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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:
2017-09-08 to 2017-12-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
17 July 1992
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Product name: Vinylcyclohexane
Chemical name: Ethenylcyclohexane
Batch/Lot number: VCH/7/17K1
Purity: 99.8 %
Stability: Stable
Water solubility: 15 mg/L (Analysis Report N0. 3612/17-e, 01.09.2017)
Appearance: Liquid
Colour: Colourless
Storage conditions: Keep containere tightly closed. Keep in a cool, well-ventilated place. Keep away from ignition source.
Expiration date: 30 March 2019
Analytical monitoring:
yes
Details on sampling:
Chemical analysis of the test item concentration in the test solutions was performed to determine exposure concentration and stability of the test item during exposure. Substance-specific analysis was performed at the test site Analytisches Zentrum Biopharm GmbH.
The samples were filled into 5 mL glass vials and stored in the fridge (5 °C ± 3 °C). The sampling was conducted according to the following specification:
> After 0 h exposure, NC (negative control) and A (100 mg test item/L) were sampled; 3 samples of 5 mL per treatment group.
> After 24 h, 48 h and 72 h exposure:
> Before renewal of the test solutions, NC and A were sampled; 3 samples of 5 mL per treatment group.
> After renewal of the test solutions, NC and A were sampled; 3 samples of 5 mL per treatment group.
> After 96 h exposure, NC and A were sampled; 3 samples of 5 mL per treatment group.
Of each sampled treatment, one of the samples from O h, 24 h, 48 h, 72 h (before and after renewal) and 96 h was sent to the analytical laboratory at the test site Analytisches Zentrum Biopharm GmbH for chemical analysis, using overnight shipping with an insulated box and thermal packs. The remaining samples were stored as retain samples in the fridge (5 °C ± 3 °C) until finalisation of the study.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solutions were prepared as Water Soluble Fractions (WSF) by adding 700.6 - 700.9 mg test item to 7 L test medium and stirring for 48 h using a magnetic stirrer at room temperature. The WSFs were filtered through a fibre glass filter with a retaining range till 0.6 µm (MN 85/70 BF, Marchery-Nagel, Düren, Germany). The filter was prepared by rinsing with ca. 100 mL purified water and preconditioning with ca. 100 mL WSF (which was discarded), to reduce adsorption of the test item. This filtered stock solution was used as single test item loading rate in the test.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: in-house breeding of Hydrotox GmbH
- Age at study initiation (mean and range, SD): ca. 9 months
- Length at study initiation (length definition, mean, range and SD): ca. 2.5 cm
- Weight at study initiation (mean and range, SD): not reported
- Method of breeding: not reported
- Maintenance of the brood fish: 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, ≥ 1/3 of the tank volume is replaced with fresh charcoal filtered tap water.

ACCLIMATION
- Acclimation period: Seven days before the start of the test, the fish were kept under test conditions in test medium. They were fed every working day with commercial fish food (TetraMin) until 24 h before test start, when feeding was stopped.


FEEDING DURING TEST
- Food type: not fed during the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Test temperature:
The temperature during the exposure was 22.8 — 23.2 °C in the control and 23.2 — 23.8 °C (required 21 — 25 °C).
pH:
The pH was 7.7 — 7.8 in the control and 7.7 — 7.8 in the test item treatment.
Dissolved oxygen:
The dissolved oxygen concentration was 7.8 — 8.4 mg/L in the control and 8.0 — 8.2 mg/L in the test item treatment (>= 60 % of the air saturation).
Conductivity:
The conductivity was 681 — 734 µS/cm in the control and 680 — 692 µS/cm in the test item treatment.
Nominal and measured concentrations:
0, 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass tanks 8 L
- Type (delete if not applicable): open
- Aeration: not reported. Because of the dissolved oxygen concentration in the control and the test item treatment throughout the test (≥ 60 % of the air saturation), aeration with air pumps during the test is assumed.
- Renewal rate of test solution (frequency/flow rate): A semi-static test design was applied. 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).
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: ≥ 1fish/L
- The vitality of the fish was assessed after 24 h, 48 h, 72 h and 96 h exposure

