1,6-Bis(methoxybenzoyloxy)hexane

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Start: 26 September 2001 Completion : 25 October 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study includes data generated according to generally valid and internationally accepted testing guidelines and performed according to GLP. The study procedure was based on EEC directive 92/69, C.4-C, December 1992, and OECD guideline No. 301 B July 17, 1992.

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

GLP compliance:

yes

Remarks:

GLP STATEMENT DATES: 23/11/2001 and 26/11/2001

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):
Activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands.

- Treatment
The sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 3.6 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (30-90 minutes) and the liquid decanted for use as inoculum at the amount of 10 ml/I of mineral medium.

Duration of test (contact time):

28 d

Initial conc.:

ca. 17.5 mg/L

Based on:

other: test material (corresponding to 12 mg/L of TOC based on the molecular formula)

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ionexchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).

A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H20; 0.50 g NH4CI dissolved in 1 L Milli-Q water, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in 1 L Milli-Q water
C) 36.40 g CaCl2.2H2O dissolved in 1 L Milli-Q water
D) 0.25 g FeCl3.6H2O dissolved in 1 L Milli-Q water

1 L mineral medium contains: 1 O ml of solution (A),
1 mL of solutions (B) to (D) and Milli-RO water.

- Test temperature: 21.5 - 23°C.
- pH: 7.4 - 7.6
- Suspended solids concentration: 3.6 g/L
- Aeration of dilution water: Yes
- Continuous darkness: Yes (dark brown-coloured bottles used)


TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles.
- Number of culture flasks/concentration:
containing test substance and inoculum (2 bottles).

- Method used to create aerobic conditions:
A mixture of oxygen (21%) and nitrogen (79%) was led 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 CO2-free air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).

- Test performed in closed vessels: sealed vessel to prevent absorption of C02 from the air.

- Details of trap for CO2 used:
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.

DETERMINATION OF CO2:
- Experimental CO2 production:
The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCI. Titrations were made every second or thirdday during the first 10 days, and thereafter at least every fifth day until the 28th day.

- Theoretical CO2 production:
The theoretical CO2 production was calculated from the molecular formula.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Containing only inoculum (2 bottles)
- Positive control: containing reference substance (ca. 40 mg/I sodium acetate, TOC= 12 mg/I) and inoculum (1 bottle).
- Toxicity control: containing test substance, reference substance and inoculum (1 bottle).

Reference substance:

acetic acid, sodium salt

Test performance:

The results of the biodegradation test were considered to be valid when:
- the total CO2 evolution in the inoculum blank at the end of the test did not normally exceed 40 mg/I. If values greater than 70 mg C02/L are obtained, the data and experimental technique should be examined critically.
For the calculation of the total CO2 evolution in the inoculum blank the volume of HCI needed for the titrating of the remaining Ba(OH)2 of the inoculum blanksand for fresh Ba(OH)2 was used.

- the difference of duplicate values for the %-degradation of the test substance at the plateau, at the end of 1the test or at the end of the 10-day window, as appropriate, was less than 20.

- the percentage degradation of the reference substance reached the level for ready biodegradability (60%) by14 days.

Acceptability of the test:
- The positive control substance was degraded at least 60% within 7 days
- The total CO2 release in the blank reached a total value of 49 mg CO2 per 2 litres of medium
- The difference of duplicate values for %-degradation of SET AFIX X 11342 was always less than 20

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

Parameter:

% degradation (CO2 evolution)

Value:

12

Sampling time:

28 d

Details on results:

Biodegradation:
The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of SET AFIX X 11342 in test bottle A and 12% degradation in test bottle B. Thus, the criterion for ready biodegradability (at least 60% degradation within 28 days, reached within 10 days of biodegradation exceeding 10%) was not met.

SET AFIX X 11342 was not readily biodegradable under the conditions of the modified Sturm test presently performed.

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

Results with reference substance:

The positive control contained 80.3 mg sodium acetate (ThCO2= 1.07 mg CO2/mg) resulting in a theoretical CO2 production following complete degradation of 85.9 mg per 2 litres.

The toxicity control contained 80.3 mg sodium acetate and 35.3 mg SETAFIX X 11342 in 2 litres of test medium. Hence, the theoretical CO2 production following complete degradation of SET AFIX X 11342 plus sodium acetate was 17 4.5 mg per 2 litres.

