Graphene

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not given

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

as of 2006

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

as of 2004

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Graphistrength® C100, Arkema, Colombes, France

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): suspensions in ultrapure water were prepared by sonication for 10 min at 45 kHz, 80 W (USC 300T, VWR, Fontenay sous Bois, France) just before each bioassay

INFORMATION ON NANOMATERIALS
- Chemical Composition: pure carbon in form of multi-walled carbon nanotubes (MWCNT)

Analytical monitoring:

not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: MWNTs were dispersed in water (EN ISO 8692, 2004) in different concentrations (mentioned below)
- Differential loading: (1) 100, 50, 10, 5, and 1 mg/L in the preliminary test, and (2) 1000, 500, 230, 105, 48, 22, and 10 mg/L for the definitive test.
- Controls: yes, one negative control
- Test concentration separation factor: ca. 2.2 in the definite test
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): It was observed that the majority of the particles did not remain in suspension between the beginning and the end of the tests but gathered at the lower part of each flask.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4 stock
- Source (laboratory, culture collection): Culture Centre of Algae and Protozoa (Ambleside, UK)
- Age of inoculum (at test initiation): not specified
- Method of cultivation: not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

not specified

pH:

preliminary test: pH 7.89 - 7.96 (0h); pH 7.91 in control and pH 8.62 - 9.42 in the test concentrations (72h)
definite test: pH 7.89 - 8.03 (0h); pH 7.98 in control and pH 7.97 - 8.90 in the test concentrations (72h)

Dissolved oxygen:

preliminary test: 9.0-9.1 mg/L (0h); 9.3-9.7 mg/L (72h)
definite test: 8.8-9.0 mg/L (0h); 8.4-8.9 (72h)

Nominal and measured concentrations:

nominal: 10, 22, 48, 105, 230, 500 and 1000 mg/L and a control

Details on test conditions:

TEST SYSTEM
- Test vessel: not specified
- Initial cells density: 1.21 x 10^-6 cells/mL (preliminary test), 1.09 x 10^-6 cells/mL (definite test)
- Control end cells density: Not specified, but biomass in the control cultures increased exponentially by a factor of 102 higher.
- No. of vessels per concentration (replicates): not specified
- No. of vessels per control (replicates): not specified

GROWTH MEDIUM
Standard growth medium was used, prepared according to EN ISO 8692, 2004.

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: not specified
- Light intensity and quality: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was derived after 24, 28 and 72 h for the control and treatment groups by measured fluorescence (Cytofluor 2350, Millipore, Molsheim, France).
- Other measurements: microscopic observations

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 100, 50, 10, 5 and 1 mg/L plus control (0 mg/L)
- Results used to determine the conditions for the definitive study: not specified

Reference substance (positive control):

yes

Remarks:

The sensitivity of the test system and the method were evaluated by performing an algal growth inhibition test on K2Cr2O7 (Sigma, Lyon, France). Results of this tests are not given.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

120 mg/L

95% CI:

>= 87 - <= 160

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34 mg/L

95% CI:

>= 23 - <= 47

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Unusual cell shape: no, the algae appeared normal at the end of the test: the normal shape
of the tested algal species is a crescent-shaped cell with an average length of 5-10 µm.
- Colour differences: no
- Any stimulation of growth found in any treatment: no

Results with reference substance (positive control):

not given

Reported statistics and error estimates:

The growth inhibition data were analysed using an Microsoft Excel sheet to calculate the effective concentration (EC50 value) and the 95% confidence interval. Probit analysis was used to calculate the 24-, 48-, and 72-h EC50 values. The no-observed effect concentration (NOEC), the highest tested concentration at which no significant inhibition of growth is observed relative to the control, was estimated by Dunnett’s test.

Table 1: Average percentage inhibition of cell growth (biomass) and growth rate of the freshwater algae Raphidocelis subcapitata(formerly known as Pseudokirchneriella subcapitata) exposed to graphene as MWCNTs for 72 h

Nominal conc. of MWCNTs [mg/L]	Av. percentage inhibition of biomass [%]	Av. percentage inhibition of growth rate [%]
0	0	0
10	13	2
22	26	6
48	54	14
105	90	44
230	99	73
500	100	82
1000	100	102

Validity criteria fulfilled:

yes

Remarks:

1) biomass in control increased exponentially by factor 102 (specific growth rate:0.92 d^-1) 2) mean CoV for section-by-section specific growth rates in control did not exceed 35% 3) CoV of av. specific growth rate in control replicates did not exceed 7%

Conclusions:

The present study was conducted according to EN ISO 8692 and OECD TG 201 with graphene as multi-walled carbon nanotubes (MWCNT) as test material. The method is to be considered scientifically reasonable with acceptable restrictions and no significant deviations from the guidelines. The results for inhibition of growth show a good dose-response-relationship. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards aquatic algae.

