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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
Study period:
August - September 2017
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)
Version / remarks:
2011
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Version / remarks:
1996
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- For determination of the test item concentrations, three replicate samples (5 mL per replicate) were taken from each test concentration and six replicates from the control at the start of the test, after 24 h and 48 h and at the end of the test.
- Sampling method: HPLC method with UV detection
- Sample storage conditions before analysis: no storage
Vehicle:
no
Details on test solutions:
The test solutions used in the test was prepared by mechanical dispersion without using any solubilising agents.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum: 3 days

ACCLIMATION
- Acclimation period: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.2 – 22.8°C
pH:
8.07 – 8.94
Nominal and measured concentrations:
nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
calculated geometric mean of measured concentrations: 1.67, 2.34, 3.28, 4.89 and 7.01 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, fill volume: glass, ~250 mL volume, 100 mL test medium
- Aeration: no
- Initial cells density: 1 × 10E4 cells/mL
- Control end cells density: 72.83 × 10E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD Medium

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water. Separate stock solutions were prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX 3 water purification system). Then, the growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, with 1N NaOH adjusted to pH of culture medium
- Photoperiod: continuous illumination
- Light intensity and quality: 8009 lux, fluorescent lamps (400-700 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at 24, 48 and 72 h manual cell counting using microscope with counting chamber

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: results of range finding study
- Range finding study: toxic effects observed starting from 10 mg/L
- Test concentrations: 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes

STATISTICAL ANALYSIS
Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using Independent Samples T-Test (2-tailed; α = 0.05) by SPSS software.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
31.19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. limits: 19.43 - 74.70
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
6.08 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. limits: 5.23 - 7.55
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.28 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
4.89 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.93 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % conf. limits: 7.18 - 12.89
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % conf. limits: 1.93 - 2.95
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.34 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.28 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid - yes
- ErC50: 0.99 mg/L; EyC50: 0.59 mg/L
Reported statistics and error estimates:
For the determination of the LOEC and NOEC, ANOVA and Dunnett’s test (α = 0.05; 2-sided) was used and for the determination of the ECx values Probit analysis was used.
Therefore, the predicted EC50 values are above the measured concentrations.
Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 31.19 mg/L and 8.93 mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 3.28 mg/L and 2.34 mg/L, respectively.
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3. The study was conducted under static conditions. The definitive test was carried out with 5 concentrations: 6.25, 12.5, 25, 50 and 100 mg/L. As the test item is not stable in the test medium, the calculated geometric mean measured concentrations were used for calculation of the effect values. In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values: The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 31.19 mg/L and 8.93 mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 3.28 mg/L and 2.34 mg/L, respectively.

Description of key information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3 (reference 6.1.5 -1). The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 31.19 mg/L and 8.93 mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 3.28 mg/L and 2.34 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
31.19 mg/L
EC10 or NOEC for freshwater algae:
3.28 mg/L

Additional information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3 (reference 6.1.5 -1). The study was conducted under static conditions. The definitive test was carried out with 5 concentration: 6.25, 12.5, 25, 50 and 100 mg/L. As the test item is not stable in the test medium, the calculated geometric mean measured concnetrations were used for calculation of the effect values. In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values: The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 31.19 mg/L and 8.93 mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 3.28 mg/L and 2.34 mg/L, respectively.