Di-tert-butyl sec-butylidene diperoxide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE
Batch no.: 41335 512306-109

Analytical monitoring:

yes

Details on sampling:

samples were taken at the start and the end of the test of the test bottles and of parallel vessels without inoculum.

Vehicle:

no

Details on test solutions:

2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE is not soluble in water dilution. WAF are thus prepared at different concentrations, 3 days before the
beginning of the test (time 0) for the preliminary and definitive test.
MILLIPORE filter (0.45}lm) for the preliminary and definitive test of allowing to obtain a limpid solution.
Since the test was performed using hermetically closed flasks, NaHC03 0.3 gil and HEPES buffer 6 mM (4-(2-Hydroxyethyl)-1 piperazineethanesulfonic acid, sodium salt)
were added to dilution water as described in ISOIFDIS 14442: Water Quality - Guidance for algal growth inhibition tests with poorly soluble organic and
inorganic solids, volatile compounds, heavy metals and waste water.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

unicellular green fresh-water algae Pseudokirchneriella subcapitata, CCAP 278/4 stock (previously named Raphidocelis subcapitata and Selenastrum capricornutum) obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK).

The conditions used for culturing algae are described in annex 1 of the method C3. Two flasks, each containing approximately 100 ml of axenic stock culture of algae are incubated at 23 ± 1 °C under lighting (photoperiod: 16 hours of illumination, 8 hours of darkness), and continuous aeration (air filtered at 0.22 µm).
These stock cultures are renewed every week, using two new cultures.
The quality of the stock culture was verified for the absence of microorganisms and deformed cells under microscopic observation before use.
3 to 5 days before the beginning of the study two pre-cultures were prepared by inoculating each stock suspension of algae (5 ml) into sterile dilution water (500 ml). The pre-cultures were incubated under the same conditions as those used for the stock cultures. Only one of the two pre-cultures was used to inoculate the test flasks; the second one was to be used only if the first one was damaged.
At the beginning of the test, the cell concentration of the pre-culture was determined. The result was used to calculate the volume needed and introduced into each test flask in order to get an initial cell concentration equal to 10^4 cells/ml.
The cell density of the pre-culture was about 3.52 10^6 cells/ml for the preliminary test and about 4.453 10^6 cells/ml for the definitive test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23.5 +/- 1 degree C

pH:

7.6-8.3

Dissolved oxygen:

8..5-10.4 mg O2/l

Nominal and measured concentrations:

100 mg/L loading
geometric mean measured: 6.2 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 120 ml glass bottles
- Type (delete if not applicable): closed, Flasks were stoppered with PTFE bungs and sealed with aluminium caps
- Initial cells density: 10E+04 cells/ml
- Control end cells density: 9.80E+05 cells/ml

GROWTH MEDIUM
- Standard medium used: yes, acvcording to 92/69/EEC C.3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Determination of algae concentration was carried out using an optical microscope equipped with an haemacytometer (Malassez cell counter)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N.A.
- Justification for using less concentrations than requested by guideline:limit test
- Range finding study
- Test concentrations: 1, 5, 10, 50, 100 mg/l
- Results used to determine the conditions for the definitive study: no significant toxicity

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading

Basis for effect:

growth rate

Results with reference substance (positive control):

the sensitivity of the test system and the methodology were evaluated every two months by performing an algal growth inhibition test on sodium dichromate. The most recent values of ErC50 and EbC50 obtained on January 29 2004 were respectively 1.20 mg/l and 0.71 mg/I. For information, ISO 8692 reports the following results for an inter-laboratory exercise on sodium dichromate: ErC50: 0.60 to 1.03 mg/l ; EbC50: 0.20 to 0.75 mg/I.

Reported statistics and error estimates:

The NOEC (No Observed Effect Concentration), is determined by statistic multisample analysis using the Dunnett method (US EPA vs 1.5 software).

Validity criteria fulfilled:

yes

Conclusions:

No effects were found up to a loading of 100 mg/L. This shows that the test substance does not give an effect up to its maximum water solubility.
The study is valid without restrictions, therefore this endpoint can be used for C&L and Risk Assessment.

Executive summary:

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata (previously known as Raphidocelis subcapitata and Selenastrum capricornutum) exposed to the test item 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE for a duration of 72 hours was performed following the method C3 described in Directive 92/69/EEC of the European Commission and in the Guideline 201 of the OECD. Algae were exposed to a range of concentrations of 2,2- DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours. The study was performed using 120 ml glass bottles stoppered with PTFE bungs and sealed with aluminium caps, containing 50 ml of test solution inoculated with an algal suspension so that the initial cell concentration was equal to Ix104 cells/ml. Tests flasks were incubated at 23 ± 1 °c continuously shaken and constantly illuminated. The cell density was measured daily. Analytical chemistry and physico-chemical measurements were carried out at the beginning and the end of the test. The concentration and loading of test item causing a 50 % reduction in biomass (EbC50 and EbL50) or in growth rate (ErC50 and ErL50) were determined. EL50-72h and NOEL derived from loading rate of the WAF (test item). EC50-72h and NOEC corresponding to the peroxide component are also reported, for information. The results were as follows:

Effective concentrations and loadings, NOEC and NOEL (mg/l):

EbL50-72h > 100 (test item)

NOELb = 100 (test item) - EbC50-72h > 3.1 (peroxide component)

NOECb = 3.1 (peroxide component)

ErL50-72h > 100 (test item)

NOELr = 100 (test item)

ErC50-72h > 3.1 (peroxide component)

NOECr = 3.1 (peroxide component)

At the limit of solubility of item: 6.21 mg/l, these average percentage of inhibition are 12.6 % for the cell growth (lAi%) and 3.3 % for the growth rate (lµ.i%).

Effective concentrations and NOEC shown in the previous table have been calculated using geometric average value of initial and final concentrations of 2,2- DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE. Concentrations of 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE were measured by liquid chromatography coupled with mass spectrometry according to the method described in annex to this report. Initial and final concentrations were not equivalent to nominal concentrations. The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, colourless and not cloudy over the period of the test. No precipitation was observed at the end of the test. Microscopic observation confirmed that the algae appeared normal at the end of the test: the normal form of the unicellular algae is a crescent shaped cell with an average length of 5-10 µm. During the test, the control pH varied by 0.42 units. The validity criterion of the study related to the growth of algae was respected : the increase in cell density (R), measured in the control solution between the end and the beginning of the test, was greater than a factor of 16 (R = 98). The validity criterion specific to C3 92/69EEC method and related to the test item stability during the test was not respected: the final concentrations of 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE were not maintained within the designated limit of 80 % of the initial concentrations. The study was performed according to the OECD principles of Good Laboratory Practice (GLP).

Description of key information

One valid study performed with the freshwater alga P. subcapitata is available. The study was performed as a limit test at 100 mg/L loading according to OECD 201 under GLP.

No significant effects were found, therefore ErL10 and ErL50 are both greater than 100 mg/L loading rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L