Ditolyl ether

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:

key study

Study period:

2010-06-08 till 2010-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

Test organisms
- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: Aeration tank of a wastewater treatment plant treating predominantly domestic sewage (Wupper area water authority, STP Odenthal)
- Date of collection: 2010-06-08
- Concentration of inoculum : 30 mg/L suspended solids

Pre-treatment of the inoculum
- The sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm and 20 °C and decanting off the supernatant.
- 1.049 g of the wet sludge was dried in order to determine the wet weight / dry weight ratio of the sludge and to prepare a stock suspension (activated sludge) of 3 g dw/L.
- The calculated amount of sludge, needed to achieve 300 mL of this stock suspension, was dissolved in mineral medium and then filled up to a defined end volume.
- Before use, the inoculum was stored for one day at room temperature under continuous shaking with aeration.

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:

TEST CONDITIONS
- Composition of medium: The mineral medium was prepared from stock solutions of mineral components (i.e. mineral salt solution, magnesium sulphate solution, calcium chloride solution and Iron (III) chloride solution) in accordance with the guideline. To prepare the mineral medium 10 mL of mineral salt solution were mixed with 800 mL deionised water. Then 1 mL each of the magnesium sulphate-, calcium chloride- and the iron (III) chloride solution were added and filled up to 1 litre with deionised water.
- Solubilising agent (type and concentration if used): No solubilising agent was used. 25 mg of the test item were weighed out and directly added to the test flasks, filled with 200 mL of mineral medium. Afterwards the volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg/L.
- Test temperature: 22+/- 1°C
- pH: 7.5 - 8.0 (measured in the test flasks at the end of the test)
- pH adjusted: no
- Aeration of dilution water: no
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: closed flasks with a test volume of 250 mL
- Number of culture flasks/concentration: 2 (test item & reference compound), 3 (inoculum blank)
- Measuring equipment: OxiTopControl System (WTW)
- Test performed in closed vessels: Yes
- Test performed in open system: not applicable
- Details of trap for CO2 and volatile organics if used: not applicable

SAMPLING
- Sampling frequency: The BOD (in mg O2/L) was measured daily

CONTROL AND BLANK SYSTEM
- Inoculum blank: Blanks with inoculum and without test item were run in parallel (3 replicates)
- Reference substance: a reference compound (sodium benzoate) was run in parallel, to check the operations of the procedures. The concentration of the reference substance used in the test was 100 mg/L
- Toxicity control: 25 mg of the test item and 25 mg of the reference compound were weighed out and added to the test flasks, filled with 200 mL of mineral medium. Afterwards the flask volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg test item and reference compound.

STATISTICAL METHODS:
The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given my manufacturer of equipment. At the end of incubation, the pH of the contents of the flasks was measured. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum) was expressed as a percentage of theoretical oxygen demand.

Reference substance:

other: sodium benzoate (Sigma-Aldrich), purity: 99.7 %, Batch no.: 1438955

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Details on results:

Within 28 days, a degradation rate of 2 % was determined for the substance. The substance is considered as not readily biodegradable under the conditions of the test.

All validity criteria of the test method are fulfilled:
- The toxicity control exhibited degradation rates >= 25% within 14 days.
- The reference compound reached the level of >= 60% for ready biodegradability within 14 days
- At the end of the test, at the plateau, or the end of the 10-day window, biodegradation in parallels with test item did not differ by more than 20 percentage points.
- The oxygen uptake of the inoculum blank was <= 60 mg/L.
In cases where the degradation was <= 60 %, the pH values were in between 6.0 and 8.5 at the end of the test (and thus poor degradation was not caused by pH influences).

