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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes
Parameter:
% degradation (O2 consumption)
Value:
75.3
Sampling time:
28 d
Remarks on result:
other: Source: CAS 17671-27-1, METI, 1992
Parameter:
% degradation (test mat. analysis)
Value:
86
Sampling time:
28 d
Remarks on result:
other: Source: CAS 17671-27-1, METI, 1992
Parameter:
% degradation (CO2 evolution)
Value:
68.1
Sampling time:
29 d
Remarks on result:
other: Source: CAS 93803-87-3, Croda, 1998. Bottle A
Parameter:
% degradation (CO2 evolution)
Value:
71.7
Sampling time:
29 d
Remarks on result:
other: Source: CAS 93803-87-3, Croda, 1998. Bottle B
Interpretation of results:
readily biodegradable
Conclusions:
Based on the available data, the target substance is considered readily biodegradable according to OECD guideline criteria (68 - 86% after 28/29 d, OECD 301 B and C).
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Oct - 19 Nov 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study with acceptable restrictions (The degradation in one replicate was below the pass level and differed by > 20%. However, the mean biodegradation based on BOD was > 70% (replicate 1: 88.5%; replicate 2: 51.56%; replicate 3: 84.79%). Moreover, the primary degradation measured by HPLC was 83, 74 and 100% in the three test vessels supports the assumption that the substance is readily biodegradable according to the OECD criteria. Therefore, the study is considered to be valid.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
yes
Remarks:
pH was not measured at the end of the study
Qualifier:
according to guideline
Guideline:
other: Circular on Test Methods of New Chemical Substances (Japan), Biodegradation test
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: mixture of activated sludge from STP with freshwater from rivers and lakes, marine water and marine sediment
Details on inoculum:
- Source of inoculum/activated sludge: location: 10 sites (4 sewage plants, 3 rivers, 1 lake and 2 bays) around Japan; Sampling depth: return sludge in sewage plants, surface water and surface soil of foreshore.
- Laboratory culture: Thirty minutes after stopping of aeration, discard about one third of the whole volume of supernatant and add an equal volume of a solution (pH adapted to 7.0 with NaOH) containing 0.1% each of glucose, peptone and pottasium orthophosphate, to the settled material and re-commence aeration. This procedure was repeated once per day.
- Temperature: 25 ± 2 °C
- Storage conditions: observed appearance and general condition of sludge, determined sediments, pH, temperature and dissolved oxygen and observed with a light microscope, if it is normal.
- Storage length: started to use: 15 Oct 1991
- Concentration of sludge: 3900 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: recommended medium in the guideline
- Test temperature: 25 ± 1 °C
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: closed oxygen consumption measuring machine with 300 mL culture bottle
- Number of culture flasks/concentration: 1 (water + test substance), 3 (sludge + test sub.), 1 (sludge + reference sub.), 1 (sludge)
- Measuring equipment: Coulometer
- Details of trap for CO2 and volatile organics if used: soda lime

SAMPLING
- Sampling frequency: Continuously
- Sterility check if applicable: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 1 bottle
- Abiotic sterile control: no
- Toxicity control: no
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
75.3
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Value:
86
Sampling time:
28 d
Details on results:
No 10-day window required. Substance was readily biodegradable.
Results with reference substance:
The oxygen consumption for aniline in the above used test system reached 60% and 72% degradation after 7 days and 14 days.

The degradation in one replicate was below the pass level (51.56%). However, the mean biodegradation based on BOD was > 70%. Moreover, the primary degradation measured by HPLC was 83, 74 and 100% in the three test vessels supports the assumption that the substance is readily biodegradable according to the OECD criteria.

Table 1. Result of BOD, DOC and HPLC after 28 days

 

water + test sub.

sludge + test sub.

Theoretical Amount

sample No. 6

sample No. 3

sample No. 4

sample No. 5

BOD (*1)

mg

0.0

85.0

49.5

81.4

96.0

DOC

mg

0.0

1.1

0.5

1.0

24.4

HPLC

mg

30.2

5.0

7.7

0.0

30

% (*2)

101

17

26

0

-

(*1) (sludge + test sub.) was calculated by subtracting blank values.

(*2) Residue percentage was calculated as followed.

