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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no details given

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

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:

Graphene oxide (GO) was synthesized according to the modified Hummers methodology. GO sheets exhibited an average planar dimension of ca. 120-200 and a thickness around 3.5 nm, as revealed by Atomic Force Microscopy. Dynamic Light Scattering analysis presented a narrow size distribution centered at 110nm, in corroboration with the AFM measurements.

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Oligo medium

Test organisms (species):

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

Details on test organisms:

Unicellular green alga, R. subcapitata, obtained from algal stock culture of Federal University of Sao Carlos (microalgae biotechnology laboratory) was cultured in a 25 mL freshwater medium Oligo (AFNOR, 1980) in acid-cleaned polycarbonate bottles and in a growth chamber with a 12:12 h light:dark cycle and a Temperature of 19 ± 0.1 °C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

not applicable

Post exposure observation period:

none

Hardness:

no details given

Test temperature:

22 ± 2 °C

pH:

no details given

Dissolved oxygen:

no details given

Salinity:

no details given

Conductivity:

no details given

Nominal and measured concentrations:

0, 0.5, 2.0, 5.0, 10.0, 20.0, 50.0, 70.0 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: acid-cleaned polycarbonate bottles
- Initial cells density: 1 x 10^5 cells/mL in exponential phase
- No. of vessels per concentration (replicates): 3

GROWTH MEDIUM
- Standard medium used: Oligo medium (AFNOR, 1980).

OTHER TEST CONDITIONS
- Photoperiod: 1,12:12 h light:dark cycle
- Light intensity and quality: 120 µEm^-2/ s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Algal growth was monitored daily for 96h by direct cell counting. Cell density was determined by counting at least two counts per replicate in a Fuchs-Rosenthal chamber.

TEST CONCENTRATIONS
- Test concentrations: 0, 0.5, 2.0, 5.0, 10.0, 20.0, 50.0, 70.0, 100.0 mg/L

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: determined by cell count

Reported statistics and error estimates:

Student's t-test was used to compare toxicity effects between control and graphene concentrations. The algal density curves in different concentrations of graphene oxide were used to obtain 96 h IC 50 (inhibition concentration of graphene to 50% of the organisms under a specific exposure time).

GO was shown to be stable in the algal medium during 96 h (experimental time). The zeta potential of GO in algal medium during 96 h did not vary significantly, also indicating a high stability.

Exposure of the algae to GO at concentrations starting at 20 mg/L caused 50% of growth inhibition.

Validity criteria fulfilled:

not specified

Conclusions:

The 96 -hour IC 50 (inhibition concentration of graphene to 50% of the organisms) was 20 mg/L.

Executive summary:

In this study similar to OECD Guideline 201, the effects of graphene oxide (GO) on green algae Raphidocelis subcapitata were evaluated. The algae were exposed to different concentrations of GO pre-equilibrated for 24 h with oligotrophic freshwater medium (20 mL) during incubation in a growth chamber under controlled conditions. Algal growth was monitored daily for 96 h by direct cell counting. The toxic effects from GO, as observed in algal density started at concentrations from 20 mg/L. The 96 -hour IC 50 (inhibition concentration of graphene to 50% of the organisms) was 20 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no details given

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

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:

see Table 1: Selected physicochemical properties of Graphene oxide (GO)

Analytical monitoring:

not specified

Details on sampling:

no details given

Vehicle:

no

Test organisms (species):

Chlorella pyrenoidosa

Details on test organisms:

no details given

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

no details given

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:

see Details on test conditions

Details on test conditions:

Effect of light shading by graphene oxide on algal growth inhibition
250-mL conical flasks with exponentially growing algal cells were placed into 1-L beakers which already contained GO suspension (50 mg/L) prepared in algal medium. The level of GO suspension in the beaker was kept in the same height with that of algal suspension in the conical flask. The algal cell numbers (A1) in the conical flasks were counted after shading in the GO suspension for 96 h. A treatment with algal medium in the beaker was conducted as a control, and the algal cells (A0) in the conical flask were also counted after culturing for 96 h. The contribution (%) of shading effect on algal growth inhibition was then calculated as follows: (A0-A1)/A0 x 100.

Membrane integrity and leakage of intracellular substances after GO exposure
Membrane integrity of algal cells after GO exposure was assessed using flow cytometry (BD Biosciences, San Jose, USA). Algal cells (1 x 10E6 cells/mL) were treated with 50 mg/L GO for 96 h. The collected algal cells were dyed with propidium iodide (PI, 50 mg/L) for 20 min in the dark. The fluorescence intensity (FI) of dyed algal cells was then determined by flow cytometry with a FL2 detector. Each sample was analyzed for at least 20,000 cells.
The measurement of electrolyte (K+) leakage from algal cells was also determined in this study. The algal cells after GO exposure for 96 h were collected and washed with phosphate buffer solution (PBS) (0.1 M, pH 7.2) for three times. For each treatment, the washed algal cells were diluted with PBS (0.1 M, pH 7.2) to the same algal cells density (1 x 10E6 cells/mL), and then shaken (220 rpm) at 25 °C for 4 h. K+ content in algal medium was then determined by atomic absorption spectrophotometry.
The leakage of DNA from algal cells was also assessed. After exposure to GO for 96 h, the cell suspension was centrifuged at 3500 rpm for 10 min. The obtained supernatant was filtered through a 0.22 µm membrane. The DNA fraction in the filtrate was then isolated using a DNA purification kit and measured with fluorescence spectroscopy using PI as a fluorescent dye (excitation 535 nm, emission 615 nm).

