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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: screening test, other
Type of information:
(Q)SAR
Adequacy of study:
key study
Study period:
October 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
The individual estimation program BIOWIN and/or its underlying predictive methods and equations have been described in numerous journal articles in peer-reviewed technical journals. Due to limited laboratory space to conduct this testing, it is not possible to complete the testing by the required 2018 deadline. As a worst case, the substance is deemed to not be “readily biodegradable” on the basis of QSAR modelling.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The Biodegradation Probability Program (BIOWIN) estimates the probability for the rapid aerobic biodegradation of an organic chemical in the presence of mixed populations of environmental microorganisms. Estimates are based upon fragment constants that were developed using multiple linear and non-linear regression analyses. Experimental biodegradation data for the multiple linear and non-linear regressions were obtained from Syracuse Research Corporation's (SRC) data base of evaluated biodegradation data (Howard et. al., 1987).
GLP compliance:
no
Inoculum or test system:
other: Not applicable; QSAR derivation.
Details on inoculum:
Not applicable
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction
Details on results:
Biowin1 (Linear Model Prediction): 1.1104 - Biodegrades Fast
Biowin2 (Non-Linear Model Prediction): 0.9699 - Biodegrades Fast
Biowin3 (Ultimate Biodegradation Timeframe): 2.2782 - Weeks-Months
Biowin4 (Primary Biodegradation Timeframe): 3.1539 - Weeks
Biowin5 (MITI Linear Model Prediction): 0.0919 - Not Readily Degradable
Biowin6 (MITI Non-Linear Model Prediction): 0.0391 - Not Readily Degradable
Biowin7 (Anaerobic Model Prediction): -1.2776 - Does Not Biodegrade Fast

On the basis of weight of evidence from the derived QSAR Results, the substance is proposed to be not readily biodegradable.

No other information available.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The outcome generated from the seven models used in the BIOWIN program results in the conclusion that LOWINOX® 22IB46 is not readily biodegradable.
Executive summary:

The ready biodegradablity of the the substance was estimated by inputting the SMILES code into the QSAR tool, US EPA On-Line EPI Suite™ BIOWIN™ v4.10. On the basis of weight of evidence from the derived QSAR Results, the substance is proposed to be not readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
3 substances are available for read across
Adequacy of study:
weight of evidence
Justification for type of information:
The substance is a member of a group of substituted phenols which share similar characteristics across the group. Please see attached justification for read across.
Reason / purpose for cross-reference:
other: QSAR data
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
Read Across to Category source.
Deviations:
no
Key result
Parameter:
% degradation (CO2 evolution)
Value:
<= 12
Sampling time:
28 d
Remarks on result:
other: Read across to similar substances.
Remarks:
Maximum % degradation of read across substances is 12%; therefore <=12% is assigned
Details on results:
Substance is read across to other similar substances. The highest % degradation of these substances is 12%; hence it is proposed that the substance would show <=12% degradation. The substance is not considered to be readily biodegradable.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
On the basis of read across to similar substances, the substance is deemed to be not readily biodegradable.
Executive summary:

On the basis of read across to similar substances, the substance is deemed to be not readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 29, 1995 to July 28, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP and reported with a GLP certificate.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
STUDY SYSTEM: Inoculum of the aqueous phase of non adapted activated sludge
Reasons for the selection of the study system: Activated sludge from the sewage plant at Hildesheim is well suited as it comprises mostly municipal sewage and hardly industrial chemical waste.
Source: Municipal sewage treatment plant, Hildesheim
Pre-treatment: The activated sludge is maintained in an aerobic condition by aeration for four hours and then homogenized with a mixer.
The sludge is filtered and the filtrate is subsequently used to initiate inoculation.
Duration of test (contact time):
28 h
Initial conc.:
35 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
FUNCTIONAL CONTROL: sodium acetate, puriss. (Fa. Flukal. Batch 317053/1993
Replicates: single
Test concentration: 35 (mg/L)
ThCO2: 1.07 (mg CO2/mg)
TOC (calculated): 10.2 (mg C/L)
Purity: > 99 (%) (acc. safety data sheet of the manufacturer)
Test of identity: aggregate state, consistency and colour of the substance according to the information of the manufacturer. The substance was identified on February 24. 1995.