Validity criteria fulfilled:

yes

Remarks:

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

Interpretation of results:

other: Not readily biodegradable under the conditions

Conclusions:

SET AFIX X 11342 was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

SET AFIX X 11342 was tested for its ready biodegradability in the carbon dioxide (C02) evolution test (modified Sturm test) at 35 mg per 2 litres, corresponding to 12 mg TOC/L.

The study procedure was based on EEC directive 92/69, C.4-C, December 1992, and OECD guideline No. 301 B July 17, 1992.

The Theoretical CO2 production (ThCO2) of SETAFIX X 11342 was calculated to be 2.51 mg CO2/mg.

SET AFIX X 11 342 appeared to be hardly soluble in water in a pre-test at 1 g/L. Therefore, weighed amounts of SETAFIX X 11342 were added to weighing bottles (test substance bottle A: 34.9 mg; test substance bottle B: 35.1 mg and toxicity control bottle: 35.3 mg). Approximately 10 ml of milli-RO water was added to each weighing bottle and after vigorous shaking the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test.

The relative degradation values calculated from the measurements performed during the test period revealed rio significant degradation of SET AFIX X 11342 in test bottle A and 12% degradation in test bottle B. Thus, the criterion for ready biodegradability (at least 60%

degradation within 28 days, reached within 10 days of biodegradation exceeding 10%) was not met.

In the toxicity control SET AFIX X 11342 was found to be not inhibitory on microbial activity.

Since all acceptability criteria prescribed by the protocol were met, this study was considered to be valid.

In conclusion, SETAFIX X 11342 was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

other: Brief Memorundum

Adequacy of study:

supporting study

Study period:

Not available

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Only a brief memorandum discussing data. No sufficient experimental details are described

Executive summary:

This memo discusses strong indications that the biodegradation of Setafix X 11342 in ready biodegradability tests is determined by the rate of dissolution of the test substance.

Reference 3

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 February 2011 - 18 July 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study includes data generated according to generally valid and internationally accepted testing guidelines and performed according to GLP. The study was based on the OECD Guideline for Testing of Chemicals No. 302 C: "Inherent Biodegradability: Modified MITI Test (II}", adopted May 12, 1981.

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Deviations:

yes

Remarks:

: Did not affect integrity of results

GLP compliance:

yes (incl. QA statement)

Remarks:

Dates of Inspection: 20 April 2009 - 5 August 2009 Date of Signature: 14 December 2009

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Activated sludge, micro organisms from a domestic waste water treatment plant was supplied by the sewage plant Darmstadt, Germany.

- Conditioning (Pretreatment):
The 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 again centrifuged. 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 determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 1.8 g dry material per litre was mixed with test water and then aerated until use.

- Concentration of sludge: 100 mg/L
[i.e. activated sludge 30 mg (dry base) concentration in the test vessel (approximately 5 mL of a stock suspension of 1.8 g/L suspended solids); the final volume was 300 mL]

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

: BOD Analysis

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

: HPLC Analysis

Details on study design:

TEST CONDITIONS
- Type and Size of test Unit: Manometric test system with test flasks of approximately 510 mL volume containing 300 mL test liquid

- Composition of medium(Test Water/Basal Culture Medium):
In deionised water analytical grade salts were added to prepare the following stock solutions:
a) 8.5 g KH2P04, 21.75 g K2HP04, 22.2 g Na2HP04 x 2 H20, 1. 7 g NH4Cl filled up with deionised water to 1000 mL volume.
b) 22.5 g MgS04 x 7 H20 filled up with deionised water to 1000 mL volume
c) 27.5 g CaCli filled up with deionised water to 1000 mL volume
d) 0.25 g FeCh x 6 H20 filled up with deionised water to 1000 mL volume

- Solubilising agent (type and concentration if used): In order to avoid precipitation of iron hydroxide in the stock solution d) immediately before use, one drop of concentrated HCl per litre was added.

