tert-butyl peroxypivalate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-12-06 to 2012-12-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

July 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

April 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.07, 0.15, 0.35, 0.8, 1.8, 4.1, 9.5, 21.7 and 50.0 mg/L
- Sampling method: Three replicates samples were taken each day of study from the test solutions, and one sample was taken from the control samples. The samples were concentrated or diluted with acetonitrile depens on their concentration.
Extraction procedure:
The cartridges were conditioned with 3 mL of acetonitrile and 2 mL of acidic water. The volume of samples, 20 mL was brought onto the column. After that, the cartridges were dried. The cartridges were eluted into volumetric flasks with 1 mL acetonitrile and the extracts were measured by HPLC.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Experiment I: A stock solution of nominally 50 mg/L was first prepared by dissolving an amount of 0.05 g test item in 1000 mL OECD medium. The test solutions of the chosen test concentrations were prepared by appropriate diluting of this stock solution. The test solutions were freshly prepared just before the start of the tests.
Experiment II: A stock solution of nominally 50 mg/L was first prepared by dissolving an amount of 0.025 g test item in 500 mL OECD medium. The test solutions of the chosen test concentrations were prepared by appropriate diluting of this stock solution. The test solutions were freshly prepared just before the start of the test.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany

ACCLIMATION
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature was in the range of 22.0 – 23.1 °C measured in the flask and between 22.0 and 24.0 °C measured within the climate chamber (Experiment I and II).

pH:

7.78 to 9.32

Nominal and measured concentrations:

Experiment I: 0.8; 1.8; 4.1; 9.5; 21.7 and 50.0 mg/L (nominal concentration); 0.414, 0.779, 2.383, 5.471, 12.301 and 26.531 mg/L (measured concentration)
Experiment II: 0.07, 0.15, 0.35 and 0.80 mg/L (nominal concentration); 0.053, 0.096, 0.89 and 0.462 mg/L (measured concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume with 100 mL test medium
- Initial cells density: 1E04 algal cells per mL
- Control end cells density: cell density in the control cultures increased by a factor of more than 16 (by a factor of 77.83 at Experiment I and 63.67 at Experiment II) within 72 hours.
- No. of vessels per concentration: three replicates
- No. of vessels per control: six replicates

TEST MEDIUM / WATER PARAMETERS
- in accordance to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously illumination
- Light intensity and quality: 7965 lux

EFFECT PARAMETERS MEASURED:
Determination of Cell Number: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
Morphological Changes of Algal Cells: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.3
- Test concentrations:
Based on the results of the preliminary range-finding test, six test concentrations in a geometric series with a separation factor of 2.3 and one control will be used in the main test (Experiment I). The following nominal concentrations plus a control were tested in the Experiment I: 0.8; 1.8; 4.1; 9.5; 21.7 and 50.0 mg/L. Because significant growth inhibition was observed in the Experiment I at the lowest tested concentration level (0.8 mg/L nominal), the obtained data were not sufficient to determine NOEC. In order to find the concentration without significant toxic effect, further concentrations (at lower levels) were tested in a complementary experiment (Experiment II). The following nominal concentrations plus a control were tested in the Experiment II: 0.07, 0.15, 0.35 and 0.80 mg/L. The measured concentrations deviated more than 20 % from the nominal during the experiments therefore the geometric mean of the measured concentrations were calculated in order to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings:
Experiment I: 0.414, 0.779, 2.383, 5.471, 12.301 and 26.531 mg/L
Experiment II: 0.053, 0.096, 0.89 and 0.462 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.417 mg/L

95% CI:

1.235 - 1.635

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.293 mg/L

95% CI:

0.238 - 0.351

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.422 mg/L

95% CI:

0.377 - 0.474

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.119 mg/L

95% CI:

0.097 - 0.141

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.096 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.189 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: Some swollen cells were observed at 48 h at the concentration of 5.47 mg/L and some thin cells were detected at 72 h at the concentration of 0.78 mg/L (Experiment I). Normal cells were also present at these concentrations. At the complementary experiment (Experiment II) abnormal appearance of algae was not observed.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: The 72-h ErC50 : 0.82 mg/L, (95 % confidence limits: 0.77 – 0.87 mg/L). The 72-h EyC50 : 0.61 mg/L, (95 % confidence limits: 0.58 – 0.65 mg/L)

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EC50 value based on growth rate was determined as 1.417 mg/L and based on yield was determined as 0.422 mg/L. The 72-h EC10 value based on growth rate was determined as 0.293 mg/L and based on yield was determined as 0.119 mg/L. The overall NOEC was determined to be 0.096 mg/L. The results are based on the measured geometric mean concentrations.

