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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:
19 May 2011 - 09 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant. Guideline study. Acceptable without restrictions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Duplicate samples were taken from the test media of all test concentrations and the control at the start of the test (without algae) and at the end of the test (containing algae).
- Sampling method: Not stated. For sampling at the end of the test from the filtrate 1:10 to 1:2.2 and from the undiluted filtrate, the test medium of the treatment replicates was pooled. For sampling from the filtrate 1:22 and from the control at the end of the test, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates was too small for analytical requirements.
- Sample storage conditions before analysis: Samples were deep-frozen immediately after sampling and stored at about -20˚C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test substance in test water, a dispersion with the loading rate of 100 mg/L was prepared by dispersing 100.07 mg of the test item in 1000 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 96 hours at room temperature, to dissolve a maximum amount of the test substance in the test water. After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (pore size 0.45 μm). The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. The test media were prepared just before the start of the test (= start of exposure).
- Controls: Reconstituted test water (OECD medium). Potassium dichromate is tested as a positive control twice a year.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported after filtration.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): The algae were cultivated at the test laboratory under standardized conditions according to the test guidelines.
- Age of inoculum (at test initiation): 3 day old culture in exponential growth phase


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
24 mg/L as CaCO3
Test temperature:
21˚C
pH:
Test start range = 8.3 - 8.4
End test range = 8.6 - 8.9
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
The following concentrations of the test substance were tested: undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2.2, 1:4.6, 1:10 and 1:22 of the undiluted filtrate. Additionally, a control (test water without test item) was tested in parallel. Measured concentrations at day 0 were: 0, 0.113, 0.183, 0.552, 1.3 and 2.91 mg test substance/L respectively. Measured concentrations at day 4 were: 0, 0.133, 0.276, 0.566, 0.991 and 2.02 mg test substance/L respectively. At the end of the test measured concentration were 69 to 151 % of the initial measured concentrations. The results are based on the mean measured test item concentration at the start and end of the test. These were 0, 0.12, 0.23, 0.56, 1.1 and 2.4 mg/L test item respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flask
- Type (delete if not applicable): closed, covered with a glass dish
- Material, size, headspace, fill volume: 50 mL flask containing 15 mL of test medium.
- Initial cells density: 5000 cells/mL
- Aeration: None, but solution continuously stirred
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (OECD medium)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature measured daily, pH at start and end of test

OTHER TEST CONDITIONS

- Photoperiod: Constant illumination
- Light intensity and quality: Approximate mean 4700 Lux (range: 4520 - 4980 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Fluorescence measurement. The measurements were performed at least in duplicate.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: Yes
- Test concentrations: Measured mean concentrations: 0.12, 0.23, 0.56, 1.1 and 2.4 mg test substance/L

Reference substance (positive control):
yes
Remarks:
Potassium dichromate (tested twice per year)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 1.5-1.9 mg test substance/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 2.8 - 3.2 mg test substance/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CL: 1.7 - 2.0 mg test substance/L.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 1.9 - 2.1 mg test substance/L.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: not determined. EC50 value above solubility limit of test substance
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 2.2 - 2.3 mg test substance/L.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities were observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none observed.
- Effect concentrations exceeding solubility of substance in test medium: Yes, 96-hour EC50 based on growth rate.

Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 72 hour growth rate (April 2011): 1.3 mg potassium dichromate/L
- Other: Results from Test Laboratory study D25712, range of the 72-hour EC50 for growth rate from 2000 to 2011: 0.71-1.7 mg/L
Reported statistics and error estimates:
72 and 96 hour EC50 values were calculated by probit analysis. The biomass, average growth rate and yield at the test concentrations were compared to the control values by Williams t-test..

