Zinc di(benzimidazol-2-yl) disulphide

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:

2017-07-13 to 2017-08-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

adopted on 17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

January 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No. of test material: OUCHI SHINKO CHEMICAL INDUSTRIAL CO., LTD.; 608011
- Expiration date of the lot/batch: 2017-08-31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep to the storeroom with suitable ventilation. Avoid fire, direct sunlight and moisture. Store at room temperature.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: from sewage plant for domestic sewage in Balatonfüred, Hungary
- Preparation of inoculum for exposure: washed by centrifugation, supernatant liquid phase decanted, solid material re-suspended in isotonic saline solution and again centrifuged (procedure repeated twice); aliquot of final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined; washed sludge suspension, corresponding to 5 g dry material per litre mixed with test water (mineral medium) and then aerated under test conditions (for 7 days) until use; pH adjustment of activated sludge inoculum was not performed.
- Pre-conditioning (07-13 July 2017) consisted of aerating (2 L/minute) activated sludge (in mineral medium) for 6 days at the test temperature
- Concentration of sludge: 2.0 mL per litre test medium
- Initial cell/biomass concentration: 104 - 106 cells/L
- Sludge filtered: Yes (after preparation)
- Type of filter used: Cotton wool

Duration of test (contact time):

28 d

Initial conc.:

6 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 301 D
- Additional substrate: No
- Test temperature: 20.0 - 20.4 °C
- pH: 7.31
- pH adjusted: No
- Aeration of dilution water: No, but aeration of mineral medium
- Continuous darkness: yes
- Oxygen concentration of the test water: 8 - 9 mg/L

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL, coded) with special neck and glass stoppers
- Number of culture flasks/concentration:
10 (+ 2 reserve) bottles containing the test item and inoculum;
14 (+ 2 reserve) bottles containing the reference item and inoculum (procedure control)
- Measuring equipment: The oxygen concentration was measured with an O2 electrode

SAMPLING
- Sampling frequency: on days: 0, 1, 4, 7, 14, 21, 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, 14 (+ 2 reserve) bottles
- Toxicity control: Yes, 10 (+ 2 reserve) bottles

Reference substance:

benzoic acid, sodium salt

Preliminary study:

No toxic effect of the test substance was found at the investigated concentration of 4 mg/L.

Key result

Parameter:

% degradation (O2 consumption)

Value:

12.3

Sampling time:

28 d

Details on results:

Under the test conditions the percentage biodegradation of the test substance reached a mean of 12.3 % after 28 days based on its ThODNH4.
The pass level for ready biodegradability is the removal of 60 % ThODNH4 in a 10-day window. Minimal biodegradation of the test item occurred in this study; the value remained far below the pass level; therefore, the test item was considered to be not ready biodegradable.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to a mean of 76.0 % after 14 days, and to a mean of 87.8% after 28 days of incubation based on its ThODNH4. The biodegradability reached its plateau on about study day 7 and from this day the slight changes were considered as being within the biological variability range of the applied test system.

Correction for Oxygen Uptake for Interference with Nitrification

Because of the N-containing test item, the oxidised nitrogen (nitrate and nitrite) concentrations were determined following each oxygen measurement with photometric method using nitrite and nitrate cell tests. The LOQ (Limit Of Quantification) of the measurements was 0.03 mg NO2/L and 0.4 mg NO3/L, respectively. The measured quantities of nitrite in the test item and toxicity control samples were below the LOQ in the 0-, 7-, 14-, 21- and 28-day samples. The nitrite concentration of the 28-day inoculum control samples was in average 0.06 mg/L. The nitrate concentration of the different samples was less than 0.4 mg/L throughout the test. Based on the results of nitrite and nitrate determinations any correction of the oxygen consumption values was considered as not necessary. The oxygen uptake resulting from a possible ammonium oxidation did not influence the amount of oxygen taken up by microbial population.

The measured nitrite concentration value in the 28-day inoculum control samples was caused likely by a technical effect (possible discoloration of the solutions and/or turbidity).

