.21–7.58 | |
1.81 mg/L HA | 1.89a | 0.82–5.66 | |
3.64 mg/L HA | 1.80a | 1.10–6.02 | |
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Oxidised | 0 (control reconstituted water) M Na | 4.48a | 2.09–7.49 |
0.9 mg/L HA | 4.07a | 1.88–6.75 | |
1.81 mg/L HA | 3.25a | 1.71–7.05 | |
3.64 mg/L HA | 3.07a | 1.52–6.87 |
Oxidised DWCNTs were generally less toxic to
The exposed organisms were studied under a light microscope to visualise how the organisms interact with the DWCNTs.
Changes in the density of oxidised DWCNTs in the gut tract of
Visual inspection of
Pristine DWCNTs were found to be more toxic to
The increase in ionic strength decreased the toxicity of both pristine and oxidised DWCNTs. Increase of Na+ did not have an effect on the toxicity of pristine DWCNTs but reduced the average LC50 of oxidised DWCNTs. Data on hydrodynamic size results show that increased ionic strength increased the hydrodynamic size of both pristine and oxidised DWCNTs with increased Ca2+ having a greater effect than increased Na+. The concentration of DWCNTs in these suspensions per unit volume is decreased and thus the amount of DWCNTs ingested by daphnia in such exposure conditions is low and results in lower biological effect. It is possible that the DWCNT concentration per unit volume did not decrease when Na+ was increased as it did in the case of Ca2+.
In a study carried out on
Moreover, the presence of DWCNTs in the gut tract of
Pristine DWCNTs were found to be more toxic to
In experiments on the effect of humic acid, the toxicity of DWCNTs was increased as the concentration of humic acid increased. For pristine DWCNTs, the LC50 at 0 mg/L humic acid was 113.64 mg/L and at the highest humic acid concentration, it was 3.64 mg/L (Table
Lethal concentrations (LC50s and 95% confidence intervals) for
Exposure | Physicochemical characteristics | EC50 (mg/L) | Confidence limits (mg/L) |
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Pristine | 0.00335 (control reconstituted water) M Na | 113.64a | 43.34–267.66 |
0.00585 M Na | 261.0 | 110.7–447.20 | |
0.00835 M Na | >500 | ||
0.0139 M Na | >500 | ||
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Oxidised | 0.00335 (control reconstituted water) M Na | 214a | 89.9–269 |
0.0083585 M Na | >500 | ||
0.00835 M Na | >500 | ||
0.0139 M Na | >500 | ||
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Pristine | 0.00449 M Ca | 112.53a | 50.60–316.60 |
0.0226 M Ca | 113.64a | 43.66–113.64 | |
0.03395 M Ca | >500 | ||
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Oxidised | 0.00449 M Ca | 274.17a | 113–439 |
0.0226 M Ca | 214a | 89.9–269.1 | |
0.03395 M Ca | >500 | ||
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Pristine | 0 (control reconstituted water) M Na | 113.64a | 43.34–267.66 |
0.9 mg/L HA | 106.93a | 66.88–139.03 | |
1.81 mg/L HA | 84.15a | 28.00–171.27 | |
3.64 mg/L HA | 64.56a | 19.23–122.02 | |
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Oxidised | 0 (control reconstituted water) M Na | 214.00a | 89.90–269.1 |
0.9 mg/L HA | 193.77b | 97.44–287.11 | |
1.81 mg/L HA | 195.33b | 91.7–322.30 | |
3.64 mg/L HA | 184.0b | 100.3–316.10 |
Fish have been reported to ingest CNTs through the gastric tract. The amount of ingested CNTs depends on the colloidal stability of ENMs in solution [
Pictures illustrating the gut of fish exposed to fish medium only (a), to 62.5 mg/L DWCNTs (b), and to 250 mg/L DWCNTs (c).
Freshwater fish such as
The stabilisation of DWCNTs in the water column by humic acid increases the probability of contact between fish and nanoparticles through diet, hence the slight increase in toxicity [
The inverse was observed in the case of increased ionic strengths where LC50s were found to be higher than 500 mg/L. This is because DWCNTs in these conditions formed larger aggregates that settled at the bottom of the flask and
Acute toxicity can also be expressed using cumulative mortalities/survival plotted over time. These plots often illustrate whether a toxicant is toxic to part of the population within a certain period during the exposure experiment or whether mortality occurs at different rates within the exposure period.
The cumulative survival of
Survival of
At the beginning of the exposures, the percentage survival of
The
Survival of
The algal growth rates for all treatments and negative control reached 50% after 40 h (Figure
Growth rate
Time-response acute toxicity plots are essential in highlighting the effect of a particular toxicant over the set period of exposure. These plots, however, are not common in nanoecotoxicity tests. The time-response plots for the three organisms show that toxicity of DWCNTs proceeds at varying rates throughout the exposure periods. None of the plots showed point of inflection which further illustrates that DWCNTs are not just toxic to that part of the test population that was exposed.
The toxicity of DWCNTs to the three organisms was found to be significantly different, with
The authors declare that there is no conflict of interests regarding the publication of this paper.