Graphene

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Sep 2019 to 11 Nov 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD. OECD Guidelines for the Testing of Chemicals, 203 Fish Acute Toxicity Test [S]. Paris: OECD, Adopted June 18, 2019.

Deviations:

yes

Remarks:

- measurements for chlorine and nitrate and ammonium in the test water are missing - measured values for aluminium, copper, iron and zinc above the thresholds in annex 3

Qualifier:

according to guideline

Guideline:

other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 203 Fish Acute toxicity test [M]. Second Edition. Beijing: China Environmental Press. 2013: 30-36

Version / remarks:

Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 203 Fish Acute toxicity test [M]. Second Edition. Beijing: China Environmental Press. 2013: 30-36.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 29763-2013, Chemicals-Rare Minnow (Gobiocypris rams) acute toxicity test [S]. Beijing: Standards Press of China, 2013

Version / remarks:

China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 29763-2013, Chernicals-Rare Minnow (Gobiocypris rams) acute toxicity test [S]. Beijing: Standards Press of China, 2013.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 27861-2011, Chemicals Fish Acute toxicity test [S]. Beijing: Standards Press of China, 2012.

Version / remarks:

China National Technical Committee for Standardization of Dangerous Chemicals Management. GB/T 27861-2011, Chemicals Fish Acute toxicity test [S]. Beijing: Standards Press of China, 2012.

Deviations:

not specified

GLP compliance:

yes

Remarks:

no certificate included in report

Analytical monitoring:

yes

Remarks:

TOC analysis

Details on sampling:

- Concentrations: 100 mg/L and blank control
- Sampling method: The test solution of all groups were filtered through 0.45 µm aqueous phase polyether sulfone (PES) filter membrane (the first 5 mL - 10 mL filtrate was discarded) before analysis.
- Sample storage conditions before analysis: The samples were analyzed immediately, if not, the samples were stored at -20°C ~ -25°C, and the measurement was carried out within 7 d.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test substance is poorly soluble in test water, so according to “Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals” (OECD number 23 Second Edition, February 8, 2019) and “Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances” (ECETOC Monograph No. 26 1996), the limit test was conducted by method of preparing water soluble fraction as test solution. The specific preparation for test solution was as follows:
0.500 g test substance was weighed into a flask and mixed with small amount of test water. After ultrasonic treatment for 15 min, the mixture was transferred into a 5 L-vessel and diluted with test water to 5000 mL to obtain the nominal concentration of 100 mg/L test suspension. The suspension was stirred with a magnetic stirrer for about 48 hours. After stirring, the test suspension was filtered through 0.45 µm PES filter membrane and discarded insoluble parts. Thus, the nominal loading rate of 100 mg/L WSF was obtained and used as test solution.
- Controls: A blank control (test water without test substance) was tested in parallel.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Test solutions of the blank control and the 100 mg/L WSF test group were observed to be clear and colorless.

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

TEST ORGANISM
- Common name: Rare Minnow
- Source: Reproduction by the test laboratory
- Length at study initiation (length definition, mean, range and SD): total length 29.00 +/- 1.97 mm
- Weight at study initiation (mean and range, SD): 0.161 +/- 0.031 g

ACCLIMATION
- Acclimation period: 270 days
- Acclimation conditions (same as test or not): same as test
- Feeding frequency during acclimation: on each workday until 24 hours before the test was started
- Health during acclimation (any mortality observed): The mortality was 0% during the 7 days prior to the start of the test, which met the requirement of mortalities being less than 5% of the population.

FEEDING DURING TEST
The fish were not fed during the exposure period.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

no details given

Post exposure observation period:

no details given

Hardness:

115 mg/l CaCO3

Test temperature:

22.8 to 23.3°C

pH:

7.87 to 8.38 pH during test
8.21 pH of test water

Dissolved oxygen:

68% to 99% of air saturation value

Salinity:

no details given

Conductivity:

no details given

Nominal and measured concentrations:

nominal: 100 mg/L
measured: not given

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria
- Type (delete if not applicable): open
- Aeration: no aeration during exposure
- Renewal rate of test solution (frequency/flow rate): Semi-static test, with renewals at 24 h intervals
- No. of organisms per vessel: 7 fish / test vessel
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.235 g fish / L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water dechlorinated
- Total organic carbon: 1.32 mg/L
- Particulate matter: < 4 mg/L
- Metals: Al = 55.5 ug/L, Cu = 1.25 ug/L, Fe = 3.46 pg/L, Zn = 37.4 ug/L
- Alkalinity: pH 8.21

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 14 h light / 10 h darkness
- Light intensity: The average illumination intensity of the test environment at the beginning and end of the test was respectively 808 lux and 776 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortalities and visible abnormalities of the test fish were recorded at 24 h, 48 h, 72 h and 96 h after the start of the test during test period.

