Single Wall Carbon Nanotubes (SWCNT)

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The subjects of investigation were bacteria of the genetically engineered strain Escherichia coli K12TG1 with the lux operon of the luminescent marine bacterium Photobacterium leiognathi cloned into it. The E. coli strain K12 TG1, with a luminescent phenotype, was obtained and is being stored at the Laboratory of Biologically Active Substances of the Chair of Microbiology of Moscow State University. These bacteria are being used as a bioluminescent sensor of the Ecolum test system for evaluating the toxicity of various substances (V. S. Danilov, A. P. Zarubina, G. E. Eroshnikov, et al., Vestn. Mosk. Un-ta, Ser. 16. Biologiya, No. 3, 20 (2002)). An overnight E. coli culture grown in an LB broth at 28 °C under subsurface conditions in a shake-flask propagator operating at 220 rpm was used in the analyses was.

GLP compliance:

not specified

Remarks:

The publication does not mention, whether the experimental study was performed under GLP; the study has been performed at the MOscow State University and thus adherence to scientific quality standards is assumed.

Analytical monitoring:

yes

Details on sampling:

A suspension of an overnight bacterial culture spun down to a 1-ml volume at 8000 g for 5 min in centrifuge tubes (ependorf-type) 1.5 ml in volume was used in the experiments. The excess liquid was decanted. Sterile distilled water was added to the bacterial biomass in the control specimens, and aqueous carbon nanotube suspensions providing for various degrees of contact between the bacterial cells and carbon nanotubes were added to the biomass in the test specimens; the concentration ratios used were as follows:
1. An aqueous bacterial cell suspension (with a concentration of 1 × 10E9 cells per milliliter) was mixed with a SWCNT suspension (with a concentration of 0.2 mg/l);
2. A much greater amount of SWCNTs (2 milligrams per milliliter) was added to the E.coli suspension;
3. Concentrated paste-like bacterial (5.6 × 10E12 cells per milliliter) and carbon nanotube (over 5 mg/ml) suspensions were mixed together.
The control and test specimens were held at room temperature (22 °C) for 14 days, and samples were taken from them a day or two later.

Vehicle:

no

Details on test solutions:

For preparation of the nanotube suspension used in the experiments, the SWCNTs powder was placed in distilled water. Immediately prior to the experiment, the SWCNT suspension was exposed to ultrasound for an hour to break down large aggregates.
A suspension of an overnight bacterial culture spun down to a 1 ml volume at 8000 g for 5 min in centrifuge tubes; 1.5 ml in volume was used in the experiments. The excess liquid was decanted. Sterile distilled water was added to the bacterial biomass in the control specimens, and aqueous carbon nanotube suspensions providing for various degrees of contact between the bacterial cells and carbon nanotubes were added to the biomass in the test specimens; the concentration ratios used were as follows:
1. An aqueous bacterial cell suspension (with a concentration of 1 × 10E9 cells per milliliter) was mixed with a SWCNT suspension (with a concentration of 0.2 mg/l);
2. A much greater amount of SWCNTs (2 milligrams per milliliter) was added to the E.coli suspension;
3. Concentrated paste-like bacterial (5.6 × 10E12 cells per milliliter) and carbon nanotube (over 5 mg/ml) suspensions were mixed together.

Test organisms (species):

Escherichia coli

Details on inoculum:

A suspension of an overnight bacterial culture spun down to a 1 ml volume at 8000 g for 5 min in centrifuge tubes; 1.5 ml in volume was used in the experiments. The excess liquid was decanted. Sterile distilled water was added to the bacterial biomass in the control specimens, and aqueous carbon nanotube suspensions providing for various degrees of contact between the bacterial cells and carbon nanotubes were added to the biomass in the test specimens.

Test type:

static

Water media type:

freshwater

Total exposure duration:

14 d

Test temperature:

22 °C

Nominal and measured concentrations:

0.2 mg/L, 2 mg/L and >5 mg/L

Details on test conditions:

Samples were exposed and measured for 14 days

Reference substance (positive control):

no

Duration:

4 d

Dose descriptor:

other: EC30

Effect conc.:

ca. 0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: bioluminescence/bacterial damage

Duration:

4 d

Dose descriptor:

other: EC80

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: bioluminescence/bacterial damage

Details on results:

The adverse effect of SWCNTs on the bacterial cells E. coli was observed in investigations into the bioluminescence of the bacteria with the luminous phenotype used in our experiments. It is well known that the test system based on bacterial luminescence is highly sensitive to various factors; its measurements are easy to implement and take a short time and tests can be conducted in micro-volumes (from 0.1 to 1.0 ml); the results usually correlate with those obtained with the other generally accepted test systems. The bioluminescent system in the damaged bacteria ceases to function. It was observed that by the fourth day of incubation in the SWCNT-bearing suspensions prepared by all the above three methods (0.2, 2 and > 5 mg/L) the bioluminescence of the cells of the E. coli strain used dropped sharply, with the toxicity index ranging between 30 and 80, depending on the bacterium–nanotube concentration ratio, which testified to the death of the cells.