Residue percentage = Residue amount (mg) / Theoretical amount (mg) x 100

Validity criteria fulfilled:
no
Remarks:
Biodegradation in one replicate differed by more then 20%.
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Apr - 14 May 1998
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
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Obtained from a municipal sewage treatment plant ‘Waterschap de Maaskant’, ‘s-Hertogenbosch, The Netherlands.
- Laboratory culture: No
- Storage conditions: The sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle for at least 30 minutes and the liquid decanted for use as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: Suspended solid concentration was 5.1 g/L
- Water filtered: No
Duration of test (contact time):
28 d
Initial conc.:
ca. 14.5 mg/L
Based on:
test mat.
Initial conc.:
ca. 12 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: According to OECD Guideline 301B using tap-water purified by reverse osmosis and subsequently passed over activated carbon and ion-exchange cartridges.
- Test temperature: 20 - 22 °C
- pH: 7.4 - 7.6
- pH adjusted: no
- Aeration of dilution water: A mixture of oxygen (21%) and nitrogen (79%) was led through a bottle, containing 0.5 – 1 L 0.0125 M Ba(OH)2 solution to trap CO2. The CO2-free air was sparged through the scrubbing solutions at a constant rate. During incubation the rate was 1-2 bubbles/sec (ca. 30 - 100 mL/min)

TEST SYSTEM
- Culturing apparatus: 2 L all-glass brown coloured bottles
- Number of culture flasks/concentration: 2 bottles
- Method used to create aerobic conditions: Bottles were sparged with CO2-free air.
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: Three CO2-absorbers containing 100 mL of a 0.0125 M barium hydroxide (Ba(OH)2) solution.

SAMPLING
- Sampling frequency: Every second or third day during the first 10 days, and thereafte at least every fifth day.
- Sampling method: On the days of CO2 measurement, the CO2 absorber closest to the bioreactor was disconnected and the barium hydroxide solution titrated with 0.05 M HCl using phenolphthalein as the indicator.
- Other: On day 28, the pH of the test suspensions was measured and 1 mL of concentrated HCl was added to each bottle. The bottles were aerated overnight to drive off the present CO2. The final titration was made on day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 bottles
- Toxicity control: yes, 1 bottle
- Other: Positive control: 1 bottle
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
68.1
Sampling time:
29 d
Remarks on result:
other: bottle A
Parameter:
% degradation (CO2 evolution)
Value:
71.7
Sampling time:
29 d
Remarks on result:
other: bottle B
Results with reference substance:
78.1% after 9 days, 60% pass level was reached.

In the toxicity control more than 25% degradation occurred within 14 d. Therefore, the test substance was assumed to be not inhibitory. According to “OECD Guidelines for the testing of chemicals – Revised introduction to the OECD guidelines for testing of chemicals, Section 3 – Part 1: Principles and strategies related to the testing of degradation of organic chemicals degradation” published by OECD (2006) the ready biodegradability data for mixtures of structurally similar chemicals should be evaluated carefully. For substances with different chain lengths the 10-day window should not be used for the interpretation of results. It is possible that a sequential biodegradation occurred which might have led to a prolonged lag-Phase and therefore a slower biodegradation period although the test substance was biodegraded in a large amount after the test duration. In this case the pass level can be applied at 28 days and is therefore readily biodegradable (68.1 - 71.7% after 28 days).

Table 1: % degradation ot the test substance in bottle A and bottle B.

day

% degradation

bottle A

bottle B

2

0.0

0.0

5

4.1

4.3

7

12.4

12.5

9

23.0

24.0

14

38.7

39.2

19

51.7

50.9

23

58.4

59.5

27

65.0

66.8

29

68.1

70.2

29

68.1

70.2

29

68.1

71.7

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
Vega version 1.1.3

2. MODEL (incl. version number)
Ready Biodegradability Model (IRFMN) v. 1.0.9

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
See “Test material information”

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached information on the model provided by the developer. Further information on the OECD criteria as outlined by the applicant is provided below under "Any other information of materials and methods incl. tables"

5. APPLICABILITY DOMAIN
See attached information and information as provided in "Any other information on results incl. tables".