Biodegradation of Toxicity Control

In the toxicity control containing both, the test item and the reference item, a mean of 38.1 % biodegradation was noted within 14 days and a mean of 39.3 % biodegradation was determined after 28 days of incubation (from day7 of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system).

Validity of the study

Inoculum Control:

The oxygen depletion in the inoculum control did not exceed 1.5 mg O2/L after 28 days. It was 1.36 mg O2/L in average.

Oxygen Concentration:

The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any time. (The lowest value was 2.40 mg O2/L and it was measured on day 28 in the procedure control.)

Parallels:

The difference of duplicate values for the degradation at the plateau, at the end of the test or at the end of the 10-d window was not greater than 20 %.

The highest difference (13.3 %) between the duplicate values from the 7th to the 28th day of the test (this period was taken into consideration as biodegradation plateau) or at the end of the test (test item, procedure control, and toxicity control groups) for degradation was calculated in the procedure control group. It was observed on the 7th day.

Remark: When seeing the whole incubation period, 46.5 % difference was noticed between the duplicate calculated biodegradation values in the test item group on the 7th day. This relatively high percentage was considered not contradictive with the validity of the study since on the 7th day the minimal test item biodegradability just reached a plateau. The difference between the corresponding measured dissolved oxygen concentration values (6.55 and 6.72 mg/L, see Table 2 of Appendix I) was minimal with about 2.6 %; therefore, this result does not affect the validity of the study.

Reference Item:

The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. (The percentage degradation of the reference item was 76.0 % on the 14th day.)

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance was considered to be not ready biodegradable.

Executive summary:

The ready biodegradability of the test item was determined with a non adapted activated sludge over a test period of 28 days in the closed bottle test according to OECD 301D (TOXI-COOP, 2017). The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item Zinc di(benzimidazol-2-yl) disulphide was investigated at the concentration of 4.0 mg/L.

The test item concentration was chosen based on the preliminary test results and based on its theoretical oxygen demand (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 1.50 mg O2/mg. In parallel (under the same conditions as the test item), positive reference item sodium benzoate was investigated at a concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control. All validity criteria of the study were met.

Under the applied test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of Zinc di(benzimidazol-2-yl) disulphide reached a mean of 12.3 % after 28 days based on its theoretical oxygen demand. The parallel running analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred (the changes in nitrite concentrations in the 28-day inoculum control samples were caused likely by a technical effect: turbidity and/or discoloration). The biodegradability value of the test item was calculated based on its ThODNH4.

The reference item sodium benzoate was sufficiently degraded to a mean of 76.0 % after 14 days, and to a mean of 87.8 % after 28 days of incubation based on ThODNH4. (The biodegradability reached its plateau on about day 7 and from this day onwards the slight changes were considered as being within the biological variability range of the applied test system.)

Description of key information

The test substance was considered to be not ready biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test item was determined with a non adapted activated sludge over a test period of 28 days in the closed bottle test according to OECD 301D (TOXI-COOP, 2017).The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item Zinc di(benzimidazol-2-yl) disulphide was investigated at the concentration of 4.0 mg/L.

The test item concentration was chosen based on the preliminary test results and based on its theoretical oxygen demand (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 1.50 mg O2/mg. In parallel (under the same conditions as the test item), positive reference item sodium benzoate was investigated at a concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control. All validity criteria of the study were met.

Under the applied test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of Zinc di(benzimidazol-2-yl) disulphide reached a mean of 12.3 % after 28 days based on its theoretical oxygen demand. The parallel running analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred (the changes in nitrite concentrations in the 28-day inoculum control samples were caused likely by a technical effect: turbidity and/or discoloration). The biodegradability value of the test item was calculated based on its ThODNH4.

The reference item sodium benzoate was sufficiently degraded to a mean of 76.0 % after 14 days, and to a mean of 87.8 % after 28 days of incubation based on ThODNH4. (The biodegradability reached its plateau on about day 7 and from this day onwards the slight changes were considered as being within the biological variability range of the applied test system.)