TEST MEDIUM / WATER PARAMETERS
- Test medium: ISO 7346 medium
- Measurement interval of water parameter: The pH, conductivity and O2-concentrations were measured daily. The temperature was measured continuously.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 14 h light: 10 h dark
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality, appearance and behavior

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test
- No range finding study
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 231 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 231 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Results with reference substance (positive control):
- Behavioural abnormalities: 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.
- Mortality of control: As no fish died until the end of the exposure, the mortality in the control as well as in the test item treatment was 0 %.
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The substance is volatile, hence the derivation from the nominal concentration.
- Effect concentrations exceeding solubility of substance in test medium: no
Reported statistics and error estimates:
As no effect on the test organisms was observable, no statistical data evaluation was performed.
Sublethal observations / clinical signs:

Results of the chemical analysis

ln the control, the test item was not detected. The initial measured test item concentrations in the test item treatment were 2.09 — 3.86 % (new test solutions) of the nominal loading rate. Until the

renewal of the test solutions and the end of the exposure period, the measured test item concentrations decreased to < LOQ — 0.02 % (old test solutions) of the nominal loading rate. As the measured test item concentrations are not within ± 20 % of the nominal loading rate, according to OECD 203 (1992) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations. Therefore, the geometric mean of all eight measurements (0 h, 24 h old/new, 48 old/new, 72 h old/new and 96 h) was calculated. For measurements which are < LOQ, the value of LOQ, i.e. 20 µg/L, was used for calculation.

Criteria of validity

> No mortality of test organisms occurred in the control until the end of the test and was therefore 1 fish.

> Dissolved oxygen concentration in the control and the test item treatment throughout the test was 7.8 mg/L and therefore ≥ 60 % of the air saturation.

The test is valid according to OECD Test Guideline 203 (17 July 1992).

Validity criteria fulfilled:
yes
Conclusions:
For the conducted semi-static acute toxicity test with Danio rerio, applying a nominal concentration of 100 mg/L vinylcyclohexane (limit test), no statistical evaluation of the effect concentrations was performed, as no effect on the test organisms was observable. Based on the geometric mean of the measured concentrations the NOEC and LOEC were determined to equal or be above 231 µg/L and to be above 231 µg/L, respectively.
Executive summary:

In a 96-h acute toxicity study, Danio rerio (zebrafish) were exposed to vinylcyclohexane at nominal concentration of 0 (control) and 100 mg a.i./L (231 µg a.i./L geometric mean of measured concentrations) under semi-static conditions.  As the measured test item concentrations are not within ± 20 % of the nominal loading rate, according to OECD test guideline 203 (1992), all results are given in relation to the analytically measured test item concentrations (geometric mean concentrations). As no effect on the test organisms was observable, no statistical data evaluation was performed. The LOEC and NOEC values, based on mortality and sublethal effects, were > 0.231 and ≥ 0.231 mg a.i./L, respectively.  No obvious effect on appearance and behaviour of the fish in the test item treatment was observable. Based on the results of this study, vinylcyclohexane does not have to be classified as hazardous to fish in accordance with Section 4.1.2.6., Annex I of Regulation 1272/2008/EC.

This toxicity study is classified as acceptable and satisfies the guideline requirement for fish acute toxicity studies with D. rerio.

Results Synopsis

 

Test organism size/age (mean wet weight or length): 9 months

Test Type (Flowthrough, Static, Static Renewal): Semi-static

 

NOEC ≥ 0.231 mg a.i./L                            

LOEC > 0.231 mg a.i./L

Endpoint(s) Effected: mortality, behaviour

Description of key information

A 96-h semi-static acute toxicity limit test with Danio rerio was conducted according to the OECD test guideline 203 and in accordance with the principles of GLP. No effects were observed. Hence, the NOEC equals or is above 231 µg/L and the LOEC is above 231 µg/L. 

Key value for chemical safety assessment

Additional information