- Test temperature: 25 °C
- pH: 6.9 - 7.5 (measured at the start of the test); 7.0-8.6 (measured at the end of the test)
- pH adjusted: No
- Aeration of dilution water: Yes
- Suspended solids concentration: 30 mg/L
- Continuous darkness: Yes

TEST SYSTEM, CONTROL AND BLANK SYSTEMs (Test Solutions)

- Test Item:
Basal culture medium - 295mL
Activated sludge - 30 mg (dry basis)
Test item - 9 mg

- lnoculum Control:
Basal culture medium - 295mL
Activated sludge - 30 mg (dry basis)

- Procedure Control:
Basal culture medium - 295mL
Activated sludge - 30 mg (dry basis)
Sodium Benzoate - 30 mg

- Abiotic Control:
Deionised water - 300mL
Test item - 9mg

- Toxicity Control:
Basal culture medium - 295mL
Activated sludge - 30 mg (dry basis)
Test item - 9 mg
Sodium Benzoate - 30 mg

- Measuring equipment:
Measurements of Oxygen Depletion (BOD-Meter): The change of pressure in the test flasks was measured automatically by means of a manometric method (BSB/BODSensor-System, Aqualytic Dortmund, Germany) about 12 times a day in a 28 day incubation period. The device consists of reaction vessels with CO2 absorber

- Test performed in closed vessels due to O2 consumed and CO2 released: Closed System

- Principle:
The sealed measuring system is not affected by barometric air-pressure fluctuations. The carbon dioxide which is released as a result of the metabolic processes of the microorganisms enters the gaseous atmosphere and is absorbed by a CO2 absorber. CO2 is produced and absorbed while O2 is
consumed and therefore pressure decreases in the vessels and this decrease is recorded by the measuring heads.
CO2 produced during test item breakdown can be calculated from the consumed O2; based on the stoichiometry of O2 consumption and CO2 production during respiration (1 mg of consumed O2 corresponds to 1.3 7 5 mg of respired CO2).

SAMPLING
- Sampling frequency: Days 0, 7, 14, 21 and 28
- Sample storage before analysis: The samples of the aqueous phase and solid phase we stored in the refrigerator at 4 ± 4°C in the dark.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

None

Test performance:

The degradation of the reference item sodium benzoate was 68.4 % within 4 days of exposure and therefore within the limit (>40% after 7 days and >65% after 14 days) given by the test guideline.

The experiment is considered to be valid since the reference item sodium benzoate fulfilled the validity criterion of biodegradation.

The recovery of the abiotic control was 76.6% at test start and therefore higher than 10% and therefore within the limit given by the test guideline.

Parameter:

% degradation (O2 consumption)

Value:

40.8

Sampling time:

28 d

Remarks on result:

other: (mean value) Based on BOD-measurements

Parameter:

% degradation (test mat. analysis)

Value:

59.7

Sampling time:

14 d

Remarks on result:

other: HPLC-analysis of Setafix Z1077 content

Parameter:

% degradation (test mat. analysis)

Value:

65.3

Sampling time:

28 d

Remarks on result:

other: HPLC-analysis of Setafix Z1077 content

Details on results:

Results of the BOD-Measurement

Degradation ofSetafix Zl077:
Based on BOD-measurements, Setafix Zl077 was degraded to 40.8% (ThOD(NH4) after 28 days of incubation.

Degradation of Toxicity Control (with Sodium Benzoate):
In the toxicity control, containing both the test item and the reference item sodium benzoate, the degradation was 68.9% after 14 days and 68.4% after 28 days of incubation.

Degradation of Abiotic Control:
In the abiotic control, no important biodegradation was determined.


Results of the HPLC Analysis of Setafix Zl 077

Primary Degradation of Setafix Z 1077:
The test item was found in the solid phase at all samplings days. After 7 and 14 days, about 50.3% and 40.3% of the initial concentration was found in the solid phase. At day 28 after test start, about 34.8% of Setafix Z1077 was still found in the solid phase of the sludge. The HPLC-analysis of Setafix Z1077 content showed a primary degradation of 59.7% after 14 days and 65 .3 % after 28 days of incubation.

Primary Degradation of Abiotic Control:
The primary degradation of the abiotic control was 37.6% after 28 days of incubation.

Primary Degradation of Toxicity Control (with Sodium Benzoate):
In combination with sodium benzoate the primary degradation of Setafix Z I 077 was found to be about 88.6% after 28 days.

Results with reference substance:

Degradation of Reference Item Sodium Benzoate:

The reference item sodium benzoate was degraded to 78.0% after 14 days and 80.4% after 28 days of incubation. The validity criterion of more than 40% degradation within 7 days respectively more than 65% degradation within 14 days was fulfilled.

