Vinylcyclohexane

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-08 to 2017-10-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

July 22nd 2010

Principles of method if other than guideline:

Because of the low viscosity the test item was added directly via pipette. Therefore 62 µL of the test item were added to the five test vessels — this is corresponding with a concentration of 100 mg/L. The density of 0.806 g/cm3 was considered in the calculation.

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 under normal temperature and pressure
Water solubility: Insoluble
Appearance: Liquid
Colour: Colourless
Storage conditions: Keep container tightly closed. Keep away from ignition sources — no smoking. The test item will be stored at ambient temperature.
Expiration date: 30 March 2019

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test item
62 µL of the test item were added directly into the test vessels with a test volume of 500 mL to obtain a final concentration of 100 mg/L. The pH of the test solution was measured before addition of inoculum.
Reference item
25.8 mg of 3,5-dichlorophenol were dissolved in deionised water and filled up to 25.8 mL using a magnetic stirrer to get a stock solution with 1.0 g/L. 0.5, 5 and 15 mL of the stock solution were added to the reference vessels in order to obtain final concentrations of 1, 10 and 30 mg/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from the municipal wastewater treatment plant Staufener Bucht was used as test system. The treatment plant has a capacity of 140 000 inhabitant equivalents and clarifies predominantly domestic wastewater. Sampling date of activated sludge was 21 June 2017.
- Method of cultivation: The activated sludge was kept aerated until use.
- Preparation of inoculum for exposure: Dry solid of the activated sludge was determined as 4.3 g/L by weight measurements before and after 3.5 h drying at 105 °C (mean of triplicate measurements). The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in aerated tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.
- Initial biomass concentration: 1.5 g/l dry solids in the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20.0 - 21.3

pH:

test item: 7.3
reference item: 7.4
blank: 7.4

Nominal and measured concentrations:

0, 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 600 mL serum bottles, Geratebau Ochs, Bovenden-Lenglern, Germany, 250 - 300 mL BOD bottles with adapter for oxygen electrode
- Aeration: All vessels were stirred and aerated throughout the contact time with room air to keep the dissolved oxygen concentration above 60 – 70 % saturation. Before starting the test, the aeration rate was measured in all test vessels. After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1.5 g/L dry solids
- Nutrients provided for bacteria: yes, synthetic sewage feed, 16 mL added to each test vessel
(1 L synthetic sewage feed contains: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2*2H2O, 0.2 g MgSO4*7H2O, 2.8 g K2HPO4)
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralized water
OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: not reported
- Light intensity: not reported
- Details on termination of incubation: After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer. This determination was repeated for each vessel at 15-min intervals, ensuring that the contact time in each vessel was 3 h.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration rate
TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test
- Test concentrations: 100 mg/L

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenol (97 % purity)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: low viscosity of test substance, volatile
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption (e.g. of test material to the walls of the test container): not reported
- Blank controls oxygen uptake rate: The respiration rate of six blanks were within 26 and 35 mg/ (L*h) which leads to a consumption rate of 20 mg oxygen per 1 g sludge (mean of six replicates).
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation (CV) was 10 % and therefore less than 30 %.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorphenol was calculated as 2.57 mg/L and was therefore within the acceptable range of 2 to 25 mg/L.

Reported statistics and error estimates:

The EC50 calculation was performed with the ToxRat software by probit analysis using linear max. likelihood regression (ToxRat Standard Version 2.09, © ToxRat Solutions GmbH). The EC50 is calculated from the slope of the curve.

Validity criteria fulfilled:

yes

Conclusions:

There was no inhibition observed in the test vessels with 100 mglL test concentration (inhibition range from -19 to 14%). The respiration rates in the test vessels were not significant lower than the respiration rate of the controls (STUDENT t-test).
EC50 > 100 mg/L
NOEC ≥ 100 mg/L

Executive summary:

The influence of vinylcyclohexane on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study was conducted according to the OECD test guideline 209 (July 22^nd, 2010).

A concentration of 100 mg/L was tested. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring.

No significant inhibition of respiration rate of the sludge was recorded at 100 mg vinylcyclohexane per litre. No further testing was needed.

Since all criteria for acceptability of the test were met, this study was considered to be valid:

> The coefficient of variation (CV) of the blank respiration rates was less than 30 %.

> The blank controls oxygen uptake rate was 20 mg oxygen per one gram of activated sludge per hour.

> The EC50 of the reference item was within the acceptable range of 2 to 25 mg/L.

In conclusion, under the conditions of the present test, vinylcyclohexane was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 100 mg/L.

Description of key information

The toxicity of vinylcyclohexane towards microorganisms was assessed via a test conducted according to the OECD test guideline 209 and in accordance with the principles of GLP. The effect on microorganisms was measured via measurement of the total respiration after 3 h. The test was conducted as limit test with a loading rate of 100 mg/L. There was no inhibition observed in the test vessels with 100 mg/L test concentration (-19 to 14 %). The respiration rates in the test vessels were not significant lower than the respiration rate of the controls. Therefore, the EC50 is above 100 mg/L and the NOEC equals or is above 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L