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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 July 2008 to 27 August 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge, microorganisms from a domestic waste water treatment plant.
The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
The activated sludge used for this study was washed by centrifugation (10 min) and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged (10 min). 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 4 g dry material per litre were mixed with test water (see below) and then aerated until use. Before use the sludge was filtered through cotton wool.
Duration of test (contact time):
>= 27 - <= 29 d
Initial conc.:
>= 2.5 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Preparation of the Test Solutions
The components were applied in the following ratio in the test flasks:
The preparation of the respective test solutions with TODI was performed according to the followings: The respective amount of TODI was weighed in directly to reach the required test item concentration of 2.5 mg/L. During the performance of the test the test solutions were ultrasonicated under continuous cooling for < 10 min. to ensure a good dispersion.

Test Item (flasks 1a and 1b):
Based on the theoretical oxygen demand (ThODNO3) of 2.48 mg O2/mg test item (calculated according to equation given in the guidelines), 8.7 mg of TODI was thoroughly mixed into 3.48 litres of aqueous test medium (corresponding to 2.5 mg/L test item, respectively a ThODNO3 of about 6.2 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), stock solution* corresponding to 24.192 mg of Sodium benzoate was mixed into 6.72 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 of about 6.012 mg O2/L). * the concentration of the stock solution was 360 mg/L

Inoculum Control (flasks 3a and 3b):
Only filtered inoculum was added to 6.80 litres of aqueous test medium.

Toxicity Control (flasks 4a and 4b):
8.7 mg of TODI and reference item stock solution* (34.8 mL) were mixed into 3.48 litres of aqueous test medium corresponding to 2.5 mg/L test item (ThODNO3 of 6.2 mg O2/L) and 3.6 mg/L reference item (ThODNH4 of 6.012 mg O2/L). * the concentration of the stock solution was 360 mg/L

General:
Microbial inoculum (2.0 mL per litre) was added to each preparation bottle.

Course of the Test
Preparation of Test Flasks
Sufficient number of BOD flasks was cleaned with 5-10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly stoppered.
The following bottles were prepared: 10 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 bottles containing the test item, reference item and inoculum (toxicity control)

Incubation Period:
28 days
Preliminary study:
The chosen test item concentration is based on the theoretical oxygen demand (ThODNO3) of 2.48 mg O2/mg test item (calculated according to equation given in the guidelines) and on the performed 14-d preliminary test. During the performance of the test the test solutions will be mixed e.g. by mechanical stirring to ensure a good dispersion.
Test performance:
No unusual observations reported during the test.
Key result
Parameter:
% degradation (O2 consumption)
Value:
-4.8
Sampling time:
28 d
Details on results:
The total oxidised nitrogen concentration was below the LOQ in the measured samples during the experiment. Therefore, the calculated BOD values were not corrected for nitrification.
Under the test conditions the percentage biodegradation of TODI reached a mean of -4.8 % after 28 days based on ThODNO3.
The test item can be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is remvoval of 60% ThODNO3.
Key result
Parameter:
BOD5
Value:
>= 0 - <= 0.04 other: mg O2/mg test item (after 7 days, not 5 days)
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Under the test conditions the percentage biodegradation of TODI reached a mean of -4.8 % after 28 days based on ThODNO3. Therefore the test item can be considered to be not ready biodegradable.
Executive summary:

The ready biodegradability of test item TODI in a Closed Bottle Test over a period of 28 days was investigated. 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.

Under the test conditions the percentage biodegradation of TODI reached a mean of -4.8 % after 28 days based on ThODNO3. Therefore the test item can be considered to be not ready biodegradable.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
GLP compliance:
not specified
Test material information:
Composition 1
Key result
Parameter:
other: BOD
Value:
> 0 - <= 6.4
Remarks on result:
other: The average percent biodegradability of estimated from BOD and residual test substance were respectively of 0% and 6.4%. MDI and its reaction products with water are not degraded by microorganisms.
Validity criteria fulfilled:
not specified
Interpretation of results:
not inherently biodegradable
Conclusions:
MDI and its reaction products with water are not degraded by microorganisms. This is in line with the results found for TODI.
Executive summary:

MDI and its reaction products with water are not degraded by microorganisms. This is in line with the results found for TODI.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1981-10-16 to 1981-12-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
other: Japan: Guidelines for Biodegradation Testing of Chemicals by microorganisms etc.
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
Principles of method if other than guideline:
This test was conducted as Existing Chemicals Survey Program by the Japanese Government.
GLP compliance:
no
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Standard activated sludge: Sludge was collected from no fewer than ten sites in Japan. The sludge was prepared and controlled in the testing laboratory according to the parameters (preparation method, temperature, pH, etc.) stated in the guideline.
Duration of test (contact time):
28 d
Initial conc.:
100 other: ppm
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Test temperature: 25 °C
- No. / sample / sludge (ppm)
1 / control blank ( - ppm) / 30
2 / sludge+sample (100 ppm) / 30
3 / sludge+sample (100 ppm) / 30
4/ water+sample (100 ppm) / -
5 / sludge+sample (100 ppm) / 30
6 / aniline (100 ppm) / 30

