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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-08 to 2018-02-23 (experimental phase: 2017-08-12 to 2017-12-12)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
17 July 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 190917
- Expiration date of the lot/batch: 19 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep only in the original container at < -18 °C, control temperature: -10 °C, emergency temperature: 0 °C. Self-accelerating decomposition temperature (SADT): + 10 °C
- Stability under test conditions: Not stable


Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.0 (Sample code: NC), 5 (Sample code: A)
- Sampling method: The samples were filled into 2 mL microcentrifuge tubes. To each vial, 1 mL acetonitrile was added and samples were stored in the freezer (≤ -18 °C).
The sampling was conducted according to the following specification:
- At the start of the test (0 h), treatment NC and A were sampled (each group: 3 samples of 1 mL).
- After 24 h, 48 h and 72 h exposure:
- Before renewal of the test solutions, treatment NC and A were sampled (each group: 3 samples of 1 mL).
- After renewal of the test solutions, treatment NC and A were sampled (each group: 3 samples of 1 mL).
- At the end of the test (96 h), treatment NC and A were sampled (each group: 3 samples of 1 mL).

Of each sampled treatment, one of the samples from 0 h, 24 h, 48 h, 72 h (before and after renewal) and 96 h was sent to the analytical laboratory at the test site biochemA GmbH for chemical analysis. The remaining samples were stored as retain samples in the freezer (≤ -18 °C) until finalisation of the study.
Chemical analysis of the test solutions was performed to determine exposure concentration of the test item and the decomposition product neodecanoic acid (C10H20O2; CAS no.: 26896-20-8) during exposure, respectively.
Vehicle:
no
Details on test solutions:
The stock solutions were prepared similar to the procedures applied for the ecotoxicity testing in daphnia and algae to ensure comparability of test results. For the daphnia and algae testing, Water Accommodated Fractions (WAF) were prepared by stirring for 66 h and subsequent filtration (Millipore membranes). For testing of fish, the stock solutions were prepared as WAF by adding 41.6 μL test item (corresponds to 40 mg; density 0.962 g/cm3) to 8 L test medium and stirring for 48 h using a magnetic stirrer at 21.1 – 23.0 °C. A reduced stirring time was applied compared to daphnia and algae testing as pretests demonstrated stable test solutions after 48 h.
The WAF was filtered through a fibre glass filter with a retaining range till 0.6 μm (MN 85/70 BF, Macherey-Nagel GmbH, Düren, Germany). The filter was prepared by rinsing with ca. 100 mL purified water and preconditioning with ca. 100 mL WAF (which was discarded), to reduce adsorption of the test item.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The test organism Danio rerio originates from the in-house breeding of Hydrotox GmbH. The zebrafish are cultured at 26 ± 2 °C with 14 h light : 10 h dark and aeration with air pumps. They are kept in glass tanks, equipped with external filters, at ≤ 1 fish/L. On workdays, they are fed with commercial fish food (TetraMin flake food, Tetra GmbH, Melle, Germany) and two times per week with freshly hatched Artemia salina larvae. At least every two weeks, ≥ ⅓ of the tank volume is replaced with fresh charcoal filtered tap water. The used test animals had a total length of ca. 2.5 cm and were ca. 11 months old.

Seven days before the start of the test, the fish were kept under test conditions in test medium at
23.2 – 24.3 °C. They were fed every working day with commercial fish food (TetraMin) until 24 h
before test start, when feeding was stopped.

The test was performed with ISO 7346 medium according to EN ISO 7346 (1997), prepared as following:
ISO 1 Calcium chloride dihydrate p.a. CaCl2 × 2 H2O: 29.4 g/L
ISO 2 Magnesium sulfate heptahydrate p.a. MgSO 4 × 7 H2O: 12.33 g/L
ISO 3 Sodium hydrogene carbonate p.a. NaHCO3: 6.3 g/L
ISO 4 Potassium chloride p.a. KCl: 0.55 g/L
After addition of 500 mL ISO 1, ISO 2, ISO 3 and ISO 4, the medium was filled up to 50 L, continuously aerated and the pH (7.7) as well as oxygen saturation (99 %) measured.

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
Renewal of the test solutions after 24 h, 48 h and 72 h with a substance-specific accompanying chemical analysis. The vitality of the fish was assessed after 24 h, 48 h, 72 h and 96 h exposure.
Test temperature:
22.2 – 22.7 °C in the control
22.6 – 23.4 °C in the test item treatment (required 21 – 25 °C).
pH:
7.8
Dissolved oxygen:
8.1 – 8.4 mg/L
Nominal and measured concentrations:
5 mg test item/L (nominal) - corresponding to 3.78 mg/L cumylperoxyneodecanoate
1.15 mg neodacanoic acid/L (measured)
In the present study, the Threshold Approach for Acute Fish Toxicity according to OECD Series on Testing and Assessment No. 126 was applied.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass tanks 8 L
Seven days before the start of the test, the fish were kept under test conditions in test medium at 23.2 — 24.3 °C. They were fed every working day with commercial fish food (TetraMin) until 24 h before test start, when feeding was stopped. The test vessels were set up in a climatised room with 14 h light : 10 h dark. A semi-static test design was applied with seven fish in the control as well as in the test item treatment. After 24 h, 48 h and 72 h exposure, the test organisms were transferred to a new set of test vessels with new test solutions by means of a net (renewal of test solutions). Mortality as well as appearance and behaviour of the animals were assessed after 24 h, 48 h, 72 h and 96 h exposure.
The pH, conductivity and O2-concentrations were measured daily. The temperature was measured continuously.
1 replicate for the control (7 fish)
1 replicate for the test item (7 fish)

