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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:
2013-12-09 to 2014-01-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material: Not applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis.
- Sampling method: Not reported
- Sample storage conditions before analysis: All 0-Hour samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Samples were taken at 0, 23, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): CCAP of SAMS Research Services, Scottish Marine Institute, Oban, Argyll, UK
- Age of inoculum (at test initiation): Not reported
- Method of cultivation: master culture were maintained in the laboratory under constant aeration and illumination at 21°C

ACCLIMATION
- Acclimation period: Not reported
- Culturing media and conditions (same as test or not): Same medium was used for stock culture as well as for range-finding test and main test
- Any deformed or abnormal cells observed: None
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not reported
Test temperature:
Temperature was maintained at 24 +/- 1°C throughout the test
pH:
The pH value of the control cultures (see Table 2 of report) was observed to increase from pH 7.5 at 0 hours to pH 8.0 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
Dissolved oxygen:
Not reported
Salinity:
Not reported
Nominal and measured concentrations:
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 92% to 112% of nominal and so the results are based on nominal test concentrations only.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): plugged with PU foam bungs to reduce evaporation
- Material, size, headspace, fill volume: glass, 100 mL fill volume
- Aeration: plugged with PU foam bungs to reduce evaporation
- Initial cells density: initial cell density 1000 (10E3) per mL
- No. of vessels per concentration (replicates): two
- No. of vessels per control (replicates): two

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH:
- Photoperiod: continious illumination (ca. 7000 lux)
- Light intensity and quality: warm white lightning (380 to 730 nm), constantly shaken at approximately 150 rpm for 72 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at 0, 23, 48 and 72 hours

TEST CONCENTRATIONS
- Range finding study: yes,
- Test concentrations: 0.1, 1.0, 10, 100 mg/L (range-finding test), and 10, 20, 40, 80 160 mg/L (main definitive test)
- Results used to determine the conditions for the definitive study: yes (highest test concentration was shifted)
Reference substance (positive control):
yes
Remarks:
potassium dichromate served as positive control
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
80 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
110 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: Calculation of EC50 value not possible, as no concentration tested resulted in greater than 50 % inhibition of growth rate.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: described as: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
80 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: described as: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
58 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: described as : yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
130 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: described as: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: 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 medium: Analysis of the control at 72 hours showed a measured concentration of 0.0016 mg/L was obtained, whilst analysis of the duplicate sample taken showed a measured concentration of 0.0019 mg/L was obtained. Given that the measured concentrations at 72 hours were just above the LOQ, and that no test item was detected at 0 hours it was considered that post study contamination had occurred which had no impact on the study itself.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 92% to 112% of nominal and so the results are based on nominal test concentrations only.

Validity criteria fulfilled:
yes
Conclusions:
Inhibition of Growth rate (72 h): NOEC = 40 mg/L, LOEC = 80 mg/L, ErC10 = 110 mg/L, ErC50 >160 mg/L
Inhibition of Yield (72 h): NOEC = 40 mg/L, LOEC = 80 mg/L, EyC10 = 58 mg/L, EyC50 = 130 mg/L
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 10, 20, 40, 80 and 160 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 92% to 112% of nominal and so the results are based on nominal test concentrations only.

Results:

Inhibition of Growth rate (72 h): NOEC = 40 mg/L, LOEC = 80 mg/L, ErC10 = 110 mg/L, ErC50 >160 mg/L

Inhibition of Yield (72 h): NOEC = 40 mg/L, LOEC = 80 mg/L, EyC10 = 58 mg/L, EyC50 = 130 mg/L

Description of key information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 10, 20, 40, 80 and 160 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 92 % to 112 % of nominal and so the results are based on nominal test concentrations only.

Results:

Inhibition of Growth rate (72 h): NOEC = 40 mg/L, LOEC = 80 mg/L, ErC10 = 110 mg/L, ErC50 >160 mg/L

Inhibition of Yield (72 h): NOEC = 40 mg/L, LOEC = 80 mg/L, EyC10 = 58 mg/L, EyC50 = 130 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
130 mg/L
EC10 or NOEC for freshwater algae:
40 mg/L

Additional information