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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-03-18 to 1999-04-23 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to ISO Method 14593 with some modifications of OECD 301 B
Qualifier:
according to
Guideline:
other: ISO 14593
Deviations:
no
Principles of method if other than guideline:
The method encompasses a modified version of the EU CO2 evolution test C.4-C and OECD TG 301-B (according to Birch and Fletcher 1991, Chemosphere 23, 507-524 and Battersby 1977, Chemosphere 34, 1813-1822)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: secondary effluent from a laboratory rolling tube treatment unit
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): laboratory rolling tube treatment unit which is fed 100% settled sewage obtained from Newton Abbot sewage treatment works
- Storage conditions: sparged with carbon dioxide free air
- Storage length: storage overnight
- Preparation of inoculum for exposure: filtered through a GF/C filter paper
- Pretreatment: sparging with carbon dioxide free air to reduce dissolved inorganic carbon content to less than 1 mg/L, pH adjustment to 6.5 ± 0.2 using mineral acid and alkali, final adjustment after sparging to 7.0 ± 0.2
- Concentration of sludge: not applicable since all solids were removed
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: test medium was made up according to ISO 14593 guideline and contained 85 mg KH2PO4, 217.5 mg K2HPO4, 334 mg Na2HPO4 x 2 H2O, 22.5 mg MgSO4 x 7 H20, 0.25 mg FeCl3 x 6 H2O, 36.4 mg CaCl2 x 2 H2O, 5 mg NH4Cl and 0.4 mg EDTA disodium salt per litre of deionised water.
- Additional substrate: no
- Solubilising agent (type and concentration if used): not applicable
- Test temperature: 20 ± 2 °C
- pH: 7 ± 0.2
- pH adjusted: yes, effluent was adjusted to 7.0 ± 0.2 after purging and added to the test medium
- Aeration of dilution water: no
- Suspended solids concentration: not applicable, solids were removed by filtration
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: sufficient bottles were prepared to provide triplicate sets for carbon analysis on 5 separate occasions for each of the reference and test substances
- Method used to create aerobic conditions: not reported
- Measuring equipment: Dohrman DC 190 analyser
- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: evolving CO2 was trapped in the headspace of the closed bottles (defined volume of 56 mL)

SAMPLING
- Sampling frequency: triplicates were analysed on days 4, 7, 14, 21 and duplicates on day 28
- Sampling method: testing bottles were sacrificed on each occasion
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
- Other: positive control with reference substance aniline

STATISTICAL METHODS: not applicable
Reference substance:
aniline
Parameter:
% degradation (CO2 evolution)
Value:
< 5
Sampling time:
28 d
Details on results:
After 28 days ethoxymethyl cyclododecylether had undergone less than 5% biodegradation, based on theoretical carbon concentration.
Results with reference substance:
The reference substance, aniline, degraded by 80% based on theoretical carbon concentrations during the 28 days of the test, confirming the inoculum contained viable organisms.

Inorganic carbon measurements and biodegradation results

Bottle contents

Inorganic carbon in mg C/L

Day 4

Day 7

Day 14

Day 21

Day 28

Blank control

1.02

1.29

1.37

1.75

1.77

1.08

1.12

1.91

1.67

2.02

1.17

1.44

1.79

1.41

 

Mean inorganic carbon in liquid phase (mg C/L)

1.09

1.28

1.69

1.61

1.90

Aniline at 10 mg C/L

6.60

8.78

9.88

9.92

9.76

6.49

8.84

9.82

9.45

9.80

6.43

8.38

9.86

9.52

10.0

Mean inorganic carbon in liquid phase (mg C/L), blank corrected

6.51

8.67

9.85

9.63

9.85

Total inorganic carbon in test bottle (mg C)

0.54

0.74

0.82

0.80

0.80

% biodegradation

54

74

82

80

80

Ethoxymethyl cyclododecylether at 10 mg C/L

1.20

1.57

1.85

1.88

2.02

1.32

1.56

1.76

2.03

1.94

1.21

1.45

1.23

2.11

 

Mean inorganic carbon in liquid phase (mg C/L)

