Graphene

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD Guideline for Testing of Chemicals, No. 202, Daphnia sp., Acute Immobilization Test and Reproduction Test, Part 1,1984

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

EU Commission Directive 92/69/EEC, C.2, Acute Toxicity for Daphnia, 1992

Deviations:

not specified

GLP compliance:

not specified

Remarks:

no information on GLP compliance available in this publication

Specific details on test material used for the study:

Diameter (nm): (no information)
Length (µm): (no information)
Lateral size (µm): 0.5 – 2.0
Purity (%) 99
Company where purchased Nanjing XFNANO Materials Tech Co., Ltd.
Atomic ratio: 95.4 % C, 3.5 % O, 1.1 % Si
BET: 802.88 ± 3.76 m2/g
Hydrodynamic diameter (nm): 1087.5 ± 194.5

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

nonionic surface-active agent polyvinylpyrrolidone K30 (PVP)

Details on test solutions:

The graphene (GN) suspension was prepared using a simplified SM7 medium, a mixture solution of CaCl2 (293.8 mg /L), MgSO4 (123.3 mg/L), K2HPO4 [0.184 mg/L), NaNO3 (0.274 mg/L), NaHCO3 (64.8 mg/L), Na2SiO3 (10 mg/L), H3BO3 (0.175 mg/L), KCl (5.8 mg/L), and KH2PO4 (0.143 mg/L), with a water hardness of 250 mg CaCO3 per L and pH of 7.8 - 8.2 as recommended in the Organization for Economic Cooperation and Development (OECD) Guideline 211.
The nonionic surface-active agent polyvinylpyrrolidone K30 (PVP) was selected as a non-covalent dispersant. In the preliminary experiment, it was shown that PVP at a concentration as high as 10 mg/L was not toxic and did not affect the growth and reproduction of 24 h old daphnia compared with controls after 21 days of chronic exposure.
The GN stock suspension was obtained by adding 10 mg of GN to 100 mL of a 1 g/L PVP solution (PVP dissolved in SM7 medium) followed by sonication using a sonication bath (ultrasonic bath KQ-500DB, Kun Shan, China) for 2 h at room temperature to obtain optimal particle dispersion. Most of the GN were suspended and no precipitate was observed after sonication. The nominal concentration (100 mg/L) was considered as the concentration of the GN stock suspension, although it was difficult to reach the full extent of exfoliation by sonication. The GN suspension with different concentrations used for acute and chronic tests was prepared by diluting the GN stock suspension followed by sonication for 20 min before exposure.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Age at study initiation (mean and range, SD): neonates less than 24h old
- Method of breeding: Daphnia were cultured in freshwater under normal environmental conditions under a 16:8 light-dark cycle at 23.5 °C. The freshwater sample was obtained from the Huo Qi Ying Bridge area, China (latitude 39°58'5.58", longitude 116° 16'53.3"), filtered through glass fiber filters before use, and renewed every 2 d.
- Feeding during test: not specified
- Food type: green alga Chlamydomonas reinhardtii at a cell density of 5 x 10,000 cells per mL (for daphnids <3 days old) and 100,000 cells per mL (for daphnids >3 days old) every day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

no details given

Test temperature:

no details given

pH:

no details given

Dissolved oxygen:

no details given

Salinity:

no details given

Conductivity:

no details given

Nominal and measured concentrations:

0.2 - 16 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour darkness photoperiod (with a 30 minute transition period).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The mortality of the daphnids was assessed per beaker by calculating the percentage of daphnids remaining alive.

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

yes

Remarks:

A 24h acute toxicity test was performed every month using the reference toxicant K2Cr2O7, to check the sensitivity of the daphnia as specified in the GB/T 13266 guideline (EC50 24h = 0.5-1.2 mg/L) and to reduce the risk of experimental failure.

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: mortality = 40% derived from graph

Duration:

48 h

Dose descriptor:

other: LC20

Effect conc.:

> 4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: derived from graph

Details on results:

- Behavioural abnormalities: none stated
- Mortality of control: none
- Other adverse effects control: none stated
- Abnormal responses: none stated
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none stated

Reported statistics and error estimates:

All data were represented as mean values +/- standard deviation. All statistics were performed using SPSS Statistics 19.0 for Windows. Values were considered to be significant if p < 0.05.