In a static test the freshwater algae Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was exposed to test substance concentrations in the range of nominal 10 to 1000 mg/L for a period of 72 hours under defined conditions. Based on the obtained results the 72h EC50 for growth rate was determined to be nominal 120 mg/L and the 72h EC50 for biomass to be 34 mg/L. The 72h NOEC was estimated at nominal 10 mg/L for growth rate and less than 10 mg/L for the parameter biomass.

As growth rate is the endpoint relevant for classification, it can be concluded that graphene in the form of MWCNT inhibited growth of the freshwater green algal species Raphidocelis subcapitata with an ErC50 (72h) of 120 mg/L and a NOErC (72h) of 10 mg/L.

Executive summary:

Study design

The effect of graphene (in the form of MWCNT) on the freshwater green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was investigated by Mouchet et al (2016). In a 72h study according to EN ISO 8692 and OECD TG 201, cultures of Raphidocelis subcapitata were exposed to nominal concentrations in the range of 10 to 1000 mg/L under static conditions.

The unicellular freshwater green alga Raphidocelis subcapitata was purchased from the Culture Centre of Algae and Protozoa (Ambleside, UK). The test material was pre-suspended in ultrapure water, sonicated for 10 min at 45 kHz, 80 W and then used to prepare the test concentrations. Algal cell cultures with an exponential growth of 1.09×10⁶ cells/mL were exposed to MWNTs dispersed in standard growth medium in concentrations of nominal 10 to 1000 mg/L. The test concentrations resulting in 0% and 100% inhibition of cell growth rate, and inhibition causing a 50% reduction in biomass within 72h (E_bC50-72 h) and in growth rate (E_rC50-72h) were estimated. The EC50 values were calculated by Probit analysis and the highest tested concentration at which no significant inhibition of growth was observed relative to the control (NOEC) was estimated by Dunnett’s test.

Results

The 72h-EC₅₀ value of graphene in form of MWCNT to freshwater algae (Raphidocelis subcapitata) was examined to be 120 mg/L based on growth rate. The 72h NOErC was estimated at 10 mg/L. The test material showed no shading effect on algal growth due to the poor dispersibility. Furthermore, the MWCNTs could adsorb micronutrients (Fe and Zn) from the algal medium, thus leading to nutrient depletion-induced inhibition of growth. ICP-MS measurement results showed significant decrease of Fe and Zn in the presence of MWCNT in algal medium.

Conclusion

Graphene in form of MWCNTs inhibited algal growth with an ErC₅₀ (72h) of nominal 120 mg/L and a NOErC (72h) of 10 mg/L.

Although there is no real structural parallel or similarity between graphene platelets with the fibre-shaped cylindrical carbon nanotubes, this study is considered as a worst-case scenario (MWCNTs are facing higher safety concerns than graphene sheets) and as such reliable for the assessment of toxic effects of graphene on freshwater green algae. It has been shown in literature (e.g. Ali-Boucetta et al. 2013) that shortening of high risk carbon nanotubes leads to dramatic reduction of adverse reactions, therefore it might be possible that the EC50 for graphene sheets is higher than the results reported here for graphene as MWCNTs. That is why this study is taken as a worst-case scenario for assessing the effect of graphene on freshwater green algae.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

The experimental design is comparable to the OECD TG 201 adn EPA OPPTS 850.5400, but it is not mentioned whether the study is GLP-compliant.

Specific details on test material used for the study:

see Table 1 section "Overall remarks, attachments": Selected physicochemical properties of Graphene (MG)

Analytical monitoring:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was pre-suspended in algal medium and then added to exponentially growing algal cells to reach the final test concentration of nominal 50 mg/L.

Test organisms (species):

Chlorella pyrenoidosa

Details on test organisms:

TEST ORGANISM
- Common name: Chlorella pyrenoidesa
- Source (laboratory, culture collection): The unicellular green alga was purchased from the institute of Hydrobiology, Chinese Academy of Science, China.

ACCLIMATION
- Acclimation period: not specified
- Culturing media and conditions (same as test or not): not specified

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

no details given

Test temperature:

25°C

Nominal and measured concentrations:

nominal: 50 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: not specified
- Initial cells density: 1×10E6 cells/mL
- No. of vessels per concentration (replicates): 3 replicates

GROWTH MEDIUM
- Standard medium used: The modified SE medium (one-tenth SE medium) was used as growth medium.
- Detailed composition if non-standard medium was used: The detailed composition is presented in section "Any other information on materials and methods incl. tables".