Validity criteria fulfilled:

yes

Remarks:

Fullfilled Validity Criteria of the Study

Interpretation of results:

inherently biodegradable

Conclusions:

It can be concluded that Setafix Z1077 will be easily transformed in an aqueous system to hydrolysis products and not used in bacterial metabolism because of an inherent biodegradation.
The potential of the activated sludge is present because Setafix Z1077 was ultimate biodegraded by 40.8% at the end of the study. Setafix is considered to be partially biodegradable.

Executive summary:

The purpose of this study was to determine the biodegradability of the test item Setafix Z1077 in accordance with the OECD Guideline for Testing of Chemicals No. 302 C: "Inherent Biodegradability: Modified MITI Test (II)", adopted May 12, 1981.

After 28 days of incubation, the test item Setafix Z1077 was found to be biodegradable by about 40.8% (mean value) based on BOD-measurement. The biodegradation of the reference item sodium benzoate was clearly in the range given by the test guideline. In the toxicity control, the degradation of the reference item was not affected by the test item.

The test item was analysed in the complete test solution using the aqueous phase and a acetonitrile-extraction for the solid phase. A primary degradation of Setafix Z1077 of 65.3% was found after 28 days of incubation in test item groups. In the toxicity controls the primary degradation 88.6% after 28 days. In the abiotic controls there was about 37.6% primary degradation and therefore an indication for a partial hydrolysis due to abiotic processes. The test item is considered to hydrolyse easily and all of the inserted amount

was found in the solid phase.

The presence of the reference item sodium benzoate did not affect the degradation of Setafix Z1077, and Setafix Z1077 also did not affect the breakdown of the reference item.

It can be concluded that Setafix Z1077 will be easily transformed in an aqueous system to hydrolysis products and not

used in bacterial metabolism because of an inherent biodegradation.

The potential of the activated sludge is present because Setafix Z1077 was ultimate biodegraded by 40.8% at the end of the study. Setafix is considered to be partially biodegradable.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Initiation date of the study: 01-09-2004 Completion date of the study: 06-10-2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This test has been performed according to slightly modified EEC, OECD and ISO Test Guidelines (OECD, 1992; EEC 1984; ISO, 1994) in compliance with the OECD principles of Good Laboratory Practice.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

yes

Qualifier:

according to guideline

Guideline:

other: ISO 10707:1994: Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)

Deviations:

yes

GLP compliance:

yes

Remarks:

GLP Compliance Statement Dates: 01/11/2004 and 02/11/2004

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Secondary activated sludge was obtained from the WWTP Nieuwgraaf in Duiven, The Netherlands (01.09.2004). The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic wastewater.

A minor deviation of the test procedures described in the guidelines was introduced: instead of an effluent/extract/mixture, activated sludge was used as an inoculum.

- Pretreatment:
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to a concentration of 2 mg DW/L in the BOD bottles.

- Type and size of filter used:
The dry weight (DW) of the inoculum was determined by filtrating 50 ml of the activated sludge over a preweighed 12 μm Schleicher and Schull filter. This filter was dried for 1.5 hours at 104°C and weighed after cooling. DW was calculated by subtracting the weighed filters and by dividing this difference by the filtered volume.

Duration of test (contact time):

28 d

Initial conc.:

0.75 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

: By HPLC analysis

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

: (BOD/ThOD) calculations

Details on study design:

TEST CONDITIONS
- Composition of medium:
The nutrient medium of the Closed Bottle test contained per liter of deionized water: 8.5 mg KH2P04, 21.75 mg K2HP04, 33.3 mg Na2HP04·2H20, 22.5 mg MgS04·7H20, 27.5 mg CaCl2, 0.25 mg FeCl3·6H20.

- Solubilising agent (type and concentration if used):
Setafix X 11342 is a poorly soluble substance in water and therefore the test substance was first dissolved in dichloromethane (1 g/L). The test substance in dichloromethane was added directly in the bottles. The solvent was allowed to evaporate in a ventilated hood for at least three hours

- Test temperature: 19 to 21°C
- pH: 6.7 - 6.8
- pH adjusted: No
Ammonium chloride was omitted from the medium to prevent nitrification. Due to this omission the pH of the medium decreased slightly. The
decrease of the pH does not effect the biodegradation in the Closed Bottle test.

