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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
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:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Principles of method if other than guideline:
no details given
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 (MG)
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:
The effect of light shading and agglomeration by graphene on algal growth inhibition was also investigated.
250-mL conical flasks with exponentially growing algal cells were placed into 1-L beakers which already contained Graphene suspension (50 mg/L) prepared in algal medium. The level of graphene 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 graphene 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.
The heteroagglomeration of algae with graphene was investigated following the approach of Schwab et al. (2011). Briefly, the suspended algal cells and graphene suspension (final concentration, 50 mg/L) were mixed in 250-mL flask. After settled down for 2 h, algal cells in the aqueous phase were counted. Growth of algal cells was not significantly inhibited after inculcated with graphene for 2 h. Therefore, the reduction of algae number in aquatic phase after 2-h settling was attributed to their agglomeration with graphene.

Membrane integrity and leakage of intracellular substances after graphene exposure
Membrane integrity of algal cells after graphene exposure was assessed using flow cytometry (BD Biosciences, San Jose, USA). Algal cells (1 x 106 cells/mL) were treated with 50 mg/L graphene for 96 h. The collected algal cells were dyed with propidium iodide (PI, 50 mg/L) for 20 min in 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 graphene 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 E6 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 graphene 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 graphene exposure. MDA content of the algal cells after exposure to graphene 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 graphene-algae contact and possible physical damage of algal cells after graphene exposure were observed with scanning electron microscopy (SEM). Algal cells pretreated with graphene 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 graphene on algal growth
Algal growth reduced by adsorption and removal of nutrients by graphene from algal medium was conducted. Graphene was added to algal medium to reach the final concentration at 50 mg/L, and then shaken under 25 °C for 96 h. Graphene 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.:
62.2 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 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 (Table 1) could readily form big aggregates, thus settling down in aqueous phase.
After settling for 2 h 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 %.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
All the experiments were run 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:
Multi-layer Graphene inhibited algae growth with an EC50 (96h) of 62.2 mg/L.
Executive summary:

The effect of graphene on Chlorella pyrenoidosa was investigated by Zhao et al (2017). In a 96 h study according to OECD TG 201, cultures of Chlorella pyrenoidosa were exposed to up to 200 mg/L graphene under static conditions.

The EC50 value of graphene to freshwater algae (Chlorella pyrenoidosa) was examined as 62.2 mg/L.

Graphene showed no shading effect on algal growth due to the poor dispersibility while it more readily heteroagglomerated with algae 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.

Description of key information

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

Key value for chemical safety assessment

EC50 for freshwater algae:
62.2 mg/L

Additional information