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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:
supporting study
Study period:
2012-10-16 to 2012-11-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Species/Origin:
Aerobic activated sludge microorganisms from a domestic waste water treatment plant were supplied by a municipal sewage treatment plant Rossdorf, Germany.
Conditioning:
The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap 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 1.5 g dry material per litre were mixed with test water (see 6.5). The activated sludge was preconditioned overnight. This suspension was used for the experiment.

Duration of test (contact time):
28 d
Initial conc.:
102.4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The purpose of this study was to determine the ready biodegradability of the test item 4,4`–Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The bio-degradation 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.
This type of study is recognized by the international test guidelines and should provide a rational basis to assess the ready biodegradation properties of the test item when incubated with aerobic activated sludge.

The test flasks were incubated at 22±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. 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.

Preparation of Test Flasks:
A stock solution was prepared by dissolving 1.2495 g of test item in 5ml acetone. 100 µl of this stock solution were dropped onto silica gel (100 mg). After evaporation of the solvent (for about 24 hours) the treated silica gel (230 – 400 mesh) was directly dosed into the flasks (ISO 10634; Handley et al., 2002). The inoculum control contained untreated silica gel.

Incubation:
The closed test flasks were incubated in an acclimatised room under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

Test Duration:
28 days

Measurement of Oxygen:
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.

Temperature:
The temperature was recorded by RMT Wincontrol automatically and documented over the course of the test.

pH-Value:
pH-values were measured in toxicity control, a separately prepared test flask with test item and in separately prepared test flasks without test item (control) at test start (to prevent loss of test item in the test flasks) and in the test item and toxicity flasks at the end of the test using a pH-electrode WTW pH 340i.

Reconstituted Test Water:
Analytical grade salts were added to deionised 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 deionised water to 1000 ml volume
b) 22.5 g MgSO4 x 7 H2O filled up with deionised water to 1000 ml volume
c) 36.4 g CaCl2 x 2 H2O filled up with deionised water to 1000 ml volume
d) 0.25 g FeCl3 x 6 H2O filled up with deionised water to 1000 ml volume
In order to avoid precipitation of iron hydroxide in the stock solution d) after storage and before use, one drop of concentrated HCl per litre was added.
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 deionised water. The pH-value was 7.5 and therefore no adjustment was necessary.


Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
Biodegradation of 4,4`–Isopropylidenedi-phenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-amino-methyl-3,5,5-trimethylcyclohexylamine:
The criterion for ready biodegradability under the condi-tions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days.
The mean biodegradation after 28 days of 4,4`–Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine was 0%; the 10 day window criterion was not passed.
The mean biodegradation percentage at the end of the 28-day exposure period was 0 %.
Therefore, 4,4`–Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine is considered to be not readily biodegradable.

Validity Criteria of the Study
Inoculum Control:
The oxygen demand of the inoculum control (medium and inoculum) was 22.5 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.
pH-Value:
The pH-value of the test item flasks at the end of the test was 8.2 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.
Reference Item:
The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline.
The reference item sodium benzoate was degraded to more than 60% after 3 days of incubation.
Test Item:
Since the biodegradation did not reach the pass criterion of 60% degradation and no 10 day-window could be determined, the validity criterion was not applicable. At the end of the experiment, the difference in replicate variation was 0%.
Parameter:
COD
Value:
2.791 other: mg O2/mg test item
Results with reference substance:
Biodegradation of Sodium Benzoate:
The reference item sodium benzoate was sufficiently degraded to 84% after 14 days and to 87% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThODNH4

Time

Percentage Biodegradation1

(Days)

4,4`–Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine1

Sodium Benzoate2

Toxicity control1, 2

 

