Vinylcyclohexane

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-22 to 2017-10-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

July 1992

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: Vinylcyclohexane
Chemical name:: Ethenylcyclohexane
Molecular formula: C8H14
CAS number: 695-12-5
Lot number: VCH/7/17K1
Purity: 99.8 %
ThOD: 3.34 mg O2/mg test substance (calculated by Hydrotox on the basis of the molecular formula)
Water solubility: Insoluble

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge, municipal wastewater treatment plant Breisgauer Bucht, 30 mg dry solids per litre. The treatment plant clarifies predominantly domestic wastewater and has a capacity of 600.000 inhabitant equivalents. Sampling date of activated sludge was on 26 June 2017. The dry solid content of the activated sludge was 3.5 g/L. It was determined by weight measurements after drying at 105°C for 4 hours (mean of triplicate measurements). The activated sludge was washed twice with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

ThOD

Initial conc.:

3.34 other: mg O2/mg test substance

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- Method: The Manometric Respirometry Test determines the ready biodegradability of test items following their oxygen consumption in closed flasks at a constant temperature (±1°C) in a range of 20 – 24°C. The consumption of oxygen is determined by measuring the negative pressure in the flasks after absorption of the evolved carbon dioxide in sodium hydroxide. A solution or suspension of the test item in a mineral medium, corresponding to 100 mg/L test item giving at least 50 – 100 mg/L of theoretical oxygen demand (ThOD), is inoculated with activated sludge (30 mg d.s./L) for 28 days. The amount of oxygen taken up by the microbial population during the biodegradation of the test item (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of the ThOD or the chemical oxygen demand (COD). The pass level for ready biodegradability is 60% of ThOD and must be reached within a 10-d window. The 10-d window begins when the degree of biodegradation reaches 10%.

TEST CONDITIONS
- Composition of medium:
Mineral medium
A: Potassium dihydrogenphosphate KH2PO4 = 8.50 g
Dipotassium hydrogenphosphate K2HPO4 = 21.75 g
Disodium hydrogenphosphate dihydrate Na2HPO4 * 2 H2O = 33.40 g
Ammonium chloride NH4Cl = 0.50 g
are dissolved in demineralised water and made up to 1 litre.

B: Calcium chloride dihydrate CaCl2 * 2H2O = 36.4 g
is dissolved in demineralised water and made up to 1 litre.

C: Magnesium sulfate heptahydrate MgSO4 * 7H2O = 22.5 g
is dissolved in demineralised water and made up to 1 litre.

D: Iron (III) chloride hexahydrate FeCl3 * 6H2O = 0.25 g
is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 1 litre.

For preparation of the mineral medium 10 mL of solution (A) is mixed with 900 mL demineralised water, 1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre. Before use, the mineral medium was aerated for about half an hour, the pH was 7.6.
- Test temperature: 20 – 24°C
- pH: 7.6
- pH adjusted: no
- Continuous darkness: yes/no

Test item
The test item was added directly. Therefore 164 mL mineral medium were filled into the test vessels and the test item was pipetted directly into the bottles. 6.1 µL of the test item were added, corresponding to a concentration of 100.1 mg ThOD/L (the density of the test item of 0.806 g/cm3 was considered). The initial pH was 7.6 and was not adjusted.

Reference substance
A stock solution of 128.2 mg/L sodium acetate in mineral medium was prepared. 164 mL of this stock solution were added into the reference vessels corresponding to a concentration of 100.0 mg ThOD/L. The initial pH was 7.6 and was not adjusted.

Toxicity control
164 mL of the stock solution with 128.2 mg/L sodium acetate (s. 5.5) and 6.1 µL of the test item were added into the toxicity control vessel, corresponding to a concentration of 200.1 mg ThOD/L. The initial pH was 7.6 and was not adjusted.

Abiotic control
6.1 µL of the test item and 2.46 g sodium azide were added into the abiotic control vessel which had been filled with 164 mL mineral medium previously. This corresponds to a test item concentration of 100.1 mg ThOD/L and sodium azide concentration of 15 g/L. The initial pH was 7.5 and was not adjusted. No inoculum was added.

Blank
164 mL of the pre-aerated mineral medium with a pH of 7.6 were filled into the blank vessels. The initial pH was 7.6 and was not adjusted.

Inoculum
Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht was used as inoculum with a concentration corresponding to 30 mg dry solids per litre. The treatment plant clarifies predominantly domestic wastewater and has a capacity of 600.000 inhabitant equivalents. Sampling date of activated sludge was on 26 June 2017. The dry solid content of the activated sludge was 3.5 g/L. It was determined by weight measurements after drying at 105°C for 4 hours (mean of triplicate measurements). The activated sludge was washed twice with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.