Results with reference substance:

The reference compound showed a degradation of 79 % after 14 days and 92 % after 28 days.
Kinetic of reference substance (degradation in %):
69 after 7 days
79 after 14 days
87 after 21 days
92 after 28 days

Table 1: BOD values (mg O2/L) after n days

	test item		blank			blankmean	reference compound		toxicity control
flask no.	15	16	1	2	3		4	5	17
code*	a1	a2	b1	b2	b3	bm	r1	r2	t1
time [d]
0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
1	7.0	7.0	8.4	5.6	7.0	7.0	35.0	35.0	39.2
2	11.2	9.8	11.2	9.8	9.8	10.3	82.5	78.3	82.5
3	15.4	14.0	18.2	16.8	16.8	17.3	99.3	99.3	102.0
4	19.6	18.2	19.6	18.2	18.2	18.7	109.0	109.0	120.0
5	22.4	19.6	21.0	19.6	21.0	20.5	119.0	123.0	130.0
6	23.8	21.0	22.4	19.6	22.4	21.5	131.0	133.0	137.0
7	25.2	22.4	23.8	21.0	23.8	22.9	140.0	137.0	141.0
8	25.2	23.8	23.8	21.0	25.2	23.3	144.0	141.0	144.0
9	28.0	23.8	22.4	19.6	23.8	21.9	147.0	143.0	147.0
10	29.4	25.2	23.8	22.4	26.6	24.3	150.0	147.0	150.0
11	29.4	26.6	25.2	22.4	28.0	25.2	154.0	151.0	151.0
12	30.8	25.2	23.8	21.0	28.0	24.3	155.0	152.0	154.0
13	30.8	26.6	26.6	22.4	29.4	26.1	158.0	157.0	157.0
14	30.8	26.6	25.2	25.2	30.8	27.1	159.0	158.0	158.0
15	32.2	26.6	25.2	22.4	30.8	26.1	162.0	161.0	161.0
16	32.2	30.8	23.8	23.8	30.8	26.1	164.0	164.0	166.0
17	32.2	30.8	26.6	23.8	32.2	27.5	168.0	166.0	171.0
18	33.6	32.2	25.2	25.2	32.2	27.5	169.0	168.0	172.0
19	33.6	32.2	26.6	23.8	33.6	28.0	171.0	171.0	175.0
20	33.6	33.6	25.2	26.6	35.0	28.9	172.0	172.0	176.0
21	35.0	32.2	26.6	25.2	35.0	28.9	172.0	175.0	178.0
22	35.0	33.6	26.6	26.6	35.0	29.4	173.0	178.0	179.0
23	35.0	35.0	29.4	26.6	39.2	31.7	176.0	182.0	182.0
24	36.4	35.0	28.0	25.2	37.8	30.3	176.0	185.0	183.0
25	37.8	35.0	28.0	26.6	39.2	31.3	178.0	186.0	185.0
26	37.8	36.4	26.6	28.0	39.2	31.3	178.0	187.0	186.0
27	39.2	36.4	29.4	28.0	40.6	32.7	179.0	190.0	189.0
28	39.2	36.4	29.4	28.0	41.9	33.1	179.0	192.0	190.0

Table 2: Degradation of the test item

Test concentration : 100 mg/L

Theoretical oxygen demand: 2.743 mg O2/mg

time [d]	blank-corrected BOD [mg O2/L]		% degradation		% degradation mean
	a1	a2	a1	a2
0	0.0	0.0	0	0	0
1	0.0	0.0	0	0	0
2	0.9	0.0	0	0	0
3	0.0	0.0	0	0	0
4	0.9	0.0	0	0	0
5	1.9	0.0	1	0	0
6	2.3	0.0	1	0	0
7	2.3	0.0	1	0	0
8	1.9	0.5	1	0	0
9	6.1	1.9	2	1	1
10	5.1	0.9	2	0	1
11	4.2	1.4	2	1	1
12	6.5	0.9	2	0	1
13	4.7	0.5	2	0	1
14	3.7	0.0	1	0	1
15	6.1	0.5	2	0	1
16	6.1	4.7	2	2	2
17	4.7	3.3	2	1	1
18	6.1	4.7	2	2	2
19	5.6	4.2	2	2	2
20	4.7	4.7	2	2	2
21	6.1	3.3	2	1	2
22	5.6	4.2	2	2	2
23	3.3	3.3	1	1	1
24	6.1	4.7	2	2	2
25	6.5	3.7	2	1	2
26	6.5	5.1	2	2	2
27	6.5	3.7	2	1	2
28	6.1	3.3	2	1	2