6. ADEQUACY OF THE RESULT
See assessment of adequacy as outlined in the "Overall remarks, attachments" section.
Principles of method if other than guideline:
- Software tool(s) used including version: Vega v1.1.3

- Model(s) used: Ready biodegradability model (version 1.0.9)
Full reference and details of the used formulas can be found in:
Meylan W.M., Howard P.H., Boethling R.S. et al. Improved Method for Estimating Bioconcentration / Bioaccumulation Factor from Octanol/Water Partition Coefficient. 1999, Environ. Toxicol. Chem. 18(4): 664-672

- Model description: see field 'Justification for non-standard information', 'Attached justification' and 'any other information on Material and methods'

- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification' and/or 'overall remarks'
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Remarks on result:
other:
Remarks:
All 3 constituents of the substance are predicted to be readily biodegradable and the results appear to be reliable.

For detailed information on the results, please refer to the attached report.

Interpretation of results:
readily biodegradable

Description of key information

Readily biodegradable: 68.1 – 71.7% CO2 within 29 d (OECD 301 B); read-across

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

There is no study available, in which the ready biodegradability of the target substance fatty acids, C14-22, C16-24-alkyl esters (CAS 92797-30-3) was assessed. Therefore, read-across to the two structurally related source substances docosyl docosanoate (CAS 17671-27-1) and 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) was conducted in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5.

The target substance is a UVCB consisting of three main ester constituents with carbon chain lengths of 38 and 40. The source substances are characterized by similar fatty acid and fatty alcohol chain lengths and are therefore considered suitable representatives for the assessment of the ready biodegradability of the target substance. A detailed read-across justification is provided in the analogue justification in IUCLID section 13.

By means of the read-across approach, two experimental studies are available, which were conducted according to internationally accepted guidelines and GLP. In addition, the experimental results are further supported by QSAR calculations that predict the ready biodegradability of the target substance by the Ready Biodegradability model IRFMN 1.0.9 (VEGA 1.1.3).

The available study with the source substance docosyl docosanoate (CAS 17671-27-1) was performed according to OECD guideline 301 C under aerobic conditions using a mixture of activated sludge from a sewage treatment plant, from freshwater (rivers and lakes), marine water and marine sediment as inoculum for a test item concentration of 100 mg/L (METI, 1992). Biodegradation was followed by measuring O2 consumption, DOC removal as well as test material analysis by HPLC. After 28 d, a biodegradation of 75.3% (BOD) was observed. Since biodegradation reached the pass level defined by OECD guideline 301 C (> 60% after 28 d, no 10-day window required) the source substance docosyl docosanoate is readily biodegradable according to the OECD criteria.

The available study with the source substance 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) was performed according to OECD 301 B under aerobic conditions using domestic activated sludge as inoculum for a test substance concentration of approx. 14.5 mg/L. After 28 d, the test item was degraded by 68.1 - 71.7%. Since 2-octyldodecyl isooctadecanoate is a UVCB substance consisting of various structurally similar constituents (with e.g. different chain-lengths, degrees and/or sites of branching or stereo-isomers), sequential (instead of concurrent) biodegradation of the individual structures can take place. But all can be considered to be readily biodegradable. Thus, with reference to Annex I of the OECD Guideline for Testing of Chemicals ‘Revised introduction to the OECD guidelines for testing of chemicals, section 3 (OECD, March 2006), the 10-day window must not be considered for this UVCB substance and, since a degradation of >60% was observed within 28 days, the substance can be regarded as readily biodegradable. Moreover, the test substance is not inhibitory to activated sludge microorganisms based on a biodegradation of > 25% in the toxicity control after 14 d.

QSAR calculations for all three main constituents provide further supporting evidence that the target substance is readily biodegradable (IRFMN 1.0.9, VEGA 1.1.3). All three constituents fall within the applicability domain of the model (global AD index = 0.916). The overall performance of the model on similar molecules is good and the results appear to be reliable for all three main constituents of the substance.

Based on the physico-chemical similarities between the target and source substances, it can be concluded that the target substance fatty acids, C14-22, C16-24-alkyl esters (CAS 92797-30-3) is readily biodegradable according to the guideline criteria, which is further supported by QSAR calculations.