1.24

1.53

1.61

2.01

1.98

Mean inorganic carbon in liquid phase (mg C/L), blank corrected

0.15

0.24

-0.08

0.40

0.09

Total inorganic carbon in test bottle (mg C)

0.02

0.02

-0.01

0.04

0.01

% biodegradation

< 5

< 5

< 5

< 5

< 5

Inorganic carbon measurements and toxicity test results

Bottle contents

Inorganic carbon (mg C/L)

Blank control + aniline (total 10 mg C/L)

8.08

Mean blank control (day 28 result)

1.90

Mean inorganic carbon in liquid phase (mg C/L), blank corrected

6.18

Total inorganic carbon in test bottle (mg C)

0.62

% biodegradation

62

Ethoxymethyl cyclododecylether + aniline (total 10 mg C/L)*

9.32

Mean blank control (day 28 result)

1.90

Mean inorganic carbon in liquid phase (mg C/L), blank corrected

7.42

Total inorganic carbon in test bottle (mg C)

0.74*

% biodegradation

74*

* as there was no evidence of biodegradation of the test substance Ethoxymethyl cyclododecylether after 28 days the carbon due to the test compound has been ignored and subsequent biodegradation is assumed to have resulted only from carbon added as aniline.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The substance was not readily biodegradable in a valid CO2 evolution test over a period of 28 days.
Executive summary:

The ready biodegradability of the test substance ethoxymethyl cyclododecylether was studied under GLP in accordance with ISO method 14593, which encompasses a modified version of the EU CO2 evolution test according to method C.4-C and OECD TG 301-B. The test medium was prepared according to the guideline. The inoculum was secondary effluent obtained from a laboratory rolling tube treatment unit which is fed 100% settled sewage from municipal sewage treatment works, which treats sewage of predominantly domestic origin. Effluent was collected overnight on the day before the start of the test, after which it was filtered to remove any solids and sparged with carbon dioxide free air to reduce the dissolved inorganic carbon content to less than 1 mg/L. During sparging the pH of the effluent was maintained at 6.5 ± 0.2 and after sparging, pH was adjusted to 7.0 ± 0.2. The concentration of effluent in final medium was 100 mL/L. The test substance was directly weighed into each testing bottle. Bottles with a total volume of 156 mL were used. The liquid volume in the test bottles was 100 mL. Bottles were closed with rubber septa. Aliquots of 95 mL of seeded medium were added to empty bottles (blank controls) or bottles containing the test substance or the reference substance aniline. The liquid volume was then made up to 100 mL by addition of deionised water. the final carbon concentration in each bottle containing substances (test or reference) was 10 mg/L. The blank controls did not received any added carbon. Sufficient bottles were made up to take triplicate samples on days 4, 7, 14, 21 and 28 of each tested concentratin. In addition, a duplicate set of abiotic controls of the test substance was prepared for carbon analysis at day 28, containing test substance, test medium and 1 mL of 10 g/L mercuric chloride to confirm that the test substance was not abiotically degraded under experimental conditions. All test bottles were sealed with butyl rubber septa and aluminium crimp caps and placed in an orbital incubator at a temperature of 20 ± 2 °C. They were incubated in the dark for 28 days whilst being shaken at 150 rpm. Biodegradation was monitored by regular analysis of sacrificial test bottles to determine the amount of inorganic carbon evolved. After 28 days incubation a 5 mL aliquot of 258 mg/L aniline solution was added to one blank control bottle and one bottle containing ethoxymethyl cyclododecylether to test for toxicity of the substance. These two bottles were incubated for a further eight days before measuring the inorganic carbon evolved. The test substance was not biodegraded over the test period of 28 days and biodegradation was < 5% at all occasions. The reference substance aniline was degraded by 80% during the 28 days of the test confirming the inoculum contained viable organisms. The substance was not toxic to the microorganisms at the concentration of 10 mg C/L corresponding to 13.5 mg/L test substance.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
August 8, 2013 - September 5, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The test was performed under GLP according to OECD 301 C (modified MITI-Test I) without deviations.
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge, different locations (specified below), non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: samples of sludge were taken from 10 locations in Japan (from surface water and surface soil of rivers, lakes, and inland sea, return sludge from sewage plants).
- Preparation of inoculum for exposure: after cultivating for 19 hours, the activated sludge was mixed with synthetic sewage and used in test.
- Composition of synthetic sewage: glucose, peptone, and potassium dihydrogenphosphate
- pH value of synthetic sewage: 7.0±1.0
- Concentration of sludge: 30 mg/L (as the concentration of suspended solid)
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: test medium was made up according to JIS K 0102-2008 section 21 and prepared as following: each 3 mL aliquot of solution A (buffer solution): 10.875 g K2HPO4, 4.25 g KH2PO4, 22.3 Na2HPO4 x 12 H2O and 0.85 g NH4Cl dissolved in purified water and made up to 500 mL, solution B (magnesium sulphate solution): 11.25 g MgSO4 x 7 H20 dissolved in purified water and made up to 500 mL, solution C (calcium chloride solution): 13.75 g CaCl2 dissolved in purified water and made up to 500 mL and solution D (Iron (III) chloride solution): 0.05 g FeCl3 x 6 H2O,dissolved in purified water and made up to 200 mL, were added in purified water and made up to 1 L of solution.
- Test temperature: 25±1 °C
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: glass vessel with lid
- Number of culture flasks/concentration: 6 vessels
- Measuring equipment: oxygen consumption measuring apparatus
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2 and volatile organics if used: Soda lime No.1