TEST SUBSTANCE: Lowinox WSP
Replicates: double
Test concentration: 15 (mg/L)
ThCO2: 3.03 (mg CO2/substance)
Molecular formula: C29H40O2
TOC (calculated): 12.4 (mg C/L); 0.827 (mgC/mg)

BLANKS: nutrient solution and inoculum
Replicates: double

TOXICITY CONTROL: not necessary, this was checked with the result: no toxicity in the test concentration (OECD 209)

PROCEDURE
Duration: 28 (d)
Frequency and duration of the application: once per setting up over 28 (d)
Test vessels: 5000 (mL), brown glass
Volume of the test medium: 3000 (mL)
Test medium: mineral nutrient solution acc. to OECD 301 B

PERFORMANCE OF THE TEST
A test of identity was made with the test substance on May 30, 1995.
The theoretical CO2 production (ThCO2) was calculated basing on the molecular formula and a test of bacterial toxicity acc. to OECD 209 was carried out on June 26, 1994 with the concentration 20 (mg/L) (NON-GLP-state).
The following test solutions were prepared in 5 L brown glass bottles as incubation vessels:
- one incubation vessel for the reference compound (R1)
- two incubation vessels for monitoring the activity of the inoculum (K1, K2)
- two incubation vessels for the test compound concentration (P l' P 2)
The necessary amounts of nutrient media and inoculum were placed in each of the incubation vessels. The vessels were then connected to the system for the production of CO2 free air and aerated for 24 h. After 24 (hl the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles.
The test and reference substance concentrations were placed in the incubation vessels, the vessels made up to 3 L with aqua bidest. and connected to the system for the production of CO2 free air.
Incubation took place over a period of 29 days in a temperature range between 22 ± 2 (°C) in a water bath.
On the 28th day 1 mL concentrated HCI was added to each of the vessels. Aeration was continued for a further 24 h and on the 29th day the quantity of CO2 released was determined.

TYPE AND FREQUENCY OF MEASUREMENTS
The temperature of the water bath was measured once every working day.
Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in a basic solution. Back titration of the residual Ba(OH)2 with 0,05 N HCI was carried out three times a week during the first ten days and thereafter twice weekly.
At the beginning and at the end of the test the COD of the test substance solutions was determined.
On the 28th day the pH of all solutions was measured prior to acidification.
Reference substance:
acetic acid, sodium salt
Test performance:
The study was performed acc. to OECD 301 8 and GLP Guidelines.
The quality criteria were full filed according to the guideline:
- The total CO2 production in the blank at the end of the test was less than 40 (mg/L).
- The percentage degradation of the functional control reached the pass level of ≥ 60 (%) before day 14.
- The test temperature remained within the range 22 ± 2 (°C).
- The difference of extremes of replicate values of the test substance at the end of the test was less than 20 (%).
Key result
Parameter:
% degradation (CO2 evolution)
Value:
10 - 12
Sampling time:
28 d
Details on results:
TEST OF IDENTIFICATION
The identity of the test substance was checked. Agreement was ascertained with regard to the state, consistency, colour, melting-point, batch number and the name of the test substance given by the sponsor.

CARBON CONTENT OF THE TEST SUBSTANCE
The ThCO2-production was calculated to be 3.03 (mgCO2/mg substance).
The carbon content was calculated on the basis of the molecular formula and the ThCO2
On this basis the test concentration of 15 (mg/l), with a carbon content of 12.4 (mgC/L) was selected.

TOXICITY TO BACTERIA
There was no bacterial toxicity in the concentration 20 (mg/L).

CO2-PRODUCTION AND BIODEGRADABILITY
The total quantity of CO2 (gross CO2 production) generated by the blank, test and reference samples is the sum of the values of all individual determinations.
For calculation the net CO2 production in test and reference samples the amount of CO2 produced was corrected for endogenous CO2 production in the control groups.
The biological degradation is calculated from the ratio theoretical CO2 production to net CO2 production.
The total amount of CO2 produced in 28 days was analysed by titration in 12 measurements.

RESULTS OF THE CONTROL GROUP
In the control group a maximum quantity of 22.6 (mg CO2/ L) was formed (quality criteria: 0: 40 (mg CO2/L) after 28 days).

RESULTS OF THE FUNCTIONAL CONTROL
The functional control came to a maximum of 100 (%) biodegradabilty after 28 days.
The pass level > 60 [%] was reached by day 6 (quality criteria: degradation > 60 [%] after 14 d).

RESULTS OF THE TEST SUBSTANCE
In the test concentration samples a mean biodegradation rate of 11 (%) was reached by day 28. The difference of extremes of replicate values of the test substance at the end of the test was less than 20 [%].

