Multi-Walled Carbon Nanotubes (MWCNT), synthetic graphite in tubular shape

Bioaccumulation: terrestrial

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: ASTM. Standard Guide for Conducting Laboratory Soil Toxicity or Bioaccumulation Test with the Lumbricid Earthworm Eisenia foetida; E1676–97

Version / remarks:

1998

Deviations:

not specified

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: synthesized at the lab
- Purity: 99 ± 1 %

RADIOLABELLING INFORMATION (if applicable)
- Specific activity: 0.122 ± 0.004 mCi/g

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Test material (0.3 or 0.03 mg/g dry soil) were dispersed by sonication in water at 262.5 W for 30 min in an ice–water bath to prevent damage to the nanotubes during the sonication process.

INFORMATION ON NANOMATERIALS
- Diameter: 30-70 nm

Radiolabelling:

yes

Remarks:

Carbon-14 labeling

Vehicle:

yes

Remarks:

water

Details on preparation and application of test substrate:

- Method of mixing into soil:
Each was air-dried and passed through a 2 mm mesh sieve, then analyzed to ensure they did not contain any traces of pyrene. Carbon-14 MWCNTs (0.3 or 0.03 mg/g dry soil) were dispersed by sonication in water at 262.5 W for 30 min in an ice–water bath to prevent damage to the nanotubes during the sonication process
- Controls: unspiked soil

Test organisms (species):

Eisenia fetida

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Carolina Biological Supply Co. (Burlington, NC)
- Life cycle stage: Adult
- Weight: 1.2 - 2.0 g
- Maintenance:
maintained on a worm bedding (Carolina Biological Supply) at 21 ± 2 °C, and kept moist with deionized water;
fed twice a week withwormfood comprised of a mixture of crude proteins and carbohydrates (Magic Worm Products, Amherst Junction, WI)

Total exposure / uptake duration:

>= 1 - <= 28 d

Total depuration duration:

> 1 - <= 7 d

Test temperature:

21 +/- 2 °C

pH:

pH (1:2 soil:deionized water extraction)
- Chelsea soils: 6.62 +/- 0.09
- Ypsilanti soils: 6.39 +/- 0.01

TOC:

Organic carbon fraction (n=4)
- Chelsea soils: 5.5 +/- 0.3%
- Ypsilanti soils: 1.7 +/- 0.1%

Moisture:

20%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 250 mL glass jar loosely closed with a cap
- Amount of soil: 30 g dry mass
- No. of organisms per container (treatment): 3
- No. of replicates per treatment group: 3
- No. of replicates per control / vehicle control: 3

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Chelsea and Ypsilanti Michigan
- Other: Soils were air-dried and passed through a 2 mm mesh sieve before spiking

VEHICLE CONTROL PERFORMED: not applicable
POSITIVE CONTROL PERFORMED
- Positive control: pyrene
- Preparation and application of positive control: Carbon-14 labeled pyrene (positions 4, 5, 9, and 10) dissolved in methanol and nonradioactive pyrene were dissolved in methylene chloride and added to the soil to give a final concentration of 0.04 mg/g dry soil. The use of methylene chloride was necessary for spiking the soil with this pyrene concentration given the limited solubility of pyrene in water. All soil samples were thoroughly tumbled and spiked with pyrene air-dried overnight to allow solvent volatilization.

TEST CONCENTRATION
- Test concentrations: 0.3 and 0.03 mg/g dry soil
- Justification: Most MWCNT uptake data points were measured using 0.3 mg/g dry soil for two principal reasons: (i) the nanotube concentrations in earthworms using 0.03 mg/g dry soil were at times below the detection limit, and (ii) the use of 0.3 mg/g dry soil resulted in similar radioactivities in soil given the differences in the specific radioactivities of the nanotubes as described below.

Carbon-14 Labeling Quantification
- Soil samples taken from the MWCNT-spiked soils were freeze-dried and combusted in the biological oxidizer(OX 500, R. J. Harvey Instrumentation Corporation). This instrument was used to burn the nanotubes at 900 °C for three minutes under a stream of oxygen gas running at 350 mL/min, and the 14CO2 released during the combustion process was captured in carbon-14 scintillation cocktail. At least four soil samples were combusted for each soil.
- Worms were removed after 1, 7, 14, and 28 d for the Chelsea soil and after 14 days for the Ypsilanti soil. After removal, the earthworms were washed with Milli-Q water, transferred to wet filter paper in Petri dishes for 24 h in the dark to allow purging of gut contents, and again rinsed with clean Milli-Q water until the radioactivity of the water had a background radioactivity concentration. The worms were then transferred to glass centrifuge tubes, freeze-dried for 24 h, weighed, combusted in a biological oxidizer, and the radioactivity determined using liquid scintillation counting.