- Continuous darkness: Yes

TEST SYSTEM
- Test vessels: The test was performed in 2SO to 300 ml BOD (biological oxygen demand) bottles with glass stoppers.
- Number of vessels:
10 bottles containing only inoculum; 10 bottles containing test substance and inoculum; 6 bottles containing sodium acetate and inoculum

- Method used to create aerobic conditions:
Activated sludge was aerated for one week
All bottles were completely filled without air bubbles.

- Measuring equipment:
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW Trioxmatic E:O 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands).
The pH was measured using a Knick 765 calimatic pH meter (Knick Germany).
The temperature was measured and recorded with a thermo couple connected to a data logger.

- Test performed in closed vessels due to significant volatility of test substance:
Closed bottles to prevent loss of dissolved oxgen

SAMPLING
- Sampling frequency: At day 7, 14, 21, and 28.

- Sampling method: Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

- Toxicity control: Yes

Reference substance:

acetic acid, sodium salt

Test performance:

The test is valid as shown by an endogenous respiration of 0.5 mg/L and by the total mineralization of the referencie compound, sodium acetate. Sodium acetate was degraded 72% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentra1tions >0.5 mg/Lin all bottles during the test period. Setafix X 11342 did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum.

Parameter:

% degradation (test mat. analysis)

Remarks:

By HPLC Analysis

Value:

81

Sampling time:

28 d

Details on results:

The calculated theoretical oxygen demand (ThOD) of Setafix X 11342 is 2.1 mg/mg

Oxygen consumption (mg/L) and the percentages biodegradation of Setafix X 11342 (BOD/ThOD) in the Closed Bottle test.

Time (days) Oxvgen Consumption (mg/L) Biodegradation (%)
0 0 0
7 0.3 19
14 0.8 50
21 0.9 56
28 1.3 81


Toxicity:
Inhibition of the endogenous respiration of the inoculum by the test substance was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected.

Test conditions:
The pH of the media was 6.7 at the start of the test. The pH of the medium at day 28 ranged from 6.7 to 6.8. Temperatures ranged from 19 to 21°C.

Results with reference substance:

The theoretical oxygen demand (ThOD) of sodium acetate is 0.8 mg/mg. Sodium acetate was degraded 72% of its theoretical oxygen demand after 14 days.
Attained > 60% biodegradation in less than 10 days.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Remarks:

Therefore, is classified as rapidly biodegradeable

Conclusions:

Setafix X 11342 is biodegraded 81 % at day 28 in the Closed Bottle test, however, as it did not meet the pass level of 60% of ThOD within a 10 day (or 14 day) window after the test system reached 10% degradation, the substance cannot be classified as readily biodegradable and should therefore be classified as "rapidly degradeable".

Executive summary:

The objective of this study is to assess the biodegradability of Setafix X 11342 using a ready biodegradability test.This test has been performed according to slightly modified EEC, OECD and ISO Test Guidelines (OECD, 1992; EEC 1984; ISO, 1994).

Setafix X 11342 is biodegraded 81 % at day 28 in the Closed Bottle test, however, as it did not meet the pass level of 60% of ThOD within a 10 day (or 14 day) window after the test system reached 10% degradation, the substance cannot be classified as readily biodegradable and should therefore be classified as "rapidly degradeable".

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 October 2009 - 21 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study includes data generated according to generally valid and internationally accepted testing guidelines and performed according to GLP. The study was based on the OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I).

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge:
Chemicals Evaluation and Research Institute, Chemical Biotesting Center, 4·25, Koraku 1 ·chome, Bunkyo·ku, Tokyo, Japan

- Method of cultivation:
After ceasing aeration of the sludge mixture for 30 minutes, container was left in still for the gravity sedimentation of sludge particles. 300 ml supernatant was removed and 300 ml synthetic sewage was added to the mixture and the container was aerated again. This procedure was repeated once every day and thus obtained sludge was incubated as activated sludge.

Synthetic sewage:
Glucose, peptone and potassium dihydrogen phosphate were dissolved in dechlorinated water (tap water filtered with commercially available activated charcoal filter cartridge ) to obtain 0.1 (w/v) % of each component and pH was adjusted with 0.1M sodium hydroxide aqueous solution to 7 .0 ± 1.0.

- Storage conditions:
The pH of incubated sludge was kept at 7.0 ± 1.0

- Suspended solid concentration of the activated sludge was 3480 mg/L.

