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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:
April 2018 to July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Chemical analysis of the test item concentrations in the test item solutions was performed to determine test item exposure concentrations. Substance specific analysis was performed.
The samples were filled into 2 ml microcentrifuge tubes and stored in the freezer (<=-18°C). The sampling was conducted according to the following specification:
After 0h; 24h and 48 h exposure, both additional vessels of control; 12.5; 25, 50, 100 and 200 mg/l were sampled, one sample per replicate: 2 samples of 0.5 ml per treatment group.
At the end of the test (72h), both additional vessels and 2 replicates of the test vessels from control; 12.5; 25; 50; 100 and 200 mg/l are samples; one sample per replicate: 4 samples of 0.5 ml per tratment group.
Vehicle:
no
Details on test solutions:
The test substance is poorly soluble in water. A stock solution was prepared as Water Soluble Fraction (WSF) by adding 222.2 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) to 1000 ml test medium and shaking for 24 h using an overhead shaker at 22.2 - 22.9 °C in the dark. The stock solution was allowed to settle for 1 h before the solution was extracted from the centre of the liquid column and filtered through a fibre glass filter with retaining range till 0.6 µm.The filter was prepared by rinsing with purified water and was preconditioned with ca. 50 ml WSF (which was discarded) to reduce adsorption of the test item. The negative control was treated in the same way as the test item solution. Into each test vessel, 50 ml test solution (including algal inoculum) was transferred. The negative control and the further test item loading rates were prepared by diluting the stock solution with test medium.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green Algae
- Strain: number: 61.81 SAG
- Source: origin University Goettingen
- Method of cultivation: At the test lab, twice a week, the stock suspension is inoculated into fresh Holm-Hansen medium under axenic conditions to keep it in exponential growth

ACCLIMATION
- Acclimation period: 4 d at 22.7 - 23.2 °C and 88.5 µE m-2 s-1 +- 8% continuous lighting

The test medium was prepared according to OECD 201
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
using standardized test medium according to OECD 201
Test temperature:
22.2 - 23.2 °C
pH:
7.7 - 7.8 in the control
7.7 - 7.9 in the test item treatment
Nominal and measured concentrations:
Nominal concentrations: control; 12.5; 25; 50; 100; 200 mg/l
Details on test conditions:
The test vessels were sealed with cellulose stoppers and kept in the light incubator (specified temperature: 21-24 °C +- 2°C). They were placed on the shaking table with adhesive mats and continuously illuminated by LEDs (warm white) with light intensity of 88.5 µE m-2 s-1 +- 8%. The ph was measured at the start (oh) and at the end (72h) of the test. The temperature was measured continuously.
The algae biomass was monitored by measuring the chlorophyll fluorescence after 0h; 24h; 48 h and 72 h. From each test vessel, 200µl test solution was transferred into a 96-well microplate and the fluorescence measured with the fluorescence microplate reader at an excitation wavelength of 465 nm and an emission wavelength of 670 nm. Each measurement was conducted in duplicate.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
other: ELR50
Remarks:
Effect Loading Rate
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Due to the very lowwater solubility of the test item, the analytical measured test item concentrations are very low at 0h and could not be detected at 24 h, 48 h and 72 h. It was not possible to calculate mean values and evaluate the results of the algae.
All results are given in relation to the nominal test item loading rates.
No concentration related effects could be observed.

Nominal test item loading rate inhibition of growth rate (%)
(mg/l) 24 h 48 h 72 h
control - - -
12.5 27.7 10.1 11.5
25 14.3 2.3 6.3
50 24.2 14.8 10.2
100 -9.7 -3.9 4.8
200 -0.7 -4.5 -3.0

Results with reference substance (positive control):
ErC50 (72h) 1.18 mg/l (CL 95%: 0.97 - 1.42 mg/l)
Reported statistics and error estimates:
Effect loading Rates (ELR): with the statistical software ToxRat Professional 3.2.1 (ToxRat Solutions GmbH, Alsdorf), ELRx and LOELR/NOELR for yield and growth rate were determined

Cell morphology: The morphology of the algal cells in the control and the test item treatment showed no obvious abnormality

Validity criteria fulfilled:
yes
Conclusions:
An ELR50 (Effect Loading Rate) of > 200 mg/l were calculated in an valid test according to OECD 201 under GLP conditions.

Description of key information

An ELR50 (Effect Loading Rate) of > 200 mg/l were calculated in an valid test according to OECD 201 under GLP conditions.

Key value for chemical safety assessment

EC50 for freshwater algae:
200 mg/L
EC10 or NOEC for freshwater algae:
12.5 mg/L

Additional information