Depuration
- After exposure for 14 d, the earthworms were removed from three containers and added to containers with clean soils to allow for depuration. After depuration for 1, 2, or 7 d, the worms were removed from their containers, and the remaining radioactivity was determined as described above.

Nominal and measured concentrations:

Nominal concentration: 0.3 and 0.03 mg/g dry soil

Key result

Type:

BSAF

Value:

ca. 0.023

Calculation basis:

other: concentration in the worm divided by that in the soil

Remarks on result:

other: Chelsea soil (0.3 mg/g)

Type:

BSAF

Value:

ca. 0.016

Calculation basis:

other: concentration in the worm divided by that in the soil

Remarks on result:

other: Chelsea soil (0.03 mg/g)

Type:

BSAF

Value:

ca. 0.014

Calculation basis:

other: concentration in the worm divided by that in the soil

Remarks on result:

other: Ypsilanti soil (0.3 mg/g)

Elimination:

yes

Parameter:

other: 1/D (elimination rate constant)

Remarks on result:

not determinable

Remarks:

The depuration behaviors of the MWCNTs did not exhibit a clear pattern. The majority of the carbon nanotubes measured were in the guts of the earthworms. The apparent uptake was suggested to be a result of sediment residing in the organism’s gut after the initial depuration period, and not of chemical absorption into the tissues of the organism.

Details on results:

- Recoveries for the MWCNT-spiked soils:
The total radioactivity spiked to the MWCNT-soil was 20.6 ± 0.7 μCi, whereas the total radioactivity in the soil estimated by using the average soil radioactivity (n=8) was 20±2 μCi thus indicating excellent recovery.

- BSAF (Mean and standard deviation values were calculated from triplicate measurements):
pyrene Chelsea soil (0.04 mg/g) 2.94 ± 0.25
pyrene Ypsilanti soil (0.04 mg/g) 14.0 ± 0.9
MWCNT Chelsea soil (0.3 mg/g) 0.023 ± 0.01
MWCNT Chelsea soil (0.03 mg/g) 0.016 ± 0.001
MWCNT Ypsilanti soil (0.3 mg/g) 0.014 ± 0.003
The low levels of MWCNT uptake, unlike pyrene, may be largely accounted for by carbon nanotubes in soil mass remaining in the worms’ guts after depuration.

Validity criteria fulfilled:

not specified

Conclusions:

The bioaccumulation of MWCNT in earthworms after 14 days of exposure was investigated. Results of a bioaccumulation factor (i.e., biota-to-soil accumulation factor (BSAF)) of 0.023 indicated a low bioaccumulation potential for MWCNT.

Executive summary:

Bioaccumulation of radio-Labeled MWCNTs by E. foetida was studied using modified ASTM E1676-97. The radio-Labeled MWCNT and a representative polynuclear hydrocarbon, pyrene, were then individually spiked to two types of soil samples; Chelsea and Ypsilanti. The uptake and depuration behaviors of the spiked materials by E. foetida were then assessed using 0.03 and 0.3 mg/g dry soil. Carbon-14 Labeling was quantified using the biological oxidizer.

The highest bioaccumulation factor (BSAF) determined for MWCNTs was 0.023 in Chelsea soil (0.3 mg/g) in the range of 0.014 to 0.023. This was almost 2 orders of magnitude smaller than those measured for pyrene, a representative PAH counterpart, indicating that MWCNTs are neither readily absorbed into organism tissues nor manifest equilibrium partitioning thereto.

The depuration behaviors of the MWCNTs did not exhibit a clear pattern. The majority of the MWCNTs measured were in the guts of the earthworms. The apparent uptake was suggested to be a result of sediment residing in the organism’s gut after the initial depuration period, and not of chemical absorption into the tissues of the organism. 

Reference 2

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 317 (Bioaccumulation in Terrestrial Oligochaetes)

Deviations:

not specified

Remarks:

Test conditions, e.g. exposure duration, sampling schedule, and treatment of results are equivalent or similar to OECD TG 317.