- Preparation of basal culture medium: Each 6 mL of solutions A, solution B, solution C and solution D were made up to 2 L with water (purified water, Kozakai Pharmaceutical Co., Ltd. JP) and then pH of this solution was adjusted to 7.2.

- Concentration of sludge:
The activated sludge of suspended solid concentration 3480 mg/L was diluted with the basal culture medium so as to make the sludge concentration of 900 mg/L

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

(HPLC Analysis)

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

(BOD Analysis)

Details on study design:

TEST CONDITIONS
- Test Vessel: 300 ml in volume (culture bottle)
- Incubation temperature: 25±1°C
- Incubation period: 28 days
- pH: The pH values of the test solutions after incubation were 6.66 in (water+ test substance) and 7.61, 7.66 and 7.60 in (sludge+ test substance).
- - pH adjusted: Yes, pH was adjusted with 0.1 M sodium hydroxide aqueous solution to 7 .0 ± 1.0.
- Aeration of dilution water: Yes
- Stirring method: Test solution was stirred by a magnetic stirrer.
- Suspended solids concentration: 3480 mg/L
- Irradiation conditions: Light-shielded

- Measuring apparatus of BOD
Closed system oxygen consumption measuring apparatus: Coulometer (No. D), Ohkura Electric Co., Ltd.
Absorbent for carbon dioxide: Soda lime (Wako Pure Chemical Industries, Ltd. No.1)

PREPARATION OF TEST SOLUTIONS
Six test vessels were prepared, and the test solutions were prepared as follows:

- Vessel #1: [water+ test substance]
30 mg of the test substance was weighted accurately and added to a test vessel containing 300 ml of purified water to make 100 mg/L as the test substance concentration.

- Vessel #2: [control blank]
290 ml of basal culture medium was added in the test vessel.

- Vessel #3: [sludge + aniline]
29.6 µL (30 mg) of aniline was added to the test vessel containing 290 ml of basal culture medium with micropipette to make 100 mg/L as aniline concentration.

- Vessel #4, Vessel #5, Vessel #6 [sludge+ test substance] - Triplicate
30 mg of the test substance was weighted accurately and added to a test vessel containing 290 ml of basal culture to make 100 mg/L as the test substance concentration.

INOCULATION OF ACTIVATED SLUDGE
10 ml of the diluted sludge (concentration 900 mg/L) was inoculated into each of the test vessels #2, #3, #4, #5 and #6 so as to make 30 mg/L as suspended solid concentration in each vessel

Reference substance:

aniline

Remarks:

(Kishida Chemical Co., Ltd., Guaranteed reagent, Lot No. C71348Y)

Preliminary study:

None performed

Test performance:

The difference between the maximum value and the minimum value for percentage biodegradation of the test substance after completion of the incubation was 1 % for by BOD and 2% for by HPLC, and the extent of degradation exceeded 20%, a criteria for confirmation of efficacy.
The percentage biodegradation of the reference substance (aniline) by BOD after 14 days was 75%, which was higher than 60%.

Parameter:

% degradation (test mat. analysis)

Remarks:

By BOD

Value:

ca. 8

St. dev.:

1

Sampling time:

28 d

Remarks on result:

other: After the completion of the incubation

Parameter:

% degradation (test mat. analysis)

Remarks:

By HPLC

Value:

ca. 5

St. dev.:

1

Sampling time:

28 d

Remarks on result:

other: After the completion of the incubation

Details on results:

BOD measurement:
The BOD in (sludge + test substance) after 28 days (the value after subtraction of the value in control blank) was 4.8, 4.8 and 5.4 mg.

Analysis of residual amount of test substance:
The residual amounts of test substance after incubation were 29.3 mg in (water + test substance) and 27.6, 28.1 and 27.4mg in (sludge+ test substance).

Percentage residue of test substance:
Percentage residue of test substance were 97% in (water + test substance) and 92%, 94% and 91 % in (sludge + test substance).

The percentage biodegradation at the 28th day were as follows:

Percentage biodegradation
Each biodegradation (%) Mean
Result by BOD 8, 8, 9 8
Result by HPLC 6, 4, 6 5

Results with reference substance:

The percentage biodegradation of the reference substance (aniline) by BOD after 14 days was 75%, which was higher than 60%.

Validity criteria fulfilled:

yes

Interpretation of results:

other: Non-biodegradable compound.

