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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study according to guideline, with experimental conditions chosen to maximise the concentration of breakdown products of the rapidly hydrolysing test substance.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
EU Method C.1 (Acute Toxicity for Fish)
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(3,5,5-trimethylhexanoyl) peroxide
EC Number:
EC Name:
Bis(3,5,5-trimethylhexanoyl) peroxide
Cas Number:
Molecular formula:
3,5,5-trimethylhexanoyl 3,5,5-trimethylhexaneperoxoate
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Name of test material (as cited in study report): Peroxan NPO (bis(3,5,5-trimehtyhexanoyl) peroxide)
- Substance type: Organic peroxide, rapidly hydrolysing
- Physical state: Liquid
- Analytical purity: See composition of test material
- Composition of test material, percentage of components: 75 % bis(3,5,5-trimethylhexanoyl) peroxide, 25 % stabiliser (isododecane)
- Isomers composition: n.a.
- Purity test date: 05 Feb 2013
- Lot/batch No.: 203033 (experiments 1-3), 301243 (experiments 4-5)
- Expiration date of the lot/batch: October 30, 2012 (batch 203033), July 31, 2013 (batch 301243)
- Stability under test conditions: Rapid hydrolysis (< 1 h) - see IUCLID section 5.1.2
- Storage conditions of test material: -8 to 0 °C

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: All
- Sampling method: To 100 mL of the respective test solution samples, 2 g NaCl and 1000 µL HCl were added and the solution was extracted two times with dichloromethane (4 mL, 3 mL). The organic phase was collected into a 10 mL flask after drying with Na2SO4. The flask was filled up to 10 mL and the solution was measured via GC/FID. Tenfold enrichment was achieved.
- Sample storage conditions before analysis: No

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was mixed with nutrient medium at a nominal loading of 10 g/L, followed by vigorous stirring at 37 ± 2 °C for 7 days. This procedure was chosen to maximise concentrations of the test material, which in itself is known to be poorly soluble, but hydrolyses rapidly, hence generating a solution of several, largely non-identifiable, breakdown products.
- Eluate: N.a.
- Differential loading: N.a.
- Controls: Blank control
- Chemical name of vehicle: N.a. - no vehicle used
- Evidence of undissolved material: Yes; in experiment 1 (limit test) the presence of undissolved test material on test vessel surfaces suggested that mortality was caused by physical interaction with the fish; therefore, the experiment was considered as invalid and had to be repeated; in all repeat trials any undissolved test material was removed by ensuring phase separtion (if necessary) and using exclusively the aqueous (upper) phase of the stock solution.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: Zebra fish (Danio rerio)
- Strain: N.a.
- Source: Umweltbundesamt (UBA), Germany
- Age at study initiation (mean and range, SD): Sexually immature young fish
- Length at study initiation (length definition, mean, range and SD): 2 ± 1 cm
- Feeding during test: No
- Food type: Warmwater fish food and daphnia (in the pre-test holding phase)
- Amount: Totalling to about 1-2 % of body weight/day (in the pre-test holding phase)
- Frequency: Three times a day (in the pre-test holding phase)

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
Post exposure observation period:

Test conditions

Test temperature:
19.1-21.8 °C (experiment 3),
21.7-22.5 °C (experiment 5)
6.6-7.0 (experiment 3),
6.8-8.7 (experiment 5)
Dissolved oxygen:
> 8.6 mg/L (95 % saturation) throughout test (experiment 3)
> 7.7 mg/L (91 % saturation) throughout test (experiment 5)
Nominal and measured concentrations:
Experiment 3 (threshold test):
Nominal: 32 mg/L
Measured: 21 mg/L (geometric mean)
Experiment 5 (full test):
Nominal: 2.5, 4.0, 6.3, 10, 16 mg/L
Measured: 1.46, 2.18, 3.44, 7.28, 11.15 mg/L (geometric mean)
Details on test conditions:
- Test vessel: Glass aquaria
- Material, size, headspace, fill volume: Maximal volume 12.5 L
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): Experiment 3 none, experiment 5 after 48 ± 1 hours
- No. of organisms per vessel: 1 fish/1.8 L
- No. of vessels per concentration (replicates): One per treatment
- No. of vessels per control (replicates): One

- Adjustment of pH: None
- Photoperiod: 12/12 hours using neon tubes

See field "nominal and measured concentrations" under "test conditions" above.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
96 h
Dose descriptor:
Effect conc.:
ca. 7.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
total dissolved test material, calculated from measured concentration of the breakdown product 2,4,4-trimethylpentan-1-ol
Basis for effect:
mortality (fish)
Remarks on result:
other: CI not calculable, due to steep concentration-response curve
96 h
Dose descriptor:
Effect conc.:
ca. 3.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
total dissolved test material, calculated from measured concentration of the breakdown product 2,4,4-trimethylpentan-1-ol
Basis for effect:
mortality (fish)
96 h
Dose descriptor:
Effect conc.:
ca. 11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
total dissolved test material, calculated from measured concentration of the breakdown product 2,4,4-trimethylpentan-1-ol
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: No abnormal behaviour of the surviving fish was observed
Reported statistics and error estimates:
EC50 estimated from dose-response curve obtained by logistic regression.
Confidence interval not calculable due to the steep dose-response curve.

Any other information on results incl. tables

Sublethal observations / clinical signs:

A general observation in both the limit and the full tests was considerable variation in chromatographic peak areas of the breakdown products, despite identical handling for preparation of the stock solutions. This indicates that hydrolytical degradation of PEROXAN NPO follows no uniform and predictable pattern.

Applicant's summary and conclusion

Validity criteria fulfilled:
Control mortality was zero; dissolved oxygen ≥ 77 % throughout the test (criterion: ≥ 60 %); pH varied by ≥ 0.8 units (criterion: ≥ 1 unit).
Executive summary:

In a 96-h acute toxicity study, zebra fish (Danio rerio) were exposed to bis(3,5,5-trimethylhexanoyl) peroxide at nominal concentrations of 0 (control), 2.5, 4.0, 6.3, 10, and 16 mg/L under semi-static conditions (renewal after 48 h). Actual concentrations (geometric means) of total dissolved test material were 0 (control), 1.46, 2.18, 3.44, 7.28, and 11.15 mg/L, determined from measured concentrations of the hydrolysis product 2,4,4-trimethylpentan-1-ol and its relative proportion among degradation products. The 96-h LC50was 7.3 mg/L (total dissolved test material). The LC100 and LC0 values, based on mortality, were 11.15 and 3.44 mg/L, respectively. Sublethal effects were not observed. Based on the results of this study, bis(3,5,5-trimethylhexanoyl) peroxide would not be classified as toxic to aquatic life in accordance with the classification system of the CLP Regulation.

This toxicity study is considered as acceptable and satisfies the guideline requirement for an acute fish toxicity study.

Results synopsis

Zebra fish (Danio rerio), sexually immature young fish, length 2 ± 1 cm

Test type static renewal

LC50: 7.3 mg/L (95 % C.I.: not determinable)

Endpoint effected: Survival/mortality