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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:
12 - 15 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and EC guidelines and according to GLP principles
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples of 2.0 mL for possible analysis were taken from all test concentrations and the control at t=0 h, t=24 h and t=72 h.
The Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at an intermediate substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 2.0 ml were taken from all test solutions for possible analysis.

On the day of analysis, the test samples were defrosted at room temperature. The samples were diluted in a 1:1 (v:v) ratio with acetonitrile and analysed. If necessary, the samples were further diluted with 50/50 (v/v) acetonitrile/water to obtain concentrations within the calibration range.
Vehicle:
no
Details on test solutions:
The test substance was completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with the highest concentration of 100 mg/L applying 14 minutes of magnetic stirring to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation):3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm) in a climate room at a temperature of 21-24°C.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
0.24 mmol (24 mg CaCO3/L)
Test temperature:
The temperature of the test medium was 22.0°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C.
pH:
Between 7.7 and 7.9
Nominal and measured concentrations:
The study was carried out as a combined limit/ range-finder test with nominal test concentrations: 0.1, 1, 10 and 100 mg/L.
Since there were no effects observed in all test concentrations, only the highest test concentration was analysed.
Analysis of the samples showed that measured concentrations were stable and in agreement with nominal throughout the test period (102-105%). Results are expressed as nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 100 mL, all-glass, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 1.0E+4 cells/mL
- Control end cells density: 2.3E+6 cells/mL
Replicates:
6 replicates of the control
6 replicates of the highest (limit) concentration
3 replicates of each lower test concentration
1 or 2 replicates of each concentration without algae
1 extra replicate of each test group for sampling purposes.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 82 to 88 µE.m-2.s-1
- 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.

EFFECT PARAMETERS MEASURED:
The cell density was determined at t = 0, 24, 48 and 72h
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm).
In order to correct for turbidity one control per concentration, without algae, was measured at each time interval.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: control and 0.1, 1, 10 and 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Nominal test substance concentrations were analytically confirmed.
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
Remarks on result:
other: Nominal test substance concentrations were analytically confirmed.
Results with reference substance (positive control):
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.9 mg/L with a 95% confidence interval ranging from 1.8 to 2.0 mg/L.
The EC50 for yield inhibition (EYC50: 0-72h) was 0.53 mg/L with a 95% confidence interval ranging from 0.43 to 1.1 mg/L.
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield.

The validity criteria were fulfilled as follows:

- The biomass in the control cultures increased exponentially by a factor of at least 16 (232) within the 72-hour test period.

- The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35% (28%).

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (2%)

Validity criteria fulfilled:
yes
Conclusions:
The substance did not significantly reduce growth rate of the fresh water algae species Pseudokirchneriella subcapitata at any of the concentrations tested.
The 72h-ErC50 exceeded an analytically confirmed nominal concentration of 100 mg/L.
The NOErC was 100 mg/L.
Executive summary:

The toxicity of the substance to the freshwater algae species Pseudokirchneriella subcapitata was determined according to OECD 201 and EU method C.3.

The substance did not significantly reduce growth rate at any of the concentrations tested.

The 72h-ErC50 exceeded an analytically confirmed nominal concentration of 100 mg/L.

The NOErC was 100 mg/L.

The study is considered reliable without restrictions.

Description of key information

The substance did not significantly reduce growth rate of the fresh water algae species Pseudokirchneriella subcapitata at any of the concentrations tested. 
The 72h-ErC50 exceeded an analytically confirmed nominal concentration of 100 mg/L.
The NOErC was 100 mg/L.

Key value for chemical safety assessment

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

Additional information

The toxicity of the substance to the freshwater algae species Pseudokirchneriella subcapitata was determined according to OECD 201 and EU method C.3.

The substance did not significantly reduce growth rate at any of the concentrations tested.

The 72h-ErC50 exceeded an analytically confirmed nominal concentration of 100 mg/L.

The NOErC was 100 mg/L.

The study is considered reliable without restrictions.