Conclusions:

In conclusion, test substance was partly biodegraded by microorganisms, but over 90% of test substance remained and it was considered that this test substance is non-biodegradable compound.

Executive summary:

The purpose of the study was to examine the degree ofbiodegradation by microorganisms of Setafix Z1077. The study was based on the OECD Test Guideline 301C.

Percentage residue of test substance were 97% in (water + test substance) and 92%, 94% and 91 % in (sludge + test substance).

The percentage biodegradation at the 28th day were as follows:

	Percentage biodegradation
	Each biodegradation	Mean
Result by BOD	8%,      8%,               9%	8%
Result by HPLC	6%,        4%               6%	5%

In conclusion, test substance was partly biodegraded by microorganisms, but over 90% of test substance remained and it was considered that this test substance is non-biodegradable compound.

Description of key information

In the ready biodegradability study (OECD 301C, MITI (I)), the test substance was partly biodegraded by microorganisms, but over 90% of test substance remained.
In the study based on EEC directive 92/69, C.4-C, December 1992, and OECD guideline No. 301 B July 17, 1992, SET AFIX X 11342 was tested for its ready biodegradability in the carbon dioxide (C02) evolution test (modified Sturm test) at 35 mg per 2 litres, corresponding to 12 mg TOC/L. SETAFIX X 11342 was not readily biodegradable under the conditions of the modified Sturm test presently performed.
However, the in the Inherent Biodegradability study Setafix Z1077 is easily transformed in an aqueous system to hydrolysis products and not used in bacterial metabolism because of an inherent biodegradation. In addition, due to the activated sludge present, Setafix Z1077 was ultimately biodegraded by 40.8% at the end of the study. Setafix is considered to be inherently biodegradable.
Based on the above studies the test substance is not considered readily biodegradable although it has been found to be inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Additional information

1) Ready Biodegradability Study:

a) In the study based on the OECD Test Guideline 301C, to examine the degree of biodegradation by microorganisms of Setafix Z1077, test substance was partly biodegraded by microorganisms, but over 90% of test substance remained and hence it was considered to be a Non-biodegradable compound.

Percentage residue of test substance were 97% in (water + test substance) and 92%, 94% and 91 % in (sludge + test substance).

The percentage biodegradation at the 28th day were as follows:

	Percentage biodegradation
	Each biodegradation	Mean
Result by BOD	8%,      8%,               9%	8%
Result by HPLC	6%,        4%               6%	5%

b) In the study based on EEC directive 92/69, C.4-C, December 1992, and OECD guideline No. 301 B July 17, 1992, SET AFIX X 11342 was tested for its ready biodegradability in the carbon dioxide (CO2) evolution test (modified Sturm test) at 35 mg per 2 litres, corresponding to 12 mg TOC/L.

SETAFIX X 11342 was not readily biodegradable under the conditions of the modified Sturm test presently performed.

2) Inherent Biodegradability Study:

The purpose of this study was to determine the biodegradability of the test item Setafix Z1077 in accordance with the OECD Guideline for Testing of Chemicals No. 302 C: "Inherent Biodegradability: Modified MITI Test (II)", adopted May 12, 1981.

After 28 days of incubation, the test item Setafix Z1077 was found to be biodegradable by about 40.8% (mean value) based on BOD-measurement. The biodegradation of the reference item sodium benzoate was clearly in the range given by the test guideline. In the toxicity control, the degradation of the reference item was not affected by the test item.

The test item was analysed in the complete test solution using the aqueous phase and a acetonitrile-extraction for the solid phase. A primary degradation of Setafix Z1077 of 65.3% was found after 28 days of incubation in test item groups. In the toxicity controls the primary degradation 88.6% after 28 days. In the abiotic controls there was about 37.6% primary degradation and therefore an indication for a partial hydrolysis due to abiotic processes. The test item is considered to hydrolyse easily and all of the inserted amount was found in the solid phase.

The presence of the reference item sodium benzoate did not affect the degradation of Setafix Z1077, and Setafix Z1077 also did not affect the breakdown of the reference item.

It can be concluded that Setafix Z1077 will be easily transformed in an aqueous system to hydrolysis products and not used in bacterial metabolism because of an inherent biodegradation.

The potential of the activated sludge is present because Setafix Z1077 was ultimately biodegraded by 40.8% at the end of the study. Setafix is considered to be partially biodegradable.