Bis(3,5,5-trimethylhexanoyl) peroxide

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31/12/2012-22/03/2013

Reliability:

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

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(3,5,5-trimethylhexanoyl) peroxide

EC Number:

223-356-0

EC Name:

Bis(3,5,5-trimethylhexanoyl) peroxide

Cas Number:

3851-87-4

Molecular formula:

C18H34O4

IUPAC Name:

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:

yes

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

Vehicle:

no

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:

TEST ORGANISM
- 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Test conditions

Test temperature:

19.1-21.8 °C (experiment 3),
21.7-22.5 °C (experiment 5)

pH:

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 SYSTEM
- 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

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

TEST CONCENTRATIONS
See field "nominal and measured concentrations" under "test conditions" above.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 7.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

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

Effect concentrations 2

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

ca. 3.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

total dissolved test material, calculated from measured concentration of the breakdown product 2,4,4-trimethylpentan-1-ol

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

ca. 11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

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:

yes

Remarks:

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