N,N'-bis(2,4,6-triisopropylphenyl)methanediimine

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-12 - 2018-07-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

Council Regulation (EC) No 440/2008, Method C.4-D “Manometric Respirometry Test“ (2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis[2,4,6-tris(propan-2-yl)phenyl]methanediimine

EC Number:

815-171-4

Cas Number:

300382-79-0

Molecular formula:

C31H46N2

IUPAC Name:

N,N'-bis[2,4,6-tris(propan-2-yl)phenyl]methanediimine

Test material form:

liquid

Remarks:

yellow to brown

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): aeration tank of a wastewater plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)
- Laboratory culture / Method of cultivation:
Before use, the inoculum was stored for three days at room temperature under continuous stirring with aeration.
The sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm and 20 °C and decanting off the supernatant.
An aliquot of the wet sludge was dried in order to determine the wet weight / dry weight ratio of the sludge and to prepare a stock suspension (activated sludge) of 3 g dw/L.
The calculated amount of sludge, needed to achieve 300 mL of this stock suspension, was dissolved in mineral medium and then filled up to a defined end volume.
- Concentration of sludge: 30 mg/L suspended solids

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

100 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

322.4 mg/L

Based on:

ThOD

Remarks:

NH4

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
Mineral medium
The mineral medium was prepared from stock solutions of mineral components described as follows.
1. Mineral salt solution: Potassium phosphate, monobasic (KH2PO4) 8.5 g
di-Potassium hydrogen phosphate (K2HPO4) 21.75 g
di-Sodium hydrogen phosphate dihydrate
(Na2HPO4 ∙ 2 H2O) 33.4 g
Ammonium chloride (NH4Cl) 0.5 g
The components were dissolved in deionised water and filled up to 1 litre.
The pH of the solution was in the recommended range of pH 7.4 ± 0.2.
2. Magnesium sulphate solution: Magnesium sulphate heptahydrate (MgSO4 ∙ 7 H2O) 22.5 g
The substance was dissolved in deionised water and filled up to 1 litre.
3. Calcium chloride solution: Calcium chloride dihydrate (CaCl2 ∙ 2 H2O) 36.4 g
The substance was dissolved in deionised water and filled up to 1 litre.
4. Iron (III) chloride solution: Iron (III) chloride hexahydrate (FeCl3 ∙ 6 H2O) 0.25 g
The substance was dissolved in deionised water and filled up to 1 litre. To stabilise the solution 1 drop of concentrated hydrochloride solution (37%) was added.
To prepare the mineral medium 10 mL of the mineral salt solution were mixed with 800 mL deionised water. Then 1 mL each of the magnesium sulphate-, calcium chloride- and the iron (III) chloride solution were added and filled up to 1 litre with deionised water.
- Test temperature: 22 ± 1 °C
- pH: 7.4 - 8.0
- pH adjusted: no
- Aeration of dilution water: yes

TEST SYSTEM
- Culturing apparatus:
Test volume: 250 mL
Test apparatus: OxiTopControl System (WTW)
Mixing: 1 magnetic stirrer per test vessel
- Number of culture flasks/concentration: 3 per test item and blank, 2 for reference compound, 1 for toxicity control

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: test item and reference compound (sodium benzoate) mixed, one replicate

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All except one; the toxicity control criterion (≥ 25 %) was not met as the biodegradation observed in that control was only 23 %.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The study was conducted under GLP according to EU method C.4D on the registered substance itself with marginal deficiencies; the available information allows to draw the conclusion that the test was conducted properly without deviation from the guideline. The method is considered reasonable, the reference substance produced the appropriate response and the validity criteria except one are met. The toxicity control criterion (≥ 25 %) was not met as the biodegradation observed in that control was only 23 %. It is possible that toxicity to sludge microorganisms caused this effect. The study was nevertheless considered reliable as from a repeated study the biodegradation was not expected to vary clearly from 0 % biodegradation after 28 days as determined in this study. Hence, the results can be considered as reliable to assess the ready biodegradability of Bis(2,4,6-triisopropylphenyl)carbodiimide. Within 28 days, a degradation rate of 0 % was determined for Bis(2,4,6-triisopropylphenyl)carbodiimide. Bis(2,4,6-triisopropylphenyl)carbodiimide is considered to be "Not Readily Biodegradable".

Executive summary:

A study was performed to assess the ready biodegradability of Bis(2,4,6-triisopropylphenyl)carbodiimide. The study was conducted in accordance with the Council Regulation (EC) No 440/2008, Method C.4-D “Manometric Respirometry Test“ (2008). This test method is in all essential parts identical with OECD Guideline 301 F (adopted July 1992). 

A suspension of Bis(2,4,6-triisopropylphenyl)carbodiimide in a mineral medium was inoculated and incubated for 28 d under aerobic conditions. During this period, degradation was followed by continuous automated BOD determinations.

 Bis(2,4,6-triisopropylphenyl)carbodiimide showed:

0 % degradation after  7 days

0 % degradation after 14 days

0 % degradation after 21 days

0 % degradation after 28 days

The reference compound sodium benzoate showed 91 % degradation after 14 days.

The validity criteria were met except that the toxicity control criterion (≥25 %) was not met as the biodegradation observed in that control was only 23 %. It is possible that toxicity to sludge microorganisms caused this effect. However, there is no data on toxicity of Bis(2,4,6-triisopropylphenyl)carbodiimide to microorganisms which would allow to conclude. The study was nevertheless considered reliable as from a repeated study the biodegradation was not expected to vary clearly from 0 % biodegradation after 28 days as determined in this study. Concluding, based on this study Bis(2,4,6-triisopropylphenyl)carbodiimide is considered not to be readily biodegradable in the environment.