Vinylcyclohexane

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-30 to 2017-12-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Product name: Vinylcyclohexane
Chemical name: Ethenylcyclohexane
Molecular formula: C8H14
CAS number: 695-12-5
Batch/Lot number: VCH/7/17K1
Purity: 99.8 %
Stability: Stable
Water solubility: 15 mg/L (Analysis Report No. 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:

- Concentrations: 0, 10, 20, 40, 80, 160
- Sampling method: Chemical analysis of the test item concentrations 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, all replicates of NC (negative control), A (10 mg test item/L, nominal), B (20 mg test item/L, nominal), C (40 mg test item/L, nominal), D (80 mg test item/L, nominal) and E (160 mg test item/L, nominal) were sampled; one sample per replicate: 4 samples of 5 mL per treatment group.
• After 24 h exposure: Before renewal of the test solutions, all replicates of NC, A, B, C, D and E were sampled; one sample per replicate: 4 samples of 5 mL per treatment group.
• After renewal of the test solutions, all replicates of NC, A, B, C, D and E were sampled; one sample per replicate: 4 samples of 5 mL per treatment group.
• After 48 h exposure, all replicates of NC, A, B, C, D and E were sampled; one sample per replicate: 4 samples of 5 mL per treatment group.
- Sample storage conditions before analysis: Of each sampled treatment, one of the samples from 0 h, 24 h (old and new test solutions) and 48 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 (one for the start and one for the renewal of the test solutions at 24 h) were prepared as Water Soluble Fractions (WSF) by adding 160.6 mg (0 h) and 160.5 mg (24 h) test item to 1000 mL test medium and shaking for 48 h using an overhead shaker at 21.7 – 25.3 °C in the dark, respectively. 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 highest test item loading rate in the test.
- Controls: The negative control (NC; test medium) was treated in the same way as the test item solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus
- Source: from the clone breeding of the Federal Environment Agency, department IV 2.4, Berlin, and cultivated at Hydrotox GmbH since October 2012
- Age of parental stock (mean and range, SD): not reported
- Feeding during test: no
- Food type: /
- Amount: /
- Frequency: /

ACCLIMATION
- Acclimation period: The daphnids are cultured at 20 ± 2 °C with 16 h light: 8 h dark and placed into fresh M4 medium twice a week.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Desmodesmus subspicatus algae corresponding to 0.1 mg C × daphnid-1 × day-1
- Feeding frequency: 10 animals / 200 mL and fed on workdays with Desmodesmus subspicatus algae corresponding to 0.1 mg C × daphnid-1 × day-1.
- Health during acclimation (any mortality observed): no abnormalities observed
Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate (Sigma, Steinheim, Germany; Lot no.: MKBF2111V, expiration date: January 2018). The recent quality testing was performed in February 2017 with EC50 (24h) = 1.32 mg/L (CL 95 %: 1.24 – 1.41 mg/L), which is in the declared range of 0.6 – 2.1 mg/L (24 h) according to OECD 202.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20.4 – 21.1 °C

pH:

7.8 – 8.2 in the control and 7.8 – 8.4 in the test item treatments

Dissolved oxygen:

7.8 – 8.2 mg/L in the control and 7.9 – 8.3 mg/L in the test item treatment

Conductivity:

690 µS/cm

Nominal and measured concentrations:

0, 10, 20, 40, 80, 160 mg/L (nominal values)
0, 74.29, 139.14, 401.89, 770.24, 3103.03 µg/L (measured, geometric mean values)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers 50 mL, Schott, Mainz
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 40 mL test solution in 50 mL glass vessel
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): change of medium after 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
The test was performed with M4 medium according to OECD 202 (2004). After addition of the stock solutions, except the vitamin stock solution S23, the medium was aerated for 1.5 h to achieve oxygen saturation and the pH (8.7), oxygen concentration (8.1 mg/L) and conductivity (690 µS/cm) was determined. Solution S23 was added directly before use of the medium.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mobility

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: Nominal loading rates of 10, 50 and 200 mg/L test item
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.965 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.685 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.77 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.402 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: not reported
- Observations on body length and weight: not reported
- Other biological observations: not reported
- Mortality of control: none
- Other adverse effects control: not reported
- Abnormal responses: not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

The effect occurrence after 24 h and 48 h exposure was calculated. With the statistical software ToxRat Professional 3.2.1 (ToxRat Solutions GmbH, Alsdorf, Germany), ECx and LOEC/NOEC were determined.