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The growth medium was used to prepare the test media.
- Culture medium different from test medium: not specified

OTHER TEST CONDITIONS
- Other: The algal suspensions were shaken twice every day.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The algal cells were counted daily under a light microscope (JNOEC, China). The growth inhibition (%) as a function of exposure times (1-4 days) was thus calculated.

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

62.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

The 96-h growth inhibition of algal cells highly depended on graphene concentration. The EC50 value of graphene was calculated as 62.2 mg/L. The growth inhibition increased with increasing exposure times (24 - 96 h).
Although suspensions of graphene were dark, no significant shading effect was observed. The reason is that carbon materials such as graphene with strong hydrophobicity (as indicated by low oxygen percentage) and low surface charge (see Table 1 in section "Overall remarks, attachments") could readily form big aggregates, thus settling down in aqueous phase.
After settling for 2h with graphene 43% of the suspended algal cells were co-settled with graphene, suggesting strong heteroagglomeration between algae and graphene. Light microscope images clearly show the attachment of algal cells on graphene sheets.
Membrane integrity of algal cells was investigated using flow cytometry (20,000 algal cells were counted). Graphene significantly caused membrane damage, K+ leakage and DNA leakage.
Graphene significantly increased the intracellular ROS level of algal cells, showing induction of oxidative stress. MDA (malondialdehyde) content can indicate the degree of lipid peroxidation, which was examined after graphene exposure for 96 h (50 mg/L).Graphene caused significant lipid peroxidation of membrane.
Graphene caused 9.4 % of nutrient depletion-induced indirect toxicity. In addition, the contribution of nutrition depletion to the total toxicity of graphene was calculated as 27 %.

Reported statistics and error estimates:

Statistical analysis of the data was performed using SPSS 16.0 by analysis of variance (ANOVA) with LSD (least significant difference) method after the verification of normality and homoscedasticity assumption. p < 0.05 for the statistical significance was used.
The median effective concentration (EC50) was calculated by nonlinear regression using the cumulative distribution function of SPSS Statistics.

Validity criteria fulfilled:

not specified

Conclusions:

The present study was conducted similar to OECD Guideline 201 and EPA OPPTS 850.5400 with multi-layer Graphene (MG) as test material. 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 aquatic algae.

In a static test the freshwater algae Chlorella pyrenoidosa was exposed to a test substance concentration of nominal 50 mg/L for a period of 96 hours under defined conditions. Based on the obtained results the 96h EC50 was determined to be nominal 62.2 mg/L.

Thus, multi-layer Graphene inhibited growth of the freshwater green algal species Chlorella pyrenoidosa with an EC50 (96h) of 62.2 mg/L.

Executive summary:

Study design

The effect of graphene on the freshwater green algal species Chlorella pyrenoidosa was investigated by Zhao et al (2017). In a 96 h study conducted similar to OECD TG 201 and EPA OPPTS 850.5400, cultures of Chlorella pyrenoidosa were exposed to a nominal concentration of 50 mg graphene/L under static conditions.

The unicellular green alga Chlorella pyrenoidesa was purchased from the institute of Hydrobiology, Chinese Academy of Science, China. The modified SE medium (one-tenth SE medium) was used for algal cells culture medium, in which the nutrient is sufficient for algal growth. The test material was pre-suspended in algal medium and then added to exponentially growing algal cells (1×10⁶ cells/mL) to reach the final test concentration of nominal 50 mg/L. The algal suspensions were shaken twice every day and the algal cells were counted daily under a light microscope (JNOEC, China). The growth inhibition (%)  as a function of exposure times (1-4 days) was thus calculated. The growth inhibition as a function of exposed concentrations after 96 h exposure was also examined, and the median effective concentration (EC₅₀) was calculated by nonlinear regression using the cumulative distribution function of SPSS Statistics. 

Results

The 96h EC₅₀ value of graphene to freshwater algae (Chlorella pyrenoidosa) was examined to be 62.2 mg/L. Graphene showed no shading effect on algal growth due to the poor dispersibility while it more readily heteroagglomerated with algae and thus, likely leading to direct contacts with algae. Furthermore the test substance could adsorb macronutrients (N, P, Mg, and Ca) from the algal medium, thus leading to nutrient depletion-induced indirect toxicity (27% of the total toxicity). Flow cytometry results showed significant decrease of membrane integrity after graphene exposure.

Conclusion

Multi-layer Graphene inhibited algal growth with an EC₅₀ (96h) of nominal 62.2 mg/L.