TEST MEDIUM / WATER PARAMETERS
The test was performed with ISO 7346 medium according to EN ISO 7346 (1997)

Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 1.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: decomposition product neodecanoic acid
Basis for effect:
mortality
Remarks on result:
other: The test item is not stable in aqueous solution at test temperature.
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: decomposition product neodecanoic acid
Basis for effect:
mortality
Remarks on result:
other: The test item is not stable in aqueous solution at test temperature.
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: The test item is not stable in aqueous solution at test temperature.
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: The test item is not stable in aqueous solution at test temperature.
Details on results:
ln the control, the test item and the decomposition product neodecanoic acid were not detected.
As the test item is not stable in aqueous solution at test temperature and was not detectable or below the limit of quantification at seven of the eight sampling times, the results are given in relation to nominal test item concentrations as well as measured concentrations (geometric mean concentrations) of the decomposition product neodecanoic acid.
No obvious effect on appearance and behaviour of the fish in the test item treatment was observable. All test organisms in the control as well as in the test item treatment appeared healthy and lively throughout the exposure.The initial measured test item concentrations in the test item treatment were 0 - 25.6 % (new test solutions) of the nominal concentration of 5 mg/L. At the renewal of the test solutions and the end of the exposure period, the measured test item concentrations were 0 % (old test solutions) of the nominal concentration. The initial measured decomposition product neodecanoic acid concentrations in the test item treatment were 0.748 — 1.85 mg/L (new test solutions). At the renewal of the test solutions and the end of the exposure period, the measured test item concentrations were 0.853 — 1.28 mg/L (new test solutions).
As the results are not maintained within ± 20 per cent of the nominal or measured initial concentration throughout the test, the results are given in relation to nominal test item concentrations as well as measured concentrations (geometric mean concentrations) of the decomposition product neodecanoic acid.
No obvious effect on appearance and behaviour of the fish in the test item treatment was observable. All test organisms in the control as well as in the test item treatment appeared healthy and lively throughout the exposure.

The purity of the test item is 75.5 %, which corresponds to a nominal concentration of 3.78 mg/L cumylperoxyneodecanoate in the test vessel with a nominal concentration of 5 mg/L test item. As the mass ratio of cumylperoxyneodecanoate (306.44 g/mol) to neodecanoic acid (172.26 g/mol) yield 56.21 %, the expected nominal concentration of neodecanoic acid was 2.21 mg/L in the test vessel, assuming a complete decomposition of the applied cumylperoxyneodecanoate. The measured concentration of neodecanoic acid was 1.15 mg/L (geometric mean) resulting in a recovery of 54.25 % in relation to the expected nominal concentration.

Validity criteria fulfilled:
yes
Conclusions:
No effect on fish was observed in the present limit test:
LOEC: > 1.15 mg/L; NOEC: ≥ 1.15 mg/L (concentrations are calculated on basis of the measured decomposition product neodecanoic acid).
Executive summary:

In a 96-h acute toxicity study according to OECD 203 (17 July 1992), zebra fish (Danio rerio) were exposed to cumylperoxyneodecanoate (75. 5 % in isododecane) at nominal concentrations of 0.0 (control) and 5 mg/L (nominal) under semi-static conditions. The test item is a mixture of 2 constituents with different solubilities and physico­chemical properties. Test solutions were prepared according to the WAF methodology (water accommodated fraction). Mortality/immobilization was observed daily. 

Concentrations of the test item were measured by LC-MS/MS. The parent substance was quantified via its degradation product neodecanoic acid. The final concentrations of the test item were not all maintained within the designated Iimit of 80 % of initial. Thus, effective concentrations were calculated using geometric means of final and initial measured concentrations of the test items degradation product.

The NOEC value, based on mortality and sublethal effects, was ≥ 1.15 mg/L, respectively. Sublethal effects were not observed.

This toxicity study is classified as acceptable and satisfies the guideline requirement for the zebra fish acute toxicity study.

 

Results Synopsis

 

Test organism size/age: Danio rerio (ca. 2.5 cm, ca. 11-month-old)

Lowest/No Observed Effect Concentration

Nominal test item concentration [mg/L]

Measured concentration of the decomposition product neodecanoic acid [mg/L]

LOEC

> 5

> 1.15

NOEC

≥ 5

 ≥ 1.15

Description of key information

In a 96-h acute toxicity study, zebra fish (Danio rerio) were exposed to 1-methyl-1-phenylethyl peroxyneodecanoate (75. 5 % in isododecane) at nominal concentrations of 0.0 (control) and 5 mg/L under semi-static conditions (limit test). The NOEC value, based on mortality and sublethal effects, was ≥ 1.15 mg/L, respectively.  The LOEC was > 1.15 mg/L. Sublethal effects were not observed.

Key value for chemical safety assessment

Additional information