tert-butyl peroxyneodecanoate

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:

2012-02-10 to 2012-04-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

adopted 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.08, 0.16, 0.30, 0.63, 1.25, 2.5 mg/L
- Sampling method: Three replicates samples were measured at the start. Two samples were analysed at the end of the study and at the first and second day of testing. The samples were extracted using solid phase extraction and analysed by a reverse phase HPLC method with UV detection on a HyperPrep HS C18 column.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item is a hydrolytically unstable material. In order to minimise hydrolysis before introduction of algae (treatment) the test solutions were prepared using water-miscible solvent (acetone) as follows: For preparation of each test concentration, individual stock solutions were prepared by dissolving an appropriate amount of test item in acetone. At first step, a work solution (concentration: 25.0 g/L) was prepared by diluting an amount of 0.625 g test item in 25 mL acetone and then the stock solutions were prepared by appropriate diluting of this work solution in acetone. The test solutions of the chosen test concentrations were prepared by appropriate diluting of these stock solutions in the dilution water (OECD medium), each immediately before analytical measurement and treatment.
- Chemical name of vehicle: acetone

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): Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.3 – 23.8 °C

pH:

7.96 – 10.13

Nominal and measured concentrations:

The following nominal concentrations of test item were tested: 0.08, 0.16, 0.3, 0.63, 1.25 and 2.50 mg/L. The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were: 0.03, 0.05, 0.11, 0.24, 0.48 and 0.82 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume with 100 mL test medium.
- Initial cells density: 10E04 cells/mL
- No. of vessels per concentration: three replicates per test concentration
- No. of vessels per control: three replicates
- No. of vessels per vehicle control: six replicates
- Additional test solution control: An additional test item solution control group (1.25 mg/L) without addition of algae was included in the experiment and analysed at the same occasions as the test concentrations in order to check the possibility of the adsorption of test item on algal biomass.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- in accordance to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: 7914 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.04 – 0.08 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.38 – 0.60 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 0.01 – 0.02 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 0.08 – 0.11 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The measured concentrations at each day showed no considerable difference compared to the measured values (at same occasions) of the test concentration of 1.25 mg/L (including algae). Therefore, it could be concluded that no adsorption of the test item on algal biomass occurred.

Reported statistics and error estimates:

Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 0 h, 24 h, 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software. Percentage inhibition of growth rate (μ) and yield (y) were calculated using EXCEL for Windows software. The ErCx, and EyCx values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software (based on the measured geometric mean concentrations). For the determination of the LOEC and NOEC, the calculated mean growth rate (μ) and yield (y) at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test using TOXSTAT software.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EC50 value based on growth rate was determined as 0.48 mg/L and based on yield was determined as 0.09 mg/L. The 72-h EC10 value based on growth rate was determined as 0.06 mg/L and based on yield was determined as 0.02 mg/L. The overall NOEC was determined to be 0.03 mg/L.

Executive summary:

The purpose of this study was to determine the effect of the test item TBPND on the growth of a unicellular green algal species Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum). Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. Based on the results of pre-experiments the following six test concentrations in a geometric series (with a separation factor of 2) were tested: 0.08, 0.16, 0.3, 0.63, 1.25 and 2.50 mg/L (nominal). The measured concentrations deviated more than 20 % from the nominal during the experiment therefore the geometric mean of the measured concentrations were calculated in order to determine exposure concentrations. The calculated geometric mean concentrations were the followings: 0.03, 0.05, 0.11, 0.24, 0.48 and 0.82 mg/L. The 72-h EC50 value based on growth rate was determined as 0.48 mg/L and based on yield was determined as 0.09 mg/L. The 72-h EC10 value based on growth rate was determined as 0.06 mg/L and based on yield was determined as 0.02 mg/L. The overall NOEC was determined to be 0.03 mg/L. The results are based on the measured geometric mean concentrations.

Description of key information

The toxicity to algae was assessed according to OECD guideline 201, EU-method C.3 and EPA OPPTS 850.4500. In this 72-hour toxicity test with green algae, the LC50 value was 0.48 mg/L (measured concentration). The no-observed-effect concentration (NOEC) was 0.03 mg/L (measured concentration). The 72-h EC10 value based on growth rate was determined as 0.06 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.48 mg/L

EC10 or NOEC for freshwater algae:

0.06 mg/L

Additional information

The purpose of this study was to determine the effect of the test item TBPND on the growth of a unicellular green algal species Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum). Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. Based on the results of pre-experiments the following six test concentrations in a geometric series (with a separation factor of 2) were tested: 0.08, 0.16, 0.3, 0.63, 1.25 and 2.50 mg/L (nominal) prepared in acetonte. The measured concentrations deviated more than 20 % from the nominal during the experiment therefore the geometric mean of the measured concentrations were calculated in order to determine exposure concentrations. The calculated geometric mean concentrations were the followings: 0.03, 0.05, 0.11, 0.24, 0.48 and 0.82 mg/L. The 72-h EC50 value based on growth rate was determined as 0.48 mg/L and based on yield was determined as 0.09 mg/L. The 72-h EC10 value based on growth rate was determined as 0.06 mg/L and based on yield was determined as 0.02 mg/L. The overall NOEC was determined to be 0.03 mg/L. The results are based on the measured geometric mean concentrations.