COURSE AND STAGES OF BIODEGRADATION OF REFERENCE AND TEST COMPOUND
In case of the test substance within 25 days only a degradation rate of 10 (%) was achieved in the test concentration 15 [mg/L].
After 28 days the biodegradability was found to be 10-12 [%].
The pass level of ≥ 60 (%) had not been reached neither in a 10 d-window nor after 28 days. The test substance must be regarded to be not ready biodegradable.
On the 28th day the pH of all solutions was measured prior to acidification (July 27, 1995).
The COD of all solutions was determined at the beginning (June 29, 1995) and at the end of the test (July 28, 1995).
The test temperature was constant at 24 [°C].
Key result
Parameter:
COD
Value:
11 - 28 other: mg/L
Results with reference substance:
In the case of the functional control. the adaptation phase transformed to a degradation phase after 2 days (degradation >10 [%]). The pass level > 60 [%J is reached by day 6.

Total CO2-production and biodegradability after 28 [d]

CO2-production after 28 [d]

Control mean value

Functional control

35 [mg/L]

Test substance

15 [mg/L]

Gros

[mg/3 L]

[mg/L]

67.8

22.6

191.4

63.8

84.3

28.1

81.3

27.1

Net

[mg/3 L]

[mg/L]

--

--

123.6

41.2

16.5

5.5

13.5

4.5

ThCO2

[mg/3 L]

[mg/L]

--

--

112.4

37.5

136.4

45.5

136.4

45.5

Biodegr.

after 28

[%]

[d]

--

100

12

10

 

CO2-production and biodegradability for all determination points in the control and functional control samples

Test day

Date

Control mv

Functional contr. 35 [mg/L]

mg CO2

mg CO2

Degr.

Gros

Net

[%]

1

30.06.

2.5

6.4

3.9

3

4

03.07.

10.2

56.2

46.0

41

6

05.07.

15.2

90.9

75.7

67

8

07.07.

19.9

121.5

101.6

90

11

10.07.

26.2

137.8

111.6

99

15

14.07.

35.3

148.7

113.4

100

18

17.07.

41.6

157.9

116.3

100

21

20.07.

47.2

165.9

118.7

100

25

24.07.

54.6

174.5

119.9

100

28

27.07.

60.7

183.1

122.4

100

29

28.07.

65.8

189.9

124.1

100

29

28.07.

67.8

191.4

123.6

100

mv = mean value

 

CO2-production and biodegradability for all determination points in the control group and test substance samples

Test day

Date

Control mv

Test substance 15 [mg/L]

Test substance 15 [mg/L]

mg CO2

Degr.

mg CO2

Degr.

mg CO2

Gros

Net

[%]

Gros

Net

[%]

1

30.06.

2.5

4.8

2.3

2

3.5

1.0

1

4

03.07.

10.2

15.7

5.5

4

12.7

2.5

2

6

05.07.

15.2

21.6

6.4

5

20.7

5.5

4

8

07.07.

19.9

26.3

6.4

5

27.0

7.1

5

11

10.07.

26.2

34.4

8.2

6

34.0

7.8

6

15

14.07.

35.3

45.2

9.9

7

43.8

8.5

6

18

17.07.

41.6

51.8

10.2

7

50.4

8.8

6

21

20.07.

47.2

58.6

11.4

8

57.7

10.5

8

25

24.07.

54.6

68.8

14.2

10

66.3

11.7

9

28

27.07.

60.7

77.1

16.4

12

74.2

13.5

10

29

28.07.

65.8

82.8

17.0

12

79.5

13.7

10

29

28.07.

67.8

84.3

16.5

12

81.3

13.5

10

Degr. = degradation; mv = mean value

 

Water Parameters, COD

 

Blanks

Functional control

Test substance

15 [mg/L]

15 [mg/L]

pH-value

7.68

7.57

8.00

7.57

7.57

COD [mg/L]

Start

End

 

<2

<2

 

<2

<2

 

19

<2

 

11*

25

 

11*

28

*= the test substance was water insoluble and was directly applicated in the test vessel.

 

Temperature of the water bath on days of titration

Date

30.06.

03.07.

05.07.

07.07.

10.07.

14.07.

17.07.

20.07.

24.07.

27.07.

28.07.

Temp. in water bath [°C]

24

24

24

24

24

24

24

24

24

24

24

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The pass level of ≥ 60 (%) had not been reached neither in a 10 d-window nor after 28 days.
The test substance must be regarded to be not ready biodegradable.
Executive summary:

The ready biodegradability was determined in the modified Sturm Test according to OECD Guideline 301 B, EU Method C.4-C and GLP principles from June 29, 1995 to July 28, 1995. The duration was therefore 28 days.

Test substance was the product Lowinox WSP, Batch No. 5E049. No carrier for dissolving the test substance was used.

Test facility was DR.U.NOACK-LABORATORIUM FÜR ANGEWANDTE BIOLOGIE in D-31137 Hildesheim.