Validity criteria fulfilled:

yes

Remarks:

(-Ready biodegradation of reference compound ≥ 60 % within 14 days. - The toxicity control exhibited degradation rates > 25 % within 14 days. - Replicates difference < 20 %. - Oxygen uptake of the inoculum blank was ≤ 60 mg/L. - No pH influence.)

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Within 28 days, a degradation rate of 2 % was determined. The test item is considered to be "Not Readily Biodegradtion". The reference compound sodium benzoate showed 79 % degradation after 14 days.

Executive summary:

The biodegradability of the substance was assessed in a "Manometric Respiratory Test" (2008) in accordance with the EU Method C.4 -D (2008) and the OECD Guideline 301 F (1992). Activated sludge of a wastewater plant treating predominantly domestic sewage was used as inoculum. A solution of 100 mg /L test item in mineral medium was inoculated and incubated at 22 +/-1 °C for 28 days under aerobic conditions in the dark. The consumption of oxygen (BOD) was determined by measuring the drop in pressure in the automated OxiTop respirometer flask. The results showed a degradation of the substance of 2 % after 28 days which indicates that the substance is not ready biodegradable under the conditions of the test.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: activated sludge, mixture of domestic and industrial (80:20); adaption not specified

Details on inoculum:

TEST ORGANISMS:
- Type : mixed population of aquatic microorganisms (activated sludge)
- Origin : Activated sludge from an aeration tank of two different wastewater treatment plants treating predominantly domestic wastewater (Wupper area water authority, WWTP Odenthal and WWTP Cologne-Stammheim); activated sludge from the aeration tanks of a wastewater treatment plant treating predominantly wastewater of industrial origin (WWTP Leverkusen Bürrig); three sludge types were mixed taking 2 parts from each of the two domestic WWTPS plus 1 part from the industrial WWTP
- Date of collections : 2014-02-03

PRE-TREATMENT OF THE INOCULUM:
− Before use, the inoculum was stored for two days at room temperature under continuous shaking with aeration
− The sludges of the three different origins were mixed as described before
− The combined sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm and 20 °C and decanting off the supernatant
− An aliquot of the wet sludge was dried in order to determine the wet weight/dry weight ratio of the sludge and to prepare a stock suspension with a defined concentration of suspended solids
− The calculated amount of sludge, needed to achieve 300 mL of this stock suspension of 5 g dw/L, was dissolved in mineral medium and then filled up to a defined end volume

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS:
- Pre-treatment of the test item: 7.5 mg of the test item were added to the test flasks, filled with 200 mL of mineral medium. Afterwards the volume was made up to 250 mL with mineral medium plus inoculum to give a final concentration of 30 mg test item/L and a final concentration of suspended solids of 100 mg/L.

TEST SYSTEM
- Culturing apparatus: closed flasks with a test volume of 250 mL
- Number of culture flasks/concentration: 2 (test item & reference compound), 3 (inoculum blank)
- Measuring equipment: OxiTop System (WTW)
- Test performed in closed vessels: Yes
- Test performed in open system: not applicable
- Details of trap for CO2 and volatile organics if used: not applicable
- Mixing : 1 magnetic stirrer per test vessel
- Incubation time : 28 days
- Incubation temperature : 25 ± 2 °C

SAMPLING:
- Sampling frequency: The BOD (in mg O2/L) was measured daily

CONTROL AND BLANK SYSTEM:
- Inoculum blank: Blanks with inoculum and without test item were run in parallel (3 replicates)
- Reference substance: a reference compound (sodium benzoate) was run in parallel, to check the operations of the procedures. The concentration of the reference substance used in the test was 30 mg test item/L
- Toxicity control: A toxicity control (test item and reference compound mixed) was not run in parallel, because the chosen concentration of the test item was not inhibitory to microorganisms

STATISTICAL METHODS:
- The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given my manufacturer of equipment. At the end of incubation, the pH of the contents of the flasks was measured. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum) was expressed as a percentage of theoretical oxygen demand.