As the measured test item concentrations are not within ± 20 % of the nominal loading rates, according to OECD 202 (2004), all results are given in relation to the analytically measured test item concentrations (geometric mean concentrations).

The test is valid according to OECD Test Guideline 202 (13 April 2004). In the control, the test item was not detected. The initial measured test item concentrations in the test item treatments were < 1.80 – 5.55 % (0 h) and 2.84 – 7.17 % (24 h new) of the nominal loading rates. Until the renewal of the test solutions and end of the exposure period, the measured test item concentrations decreased to 0.18 – 0.52 % (24 h old) and 0.16 – 0.68 % (48 h) of the

nominal loading rates.

The immobilities of the test organisms in the control (NC) and the test item treatments are presented in Table 1.

Table 1: Immobility after 24 h and 48 h exposure

Nominal test loading rate (mg/L)	Daphnia introduced				Immobile Daphnia (replicates 1-4)								Immobility [%]
	0 h				24 h				48 h				24 h	48 h
	1	2	3	4	1	2	3	4	1	2	3	4
NC	5	5	5	5	0	0	0	0	0	0	0	0	0	0
10	5	5	5	5	0	0	0	0	0	0	0	0	0	0
20	5	5	5	5	0	0	0	0	0	0	0	0	0	0
40	5	5	5	5	0	0	0	0	0	0	0	0	0	0
80	5	5	5	5	1	0	0	0	2	1	1	0	5	20
160	5	5	5	5	1	0	2	1	5	5	5	5	20	100

Validity criteria fulfilled:

yes

Conclusions:

For the conducted semi-static acute immobilization test with Daphnia magna, applying nominal concentrations of 10 to 160 mg/L vinylcyclohexane, effect concentrations were cacluated on basis of measured concentrations (geometric mean):
- 48 h EC50: 965 µg/L (95 % CL: not determined);
- 48h LOEC: 770 µg/L;
- 48h NOEC: 402 µg/L.

Executive summary:

The 48-hr-acute toxicity of vinylcyclohexane to Daphnia magna (Straus) was studied under semi-static conditions. Daphnids were exposed to control and test chemical at nominal concentrations of 10, 20, 40, 80 and 160 mg a.i./L for 48 hr. As the measured test item concentrations are not within ± 20 % of the nominal loading rates, according to OECD test guideline 202 (2004), all results are given in relation to the analytically measured test item concentrations (geometric mean concentration). Immobilisation and sublethal effects were observed daily.  The 48-hour EC50 was 965 µg a.i./L (measured). The 48-hr NOEC based on immobilization was 402 µg a.i./L. 

Based on the results of this study, vinylcyclohexane would be classified as toxic to D. magna in accordance with the EU CLP Regulation. 

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

 

Results Synopsis

 

Test Organism Age (e.g. 1stinstar): < 24 h

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

 

EC50: 965 µg a.i./L                            95 % C.L.:  not determined

NOEC: 402 µg a.i./L                      

Endpoint(s) Effected: Immobilisation

Description of key information

A 48-h semi-static acute toxicity study with Daphnia magna was conducted according to the OECD guideline 202 and in accordance with the principles of GLP. The derived 48 h EC50 was 965 µg a.i./L based on the geometric mean of measured concentrations. The 48-hr NOEC based on immobilization was 402 µg a.i./L. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

965 µg/L