The results are based on the mean measured test item concentration calculated as the geometric means of the concentrations measured at the start and end of the test.
  Mean measured concentration of the test item
Treatment/Dilution mg test substance /L
Dilution 1:22 0.12
Dilution 1:10 0.23
Dilution 1:4.6 0.56
Dilution 1:2.2 1.1
Undiluted filtrate (loading rate 100/mg test substance/L) 2.4

FULL RESULTS
  After 72 hours After 96 hours
Parameter Biomass (AUC) Growth rate Yield Biomass (AUC) Growth rate Yield
EC50 [mg/L]  1.7   2.9 1.8 2 > 2.4 2.2
95% CL 1.5-1.9 2.8 - 3.2 1.7 - 2.0 1.9 - 2.1 n.d. 2.2 - 2.3
EC20 [mg/L] 1.2 2.0 1.4 1.5 >2.4 1.8
95% CL 0.96 - 1.4 1.9 - 2.1 1.2 - 1.5 1.4 - 1.7 n.d. 1.8 - 1.9
EC10 [mg/L] 0.98 1.6 1.2 1.3 2.1 1.7
95% CL 0.75 - 1.2  1.4 - 1.8 0.98 - 1.3 1.1 - 1.5 2.1 - 2.2  1.6 - 1.7
NOEC [mg/L] 0.12 1.1 1.1 1.1 1.1 1.1
LOEC[mg/L] 0.23 2.4 2.4 2.4 2.4 2.4

n.d.: could not be determined

Validity criteria fulfilled:
yes
Conclusions:
In a guideline, 96-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the test substance exhibited 96-hour EC50 values of 2.0, 2.2 and > 2.4 mg test substance/L based on biomass (AUC), yield and growth rate, respectively. The 96-hour NOEC was 1.1 mg test substance/L for biomass, growth rate and yield. The 96-hour LOEC was 2.4 mg test substance/L for biomass, growth rate and yield. The results are based on the mean measured test item concentrations at the start and end of the test.
Executive summary:

The toxicity of the test substance to the green algae Pseudokirchneriella subcapitata was tested in an GLP compliant, guideline study. The algae were exposed for up to 96 hours to 5 concentrations of the test substance. As the test substance has low solubility in the test media a dispersion was formed by mixing the test substance in the test media for a prolonged period of time. After filtration this stock solution was used undiluted as the maximum exposure concentration and as a series of dilutions to form the other test concentrations. Analytical monitoring of the exposure concentrations was undertaken and the results are expressed as the mean measured concentration from the start and end of the exposure period. The 96-hour EC50 values are 2.0, 2.2 and > 2.4 mg test substance/L based on biomass (AUC), yield and growth rate, respectively. The 96-hour NOEC was 1.1 mg test substance/L for biomass, growth rate and yield. and the 96-hour LOEC was 2.4 mg test substance/L for biomass, growth rate and yield.

Description of key information

96-h ErC50 > 2.4 mg/l, 96-h NOErC = 1.1 mg/l, OECD 201, static, Pseudokirchneriella subcapitata, Liedtke 2011

In a guideline, 96-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the test substance exhibited 96-hour EC50 values of 2.0, 2.2 and > 2.4 mg test substance/L based on biomass (AUC), yield and growth rate, respectively. The 96-hour NOEC was 1.1 mg test substance/L for biomass, growth rate and yield. The 96-hour LOEC was 2.4 mg test substance/L for biomass, growth rate and yield. The results are based on the mean measured test item concentrations at the start and end of the test.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
1.1 mg/L

Additional information

The toxicity of the test substance to the green algae Pseudokirchneriella subcapitata was tested in a GLP compliant, guideline study. The algae were exposed for up to 96 hours to 5 concentrations of the test substance, namely 0.12, 0.23, 0.56, 1.1 and 2.4mg/l alongside a water control. The EC50 growth rate was found to be above the highest dose tested, which was restricted by the test item solubility in the test medium. The study is considered reliable and relevant for risk assessment. As no 96h EC50 could be determined for the growth rate, the result was not considered adequate for classification and labeling purposes.