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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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Test guideline under Japan Chemical Substances Control Law
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Lot No.: 140220
Analytical monitoring:
yes
Details on sampling:
Rangefinder:
A preliminary solubility trial was conducted prior to the range finder, which included prolonged stirring
(24 hours), followed by filtration (Millex 0.45 um) of a 100 mg/L stock (in triplicate). This provided a mean solubility of 5.3 mg/L, which was used as the limit concentration in the definitive test. During the range finder analytical samples were taken at 0 and 72 hours.

Definitive:
- Concentration: 5.3 mg/L
- Frequency: Samples for analysis were taken from the limit test concentration, control and solvent control at the start of the study (t = 0h Fresh), and (t = 72h Expired).
- Sampling method: At the beginning of the test, 1.0 mL was collected from each test solution (from the middle) and mixed. An aliquot (0.75 mL) of this was added to 0.75 mL acetonitrile and mixed further, prior to HPLC analysis. At 72 hours 1.0 mL of each test culture (from the middle) of all test vessels was mixed and centrifuged at 3000 rpm. An aliquot of the supernatant (0.75 mL) was collected and 0.75 mL of acetonitrile added. This was then mixed prior to HPLC analysis.
- Sample storage conditions before analysis: Not detailed, but assumed to be analysed on the day of sampling.
Vehicle:
yes
Details on test solutions:
Preparations of test medium started with the preparation of a solvent stock solution in N,N-Dimethylformamide (Vehicle) with the test material. An
aliquot of this (100 uL/L) was then added into a media bottle containing test medium and stirred for 15 minutes (magnetically) which provided the
limit concentration of 5.3 mg/L. This was then transferred to a total of 7, 300 mL test vessels and mixed. An equivalent amount of solvent was added for the solvent control (vehicle) group.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green algae
- Scientific name: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): American type culture collection (June 20, 1996).
- Method of cultivation: Periodic subculture using gorham medium under sterile conditions.

ACCLIMATION
- Acclimation period: 27 to 30 June 2014
- Culturing media and conditions (same as test or not): Same conditions as test cultures.
- Any deformed or abnormal cells observed: None confirmed.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
21.5 to 22.3°C measured in the incubation chamber
pH:
7.8 to 7.9
Nominal and measured concentrations:
Nominal: 0 (control), 0 (solvent control) and 5.3 mg/L.
Measured: 0 (control), 0 (solvent control) and 5.1 mg/L (time weighted mean).
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmyer flasks
- Material, fill volume: Glass, 100 mL
- Initial cells density (Biomass): 5 x 10^3 cells/mL
- Control end cells density: 1.85 x 10^6
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6
- Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells
were kept in suspension by continuous shaking (100 rpm).

GROWTH MEDIUM
- Standard medium used: yes (as per OECD guidelines)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Media as detailed in the relevant OECD guidance. The medium was sterilized by filtration (pore diameter
0.22 um).
- Intervals of water quality measurement: 0 and 72 hours

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 69 to 75 µE.m-2.s-1 using white fluorescent lamps.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 24, 48 and 72 hours; measurement of biomass. At 72 hours algae was
observed under a microscope for any abnormalities.
- Determination of cell concentrations: Electronic particle counter

TEST CONCENTRATIONS
- Range finding study
Test concentrations: 0.053, 0.53 and 5.3 mg/L.
- Results used to determine the conditions for the definitive study: No growth inhibition at limit of solubility (5.3 mg/L).
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 5.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No effect upto limit of solubility
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.1 mg/L
Nominal / measured:
meas. (TWA)
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): None
- Unusual cell shape: None
- Effect concentrations exceeding solubility of substance in test medium: No effect upto the limit of solubility in the test media.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 0.804 mg/L (95% confidence limits: 0.781 - 0.828 mg/L). Within historical range.

Percentage inhibition of growth rate (total test period)

 Test group (mg/L)  Mean  Std.Dev.  n  % inhibition
 Control 0.0821  0.0008 
 Solvent Control 0.0803 0.0012 
 5.3 (nominal), 5.1 (measured) 0.0790  0.0024  1.7 
Validity criteria fulfilled:
yes
Conclusions:
Due to the low solubility of the test substance in test medium, concentration levels that might be toxic for algae could not be reached.
The EC50 for growth rate inhibition (72h-ERC50) was above the solubility limit of the test substance in test medium (> 5.1 mg/L)
The 72h-NOEC for growth rate inhibition was at the solubility limit of the test substance in test medium (5.1 mg/L).

Description of key information

In a 72hr acute toxicity study, green algae (Pseudokirchneriella subcapitata) were exposed to the test substance at measured concentrations of 0 (control), 0 (solvent control), or 5.1 mg/L under semi-static conditions. The 72h-NOEC for growth rate inhibition was at the solubility limit of the substance in test medium.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
5.1 mg/L

Additional information

Due to the very low solubility of this substance in test medium, concentration levels that might be toxic for algae could not be reached.

The EC50 for growth rate inhibition (72hr ErC50) was above the solubility limit of the test substance in test medium (> 5.1 mg/L). The 72h-NOEC for growth rate inhibition was at the solubility limit of the substance in test medium (5.1 mg/L).