Graphene

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Sep 2019 to 14 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

Adopted 4th April, 1984

Deviations:

no

GLP compliance:

yes

Remarks:

no certificate included in the study report

Analytical monitoring:

not required

Remarks:

Not recommended by the relevant test guideline, since the study was performed by weighting and direct incorporation of a defined amount of test material to dry quartz sand followed by mixture with wet artificial soil.

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used):
Based on the predicted moisture content of artificial soil, approximately 30%, respectively 589 mg of test substance (purity was 98%) was weighed and mixed well with quartz sand to a total weight of about 10 g by grinding. Each mixture was then mixed with about 740 g wet artificial soil to obtain test substance concentrations of 1001 mg/kg (dry weight of artificial soil). Four replicates were prepared.
- Controls:
Blank control was prepared by mixing about 10 g of quartz sand and about 740 g wet artificial soil, with four replicates.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms (Eisenia fetida)
- Source: The earthworms were purchased from Fogang Wotu Agricultural Technology Co. Ltd. and raised in the test facility (Batch No: EEf20190312-1)
- Age at test initiation: adult (older than 2 months)
- Weight at test initiation: 450 mg - 488 mg per earthworm


ACCLIMATION
- Acclimation period: 24 h
- Acclimation conditions: The test organisms were with clitellum and were transferred into artificial soil (without test substance) approximately 24 hours prior to the start of the test.

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

10 % w/w Sphagnum peat, 20 % w/w Kaolinite clay (analytical agent), 70 % w/w quartz sand (partizle size between 50 - 200 µm)

Limit test:

yes

Total exposure duration:

14 d

Post exposure observation period:

no

Test temperature:

18.1 °C - 21.8 °C

pH:

5.72 (at the beginning of the test)
5.97 - 6.22 (at the end of the test)

Moisture:

30.1% (dry weight, at the beginning of the test)
27.8% - 28.2% (dry weight, at the end of the test)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1 liter glass vessel
- Amount of soil or substrate: 750 g wet weight of substrate
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition: 10 % w/w Sphagnum peat, 20 % w/w Kaolinite clay (analytical agent), 70 % w/w quartz sand (particle size between 50 - 200 µm)

OTHER TEST CONDITIONS
- Photoperiod: Continuous light
- Light intensity: The light intensity was 688 lx at the beginning of the test and 662 lx at the end of the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
1) at 7 d and 14 d of exposure, the test matrix inside the container was gently placed onto a flat plate and the earthworms were sorted from the artificial soil. Any abnormal behavior or pathological symptoms were checked and recorded by provoking rump of the earthworms with a probe.
2) pH value: At the beginning and end of the test, 5 g of air-dried artificial soil was sampled for pH determination.
3) Moisture content of artificial soil: At the beginning and the end of the test, appropriate amounts of artificial soils were dried to constant weight at 105 ± 5 °C to measure moisture of the artificial soil.
4) Environmental conditions: at the start and the end of the test, the light intensity of the test environment above the test container location was determined and recorded.
5) Weight of earthworms: at the beginning and the end of the test, detennined and recorded the amounts and total weights of survival earthworms in each container and calculate the mean weight.

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Range finding study: Concentrations for the main test were based on the results of the range-finding test (100 mg/kg, 301 mg/kg, 501 mg/kg and 1000 mg/kg test substance) with no mortalities in the test groups
- Test concentrations: Limit test at 1001 mg/kg (dry weight of artificial soil) was designed

Nominal and measured concentrations:

nominal: 1001 mg/kg (dry weight of artificial soil) and a blank control

Reference substance (positive control):

yes

Remarks:

The sensitivity test was carried out with the reference substance chloroacetamide (C2H4ClNO) based on the artificial soil test of the same batch Eisenia foetida.

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

>= 1 001 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: After 7 days and 14 days exposure, the mortalities of earthworms in the blank control were 0% and 0%, and were 0% and 0% in the 1001 mg/kg (dry weight of artificial soil) test group.

Reported statistics and error estimates:

Due to the test substance showed no toxicity to the experimental earthwonns, Microsoft Excel was used to analyze the mean weight of live earthworms, the mortality of earthworms and the moisture content of artificial soil.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test substance to the eartworm Eisenia fetida was determined according to OECD 207 under GLP. The method is to be considered scientifically reasonable without restrictions or deviations from the guideline. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards the soil macroorganism Eisenia fetida.
The study was conducted as a limit test at nominal 1001 mg/kg (dry weight of artificial soil) for a period of 14 days under defined conditions. Based on the obtained results the 14d LC50 was determined to be greater or equal to nominal 1001 mg/L (derived from graph).

Executive summary:

Study design

The acute toxicity of the test substance to Eisenia fetida was determined in a 14 days artificial soil test according to "Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals-Effects on Biotic Systems, 2013, 207 Earthworm, Acute Toxicity Test" and "OECD, Guidelines for the Testing of Chemicals, 1984, 207 Earthworm, Acute Toxicity Test", in compliance with GLP.

Based on the results of a range-finding test, a limit test with a concentration of 1001 mg/kg dry artificial soil weight was conducted. 1 treatment group and one control group, each consisting of four replicates with 10 worms each were included in the main test. The numbers of mortalities were determined after 7 and 14 days exposure.

Results

The mortality of the earthworms in the blank control group and solvent control group was 0% at the end of the test which fulfilled the criteria for the validity of not exceeding 10%.

Conclusion

Under the test conditions, the result for the 14 d median lethal concentration (LC50) was greater than 1001 mg/kg (dry weight of artificial soil).

Description of key information

The toxicity of the test item graphene towards soil organisms was assessed in a static test according to OECD TG 207 under GLP: Eisenia fetida LC50 (14d) > 1001 mg/kg soil dw

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 001 mg/kg soil dw

Additional information

The acute toxicity of the test substance to Eisenia fetida was determined in a 14 days artificial soil test according to "Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals-Effects on Biotic Systems, 2013, 207 Earthworm, Acute Toxicity Test" and "OECD, Guidelines for the Testing of Chemicals, 1984, 207 Earthworm, Acute Toxicity Test", in compliance with GLP.

Based on the results of a range-finding test, a limit test with a concentration of 1001 mg/kg dry artificial soil weight was conducted. 1 treatment groups and one control group, each consisting of four replicates of 10 worms per concentration were included in the main test. The numbers of mortalities were determined after 7 and 14 days exposure.

The mortalities of the earthworms in the blank control group and solvent control group were 0%, which was not more than 10 percent, i.e. the test is evaluated as valid.

Under the test conditions, the result for the 14 d median lethal concentration (LC₅₀) was greater than 1001 mg/kg (dry weight of artificial soil).