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Ecotoxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with analytical monitoring
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, 2009
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations
- Sampling method: Sampling was performed at the start of the exposure (0 h) from the inoculated replicate 7 of each concentration and the control.
At the end of the exposure (72 h) samples of the inoculated replicates 1-3 of the test concentrations and of the inoculated replicates 1- 6 of the control group were taken. The replicates were combined before sampling. Additionally, samples were collected from the uninoculated replicate 0 of each test concentration and the control at the end of the exposure (72 h).
- Sample storage conditions before analysis: Storage at room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accommodated fraction (WAF) of the test substance were prepared according to OECD 23. The test substance was homogenized by shaking the container before removal for weighing. The stock solutions were prepared by weighing the test substance on a glass coverslip then adding this to 1L of test medium in a 2 L glass flask. The stock solutions were shaken for approximately 10 min at 20 ± 2 °C. The test substance was made up with OECD medium to reach concentrations of 111% of the nominal concentration to compensate for the dilution by the later addition of inoculum.
- Differential loading: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The stock test solutions in test groups 4.6 and 10 mg/L appeared slightly turbid and test groups 22 – 100 mg/L appeared strongly turbid after preparation. Since the test substance was homogenously dispersed in the test medium, no attempt was made to separate any potentially undissolved components. The algal cells were exposed to the entire loaded mass of test substance.
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus (former name: Scenedesmus subspicatus)
- Strain: CHODAT SAG 86.81
- Source (laboratory, culture collection): Supplier: Collection of algal cultures in University of Göttingen/Germany
- Age of inoculum (at test initiation): The test algae are taken from a 3 days-old continously growing preculture
- Method of cultivation: The test strain is kept in liquid culture in the laboratory.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test medium
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.4 - 22.7 °C
pH:
8.1
Nominal and measured concentrations:
Nominal (loading rate): 4.6, 10, 22, 46 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Erlenmeyer flasks (nominal 250 ml), plugged with gas permeable silicone sponge caps, 100 ml test volume
All test vessels were saturated for at least 24-h before use with 100 mL of solution to reduce the possibility of test substance loss during the test by adsorption to the test vessel. This solution was discarded and fresh test solution was used to start the test.
- Initial cells density: 5000 cells/mL
- Control end cells density: 570000 cells/mL (72h)
- 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: according to OECD 201

OTHER TEST CONDITIONS
- Photoperiod: continous
- Light intensity and quality: Artificial light, type universal white (OSRAM L 25), average 5275 lux (within ± 15 % variability) at a wave length of 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: in vivo chlorophyll-a-fluorescence (pulsed excitation with light flashes having a wave length of 432 nm) after 0, 24, 48, and 72 hours
- pH values: after 0 and 72 hours

TEST CONCENTRATIONS
4.6, 10, 22, 46 and 100 mg/l. A control was tested in parallel.
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
85.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
39 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL90
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
46.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
17.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL90
Effect conc.:
93.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): After 72h
- 46 mg/L (loading rate): low cell density, partial clumping
- 100 mg/L (loading rate). Cells smaller than control, low cell density, mostly clumped
- Any stimulation of growth found in any treatment: No

STATISTICS:
The yield and growth rate over the exposure period was calculated for each replicate flask of each test group (ISO/TS 20281) and inhibition for each test group was determined by comparison to the control. The percent inhibition of the mean yield and growth rate compared to the control was calculated for each test group. The data are illustrated using plots of percent inhibition (response) versus concentration. ECx values
were calculated via interpolation from the concentration-response relationship.
SAS statistical software was used for the statistical evaluation of the data. The LOEC was determined by comparing the means of the calculated yield or growth rate of the various concentration levels with the control. The Dunnett’s test (one-sided) was carried out at a 95% significance level. The analyses were based on the assumption that each higher tested concentration must have at least the same or a stronger effect than the LOEC. The NOEC was the next tested concentration below the LOEC.
Results with reference substance (positive control):
In order to verify that the algal cultures are responding normally to toxic stress, tests with a reference substance (potassium dichromate) are conducted. Reference substance tests are conducted according to OECD 201 guidelines and in accordance with GLP, but without a GLP status. The results from the reference substance test are compared to potassium dichromate EC50 values published in ISO test guideline 8692, which represent the typical response range for the algal species tested. According to the test ISO guideline 8692 the EC50 values of the reference substance potassium dichromate should be in the range: ErC50 = 0.71 – 0.97 mg/L after 72 hours for Desmodesmus subspicatus.
The ErC50 (72 h) of the control substance potassium dichromate was 0.91 mg/L (Date of the last control experiment: 22 Feb 2010 , project number: 60E0063/043030). These results indicate that the algae are responding normally to toxicant stress.

The mean measured concentrations could not be calculated for all test concentrations. Since the algal cells were exposed to the entire loaded mass of test substance in each test group, the results of this study are based on loading rate. The results should be considered as the effect of the parent test substance and all degradation products.

The analysis of uninoculated 72-h samples (e.g. replicates without algae) confirms the results from the 72-h inoculated samples. Therefore the decrease in measured concentration during the test was not due to binding of the test substance to the increasing algal biomass, but rather due to loss by instability or precipitation of the test substance in the test system and under test conditions.

Validity criteria fulfilled:
yes
Remarks:
cell multiplication factor in the untreated control was 114-fold after 72h; the mean coefficient of variation for section-by-section growth rates for each test day in the control cultures was 17%; the coefficient of variation of average specific growth ra
Conclusions:
The EL50 (72 h) and the NOELR for growth rate were found to be 85.1 mg/L and 22 mg/L, respectively. In addition, the EL50 (72 h) and the NOELR for biomass were 46.9 mg/L and 10 mg/L, respectively
Executive summary:

Toxicity of ASA to Desmodesmus subspicatus was tested in a study performed according to OECD 201, where the algae was exposed for 72 h to nominal loading rates of 4.6, 10, 22, 46 and 100 mg/L as water accommodated fraction prepared in accordance with OECD 23. At 46 and 100 mg/L clumping of cells was observed. As a result, the EL50 (72 h) and the NOELR for growth rate were found to be 85.1 mg/L and 22 mg/L, respectively. In addition, the EL50 (72 h) and the NOELR for biomass were 46.9 mg/L and 10 mg/L, respectively

Description of key information

EC50 (72 h): 85.1 mg/L (growth), NOELR (72 h) 22 mg/L (growth), EC50 (72 h) 46.9 mg/L (biomass), NOELR (72 h) 10 mg/L (biomass) (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
85.1 mg/L
EC10 or NOEC for freshwater algae:
22 mg/L

Additional information

Toxicity of the substance to Desmodesmus subspicatus was tested in a study performed according to OECD 201, where the algae were exposed for 72 h to nominal loading rates of 4.6, 10, 22, 46 and 100 mg/L as water accommodated fraction prepared in accordance with OECD 23. At 46 and 100 mg/L clumping of cells was observed. As a result, the EL50 (72 h) and the NOELR for growth rate were found to be 85.1 mg/L and 22 mg/L, respectively. In addition, the EL50 (72 h) and the NOELR for biomass were 46.9 mg/L and 10 mg/L, respectively