1,4-phenylenebis(nitrilo-2,2-dimethylprop-1-yl-3-ylidene) didodecanoate

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:

2015-05-20 to 2015-06-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Species: Activated Sludge, microorganisms from a domestic waste water treatment plant.
- Source of inoculum/activated sludge: The activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 19 May 2015.
- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with reconstituted water (see above) filtered through cotton wool and then aerated under test conditions until use. For this study 200 mL inoculum was prepared one day before the test. The pH of the activated sludge inoculum after preparation was 7.70. The inoculum was not pre-adapted to the test chemical.

Duration of test (contact time):

28 d

Initial conc.:

2.5 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST UNITS
- Type and size: Winkler bottles (300 mL) with special neck and glass stoppers.
- Identification: Each test flask was uniquely identified with the study number, test group, days of measurement and replicate number.

TEST CONDITIONS
- Composition of medium: According to guideline
- Test temperature: 22 +/- 2 °C
- pH: The pH was checked prior study start and found to be 7.45. pH adjustment was considered as not necessary.
- Oxygen concentration of the test water: About 8-9 mg/L (was measured at the start of the test and found to be 8.65 mg/L).
- Continuous darkness: yes

TEST SYSTEM
- Type and size: Winkler bottles (300 mL) with special neck and glass stoppers.
- Preparation of the Test Solutions:
The preparation of the respective test solutions with SIKA Hardener LPP (SIKA Hӓrter LPP) was performed according to the following:
The respective amount, 25 mg of SIKA Hardener LPP (SIKA Hӓrter LPP) was weighed and suspended by mechanical dispersion (~ 10 min ultrasonic treatment under chilling) in 1000 mL of reconstituted water and this solution (homogeneous opalescent suspension) containing the test item was adequately stirred to ensure a good dispersion (extra care was taken for avoiding of air bubbles in the stirred solution).
- Test Item (flasks 1a and 1b): The concentration of the test item (2.5 mg/L) was chosen based on the preliminary test results and based on the on the theoretical oxygen demand (ThODNH4) of 2.68 mg O2/mg test item (calculated according to equation given in the guidelines, assuming that no nitrification occurs). At the start of the test, a suitable volume (500 mL) of the test item homogeneous opalescent suspension was thoroughly mixed into the respective volume (5000 mL) of aqueous test medium corresponding to 2.5 mg/L test item, respectively a ThODNH4 of about 2.5 x 2.68=6.7 mg O2/L.
- Procedure Control (Sodium benzoate;(flasks 2a and 2b): Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), at the start of the test a stock solution* of Sodium benzoate was mixed into the aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 of about 3.6 x 1.67=6.012 mg O2/L).
* the concentration of the stock solution was 360 mg/L
- Inoculum Control (flasks 3a and 3b): Only filtered inoculum (10 mL) was added to the aqueous test medium (5000 mL).
- Toxicity Control (flasks 4a and 4b): Test (500 mL) and reference item (50 mL) stock solutions (in the case of test item a homogeneous opalescent suspension) were mixed into the aqueous test medium (5000 mL) corresponding to the investigated test item concentration of 2.5 mg/L [chosen based on the preliminary information about the test item and based on its ThODNH4] and to 3.6 mg/L concentration of the reference item.
- General: Microbial inoculum (2.0 mL per litre test medium) was added to each preparation bottle.
- Test bottles:
10 (+2 reserve) bottles containing the test item and inoculum
10 (+2 reserve) bottles containing the reference item and inoculum (procedure control)
10 (+2 reserve) bottles containing only inoculum (inoculum control)
10 (+2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)
- Measuring equipment: The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]
- Test Parameters:
- Measurement of Oxygen: The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method].Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.
- Measurement of total oxidized N (nitrite and nitrate): Because of the nitrogen content of the test item, samples for nitrate and nitrite analysis were taken from all vessels (of test item, inoculum control and toxicity control group) and the oxidized nitrogen (nitrate and nitrite) concentrations were measured. For technical reason the nitrate and nitrite analysis of the start (day 0) samples was performed two days later, the analysis of 14-day samples was performed one week later, together with the 21-day samples. The samples were stored in freezer until determination of nitrate and nitrite. The 7-day, 21-day and 28-day samples were analysed directly after oxygen measurements.
- Measurement of Temperature: Temperature was measured continuously using min/max thermometer and noticed daily.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Only filtered inoculum (10 mL) was added to the aqueous test medium (5000 mL).
- Toxicity control: Test (500 mL) and reference item (50 mL) stock solutions (in the case of test item a homogeneous opalescent suspension) were mixed into the aqueous test medium (5000 mL) corresponding to the investigated test item concentration of 2.5 mg/L and to 3.6 mg/L concentration of the reference item.

Reference substance:

other: Sodium benzoate

Key result

Parameter:

% degradation (O2 consumption)

Value:

48.1

Sampling time:

28 d

Remarks on result:

other: based on ThODNH4

Details on results:

The parallel running analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred, therefore the biodegradability of the test item was calculated based on its ThODNH4; any correction, based on the nitrite and/or nitrate content was not performed.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to a mean of 83.8 % after 14 days, and to a mean of 85.1 % after 28 days of incubation, based on ThODNH4.

Biodegradation of the Toxicity Control:

In the toxicity control containing both, the test item and the reference item, a mean of 45.7 % biodegradation was noted within 14 days and 46.1 % biodegradation after 28 days of incubation.

Validity criteria 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 1.43 mg O2/L in average.)
- Oxygen Concentration: The residual oxygen concentration in the test flasks did not drop below 0.5 mg O2/L at any time. (The lowest value was 0.99 mg O2/L, it was measured on the 21st day in the toxicity control). 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 will not be greater than 20 %. At the biodegradation plateaus (test item, procedure control, and toxicity control groups) the highest difference (11.3 %) between duplicate values for degradation was calculated in the test item group, it was observed on the 7th day.
- 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 83.8 % on the 14th day).

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The percentage biodegradation of the test item reached a mean of 48.1 % after 28 days based on its ThODNH4. Based on this results, the test item is considered to be not ready biodegradable, since  the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window.

Executive summary:

in this study the ready biodegradbility of the the test substance, SIKA Hardener LPP was investigate in a Closed Bottle Test according to OECD guideline 301D. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure.

Under the test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of SIKA Hardener LPP (SIKA Hӓrter LPP) reached a mean of 48.1 % after 28 days based on its ThODNH4. The test item is considered to be not ready biodegradable, since  the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window.The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.

Description of key information

Biodegradation in water: Screening test

The percentage biodegradation of the test item reached a mean of 48.1 % after 28 days based on its ThODNH4. Based on this results, the test item is considered to be not ready biodegradable, since  thepass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window.

 

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

In this study the ready biodegradbility of the the test substance was investigate in a Closed Bottle Test according to OECD guideline 301D.The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure.
Under the test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of SIKA Hardener LPP reached a mean of 48.1 % after 28 days based on its ThODNH4. Thus the test item is considered to be not ready biodegradable, since  the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window.The percentage biodegradation of the reference item (sodium benzoate) confirms the suitability of the used activated sludge inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.