Tangled Multi-Walled Carbon Nanotubes

Toxicity to other aquatic organisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to other aquatic vertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: ISO 21427-1

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The exposure was performed according to the French Standard AFNOR NF T90–325 and the International Standard 21427–1.
Xenopus larvae were exposed for 12 days to 0.1, 1, 10 and 50 mg/l of MWNTs in reconstituted water (RW, distilled tap water to which nutritive
salts were added [294 mg/l CaCl2•2H2O; 123.25 mg/l MgSO4•7H2O; 64.75 mg/l NaHCO3; 5.75 mg/l KCl]).

Test organisms (species):

Xenopus laevis

Details on test organisms:

The Xenopus males were injected with 50 IU of Pregnant Mare’s Serum Gonadotrophin (PMSG 500; Intervet, France, [9002–70–4])
and the females with 750 IU of human chorionic gonadotropin (HCG; Organon, France, [9002–61–3]) to induce spawning. Viable eggs
were maintained in an aquarium also containing normal tap water filtered through active charcoal at 20–22°C, until they reached a development
stage appropriate for experimentation. Xenopus exposure began on larvae at stage 50.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

12 d

Hardness:

According to standard medium composition

Test temperature:

22 +/- 0.5 °C

pH:

no data (according to standard medium composition)

Dissolved oxygen:

no data

Salinity:

no data (according to standard medium composition)

Nominal and measured concentrations:

0.1, 1, 10 and 50 mg/l, nominal concentrations (suspensions)

Details on test conditions:

Larvae were exposed in groups of 20 animals in crystallizing dishes containing either control media (negative controls [NCs] and positive controls [PCs]) or test media (0.1, 1, 10 and 50 mg/l of MWNTs in RW).The PC was cyclophosphamide monohydrate ([6055–19–2], Sigma, France).
Mortality of larvae exposed to CNTs was examined for 12 days according to the standardized recommendations by visual inspection.
Growth inhibition was evaluated by measuring the size of each larva (n = 20) at the beginning of the exposure (t0) and at the end of the exposure (t12) using the Mesurim image analysis software.
At the end of exposure, a blood sample was obtained from each anesthetized larva (MS222, Sandoz, France). Technical procedures are well described on the standardized recommendations fascicles. The number of erythrocytes that contained one micronucleus or more (micronucleated
erythrocytes [MNE]) was determined in a total sample of 1000 erythrocytes per larva. Based on median values and quartiles, the number of micronucleated erythrocytes per thousand, MNE ‰ is presented with their 95% confidence limits expressed by the median ± 1.57 × interquartile range (IQR; upper quartile – lower quartile)/vn. The difference between the theoretical medians of the test groups and the theoretical median of the NC group is significant to within 95% certainty if there is no overlap.

Reference substance (positive control):

yes

Remarks:

cyclophosphamide monohydrate

Duration:

12 d

Dose descriptor:

LC0

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

12 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: larvae growth inhibition (size)

Duration:

12 d

Dose descriptor:

NOEC

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: micronucleated erythrocytes (MNE)

Remarks on result:

other: genotoxicity

Details on results:

The measurements of the larval size show that larvae exposed in the presence of 50 mg/l of MWNTs have significantly reduced size compared with the NC in a dose dependent manner. Larvae exposed to 10 mg/l of MWNTs showed non significant growth inhibition. Larvae exposed to 0.1 and 1 mg/l of MWNTs did not show any sign of toxicity compared with the NC.
Xenopus larvae exposed over 12 days to CNTs in water displayed a particular visual aspect compared with NC larvae (Figure 4). Gills showed no difference whatever the MWNT concentration, whereas intestinal tract from exposed larvae had black masses that were particularly visible through the thin peritoneal membrane of the larvae owing to their deep black color. Figure 5 shows that the proportion of black masses seemed to increase with the MWNT concentration.
Carbon nanotubes were tracked using their Raman signal. In the lumen, a high intensity of the G band for all concentrations ranging from 0.1 to 50 mg/l has been found, corroborating the visual and TEM observations, evidencing the presence of CNTs. In the intestinal barrier, the intensity of the G band was zero, thus no CNTs were localized in the barrier and beyond it. We observed no gradient of G-band intensity between the lumen and the intestinal cells, suggesting that CNTs do not cross the intestinal wall.
Photonic observations of larvae exposed to MWNTs indicate presence of CNTs only in the lumen and not in the gills, even at the highest concentration. By contrast, in the case of larvae exposed to DWNTs, black masses were observed in gills whatever the concentration.

Results with reference substance (positive control):

The PC (9.5 ± 1.49) showed significantly higher MNE ‰ as compared with the NC group.

Validity criteria fulfilled:

yes

Remarks:

positive control in genotoxicity

Conclusions:

For exposure duration of 12 days, Xenopus larvae did not show mortality up to the highest tested concentration 50 mg/L. No clastogenic effects were recorded (micronucleated erythrocytes). Reduction of growth was observed at 50 mg/L but not at 10 mg/L.
Observation with Raman spectroscopy indicates no absorption of MWCNTs through instestinal barrier.

Executive summary:

In this study a number of observations have been made of effects of exposure of Xenopus laevis larvae to Graphistrength C100 MWCNTs (as same authors did previously on DWCNTs), using the protocol described in the ISO 21427 -1 standard.

No mortality was registered up to the highest tested concentration of 50 mg/L, nor any significant micronucleus induction. Gills were not obstructed (differing from DWCNTs). MWCNTs present in the lumen of gastro-instestinal tract do not cross the barrier as shown by Raman spectroscopy.

Description of key information

In this study a number of observations have been made of effects of exposure of Xenopus laevis larvae to Graphistrength C100 MWCNTs using the protocol described in the ISO 21427 -1 standard.

No mortality was registered up to the highest tested concentration of 50 mg/L, nor any significant micronucleus induction. Gills were not obstructed. MWCNTs present in the lumen of gastro-instestinal tract do not cross the barrier as shown by Raman spectroscopy.