SAMPLING
- Sampling frequency: BOD values were recorded on 7th, 14th, 21st and 28th day. Concentrations of test solution were analyzed at the end of the test.
- Sampling method: BOD values were read directly from the oxygen consumption measuring apparatus.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: no
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Value:
4
Sampling time:
28 d
Details on results:
After 28 days, the test item did not undergo biodegradation under the test conditions of this study.
Results with reference substance:
The reference substance (aniline) was degraded by 77% and 93% based on theoretical oxygen demand after 7 days (specified: >40%) and 14 days (specified: >65%), respectively. The test was valid as all the values in this test met the criteria.

Results of test item:

 

Sludge + test item

Vessel 2

Vessel 3

Vessel 4

Average

% biodegradation by BOD

%

0

-6

0

0 (-2)*

% biodegradation by GC

%

4

4

3

4

*The average percent biodegradation was regard as 0% because the calculated average value shown in parent substance was negative.

Validity of test conditions:

 

Value in this test

Value of criterion

Difference between extremes of replicates % biodegradation

% biodegradation by BOD

6%

<20%

% biodegradation by GC

1%

% biodegradation of aniline by BOD

After 7 days

77%

>40%

After 7 days

93%

>65%

BOD value of control blank

After 28 days

6.1 mg

<60 mg/L (18 mg)

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item is not readily biodegradable (0% after 28 days).
Executive summary:

The ready biodegradability of the test substance was investigated under GLP conditions in accordance with OECD301 C (modified MITI test (I)). All validity criteria specified in the test guideline were fulfilled.

The test medium was prepared according to the guideline JIS K 0102-2008 section 21. The samples of activated sludge were taken from 10 locations in Japan (from surface water and surface soil of rivers, lakes, and inland sea, return sludge from sewage plants). The concentration of activated sludge was 30 mg/L (as the concentration of suspended solid). The concentration of test item or reference substance was 100 mg/L. The values of consumption of oxygen (BOD-values) were recorded on 7th, 14th, 21stand 28thday. At the end of the test, the test item in test solution was analyzed by GC and IR methods. The results showed that the test item is not readily biodegradable, as the percentage of biodegradation was 0% after 28 days. The GC- and IR-analyses confirmed that the test item was not changed after an exposure period of 28 days. Therefore, further confirmation of the degradation product was not necessary. 

Description of key information

The substance is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Two valid studies on the ready biodegradability of the substance (ethoxymethoxy)cyclododecane are available.

The key study in accordance with OECD TG 301-B (1999) revealed no biodegradation (< 5%) over the 28 days of the test. The substance was not toxic to the microorganisms in the inoculum at a concentration of 13.5 mg/L.

The supporting study according to OECD TG 301C (2013) confirms this result (no biodegradation observed, < 5% degradation).