Executive summary:

The toxicity to algae was assessed according to OECD guideline 201, EU method C.3 and OPPTS 850.5400. Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. Test concentrations were prepared by appropriate diluting of a stock solution. Based upon the results from the preliminary experiment, nominal concentrations of 0.8; 1.8; 4.1; 9.5; 21.7 and 50.0 mg/L were investigated in the main study. Because significant toxic effect was observed in the lowest tested concentration, influence of the test item was further investigated in a complementary experiment at the following nominal concentrations: 0.07, 0.15, 0.35 and 0.80 mg/L. The measured concentrations deviated more than 20 % from the nominal during the experiments therefore the geometric mean of the measured concentrations were calculated in order to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: Experiment I: 0.414, 0.779, 2.383, 5.471, 12.301 and 26.531 mg/L; Experiment II: 0.053, 0.096, 0.189 and 0.462 mg/L.

For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test using TOXSTAT software. The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate and yield of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.189 – 26.531 mg/L (Bonferroni t-Test, α = 0.05). Accordingly the 72-hour overall NOEC was determined as 0.096 mg/L.

The ErCx and EyCx values of the test item and their confidence limits were calculated using Probit analysis by SPSS PC+ software. The 72-h ErC10 was determined as 0.293 mg/L and the 72-h ErC50 was determined as 1.417 mg/L. The 72-h EyC10 was determined as 0.119 mg/L and the 72-h EyC50 was determined as 0.422 mg/L.

Description of key information

The toxicity of tert-butyl peroxypivalate to algae was assessed according to OECD guideline 201, EU method C.3 and OPPTS 850.5400. The 72-h EC50 value based on growth rate was determined as 1.417 mg/L and based on yield was determined as 0.422 mg/L. The 72-h EC10 value based on growth rate was determined as 0.293 mg/L and based on yield was determined as 0.119 mg/L. The overall NOEC was determined to be 0.096 mg/L. The results are based on the measured geometric mean concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.417 mg/L

EC10 or NOEC for freshwater algae:

0.293 mg/L

Additional information

The toxicity of tert-butyl peroxypivalate to algae was assessed according to OECD guideline 201, EU method C.3 and OPPTS 850.5400. Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. Test concentrations were prepared by appropriate diluting of a stock solution. Based upon the results from the preliminary experiment, nominal concentrations of 0.8; 1.8; 4.1; 9.5; 21.7 and 50.0 mg/L were investigated in the main study. Because significant toxic effect was observed in the lowest tested concentration, influence of the test item was further investigated in a complementary experiment at the following nominal concentrations: 0.07, 0.15, 0.35 and 0.80 mg/L. The measured concentrations deviated more than 20 % from the nominal during the experiments therefore the geometric mean of the measured concentrations were calculated in order to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: Experiment I: 0.414, 0.779, 2.383, 5.471, 12.301 and 26.531 mg/L;

Experiment II: 0.053, 0.096, 0.189 and 0.462 mg/L. For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test using TOXSTAT software. The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate and yield of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.189 – 26.531 mg/L (Bonferroni t-Test, α = 0.05). Accordingly the 72-hour overall NOEC was determined as 0.096 mg/L. The ErCx and EyCx values of the test item and their confidence limits were calculated using Probit analysis by SPSS PC+ software. The 72-h ErC10 was determined as 0.293 mg/L and the 72-h ErC50 was determined as 1.417 mg/L. The 72-h EyC10 was determined as 0.119 mg/L and the 72-h EyC50 was determined as 0.422 mg/L.