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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:
28-jul-2009 to 20-aug-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
The batch of 2 -sec-butylphenol tested was a clear colourless liquid with a purity of 99.6%.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and the control
- Sampling method: volume 2 ml, at t=0 h, t=24 and t=72 h
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation of test solutions started with the highest concentration of 100 mg/l. No special treatment other than a short period of magnetic stirring (15 minutes) was applied to completely dissolve this concentration in test medium. The lower test concentrations were prepared by subsequent dilution. 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 10E4 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): 4 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 10E4 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 in a climate room at a temperature of 21-24°C.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
21.9 and 22.2°C
pH:
At t=0 h: 8.2
At t=72 h: 8.0 - 8.1
Nominal and measured concentrations:
Combined limit/ Range finding:
Nominal concentrations: 0.1, 1, 10 and 100 mg/l
Measured concentrations, 0.10 and 10 mg/l:
At t=0 h: 96 and 93 % of nominal
At t=72 h: 71 and 69 % of initial
Final test
Nominal concentrations: 1, 3, 9, 27 and 81 mg/l
Measured concentrations:
At t=0 h: 90 to 96 % of nominal
At t=72 h: 64 to 88 % of initial
TWA concentrations: 0.82, 2.5, 7.1, 22 and 64 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all glass
- Type: open
- Material, size, headspace, fill volume: 150 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 1620000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1 replicate of each test concentration without algae.
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 75 to 96 µE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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 Varian Cary 50 single beam spectrophotometer with cuvettes (pathlength =10 mm). Algal medium was used as blank and the extra replicates without algae as background for the treated solutions.]
- Other: 72 h NOErC, 72 h NOEbC, 72 h ErC50, 72 h EbC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Test concentrations: 1, 3, 9, 27 and 81 mg/l
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.82 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
10 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval ranging from 8.8 to 12 mg/l
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50:
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.8 mg/l with a 95 % confidence interval ranging from 1.4 to 2.5 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.6 mg/l with a 95 % confidence interval ranging from 0.5 to 0.9 mg/l. Historical ranges are not yet available.
Validity criteria fulfilled:
yes
Conclusions:
2-sec-butylphenol reduced growth rate and inhibited the yield of this fresh water algae species significantly at a TWA concentration of 2.5 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) based on TWA concentrations was 10 mg/l with a 95% confidence interval ranging from 8.8 to 12 mg/l.

The EC50 for yield inhibition (EYC50: 0-72h) based on TWA concentrations was 3.4 mg/l with a 95% confidence interval ranging from 1.4 to 8.2 mg/l.

The NOEC for growth rate reduction and yield inhibition based on TWA concentrations was 0.82 mg/l.
Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with 2 -sec-butylphenol. The study procedures were based on the OECD guideline No. 201, 2006. In addition, the procedures were designed to meet the test methods of the EEC directive 92/69, Part C.3, 1992 and the ISO International Standard 8692, 2004.

The batch of 2 -sec-butylphenol tested was a clear colourless liquid with a purity of 99.6% and completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with the highest concentration of 100 mg/l. No special treatment other than a short period of magnetic stirring (15 min) was applied to completely dissolve this concentration in test medium. The lower test concentrations were prepared by subsequent dilution. The final test solutions were all clear and colourless.

A final test was performed based on the results of a preceding combined limit/range-finding test. Exponentially growing algal cultures were exposed to a control, and 1.0, 3.0, 9.0, 27 and 81 mg/l. The total test period was 72 hours and the innitial algal cell density was 104 cells/ml. samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours and the end of the test period.

At the start of the test, the actual test concentrations were in agreement with the nominal concentrations (90 -96 relative to nominal). Measured concentrations at the end of the 72 -hour period decreased by more than 20% in samples taken from the lowest test concentration with algae and the highest concentration without algae. based on these results, the Time Weight Average (TWA) exposure concentrations were calculated. The range tested based on TWA concentrations was 0.82, 2.5, 7.1, 22 and 64 mg/l.

The study met the acceptability criteria perscribed by the protocol and was considered valid.

2 -sec-butylphenol reduced growth rate and inhibited the yield of this fresh water algae species significantly at a TWA concentration of 2.5 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0 -72 h) based on TWA concentrations was 10 mg/l with a 95% confidence interval ranging from 8.8 to 12 mg/l..

The EC50 for yield inhibition (EYC50: 0 -72 h) based on TWA concentrations was 3.4 mg/l with a 95% confidence interval ranging from 1.4 to 8.2 mg/l.

The NOEC for growth rate reduction and yield inhibition based on TWA concentrations was 0.82 mg/l.

Description of key information

2 -sec-butylphenol reduced growth rate and inhibited the yield of this fresh water algae species significantly at a TWA concentration of 2.5 mg/l and higher.
The EC50 for growth rate reduction (ERC50: 0 -72 h) based on TWA concentrations was 10 mg/l with a 95% confidence interval ranging from 8.8 to 12 mg/l..
The EC50 for yield inhibition (EYC50: 0 -72 h) based on TWA concentrations was 3.4 mg/l with a 95% confidence interval ranging from 1.4 to 8.2 mg/l.
The NOEC for growth rate reduction and yield inhibition based on TWA concentrations was 0.82 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:
10 mg/L

Additional information

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with 2 -sec-butylphenol. The study procedures were based on the OECD guideline No. 201, 2006. In addition, the procedures were designed to meet the test methods of the EEC directive 92/69, Part C.3, 1992 and the ISO International Standard 8692, 2004.

The batch of 2 -sec-butylphenol tested was a clear colourless liquid with a purity of 99.6% and completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with the highest concentration of 100 mg/l. No special treatment other than a short period of magnetic stirring (15 min) was applied to completely dissolve this concentration in test medium. The lower test concentrations were prepared by subsequent dilution. The final test solutions were all clear and colourless.

A final test was performed based on the results of a preceding combined limit/range-finding test. Exponentially growing algal cultures were exposed to a control, and 1.0, 3.0, 9.0, 27 and 81 mg/l. The total test period was 72 hours and the innitial algal cell density was 104cells/ml. samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours and the end of the test period.

At the start of the test, the actual test concentrations were in agreement with the nominal concentrations (90 -96 relative to nominal). Measured concentrations at the end of the 72 -hour period decreased by more than 20% in samples taken from the lowest test concentration with algae and the highest concentration without algae. based on these results, the Time Weight Average (TWA) exposure concentrations were calculated. The range tested based on TWA concentrations was 0.82, 2.5, 7.1, 22 and 64 mg/l.

The study met the acceptability criteria perscribed by the protocol and was considered valid.