Oxidative stress- and physical penetration-induced membrane damage
The generation of intracellular reactive oxygen species (ROS) was detected using an ROS indicator, 2,7-dichlorofluorescin diacetate (H2DCFDA).
Thiobarbituric acid reactive substance (TBARS, Sigma) was used to indicate malondialdehyde (MDA) content which reflects the lipid peroxidation level of algal cells after GO exposure. MDA content of the algal cells after exposure to GO for 96 h was determined using Multiskan spectrum (Thermo, USA) at 532 nm. The algal cells exposed to 200 µM hydrogen peroxide for 2 h was set up as a positive control.
Direct GO-algae contact and possible physical damage of algal cells after GO exposure were observed with scanning electron microscopy (SEM). Algal cells pretreated with GO were prefixed in 2.5% glutaraldehyde for 12 h, washed three times with phosphate buffer (pH 7.2), and then post-fixed in 1% osmium tetroxide for 2 h. After fixation, all the samples were dehydrated with increasing concentrations of ethanol (30, 50, 70, 80, 90,100%) and permeated with tertbutyl alcohol. Finally, these samples were freeze-dried and gold-coated, and then observed using SEM (HITACHI S-4800, Japan).

Effect of nutrient adsorption by GO on algal growth
Algal growth reduced by adsorption and removal of nutrients by GO from algal medium was conducted. GO was added to algal medium to reach the final concentration at 50 mg/L, and then shaken under 25 °C for 96 h. GO suspension was then centrifuged and filtered twice with 0.22 µm membrane (millex, Millipore). The obtained supernatant and the algal medium without nutrient removal were used to culture algal cells, and the 96 h growth inhibition induced by nutrient depletion was then calculated. In addition, the concentrations of macroelements (N, P, Ca, Mg, K) and microelements (Fe, Mn, Cu, Zn) in the supernatant were quantiied.

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

37.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The 96-h growth inhibition of algal cells highly depended on GO concentration. The EC50 value of GO was calculated as 37.3 mg/L. The growth inhibition increased with increasing exposure times (24 - 96 h).
As dispersed NPs could reduce the light transmittance of NPs suspension, the indirect toxicity induced by a shading effect on algae growth of GO was investigated. GO had significant shading effect on algal growth. GO shading caused 9.02% of the total growth inhibition as calculated from the algal growth results. The contribution of shading effect to the total GO toxicity (54.9%) was then calculated as 16.4%. GO was dispersed well during 96-h exposure due to its hydrophilic surface and strong electrostatic repulsion between individual GO sheets (zeta potential, -27.3 mV), and GO sheets became dark during incubation, thus further reducing light availability for algal growth results.
Furthermore, GO significantly caused membrane damage, K+ leakage and DNA leakage. In addition, GO significantly increased the intracellular ROS level of algal cells, showing induction of oxidative stress, and GO also caused significant lipid peroxidation of membrane.
GO caused 28.7 % of nutrient depletion-induced indirect toxicity. In addition, the contribution of nutrition depletion to the total toxicity of GO was calculated as 53 %.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

All the experiments were run in at least three replicates. 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.

Validity criteria fulfilled:

not specified

Conclusions:

Graphene oxide inhibited algae growth with an EC50 (96h) of 37.3 mg/L.

Executive summary:

The effect of graphene oxide (GO) on Chlorella pyrenoidosa was investigated in a study similar to OECD Guideline 201. In a 96 h study, cultures of Chlorella pyrenoidosa were exposed to up to 200 mg/L GO under static conditions. For GO, shading effect (~16%), oxidative stress-induced membrane damage, and nutrient depletion (~53%) were responsible for the observed toxicity. Flow cytometry results showed significant decrease of membrane integrity after GO exposure.

The EC50 value of GO to freshwater algae (Chlorella pyrenoidosa) was examined as 37.3 mg/L.

Description of key information

The effect of graphene oxide (GO) on Chlorella pyrenoidosa was investigated in a study similar to OECD Guideline 201. In a 96 h study, cultures of Chlorella pyrenoidosa were exposed to up to 200 mg/L GO under static conditions.

For GO shading effect (~16%), oxidative stress-induced membrane damage, and nutrient depletion (~53%) were responsible for the observed toxicity.

Flow cytometry results showed significant decrease of membrane integrity after GO exposure. The EC50 value of GO to freshwater algae (Chlorella pyrenoidosa) was examined as 37.3 mg/L (Zhao, 2017).

In a second study similar to OECD Guideline 201, the effects of graphene oxide (GO) on green algae Raphidocelis subcapitata were evaluated. The algae were exposed to different concentrations of GO pre-equilibrated for 24 h with oligotrophic freshwater medium (20 mL) during incubation in a growth chamber under controlled conditions. Algal growth was monitored daily for 96 h by direct cell counting. The toxic effects from GO, as observed in algal density started at concentrations from 20 mg/L. The 96 -hour IC 50 (inhibition concentration of graphene to 50% of the organisms) was 20 mg/L (Nogueira, 2015).

Key value for chemical safety assessment

EC50 for marine water algae:

20 mg/L