The following six bottles were prepared.
- Bottle 4: test substance (TODA) in water at 100 mg/L
- Bottle 2, 3 and 5: test substance (TODA) in mineral medium at 100 mg/L
- Bottle 6: reference compound (aniline) in mineral medium at 100 mg/L
- Bottle 1: mineral medium only.
Bottles 2, 3 and 5 (TODA suspension), 6 (aniline) and 1 (inoculum blank) with a small volume of the inoculum to give a concentration of 30mg/L suspended solids. No inoculum was added to Bottle 4 which serves as an abiotic control.
Reference substance:
aniline
Preliminary study:
Not described.
Key result
Parameter:
% degradation (O2 consumption)
Value:
3
Sampling time:
28 d
Parameter:
other: HPLC method
Value:
6
Sampling time:
28 d
Results with reference substance:
% degradation of aniline (7th day): 61%

Under the test conditions the percentage biodegradation of TODA reached 3% after 28 days.

% degr. = (BOD – B)TOD×100= (6.1 mg - 3.7 mg)90.3 mg×100 = 2.7 % -> 3 %

Whereby,

BOD – B  = (No.2 + No.3 + No.5)3 -No.1 =  (4.4 mg +4.4 mg+9.5 mg)/3-3.7mg = 2.4 mg

TOD = 30.0×3.01= 90.3 mg

C14H16N2+ 20 O2  →14CO2+ 8H2O+2NO2

 20×O2C14H16N2 640.0/212.3 = 3.01

% degr. for aniline (7th day): 61%

 

HPLC analytical result: main test 4 weeks result

Sample name

Injection volume

(μL)

Peak height of Sample (A) (mm)

Concentration (C)

(μg/mL)

Residual volume(E)

(mg) diluted to 100mL

Residual volume(mg)

Average (F)

% degr.

Standard 300ppm

10

177.7

 

 

 

6

No.4 Water+sample

173.9

294

29.4

29.4

No.2 Sludge+sample

165.9

280

28.0

27.7

No.3 Sludge+sample

168.0

284

28.4

No.5 Sludge+sample

158.6

268

26.8

No.1 Sludge blank

 (Control blank )

N.D.

0

 

 

(A): Peak height(mm)

(C): ratio of Standard 300ppm

(E): (C)×1001000

(G): % degr.= (29.4- 27.7)29.4×100 = 6 %

 

HPLC analytical result: Recovery test

Sample name

Injection volume

(μL)

Peak height of Sample (A) (mm)

Concentration (C)(μg/mL)

Magnification

Rate (dilution concentration)

Residual volume(E)

(mg)

diluted to 100mL

Residual volume(mg)

Average (F)

% Recovery

(G)

Water+sample-1

10

158.5

275

1

27.5

28.2

94

Water+sample-2

165.8

288

1

28.8

Sludge+sample-1

156.0

271

1

27.1

27.2

91

Sludge+sample-2

156.9

272

1

27.2

Standard 300ppm

172.9

 

 

 

 

 

(A): Peak height (mm)

(C): ratio of Standard 300ppm

(E): (C)×1001000

(G): % recovery = (F)30.0×100

Under the test conditions the percentage biodegradation of TODA reached 6% by HPLC after 28 days.

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Under the test conditions the percentage biodegradation of TODA reached 3 % based on TOD and reached 6 % by HPLC after 28 days. Therefore the test item can be considered to be not ready biodegradable.
Executive summary:

The ready biodegradability of the degradation product ( 4,4'-bi-o-toluidine) of TODI was investigated over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item aniline was tested simultaneously under the same conditions as the test item.

Under the test conditions the percentage biodegradation of TODA reached 3% based on TOD and reached 6% by HPLC after 28 days. Therefore the test item can be considered to be not ready biodegradable.

 

Description of key information

TODI can be considered to be not ready biodgradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

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.

Under the test conditions the percentage biodegradation of TODI reached a mean of -4.8 % after 28 days based on ThODNO3. Therefore the test item can be considered to be not ready biodegradable.

This result is supported by a study performed with 4,4'-MDI according to OECD Guideline 302 C (Inherent Biodegradability). Here, the average percent biodegradability of MDI and resulting compounds in water were 0% and 6.4%, respectively.

Furthermore, the ready biodegradability of TODI's hydrolysis product ( 4,4'-bi-o-toluidine) TODA was investigated over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item aniline was tested simultaneously under the same conditions as the test item. Under the test conditions the percentage biodegradation of TODA reached 3% based on TOD and reached 6% by HPLC after 28 days. Therefore the test item can be considered to be not ready biodegradable.

The analogue substance, methylenediphenyl diisocyanate, was determined to be not inherently biodegradable in a modified MITI test according to OECD 302C.