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Environmental fate & pathways

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:
2017-08-03 - 2017-08-31 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
Commission Regulation 440/2008/EC, Method C.4-D of May 30, 2008: Manometric Respirometry Test (EEC Publication No. L 142/496, May 2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry Test", adopted July 17, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-diethyl-2,4-diisocyanato-5-methylbenzene
Cas Number:
2162-70-1
Molecular formula:
C13H14N2O2
IUPAC Name:
1,3-diethyl-2,4-diisocyanato-5-methylbenzene
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At 20 ± 5 °C, in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Bensheim, Germany
- Preparation of inoculum for exposure: The aerobic activated sludge used for this study was deposited for 45 minutes and afterwards washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. 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 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
>= 51.23 - <= 52.46 mg/L
Based on:
test mat.
Remarks:
Concentration given as 12.8 and 12.5 mg per flask, containing 244 ml
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
Reconstituted Test Water: Analytical grade salts were added to pure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; The pH-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage.
50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water.
- Solubilising agent (type and concentration if used): Silicone oil AR20 was used as an emulsifying agent.
- Test temperature: 22°C ± 1°C
- pH:
7.5 to 7.6 (measured at the start of the test)
7.5 to 8.1 (measured at the end of the test)
- pH adjusted: no
- Aeration of dilution water: overnight, after addition of the sludge
- Suspended solids concentration: 2 ml of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water to a final volume of 244 ml
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus:
Type and Size: Manometric Test System with test flasks containing a volume of approximately 500 mL.
Apparatus: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany
Principle: The test flasks were incubated at 22°C ± 1°C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany. The test flasks were closed gas-tight by a measuring head.
- Number of culture flasks/concentration: 2
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

SAMPLING
The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany) each day.
The temperature was recorded by means of the automated software AMR Wincontrol©.
pH-values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at the end of the test, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 flask
- Abiotic sterile control: 1 flask
- Toxicity control: 1 flask
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
ThODNH4: 1.666 mg oxygen per mg sodium benzoate (calculated)

Results and discussion

% Degradation
Key result
Parameter:
% degradation (O2 consumption)
Value:
< 10
Sampling time:
28 d
Remarks on result:
other: not readily biodegradable
Details on results:
The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.
DETDI did not reach 10% degradation during the incubation time of 28 days and therefore the 10 day window criterion was not passed. The mean biodegradation at test end after 28 days was 0% (ThODNH4, ThODNO3).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The biodegradation of the test item was performed under GLP according to OECD 301F. Hence, the results can be considered to be sufficiently reliable to assess the ready biodegradability of the test item. The degradation rate of DETDI did not reach 60% within the 10-day window or after 28 days.
Therefore, DETDI is considered to be not readily biodegradable.
Executive summary:

This study was designed to comply with the following methods:

-     Commission Regulation 440/2008/EC, Method C.4-D of: Manometric Respirometry Test (EEC Publication No. L 142/496, May 2008)

OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry Test", adopted

Aerobic activated sludge (microorganisms from a domestic waste­water treatment plant) was supplied by the sewage treatment plant of Bensheim.

The test item DETDI was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation at 22°C ± 1°C, darkness.

Test Item Loading Rate (initial concentration in medium C0) was 51.8 mg/L corresponding to an oxygen demand of about 100.9 mg/L (ThODNH4) and 129.7 mg/L (ThODNO3).

54.5 mg/L corresponding to an oxygen demand of about 90.8 mg/L (ThODNH4) of sodium benzoate served as positive control.

The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.

DETDI did not reach 10% degradation during the incubation time of 28 days and therefore the 10 day window criterion was not passed. The mean biodegradation at test end after 28 days was 0% (ThODNH4, ThODNO3).

Therefore, DETDI is considered to be not readily biodegradable based on ThODNH4.and ThODNO3.

The reference item sodium benzoate was sufficiently degraded to 85% after 14 days and to 94% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 34% biodegradation was noted within 14 days and 36% biodegradation after 28 days of incubation (based on ThODNH4). According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.