TEST SYSTEM
- Culturing apparatus and measurement equippment: OxiTop®-Control from WTW, Weilheim and the Sensomat-System from Aqualytic GmbH & Co., Langen were used as test systems. They consist of narrow-necked glass bottles with rubber sleeve inserts for NaOH pellets, in which the carbon dioxide evolved is absorbed. The liquid volume was fixed as 164 mL each. Magnetic stir bars were introduced into the bottles which were positioned on a stirrer platform. The bottles were sealed tightly with the measuring heads with a measuring range of 500 – 1350 hPa. Every 112 minutes the current pressure was measured and stored by each measuring head. At the end of the experiment pressure data were read out via an infrared interface to the controller unit and afterwards data were transferred via a RS232 interface using the Achat OC software of WTW to an Excel file where further data processing was carried out. Additionally, several measured values were randomly read out and recorded by hand and were afterwards compared with the printed excel table for quality control.
- Number of culture flasks/concentration: In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound (reference), one reactor containing reference compound and test item (toxicity control) and one reactor containing the test item and the biozid sodium azide (abiotic control) were set up. The test flasks were filled as described in paragraphs 5.4 - 5.8 and tempered to the incubation temperature for about 2 hours. Afterwards, 1.4 mL of the inoculum were added into each flask except the abiotic control vessel, one sodium hydroxide pellet was added to each rubber quiver inserted in the flasks and the flasks were sealed tightly with the measuring heads and the test was started. After 28 days the data were read out and the pH in the flasks was measured on the next day.

Reference substance:

other: sodium acetate

Key result

Parameter:

% degradation (O2 consumption)

Value:

7.5

Sampling time:

28 d

Details on results:

The biodegradation at the end of the test (day 28) was 7.5% of ThOD (mean of three replicates).

Results with reference substance:

The reference compound sodium acetate reached the pass level for ready biodegradability (60% ThOD within a 10-day window) within 8 days.

Test item

The biodegradation at the end of the test (day 28) was 7.5 % of ThOD (mean of three replicates). Therefore, the test item didn’t reach the criteria for ready biodegradability (60 % of ThOD within a 10-d window). The pH in the test vessels at the end of the test was 7.5.

Toxicity control

The degradation in the toxicity control reached 36.5 % within 8 days and was thus above the criterion for inhibition effects to the inoculum (< 25 % on day 14). The test item had no toxic effect to the inoculum according to the validity criteria of OECD 301. The pH in the toxicity control vessel at the end of the test was 9.0.

Abiotic control

No abiotic degradation could be detected in the abiotic control. Temporarily, an underflow of the admissible range occurred which means that instead of an oxygen consumption an overpressure was measured. In these cases, the degradation values were equated with 0. The pH in the abiotic control vessel at the end of the test was 8.5.

Reference item

The reference compound sodium acetate reached the pass level for ready biodegradability (60 % ThOD within a 10-day window) within 8 days. The pH in the reference vessels at the end of the test was between 9.0 and 9.1.

Blank

The oxygen consumption of the blanks was 18.8 mg/L in 28 days (mean of three replicates, see table 1). The pH in the blank vessels at the end of the test was 7.5.

Figure 1 below displays the degradation of the test item, reference item, toxicity control and abiotic control.

The temperature was 21.8 – 22.8 °C throughout the whole study.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item is not readily biodegradable.

Executive summary:

The biodegradation of vinylcyclohexane was studied in the Manometric Respirometry Test according to the OECD test guideline 301 F with activated sludge from a domestic sewage treatment plant (The dry solid content of the activated sludge was 3.5 g/L). Vinylcyclohexane was applied with 100.1 mg ThOD/L (the density of the test item of 0.806 g/cm3 was considered).  The experiment was conducted in compliance with the OECD GLP standards.

The pressure decrease in the reaction vessels was measured over the complete experimental phase of 28 days. The amount of O₂ consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

The biodegradation at the end of the test (day 28) was 7.5 % of ThOD (mean of three replicates), thus the test item did not reach the criteria for ready biodegradability (60 % ThOD within a 10-d window). The degradation of the toxicity control after 14 days was > 25 %. Thus, the test item had no inhibitory effect on the inoculum. No abiotic degradation could be detected.

Vinylcyclohexane is considered to be not readily biodegradable.

Description of key information

In a GLP test according to OECD guideline 301 F, the biodegradation of vinylcyclohexane was 7.5 % of ThOD (mean of three replicates) after 28 days. Thus, the test item is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

For further details on the assessment of the persistency of vinylcyclohexane please refer to the PBT assessment report in IUCLID section 13.