Results with reference substance (positive control):

None applied

The AFM studies into the condition of the bacterial cells exposed to SWCNT enabled to obtain and analyze their images prior to, during, and at the end of the experiment. The analysis of such images showed that the population of the original bacterial suspension was heterogeneous, both in size and morphology, and contained mainly morphologically sound cells. The substantial differences in morphology between individual bacterial cells resulted from the fact that they did not develop synchronously and were at different stages of division and growth.

The cells were of characteristic rod-like shape and measured 2.0 – 2.5 µm in length, around 1 µm in width, and 250 ±50 nm in height. As already noted, to study the action of SWCNTs on bacterial cells, use was made of three sample preparation methods, which provided for three different degrees of contact between the cells and nanotubes. In spite of the fact that the bacterium–nanotube concentration ratios in the suspensions were dissimilar, the AFM investigations showed that the action of the carbon nanotubes on the E.coli cells was similar in all experiments.

The Raman spectroscopy of the samples prepared by the different methods revealed regions therein filled with the nanotubes. Their number and size in the suspensions under study varied depending on the concentration of the nanotube material. Morphological changes in the cells were observed to occur only in regions covered with a substantial layer of SWCNTs.

In all series of experiments, morphological changes in the bacterial cells that were contacting nanotubes became noticeable by the fourth day. One can see that the cells exposed to SWCNTs are substantially flattened in comparison with the controls; repeated irregularities were observed on their surface, and their height did not exceed 70 – 80 nm. What is more, the cells observed in that case differed in their lateral dimension.

The bacteria in the area covered by SWCNTs differ in shape and have a small height, whereas those having no direct contact with the nanotubes retain their initial condition and morphology characteristic of bacteria.

We believe that this phenomenon can be explained by the mechanical action exerted by the nanotubes on the bacterial cells. When directly contacting the cells, the thin nanotubes probably damage the cell wall and membrane mechanically, causing the loss of the contents of the cytoplasm and the ensuing death of the bacteria.

The AFM analyses of the bacterial suspensions prepared with purified and unpurified nanotube material revealed no fundamental differences in the action of nanotubes on bacterial cells. This can bear witness to the fact that the phenomenon observed is due to the effect of the carbon nanotubes on the vitality of the bacterial cells, with the admixtures of the other forms of carbon and metal catalysts contained in the unpurified, as-synthesized nanotubes adding nothing to the bactericidal effect observed.

Validity criteria fulfilled:

not specified

Conclusions:

Cell damaging effects were observed beginning after 4 days of exposure of E. coli cells to SWCNT material in a sharp manner, as recorded by bioluminescence measurements over a period of 14 days. No EC50 was provided by the authors but considering the different concentrations tested and the values reported one can estimate an EC30 of 0.2 mg/L and an EC 80 of ~5 mg/L.

Executive summary:

The authors have revealed that direct contact between bacterial cells (Escherichia coli K12TG1 with the lux operon of the luminescent marine bacterium Photobacterium leiognathi cloned into it) and SWCNTs inflicts damage on the cells. This allows the authors to conclude that single-walled carbon nanotubes are capable of bactericidal action, probably causing mechanical damage to the bacterial cell wall and membrane. Interestingly, the effect only started after 4 days of exposure as measured by observing the bioluminescence which implies that a significant contact time is required supporting the assumption for a mechanical effect (damage of cell membrane), as also stipulated by other authors. The effect appear to be independent from the purity of the single-walled carbon nanotubes as no significant difference between unpurified and purified material was observed.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is highly insoluble in water, hence indicating that aquatic toxicity is unlikely to occur

Description of key information

Standard OECD 209 studies do run with an exposure period of 3 hours and in aqueous suspensions (sludge) but require the test substance to be soluble in aqueous systems, which is not the case here. Based on test data on a single E.coli strain under very specific conditions and exposure time up to 14 days, it was shown that effects only occurred at the fourth day and then was rather dramatic, implying a mechanical mode of action (damage of cell membrane causing cell death and end of bioluminescence). Although this supports that SWCNT may be toxic to microorganisms, it does not allow a prediction towards toxicity to sewage sludge microorganisms, typically being exposed for a much shorter time and non-static (permanent agitation and much more dilute exposure), whereas in this study SWCNT was settled on the bacterial surface, compared to the situation in a STP where the sludge is in permanent movement. Thus, results of this toxicity towards E.coli bacteria cannot be reliably used for STP DNEL derivation. Due to the extremely poor water solubility of the SWCNTs, testing in an OECD 209 setting is technically not feasible. Considering that SWCNT is insoluble in water and may reach STPs only in suspension, it can be assumed that such SWCNT, even if reaching the STP, would become removed by adsorptive measures at the entry of a STP, before reaching the biological treatment unit.