Reference substance:

benzoic acid, sodium salt

Remarks:

Purity: 99.7 %, Batch no.: 1438955 (Sigma-Aldrich)

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

7 d

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

1

Sampling time:

21 d

Parameter:

% degradation (O2 consumption)

Value:

3

Sampling time:

28 d

Details on results:

Within 28 days a degradation rate of 3 % was determined for ditolyl ether. Ditolyl ether is considered to be "Not Inherently Biodegradable".

Results with reference substance:

The reference compound sodium benzoate showed 78 % degradation after 7 days and 81 % degradation after 14 days.

Validity criteria fulfilled:

yes

Remarks:

The percent degradation of the reference compound sodium benzoate reached the level of ≥ 40 % after 7 days and ≥ 65 % after 14 days.

Interpretation of results:

not inherently biodegradable

Conclusions:

Within 28 days a degradation rate of 3 % was determined. Ditolyl is considered to be "Not Inherently Biodegradable". The reference compound sodium benzoate showed 78 % degradation after 7 days and 81 % degradation after 14 days.

Executive summary:

The study was conducted in accordance with the OECD Guideline 302 C “Inherent Biodegradability: Modified MITI Test (II)” (1981) (based on standard operating procedures for OECD Guideline 301F (1992) and the Council Regulation (EC) No 440/2008, method C.4-D “Manometric Respirometry Test” (2008)).

A suspension of ditolyl ether in a mineral medium was inoculated with 40 % sludge of each of two different municipal sewage treatment plants (STP) and 20 % sludge of an industrial STP and incubated under aerobic conditions. During this period, degradation was followed by continuous automated BOD determinations.  

The results showed a degradation of 3 % after 28 days which indicates that ditolyl ether is not inherently ready biodegradable under the conditions of the test.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: secondary effluent of a wastewater treatment plant treating predominantly domestic sewage

Details on inoculum:

- Type : mixed population of aquatic microorganisms
- Origin : secondary effluent of a wastewater treatment plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)
- Date of collection : 2014-02-03
- Pre-treatment : separation of coarse particles by filtration, aeration of the resulting inoculum for 3 day
- Effluent concentration of reaction mixture : 2.6 mL/L (3 mL/1.16 L)

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

PRE TREATMENT OF THE TEST ITEM:
- 2.3 mg of the test item were weighed out and added to the test flasks, filled with ca. 700 mL of mineral medium. Then all flasks were treated in an ultrasonic bath for 10 up to 20 minutes to keep off the test item from staying on the surface. Afterwards the inoculum was added and the flasks volume was made up to 1.16 L with mineral medium and the flasks were closed free from air bubbles with glass stoppers.
- Initial concentration of test item : 2 mg/L (2.3 mg/1.16 L)

TEST SYSTEM
- Culturing apparatus: closed flasks with a test volume of 250 mL
- Number of culture flasks/concentration: 2 (test item & reference compound, inoculum blank)
- Test performed in closed vessels: Yes
- Test performed in open system: not applicable
- Details of trap for CO2 and volatile organics if used: not applicable
- Incubation time : 28 days
- Incubation temperature : 22 ± 2 °C
- Continous darkness: yes

CONTROL AND BLANK SYSTEM
- Inoculum blank: Blanks with inoculum were run in parallel
- Test substance: The concentration of the test item was 2. mg/L and the test on degradation was run in parallel
- Reference substance: A reference compound (sodium benzoate) was run in parallel, to check the operations of the procedures. The concentration of the reference substance used in the test was 2.9 mg/L
- Toxicity control: A toxicity control (test item and reference compound mixed) was not run in parallel, because the chosen concentration of the test item was not inhibitory to microorganisms