Description of key information

The toxicity of the test item graphene towards aquatic freshwater algae was assessed in a study according to OECD TG 201: Chlorella pyrenoidosa EC₅₀ (96h) = 62.2 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

62.2 mg/L

Additional information

The effect of graphene on the freshwater green algal species Chlorella pyrenoidosa was investigated by Zhao et al (2017). In a 96 h study conducted similar to OECD TG 201 and EPA OPPTS 850.5400, cultures of Chlorella pyrenoidosa were exposed to a nominal concentration of 50 mg graphene/L under static conditions.

The unicellular green alga Chlorella pyrenoidesa was purchased from the institute of Hydrobiology, Chinese Academy of Science, China. The modified SE medium (one-tenth SE medium) was used for algal cells culture medium, in which the nutrient is sufficient for algal growth. The test material was pre-suspended in algal medium and then added to exponentially growing algal cells (1×10⁶ cells/mL) to reach the final test concentration of nominal 50 mg/L. The algal suspensions were shaken twice every day and the algal cells were counted daily under a light microscope (JNOEC, China). The growth inhibition (%)  as a function of exposure times (1-4 days) was thus calculated. The growth inhibition as a function of exposed concentrations after 96 h exposure was also examined, and the median effective concentration (EC₅₀) was calculated by nonlinear regression using the cumulative distribution function of SPSS Statistics. 

The 96h EC₅₀ value of graphene to freshwater algae (Chlorella pyrenoidosa) was examined to be 62.2 mg/L. Graphene showed no shading effect on algal growth due to the poor dispersibility while it more readily heteroagglomerated with algae and thus, likely leading to direct contacts with algae. Furthermore the test substance could adsorb macronutrients (N, P, Mg, and Ca) from the algal medium, thus leading to nutrient depletion-induced indirect toxicity (27% of the total toxicity). Flow cytometry results showed significant decrease of membrane integrity after graphene exposure.

In conclusion multi-layer Graphene inhibited algal growth with an EC₅₀ (96h) of nominal 62.2 mg/L.

 

The moderate hazard of the registered substance to freshwater algae is supported by a publication of Mouchet et al (2016), where the effect of graphene (in the form of MWCNT) on the freshwater green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was investigated. In a 72h study according to EN ISO 8692 and OECD TG 201, cultures of Raphidocelis subcapitata were exposed to nominal concentrations in the range of 10 to 1000 mg/L under static conditions.

The unicellular freshwater green alga Raphidocelis subcapitata was purchased from the Culture Centre of Algae and Protozoa (Ambleside, UK). The test material was pre-suspended in ultrapure water, sonicated for 10 min at 45 kHz, 80 W and then used to prepare the test concentrations. Algal cell cultures with an exponential growth of 1.09×10⁶ cells/mL were exposed to MWNTs dispersed in standard growth medium in concentrations of nominal 10 to 1000 mg/L. The test concentrations resulting in 0% and 100% inhibition of cell growth rate, and inhibition causing a 50% reduction in biomass within 72h (EbC50-72 h) and in growth rate (ErC50-72h) were estimated. The EC50 values were calculated by Probit analysis and the highest tested concentration at which no significant inhibition of growth was observed relative to the control (NOEC) was estimated by Dunnett’s test.

Based on the obtained results the 72h-EC50 value of graphene in form of MWCNT to freshwater algae (Raphidocelis subcapitata) was examined to be 120 mg/L for the parameter growth rate. The 72h NOErC was estimated at 10 mg/L for the parameter growth rate. The test material showed no shading effect on algal growth due to the poor dispersibility. Furthermore, the MWCNTs could adsorb micronutrients (Fe and Zn) from the algal medium, thus leading to nutrient depletion-induced inhibition of growth. ICP-MS measurement results showed significant decrease of Fe and Zn in the presence of MWCNT in algal medium.

In conclusion, Graphene in form of MWCNTs inhibited algal growth with an ErC50 (72h) of nominal 120 mg/L and a NOErC (72h) of 10 mg/L.

Although there is no real structural parallel or similarity between graphene platelets with the fibre-shaped cylindrical carbon nanotubes, this study is considered as a worst-case scenario (MWCNTs are facing higher safety concerns than graphene sheets) and as such reliable for the assessment of toxic effects of graphene on freshwater green algae. It has been shown in literature (e.g. Ali-Boucetta et al. 2013) that shortening of high risk carbon nanotubes leads to dramatic reduction of adverse reactions, therefore it might be possible that the EC50 for graphene sheets is higher than the results reported here for graphene as MWCNTs. That is why this study is taken as a worst-case scenario for assessing the effect of graphene on freshwater green algae.