Principles of method if other than guideline:

- Principle of test: measurement of microwave-induced heating to quantify MWCNT uptake in earthworms
- Short description of test conditions: The test consists of two phases; the uptake phase (up to 28 days) and the elimination phase (up to 7 days).
A controlled volume of MWCNT dispersion was mixed with 20 mg of earthworm powder with a micro pipette (0–10 μL) to prepare the known samples for the generation of a calibration curve.
Earthworms exposed to MWCNTs in contaminated soil were first processed into a powder by freeze drying. Then, samples were measured by utilizing 10 s exposure to 30 W microwave power. Microwave-induced heating was used to quantify MWCNTs concentrations in earthworms. The uptake of MWCNTs was correlated with the temperature rise of the samples using the calibration curve. This method showed the potential to quantitatively measure MWCNTs in earthworms at low concentrations (~0.1 μg in 20 mg of earthworm).
The uptake rate constant (ku) and the elimination rate constant (ke) were calculated from a one-compartment first-order toxicokinetic model equation. BAF was calculated based on the estimated ku and ke.
- Parameters analysed / observed: microwave-induced heat

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Cheap Tubes Inc (Brattleboro, VT)
- Purity: >95 wt.%
- Ash content: 1.5 wt.%

INFORMATION ON NANOMATERIALS
- Outer diameter: 30-50 nm
- Length: 10-20 um

Radiolabelling:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used):
Soil (20 g) in a rotating containerwas spiked with dryMWNTs by the addition of small volumes at a time. After MWNTs were completely mixed into the soil, the soil container was sealed and tumbled overnight.
- Controls: unspiked clean soil

Test organisms (species):

Eisenia fetida

Details on test organisms:

TEST ORGANISM
- Source: Yelm Earthworm and Castings Farm (Yelm, Washington)
- Age at test initiation: Adult
- Weight at test initiation: 0.8–0.9 g

Total exposure / uptake duration:

>= 1 - <= 28 d

Total depuration duration:

>= 1 - <= 7 d

Test temperature:

18–22 °C

Moisture:

30%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 8-oz amber glass jar loosely covered by Teflon-lined lids
- Amount of soil or substrate: 20 g
- No. of organisms per container (treatment): 1
- No. of replicates per treatment group: 3
- No. of replicates per control / vehicle control: 1

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Source: Sandy loam soil (Lubbock, TX)
- pH: 7.8
- organic carbon content:1.1%
- % sand: 74%
- % silt: 16%
- % clay: 10%

VEHICLE CONTROL PERFORMED: No

EXPOSURE DURATION AND SAMPLING INTERVAL
- Uptake phase: Earthworms were removed from triplicate jars containing MWNTs as well as from one control jar on days 1, 7, 14, and 28. Following a 24 h depuration period on filter paper, purged worms were washed, weighed, and stored in a refrigerator at −20 °C until freeze drying.
- Elimination phase: After exposure in soil contaminated with 3 mg/g MWNTs for 14 d, a parallel elimination experiment was also conducted in CNT-free soil. Earthworms were removed from triplicate jars on days 1, 5, and 7.

QUANTIFICATION OF MWCNT IN EARTHWORMS
Earthworms exposed to MWCNTs in contaminated soil were first processed into a powder by freeze drying. Then, samples were measured by utilizing 10 s exposure to 30 W microwave power. Microwave-induced heating was used to quantify MWCNTs concentrations in earthworms. The uptake of MWCNTs was correlated with the temperature rise of the samples using the calibration curve.

Nominal and measured concentrations:

Nominal concentration: 3 mg/g MWNTs in soil

Key result

Type:

BSAF

Value:

ca. 0.015 dimensionless

Calculation basis:

other: concentration in the worm divided by that in the soil

Kinetic parameters:

The uptake rate constant (ku) and the elimination rate constant (ke) were calculated from a one-compartment first-order toxicokinetic model equation.
- estimated ku (uptake coefficient of MWNTs from soil): 325±81 μg/g/d
- estimated ke (elimination rate constant of MWNTs from soil): 21330±100 μg/g/d

Details on results:

- MWCNT uptake
Earthworms had a tendency to take up MWCNTs, with a maximum body burden of 85±13 μg/g on day 14. However, MWCNT body burden in earthworms decreased to 33±8.3 μg/g on day 28. In order to further examine if earthworms could eliminate MWCNTs, earthworms were transferred into clean soil after a 14 d MWCNT exposure. Earthworms had the tendency to depurate MWCNTs very quickly. The MWCNT concentration in earthworms (85±13 μg/g on day 14) decreased to 21±6 μg/g by day 5 of elimination.
BAF was calculated based on estimated ku (325±81 μg/g/d) and ke (21330±100 μg/g/d) values. The calculated BAF was 0.015±0.004.