SAMPLING:
- Sampling frequency: The BOD (in mg O2/L) was measured on day 0, 6, 14, 21 and 28

EXPOSURE CONDITIONS:
- Test volume : 1.16 L
- Incubation time : 28 days
- Incubation temperature : 22 ± 2 °C

Reference substance:

benzoic acid, sodium salt

Remarks:

Purity: 99.7 %, Batch no.: 1438955 (Sigma-Aldrich)

Parameter:

% degradation (O2 consumption)

Value:

1

Sampling time:

6 d

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

21 d

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Results with reference substance:

The reference compound sodium benzoate showed 78 % degradation after 14 days.

Validity criteria fulfilled:

yes

Remarks:

O2-depletion in the blank control <= 1.5 mg dissolved O2/L after 28 days; biodegradation in parallels with test item did not differ by more than 20 percentage points; ref. compound >= 60 % for ready biodegradability within 14 days

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Ditolylether showed 2 % degradation after 28 days. Therefore, Ditolylether is considered to be “Not Readily Biodegradable“. The reference compound sodium benzoate showed 78 % degradation after 14 days.

Executive summary:

The study was conducted in accordance with the Council Regulation (EC) No 440/2008, Method C.4-E “Closed Bottle Test (2008)”.

This test method is in all essential parts identical with OECD Guideline 301 D (adopted July 1992).

The method assessed the ready biodegradability of ditolyl ether under aerobic conditions at 22 ± 2°C in the dark. The test lasted for 28 days. During this period, the biodegradation was determined on the basis of the reduction of dissolved oxygen (BOD). A measured volume of inoculated mineral medium, containing a known concentration of 2 mg test item/L (usually 2-10 mg/Litre), to give usually 5-10 mg ThOD/Litre as the nominal sole source of organic carbon in a closed flask was applied. The degree of biodegradation was calculated by the amount of oxygen taken up by ditolyl ether (corrected for that in the blank inoculum control) as a percentage of theoretical oxygen demand (ThOD).

The results showed a degradation of ditolyl ether of 2 % after 28 days which indicates that ditolyl ether is not readily biodegradable under the conditions of the test.

Description of key information

Within 28 days, a degradation rate of 2 % was determined. The test item is considered to be "Not Readily Biodegradtion". The reference compound sodium benzoate showed 79 % degradation after 14 days.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The existing study on ready biodegradation (Currenta, 2010) reported 2% degradation and, thus, indicates that ditolyl ether is not readily biodegradable. However, revising the dossier and this study it got obvious that the study result might be flawed. This GLP and OECD 301F conform study was conducted with a test concentration of 100 mg/L, which is in accordance with the OECD guideline. However, the water solubility of the substance is 2.83 mg/L and, therefore, the test concentration could have been too high, and only parts of the substance might have been available to bacteria and to biodegradation.

Furthermore, there is another study (Kanne, 1986) which is somehow difficult to interpret as the method refers to a ready biodegradation but as adapted sludge was used the study finally investigated on inherent biodegradation. The study result, therefore, has to be questioned and was scored with Klimisch 3.

Thus, the available data on biodegradation might not be appropriate to conclude on the persistence of ditolyl ether (based on screening tests), and the registrant decided to conduct new GLP and OECD conform studies on:

1) ready biodegradation (OECD 301D) and

2) inherent biodegradation (OECD 302C).

In contrast to the assumption that the existing data on biodegradation might have been flawed, the new studies confirmed that ready and inherent biodegradation of ditolyl ether are low with 2-3% after 28 days.

Concluding, existing and new studies on ready and inherent biodegradation reported only 2-3% degradation. Based on these screening studies ditolyl ether is not rated as biodegradable.