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

0

0

29

0

2

0

0

44

1

3

0

0

63

9

4

0

0

68

13

5

0

0

71

16

6

0

0

76

19

7

0

0

78

20

8

0

0

76

22

9

0

0

79

24

10

0

0

79

25

11

0

0

82

26

12

0

0

81

26

13

0

0

81

26

14

0

0

84

26

15

0

0

84

27

16

0

0

82

26

17

0

0

82

26

18

0

0

82

26

19

0

0

84

27

20

0

0

84

27

21

0

0

82

27

22

0

0

82

27

23

0

0

82

27

24

0

0

82

27

25

0

0

82

27

26

0

0

85

27

27

0

0

85

27

28

0

0

87

28

            1ThODNH4of 4,4`–Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-

            epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine:

            2.791 mg O2/mg test item
            2ThODNH4of sodium benzoate: 1.666 mg O2/mg reference item

Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily biodegradable
Conclusions:
Percentage Biodegradation:
The mean biodegradation percentage at the end of the 28-day exposure period was 0%.
Conclusion:
The degradation rate of 4,4`–Isopropylidenediphenol, oli-gomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylaminedid not reach 60% within the 10-day window and after 28 days of incubation.
4,4`–Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine is considered to be not readily biodegradable.
Executive summary:

The ready biodegradability of the test item in a manometric respirometry test was determided based on the procedures indicated by the following internationally accepted methods:

- EU method C.4 -D

- OECD method 301 F

The test item 4,4`–Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine 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.

For the test item the evaluation of biodegradation has to be based on COD determination.

The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days.

The mean biodegradation after 28 days of 4,4`–Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine was 0%; the 10 day window criterion was not passed.

Therefore, 4,4`–Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine is considered to be not readily biodegradable.

The reference item sodium benzoate was sufficiently degraded to 84% after 14 days and to 87% after 28 days of incubation, thus confirming the suitability of theaerobic activatedsludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 26% biodegradation was noted within 14 days and 28% biodegradation after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to theaerobic activatedsludge microorganisms.

This study is classified acceptable and satisfies the guideline requirements for ready biodegradability studies.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 November 2012 - 13 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • ISO Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium" (1995).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, 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 4.5 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (37 minutes) and the supernatant liquid was used as inoculum at the amount of 10 ml/l of mineral medium.

Duration of test (contact time):
28 d
Initial conc.:
16.5 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
other: Total Organic Carbon (TOC)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water
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.

- Test temperature: between 22.2 and 22.8°C.
- pH:
At t=0 d: 7.5 - 7.6
At t=28 d: 7.5 – 7.9
- pH adjusted: only before the start of the test. Adjusted using 1 N HCl (original pH 7.6-7.8)
- Aeration of dilution water: Not before the test, the test is aerated continously
- Suspended solids concentration: The concentration of suspended solids was 4.5 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (37 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium.


- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration:
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).
- Method used to create aerobic conditions:
Synthetic air (a mixture of oxygen (ca. 20%) and nitrogen (ca. 80%)) was sparged through the solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used:
CO2 was trapped in barium hydroxide solution. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul). Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 days.


SAMPLING
- Sampling frequency: Titration were made on day: 2, 5, 7, 9, 14, 19, 23, 27 and 29
- Sampling method: Titration of the whole volume of CO2-absorber

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
1
Sampling time:
29 d
Remarks on result:
other: HCl added on the 28th day (last CO2-measurement on the 29th day)
Parameter:
% degradation (CO2 evolution)
Value:
5
Sampling time:
29 d
Remarks on result:
other: HCl added on the 28th day (last CO2-measurement on the 29th day)
Details on results:
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine

In the toxicity control more than 25% biodegradation occurred within 14 days (approximately 28%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.
Results with reference substance:
The reference substance was biodegraded by at least 60% (74%) within 14 days.
Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily biodegradable (1-5%)
Conclusions:
4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Description of key information

Ready Biodegradability of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine in a Modified Sturm Test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine had a maximum biodegradation of 5% after 28 days and therefore considered to be not readily biodegradable in the modified Sturm test. In a manometric respiratory ready biodegradation test , 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine showed no biodegradation after 28 days.