Single Wall Carbon Nanotubes (SWCNT)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 June 2016 - 08 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

Deviations:

yes

Remarks:

Due to the pure and poorly water soluble nature of the test item, the test media was prepared as a saturated solution

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the uninoculated control and 100% v/v saturated solution test preparation at 0 and 72 hours.

Vehicle:

no

Details on test solutions:

Due to the pure and poorly water soluble nature of the test item, the test media was prepared as a saturated solution at an initial loading rate of 10 mg/L.
A nominal amount of test item (110 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) followed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution.
An aliquot (1 liter) of the stock solution was inoculated with 7.3 mL of algal suspension to give the required test concentration of 100% v/v saturated solution.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ±1 °C

pH:

8.1 - 8.9

Nominal and measured concentrations:

Based on the results of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration no effect on algal growth was observed.

Total Organic Carbon (TOC) analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured carbon concentration of 1.51 mg/L . A decline in the measured carbon concentration was observed at 72 hours to less than the limit of quantification (LOQ) of the analytical method which was considered to be 1.0 mg C/L.

Details on test conditions:

250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control and 100% v/v saturated solution treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.89 x 105 cells per mL. Inoculation of 1 liter of test medium with 7.3 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Total Organic Carbon (TOC) analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured carbon concentration of 1.51 mg/L was obtained. A decline in the measured carbon concentration was observed at 72 hours to less than the limit of quantification (LOQ) of the analytical method which was considered to be 1.0 mg C/L.
Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the nominal test concentration of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be 100% v/v saturated solution.
This study showed that there were no toxic effects at saturation.

Results with reference substance (positive control):

Results on potassium dichromate:
ErC50 (0 – 72 h) : 1.5 mg/L; 95% confidence limits 1.3 – 1.7 mg/L
EyC50 (0 – 72 h) : 0.79 mg/L; 95% confidence limits 0.70 – 0.89 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 1.0 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item, showing a clear inhibition of algal growth.

Validity criteria fulfilled:

yes

Conclusions:

This study showed that there were no toxic effects at saturation. EC50, considering both growth rate inhibition and yield, is greater than 100% v/v saturation. Analytical measurement of this concentration was not possible necause under the LOQ (1 mg/L ca.).

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Due to the pure and poorly water soluble nature of the test item, the test media was prepared as a saturated solution at an initial loading rate of 10 mg/L. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a solution of the test item at a nominal concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test item solution was prepared by stirring an excess (10 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 liter discarded in order to pre-condition the filter) followed by centrifugation at 40000 g for 30 minutes to produce a 100% v/v saturated solution of the test item. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Total Organic Carbon (TOC) analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured carbon concentration of 1.51 mg/L was obtained. A decline in the measured carbon concentration was observed at 72 hours to less than the limit of quantification (LOQ) of the analytical method which was considered to be 1.0 mg C/L. Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the nominal test concentration of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be 100% v/v saturated solution. This study showed that there were no toxic effects at saturation.

Description of key information

Exposure of Pseudokirchneriella subcapitata to the test item, in a study according to OECD TG 201, gave EC50 values based on the nominal test concentration of greater than 100% v/v saturated solution.

The No Observed Effect Concentration was determined to be 100% v/v saturated solution. This study showed that there were no toxic effects at saturation.

Additional information

The concentration of substance in the 100% v/v saturated solution was not possible because it was under the LOQ (1 mg/L ca.).