TEST CONCENTRATIONS
- Test concentrations: 100 mg/L

Reference substance (positive control):

yes

Remarks:

A sensitivity test with the reference substance K2Cr2O7 (performed on February 25, 2019) showed that the 96h-LC50 of K2Cr2O7 to Rare minnow (Gobiocypris rams) was 193 mg/L and the 95% confidence limits ranged from 152 mg/L to 244 mg/L.

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

WSF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

WSF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

WSF

Basis for effect:

mortality (fish)

Details on results:

- Observations on body length and weight: The average and standard deviation of body weight and total length of 7 test fish were 0.161 g +/- 0.031 g and 29.00 mm +/- 1.97 mm, respectively. So loading rate of fish in the test was 0.235 g fish/L met the requirement of =< 1.0 g fish / L and total length was within the range of 30 mm +/- 10 mm.
- Mortality of control: At the end of the test, the cumulative mortality of the blank control was 0%, which met the validity criterion that the mortality should not exceed 10%.
- Abnormal responses: No abnormal responses of test fish were observed in the blank control and the 100 mg/L WSF test group.

Results with reference substance (positive control):

no details given

Reported statistics and error estimates:

Due to the lack of effects and the performance of a limit test, a statistical analysis was not necessary for the result evaluation.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of test substance to Rare minnow (Gobiocypris rarus) was determined according to OECD 203 under GLP in a limit test with nominal loading rate 100 mg/L under semi-static conditions (24 h intervals for medium renewal). For analytical determination of the treatment total organic carbon (TOC) analysis was used. The measurement results of the treatment lay in the same range as the results for the blank control, therefore no measured concentration for the treatment could be calculated.
At the end of the test, the cumulative mortality in the treatment 100 mg/L was 0%. Hence, the results for the 96 h median lethal loading rate (LL50), the lowest loading rate causing 100% mortality (LL100), and the highest loading rate causing 0% mortality (LL0) were described based on the nominal loading rate of the test substance (100 mg/L). The test results are given as below:
96 h-LL50 > 100 mg/L (based on the nominal loading rate)
96 h-LL100 > 100 mg/L (based on the nominal loading rate)
96 h-LL0 ≥ 100 mg/L (based on the nominal loading rate)

Executive summary:

Study design

In the present study the acute toxicity of the test substance to Gobiocypris rarus was determined by a semi-static limit test according to OECD TG 203 under GLP criteria.

0.500 g of the test substance were mixed with a small amount of test water, ultrasonicated and then diluted in 5000 mL test water to prepare a stock suspension with a nominal loading rate of 100 mg/L. The suspension was stirred for about 48 hours, after stirring, the test suspension was filtered through 0.45 µm PES filter membrane to discard insoluble parts. The test duration was 96h.

Results

No mortality occurred in the control during the test and the dissolved oxygen concentration was greater than 60.0 % of the air saturation value (ASV) throughout the test duration. Hence, the test was considered to be valid.

During the test, the measured concentrations of the test substance were determined by TOC analysis, but the results of treatment and blank control were similar. The 96h LL₅₀value for the test substance to Gobiocypris rarus was based on nominal loading rate and stated as >100 mg/L. The maximum loading rate causing no mortality and the minimum loading rate causing 100 percent mortality within the period of the test were stated with ≥ 100 mg/L and > 100 mg/L, respectively.

Conclusion

96 h-LL50 > 100 mg/L (based on the nominal loading rate)

Description of key information

The short-term toxicity of the test item graphene towards fish was assessed in a semi-static test according to OECD TG 203 under GLP: Gobiocypris rarus LL₅₀(96h) > 100 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

100 mg/L

Additional information

In the present study the acute toxicity of the test substance to Gobiocypris rarus was determined by a semi-static limit test according to OECD TG 203 under GLP criteria.

0.500 g of the test substance were mixed with a small amount of test water, ultrasonicated and then diluted in 5000 mL test water to prepare a stock suspension with a nominal loading rate of 100 mg/L. The suspension was stirred for about 48 hours, after stirring, the test suspension was filtered through 0.45 µm PES filter membrane to discard insoluble parts. The test duration was 96h.

No mortality occurred in the control during the test and the dissolved oxygen concentration was greater than 60.0 % of the air saturation value (ASV) throughout the test duration. Hence, the test was considered to be valid.

During the test, the measured concentrations of the test substance were determined by TOC analysis, but the results of treatment and blank control were similar. The 96h LL₅₀value for the test substance to Gobiocypris rarus was based on nominal loading rate and stated as >100 mg/L. The maximum loading rate causing no mortality and the minimum loading rate causing 100 percent mortality within the period of the test were stated with ≥ 100 mg/L and > 100 mg/L, respectively.

96 h-LL50 > 100 mg/L (based on the nominal loading rate)