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Toxicity to aquatic algae and cyanobacteria

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Description of key information

Due to borderline effects observed and questionable test conditions taken the instability of the test item into account, the validity of the first algal growth inhibition study with tert-butyl 3,5,5-tris(methylperoxy)hexanoate was called into question (disregarded study). The reliability is categorised as Klimisch 4 (not assignable), as a final evaluation and EC50 value deduction is not possible. Thus, a further study was initiated to clarify the effects especially at the trigger value of 1 mg/L (key study).
In the disregarded study, the effect of the test item tert-Butylperoxy-3,5,5-trimethylhexanoat on the growth of fresh-water green algae Pseudokirchneriella subcapitata was assessed. The ErC50 (72 h) based on growth rate was determined to be 0.33 mg/L.
In the key study, a 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with the test item, the 72-h EC50 based on biomass was determined to be 0.3226 mg/L, based on the growth rate 0.5098 mg/L and based on the yield the 72-h EC50 was 0.2707 mg/L.
The overall NOEC was determined to be 0.125 mg/L (all calculations were based on the geometric mean of measured concentrations). The results obtained in this second study were used for the risk assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.51 mg/L
EC10 or NOEC for freshwater algae:
0.125 mg/L

Additional information

Due to borderline effects observed and questionable test conditions taken the instability of the test item into account, the validity of the first algal growth inhibition study with tert-butyl-3,5,5-tris(methylperoxy)hexanoate was called into question (disregarded study). The reliability is categorised as Klimisch 4 (not assignable), as a final evaluation and EC50 value deduction is not possible. Thus, a further study was initiated to clarify the effects especially at the trigger value of 1 mg/L (key study). The results obtained in this second study were used for the risk assessment.

In the key study, tert-Butylperoxy-3,5,5-trimethylhexanoat was tested in aquatic algae Pseudokirchneriella subcapitata according to EU-Method C.3 (2009) and OECD guideline 201 (2006). At the start of the test the following test item concentrations were measured (TISS=Test Item Stock Solution): TISS/64: 0.192 mg/L; TISS/32: 0.392 mg/L; TISS/16: 0.761 mg/L; TISS/8: 1.55 mg/L; TISS/4: 3.12 mg/L.

At the end of the test the measured concentrations were in the range of 41-46 % of the measured start concentrations. The following tert-Butylperoxy-3,5,5-trimethylhexanoat concentrations were measured at the end of the test: TISS/64: 0.081 mg/L; TISS/32: 0.160 mg/L; TISS/16: 0.350 mg/L; TISS/8: 0.71 mg/L; TISS/4: 1.32 mg/L. The test item concentrations of the test solutions were measured by reverse phase HPLC method with UV detection at the start and at the end of the test. The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.250–2.029 mg test item/L (Bonferroni t-Test (and William’s test), α=0.05).The test concentration of 0.125 mg/L was determined as the 72-hour NOEC.

In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with tert-Butylperoxy-3,5,5-trimethylhexanoat, the 72-h EC50 based on biomass was determined to be 0.3226 mg/L, based on the growth rate 0.5098 mg/L and based on the yield the 72-h EC50 was 0.2707 mg/L.

The overall NOEC was determined to be 0.125 mg/L (all calculations were based on the geometric mean of measured concentrations).

In the disregarded study, tert-Butylperoxy-3,5,5-trimethylhexanoat was tested in aquatic toxicity test to green algae Pseudokirchneriella subcapitata according to EU-Method C.3 and OECD guideline 201 (1984).

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 1x104cells/ml. Test flasks were incubated at 23 ± 0.5°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 following nominal test solution concentrations were used in the definitive test: 0.5, 1, 2.2, 4.5, 9.5, 20 mg/l. Effective concentrations and NOEC have been calculated using the geometric mean concentrations of tert-butyl-3,5,5-trimethylperoxyhexanoate.

Concentrations of tert-Butylperoxy-3,5,5-trimethylhexanoat were measured by gas chromatography and the measured concentration were used for NOEC, LC50 calculations.

In this 72 -h algal growth inhibition test the EC50 (72 h) based on growth rate was determined to be 0.33 mg/L. The 72 -h EC50 based on yield was 0.14 mg/L The overall NOEC was determined to be 0.04 mg/L. All results were based on the nominal concentrations.