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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:
2016-06-23 to 2017-01-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L
- Sampling method: The concentrations of the test item were analytically verified from all test concentrations and the control after 0 hours (test start) and 72 hours (test end). Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis:All samples were stored at room temperature before preparation and before analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution of 100 mg test item/L was prepared by dissolving 50.0 mg test item in 500 mL test water by intense stirring for 15 minutes. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.
The test media were prepared just before introduction of the algae (= start of the test).

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Pseudokirchneriella subcapitata
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Test temperature:
22.7 to 23.0 °C
pH:
8.0 to 8.1 at test start and
8.4 to 10.1 at test end
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Nominal:100, 32, 10, 3.2 and 1.0 mg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 50 mL Erlenmeyer flasks, test volume 50 mL, covered with glass dishes and incubated in a water bath.
- Aeration: Magnetic stirrer
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: 5000 algal cells per mL test medium
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted Water
(OECD Medium):

Analytical grade salts were added at the following nominal concentrations in deionised water:
Macro-nutrients: NaHCO3 50.0 mg/L
CaCl2  2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4  7 H2O 15.0 mg/L
MgCl2  6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L
Trace Elements: Na2EDTA  2 H2O 100.0 µg/L
FeCl3  6 H2O 64.0 µg/L
MnCl2  4 H2O 415.0 µg/L
H3BO3 185.0 µg/L
Na2MoO4  2 H2O 7.0 µg/L
ZnCl2 3.0 µg/L
CoCl2  6 H2O 1.5 µg/L
CuCl2  2 H2O 0.01 µg/L
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3. The pH was adjusted with 1 M HCl to pH 8.1.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: 24 h
- Light intensity and quality: 5803 lux (range: 5480 to 6440 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The cell density on each observation time and of each sample was determined by spectrophotometric measurement (absorption at 685 nm). Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae). Measured absorption was related to the cell density according to a calibration curve (linear regression based on determination of cell numbers and absorption of an algal suspension and its dilutions).
















Reference substance (positive control):
yes
Remarks:
Potassium dichromate
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
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
47.7 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
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test mediu
Results with reference substance (positive control):
Parameter
(0-72 h) Yield [mg test item/L] Growth rate [mg test item/L] Biomass [mg test item/L]
72-hour EC50 0.721 1.50 0.794
95 % conf. interval 0.588 - 0.847 1.43 - 1.58 0.668 - 0.910

72-hour EC20 0.531 0.731 0.552
95 % conf. interval 0.367 - 0.638 0.673 - 0.785 0.381 - 0.658

72-hour EC10 0.452 0.502 0.456
95 % conf. interval 0.280 - 0.562 0.447 - 0.554 0.278 - 0.569

72-hour NOEC 0.45 0.45 0.45
72-hour LOEC 1.0 1.0 1.0

Values refer to nominal test concentrations



Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Cell Density
Increase in Control Cultures:


At least by a factor of 16 within 72 hours

Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures:



Must not exceed 35 %

Coefficient of Variation of Average Growth between Control Replicates:



Must not exceed 7 %

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour ErC50 value was determined to be > 100 mg test item/L. The 72-hour NOEyC was determined to be 3.2 mg test item/L and the associated 72-hour LOEyC was 10 mg test item/L. The 72-hour NOErC was determined to be 3.2 mg test item/L and the associated 72-hour LOErC was 10 mg test item/L.
Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3. at IBACON GmbH, Rossdorf. The aim of the study was the determination of NOEC, LOEC, EC10-, EC20- and EC50-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5000 cells/mL. A stock solution of 100 mg/L was prepared by direct addition of the test item to the dilution water and shaken until the test item was completely dissolved. Five concentrations were tested in a geometrical series with a dilution factor of 3.2 (nominal): 100, 32, 10, 3.2 and 1.0 mg test item/L. Three replicates were tested for each test item concentration and six replicates for the control. The quantification of the test item was performed using liquid chromatography with MS/MS detection. At the start of the test, 86 % of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 90 % of the nominal value was determined (average of all test concentrations). The 72-hour ErC50 value was determined to be > 100 mg test item/L and the 72-hour NOErC was determined to be 3.2 mg test item/L.

Description of key information

The toxicity of the test substanceto the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 761/2009/Method C.3. at IBACON GmbH, Rossdorf. The study was conducted under static conditions with an initial cell density of 5000 cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of 3.2 (nominal): 100, 32, 10, 3.2 and 1.0 mg test item/L. The 72-hour ErC50 value was determined to be > 100 mg test item/L and the 72-hour NOErC was determined to be 3.2 mg test item/L.

Key value for chemical safety assessment

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

Additional information