The test substance concentration selected as appropriate was 15 (mg/l), corresponding to a carbon content of 12.4 (mgC/L). There was no bacterial toxicity in the test concentration.

The degradation was followed by CO2analyses, which was produced by the respiration of bacteriae.

The amount of CO2produced by the test substance is determined for the test period and calculated as the percentage of total CO2that the test substance could have theoretically produced based on carbon composition. Biodegradability is therefore expressed as a percentage ThCO2(calculated by molecular formula) and was calculated for each titration of CO2.

In order to check the activity of the study system sodium acetate was used as a functional control. The functional control reached the pass level > 60 (%) by day 6.

The biological degradation of the test substance is shown graphically in comparison with the readily degradable functional control.

Within 25 days only a mean degradation rate of 10 (%) was achieved in the test concentration 15 [mg/L].

After 28 days the biodegradability was found to be 10-12 (%).

The pass level of ≥ 60 (%) had not been reached neither in a 10 d-window nor after 28 days.

The test substance must be regarded to be not ready biodegradable.

 

Ready Biodegradability of test substance Lowinox WSP

Concentration 15 [mg/L]

Biodegradation [%]

Test duration [d]

6

15

21

28

Repl. 1

5

7

8

12

Repl. 2

4

6

8

10

 

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 June 2012 to 19 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD, EU and ISO test guidance in copliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
according to guideline
Guideline:
other: ISO 9439, 1999 and ISO 10634, 1995
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Hygroscopic: No
Volatile: No
Stability in water: Unknown
Solubility in water: Not indicated
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas','s-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.4 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (43 minutes) and the supernatant liquid was used as inoculum at the amount of 10 ml/l of mineral medium.
Reason for selection :The test has been accepted internationally for determining the 'ready' biodegradability of test substances under aerobic conditions.
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test concentration and preparation of test solutions: 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was a white powder with a purity of 99.4%. The test substance was tested in duplicate at 15 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula.
Since 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test substance bottle A: 29.0 mg; test substance bottle B: 29.1 mg and toxicity control bottle: 29.1 mg). To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and test medium, since the test substance tended to float on the water surface.

Reference Substance
Identification number: RS186
Name: Sodium acetate
Description: White powder (determined at NOTOX)
Molecular formula: CH3COONa (taken from label)
Molecular weight: 82.03 (taken from label)
Batch number: K34333668
Article number: 1.06268.0250
Purity: ≥99.0%
Expiry Date: 28 February 2015
Certified: Yes
Storage conditions: At room temperature in the dark
Supplier: Merck, Darmstadt, Germany

Reference substance concentration and preparation of test solutions: A solution of sodium acetate was prepared by dissolving 400.1 mg in Milli-RO water and making this up to a total volume of 100 ml. Volumes of 20 ml from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg sodium acetate per litre (12 mg TOC/l).

Test procedure and conditions
Test duration: 28 days (last CO2-measurement on the 29th day). During the test period the test media were aerated and stirred continuously.
Test vessels: 2 litre all-glass brown coloured bottles.
Milli-RO / Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).
Stock solutions of mineral components:
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.
Mineral medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.
Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
Synthetic air (CO2 < 1 ppm): A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).

Preparation of bottles
Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Type and number of bottles:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
Preparation: At the start of the test (day 0) test and reference substance were added to the bottles containing the microbial organisms and mineral components.
The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.
Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

Measurements and recording:
pH: At the start of the test (day 0) and on the 28th day.
Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
2 - 12
Sampling time:
28 d
Details on results:
The ThCO2 of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was calculated to be 2.99 mg CO2/mg.
The relative biodegradation values calculated from the measurements performed during the test period revealed 2 and 12% biodegradation of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.
In the toxicity control more than 25% biodegradation occurred within 14 days (33%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Results with reference substance:
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.

Notes: Except for the percentages biodegradation, all calculations are performed without rounding off. Produced CO2: negative values are expressed as 0.00 ml HCI.