Validity criteria fulfilled:

not specified

Conclusions:

The study was conducted similar to the EU Commission Directive 92/69/EEC, Part C.2 (1992) and the OECD TG 202, Part I (1984). The method is to be considered scientifically reasonable with acceptable restrictions and no significant deviations from the guidelines. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards the aquatic invertebrate Daphnia magna.
In a static test the freshwater invertebrate Daphnia magna was exposed to the test substance at concentrations of nominal 0.2 - 16 mg/L for a period of 48 hours under defined conditions. Based on the obtained results the 48h LC50 was determined to be > 16 mg/L (derived from graph).

Executive summary:

Study design

The acute toxicity of graphene (GN) to Daphnia magna was determined in a 48-hour static test similar to the EU Commission Directive 92/69/EEC, Part C.2 (1992) and the OECD TG 202, Part I (1984). This study is classified as reliable with restrictions (reliability 2) and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

The study was conducted with test concentrations of nominal 0.2 - 16 mg/L. The GN stock suspension was obtained by adding 10 mg of GN to 100 mL of a 1 g/L PVP solution (PVP dissolved in SM7 medium) followed by sonication using a sonication bath (ultrasonic bath KQ-500DB, Kun Shan, China) for 2 h at room temperature to obtain optimal particle dispersion. Most of the GN were suspended and no precipitate was observed after sonication. The nominal concentration (100 mg/L) was considered as the concentration of the GN stock suspension, although it was difficult to reach the full extent of exfoliation by sonication. The test media with different concentrations were prepared by diluting the GN stock suspension followed by sonication for 20 min before exposure. To characterize the stability of graphene in suspension, the hydrodynamic diameter, electrophoretic mobility and zeta potential of graphene were recorded on a Zetasizer Nano ZS system (Malvern Instruments, UK) during a period of 48 h.

Results Application of up to nominal 16 mg/L graphene induced < 50% daphnid mortality.

Conclusion The 48h LC₅₀ was determined to be > 16 mg/L (derived from graph).

Description of key information

The short-term toxicity of the test item graphene towards aquatic invertebrates was assessed in a semi-static test according to OECD TG 203: Daphnia magna EC₅₀(48h) > 16 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

16 mg/L

Additional information

The acute toxicity of graphene (GN) toDaphnia magnawas determined in a 48-hour static test similar to the EU Commission Directive 92/69/EEC, Part C.2 (1992) and the OECD TG 202, Part I (1984).This study is classified as reliable with restrictions (reliability2) and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

The study was conducted with test concentrations of nominal 0.2 - 16 mg/L. The GN stock suspension was obtained by adding 10 mg of GN to 100 mL of a 1 g/L PVP solution (PVP dissolved in SM7 medium) followed by sonication using a sonication bath (ultrasonic bath KQ-500DB, Kun Shan, China) for 2 h at room temperature to obtain optimal particle dispersion. Most of the GN were suspended and no precipitate was observed after sonication. The nominal concentration (100 mg/L) was considered as the concentration of the GN stock suspension, although it was difficult to reach the full extent of exfoliation by sonication. The test media with different concentrations were prepared by diluting the GN stock suspension followed by sonication for 20 min before exposure. To characterize the stability of graphene in suspension, the hydrodynamic diameter, electrophoretic mobility and zeta potential of graphene were recorded on a Zetasizer Nano ZS system (Malvern Instruments, UK) during a period of 48 h. Application of up to nominal 16 mg/L graphene induced < 50% daphnid mortality. The 48h LC₅₀was determined to be > 16 mg/L (derived from graph).

The results reported in the key study are supported by a study conducted by Guo et al., 2013. In the supporting study by Guo et al. no Daphnia were immobilized after being exposed to graphene concentrations up to 250 µg/L for 48h. Thus, this study underlines the absence of acute toxicity of graphene to Daphnia magna at low concentrations.