- Results with control:
Earthworms exposed in clean soil were also analyzed for a possible false positive reading with the microwave-induced heating method. No heat generation above the detection limit was observed for control earthworm sample on days 1, 7 and 14. However, an unexpectedly high heat response was detected in one control sample on day 28. Given the consistent lack ofMWNT detection in other control samples, it is possible hat either soil from the sample on day 28 or the sample itself was accidently contaminated with naturally occurring or engineered carbon nanomaterials.

Validity criteria fulfilled:

not applicable

Conclusions:

The bioaccumulation of MWCNT in earthworms after 28 days of exposure was investigated. Results of a bioaccumulation factor (i.e., biota-to-soil accumulation factor (BSAF)) of 0.015±0.004 indicated a low bioaccumulation potential for MWCNT.

Executive summary:

The bioaccumulation of MWCNT in earthworms after 28 days of exposure was investigated. 

A controlled volume of MWCNT dispersion was mixed with 20 mg of earthworm powder with a micro pipette (0–10 μL) to prepare the known samples for the generation of a calibration curve.

Earthworms exposed to MWCNTs in contaminated soil were first processed into a powder by freeze drying. Then, samples were measured by utilizing 10 s exposure to 30 W microwave power. Microwave-induced heating was used to quantify MWCNTs concentrations in earthworms. The uptake of MWCNTs was correlated with the temperature rise of the samples using the calibration curve.

Earthworms had a tendency to take up MWCNTs, with a maximum body burden of 85±13 μg/g on day 14. However, MWCNT body burden in earthworms decreased to 33±8.3 μg/g on day 28. In order to further examine if earthworms could eliminate MWCNTs, earthworms were transferred into clean soil after a 14 d MWCNT exposure. Earthworms had the tendency to depurate MWCNTs very quickly. The MWCNT concentration in earthworms (85±13 μg/g on day 14) decreased to 21±6 μg/g by day 5 of elimination. BAF was calculated based on estimated ku (325±81 μg/g/d) and ke (21330±100 μg/g/d) values. The calculated BAF was 0.015±0.004.

Description of key information

BSAF (earthworm) : 0.014 to 0.023 (BSAF average 0.02 ± 0.006) (Peterson et al. 2008).

BSAF (earthworm) : 0.015 ± 0.004 (Li et al. 2013).

Key value for chemical safety assessment

BCF (terrestrial species):

0.02 dimensionless

Additional information

According to ECHA Appendix R7-2 (2021), soil and sediment compartments are considered potential sinks for nanomaterials and therefore considered relevant when considering nanomaterial fate in the environment. This is consistent with our evaluation of MWCNT and its environmental fate: MWCNT was found to adsorb to sludge and not remain in the water phase as reported in the STP feasability study included in this dossier. Therefore, we consider the terrestrial compartment as vulnerable for potential concern of bioaccumulation of MWCNT rather than the aquatic environment.   

Also, ECHA appendix R7-2 (2021) for the endpoint specific assessment of nanomaterial considers the soil compartment as a potential sink for nanomaterials and therefore relevant when considering nanomaterial fate in the environment. Since the terrestrial environment was considered the relevant route of exposure to MWCNT in terms of bioaccumulation, two relevant experimental studies on bioaccumulation in earthworm after 28 days of exposure are included in this dossier.

Peterson et al. (2008)[1] investigated bioaccumulation of MWCNT by Eisenia foetida, specifically the uptake and depuration behaviours. Bioaccumulation factors (i.e., biota-to-soil accumulation factor (BSAF)) were determined in the range of 0.014 to 0.023 (BAF average 0.02 ± 0.006) covering two different types of soil. The result indicated that purified carbon nanotubes were neither readily absorbed into organism tissues nor manifested equilibrium partitioning.

Also, Li et al. (2013)[2] investigated the bioaccumulation of MWCNT in earthworms after 28 days of exposure. Results of a bioaccumulation factor (i.e., BSAF) of 0.015±0.004 covering one soil indicated a low bioaccumulation potential for MWCNT. The results were in line with earlier findings by Peterson et al. (2008).

In conclusion, the bioaccumulation potential for MWCNT was considered low for the terrestrial environment.

 

[1] Petersen, E.J., Huang, Q. and Weber, Jr, W.J., 2008. Bioaccumulation of radio-labeled carbon nanotubes by Eisenia foetida. Environmental science & technology, 42(8), pp.3090-3095.

[2] Li, S., Irin, F., Atore, F.O., Green, M.J. and Cañas-Carrell, J.E., 2013. Determination of multi-walled carbon nanotube bioaccumulation in earthworms measured by a microwave-based detection technique. Science of the total environment, 445, pp.9-13.