 

CO2production and percentage biodegradation of the positive control substance

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)(%)

Blank (mean)

Positive control

2

48.33

39.90

8.43

9.3

9.3

11

5

47.28

24.42

22.86

25.1

34.4

40

7

46.51

35.63

10.88

12.0

46.4

54

9

47.44

40.62

6.82

7.5

53.9

63

14

45.71

39.24

6.47

7.1

61.0

71

1)Calculated as the ratio between CO2produced (cumulative and the ThCO2of sodium acetate 85.6 mg CO2/2l

 

CO2production and percentage biodegradation of the test substance (bottle A)

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)(%)

Blank (mean)

Bottle A

2

48.33

48.83

0.00

0.0

0.0

0

5

47.28

47.26

0.02

0.0

0.0

0

7

46.51

46.51

0.00

0.0

0.0

0

9

47.44

47.06

0.38

0.4

0.4

1

14

45.71

45.67

0.03

0.0

0.5

1

19

44.28

44.68

0.00

0.0

0.5

1

23

43.71

44.29

0.00

0.0

0.5

1

27

43.95

44.75

0.00

0.0

0.5

1

29

46.11

45.51

0.60

0.7

1.1

1

29

48.81

48.17

0.63

0.7

1.8

2

29

50.18

49.90

0.28

0.3

2.1

2

1)Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 86.7 mg CO2/2l

 

CO2production and percentage biodegradation of the test substance (bottle B)

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)(%)

Blank (mean)

Bottle B

2

48.33

48.79

0.00

0.0

0.0

0

5

47.28

46.98

0.30

0.3

0.3

0

7

46.51

45.95

0.56

0.6

0.9

1

9

47.44

45.91

1.53

1.7

2.6

3

14

45.71

45.10

0.60

0.7

3.3

4

19

44.28

43.15

1.13

1.2

4.5

5

23

43.71

42.45

1.26

1.4

5.9

7

27

43.95

42.98

0.97

1.1

7.0

8

29

46.11

43.98

2.13

2.3

9.3

11

29

48.81

47.58

1.23

1.3

10.7

12

29

50.18

50.17

0.01

0.0

10.7

12

1)Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 87.0 mg CO2/2l

 

CO2production and percentage biodegradation of toxicity control

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)(%)

Blank (mean)

Toxicity control

2

48.33

41.84

6.49

7.1

7.1

4

5

47.28

21.68

25.60

28.2

35.3

20

7

46.51

35.05

11.46

12.6

47.9

28

9

47.44

39.53

7.91

8.7

56.6

33

14

45.71

50.00

0.00

0.0

56.6

33

1)Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test substance and positive control: 172.6 mg CO2/2l (ThCO2test substance: 87.0 mg CO2/2l + ThCO2sodium acetate: 85.6 mg CO2/2l)

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

Determination of ‘ready’ biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol.

 

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C and the ISO International Standard 9439, 1999 and ISO Standard 10634, 1995. The study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was a white powder with a purity of 99.4%. The test substance was tested in duplicate at 15 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula. The Theoretical CO2production (ThCO2) of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was calculated to be 2.99 mg CO2/mg.

 

The study consisted of six bottles:

-2 inoculum blanks (no test substance),

-2 test bottles (6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol),

-1 positive control (sodium acetate) and

-1 toxicity control (6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol plus sodium acetate).

 

Since 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and test medium, since the test substance tended to float on the water surface. Test duration was 28 days (last CO2-measurement on the 29thday).

 

The relative biodegradation values calculated from the measurements performed during the test period revealed 2 and 12% biodegradation of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met. In the toxicity control, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was found not to inhibit microbial activity.

 

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol was designated as not readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only short abstract is available, but the report was approved by the Japanese government and was cited in OECD SIDS Dossier (2000)
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
other: MITI (I) method (1974)
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
not specified
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
sludge sampling sites:
Sludge sampling were made at 10 places in Japan.
Mixing of activated sludge:
5 L of the filtrate of the supernatent of an activated sludge in the present use was mixed with 500 mL of the filtrate of the supernatent of a newly collected sludge respectively and the mixture was cultured at pH 7.0 +-1.0 under the sufficient aeration (prefiltered open air was used).
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
not reported
Reference substance:
other: Aniline was used as reference substance.
Preliminary study:
not appliable
Test performance:
not applicable
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Value:
1
Sampling time:
28 d
Remarks on result:
other: (based on HPLC analysis for the parent)
Details on results:
The test substance was not ready biodegradable.
Results with reference substance:
not reported

no remarks

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance was not readily biodegradable.

Executive summary:

DBMC was tested in a biodegradation test under the conditions of the MITI (I) method. The test resulted in 0% degradation after 28 days (1% based on HPLC analysis) and the test substance was classified as not ready biodegradable.

Reference: MITI, 1992

Description of key information

The ready biodegradability of the substance is read across to other similar substances.  The highest % degradation of these substances is 12%; hence it is proposed that the substance would show <=12% degradation.  The substance is not considered to be readily biodegradable.  This is further endorsed by estimation using the SMILES code into the QSAR tool, US EPA On-Line EPI Suite™ BIOWIN™ v4.10.On the basis of weight of evidence from the derived QSAR Results and analogous substances, the substance is proposed to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information