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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:
29 March 2016-14 May 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)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 4M413
- Expiration date of the lot/batch: 31 December 2016
- Physical state/Appearance: Clear colorless liquid
- Purity: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Disolved in culture medium and pH adjusted to 7.5
- Final dilution of a dissolved solid, stock liquid or gel: 100 mg/L

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and each tested concentration
- Sampling method: Taken from stock and pooled replicates at 0 and 72 h
- Sample storage conditions before analysis: Frozen
Vehicle:
yes
Remarks:
Culture medium
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
First range-finder:
A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.9 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The results of the initial range-finding test showed significant inhibition of growth occurred at the test concentration of 100 mg/L where the 0-Hour pH was determined to be 10.2. In order to determine whether it was the highly alkaline nature of the test item which caused the inhibitory effects observed, a second range-finding test was conducted whereby the pH of the 100 mg/L stock solution was adjusted to pH 7.5 prior to inoculating with algal cells.
A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution. The pH of the 100 mg/L stock solution was measured and adjusted to pH 7.5. An aliquot (450 mL) of the pH adjusted stock solution was inoculated with algal suspension (4.3 mL) to give the required test concentration of 100 mg/L.

Second range-finder:
A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution. The pH of the 100 mg/L stock solution was measured and adjusted to pH 7.5. An aliquot (450 mL) of the pH adjusted stock solution was inoculated with algal suspension (4.3 mL) to give the required test concentration of 100 mg/L. The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Initial experiment:
Based on the results of the range-finding tests an initial experiment was conducted using a test concentration range of 6.25, 12.5, 25, 50 and 100 mg/L. The pH of the 100 mg/L stock solution was measured and adjusted to pH 7.5 prior to performing serial dilutions and inoculating with algal cells. Significant inhibition of yield was observed to have occurred at 6.25 mg/L and hence it was considered appropriate to conduct the definitive test using a test concentration range of 1.0, 3.2, 10, 32 and 100 mg/L.

Definitive test:
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution. The pH of the 100 mg/L stock solution was measured and adjusted to pH 7.5 prior to performing a series of dilutions to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (4.1 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: CCAP278-4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP) SAMS Research Services Limited, Scottish Marine Ocean Institute, Oban, Argyll, Scotland

ACCLIMATION
- Culturing media and conditions (same as test or not): The culture medium used for the range-finding tests, initial experiment and definitive test was the same as that used to maintain the stock culture.
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
24 +/- 1 degree C
pH:
adjusted to 7.5
Salinity:
not applicable
Nominal and measured concentrations:
Range finder: 0.10, 1.0, 10 and 100 mg/L
Definitive study:
* nominal: control, 1.0, 3.2, 10, 32, 100 mg/L
* measured: 0 hrs: < LOQ, 1.03, 3.27, 9.53, 30.3, 93.9 mg/L
* measured: 72 hrs: < LOQ, 0.73/1.07, 2.72, 9.49, 29.3, 97.5 mg/L

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 85% to 107% of nominal and so the results are based on nominal test concentrations only
Details on test conditions:
TEST SYSTEM
- Test vessel: Flask
- Type (delete if not applicable): closed, foam stopper
- Material, size, headspace, fill volume: 250 ml glass conical
- Shaken, 150 rpm
- Initial cells density: 5.0 x 10^3 cells/ml
- Control end cells density: 8.93 x 10^5 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified DI
- Culture medium different from test medium: no
- Intervals of water quality measurement: 0 and 72 h

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: 72 h
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux, warm white


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell density
- Determination of cell concentrations: Coulter counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study: yes
- Test concentrations: 0.1, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
89 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Unusual cell shape: Some enlarged cells at 100 mg/L
- Other: 72-h ErC20 = > 100 mg/L

Results with reference substance (positive control):
- Results with reference substance valid? yes
- 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
- Other: The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
Dunnett's test for growth rate and yield after 72 h, performed with SAS software.
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated in an OECD 201 study. The test substance was found to have an EC50 of >100 mg/L, the ErC10 was 89 mg/L and the NOErC was 100 mg/L.
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 was designed to be compatible with 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.

Following preliminary range-finding tests and an initial experiment, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The pH of the 100 mg/L stock solution was measured and adjusted to pH 7.5 prior to performing serial dilutions and inoculating with algal cells.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 85% to 107% of nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on nominal test concentrations:

 Response variable  EC10 (mg/L) EC20 (mg/L)   EC50 (mg/L)  No Observed Effect Concentration (NOEC) (mg/L)  Lowest Observed Effect Concentration (LOEC) (mg/L)
 Growth rate  89 >100   >100  100  NA
 Yield  35 54  >100   32 100 

Description of key information

Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours at a temperature of 24 ± 1 °C.

 Response variable  EC10 (mg/L) EC20 (mg/L)   EC50 (mg/L)  No Observed Effect Concentration (NOEC) (mg/L)  Lowest Observed Effect Concentration (LOEC) (mg/L)
 Growth rate  89 >100   >100  100  NA
 Yield  35 54  >100   32 100 

The test item is not toxic for Algae.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
89 mg/L

Additional information

The test item is not toxic on green algae.