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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Sep 2014 - 27 Oct 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- A mixed population of activated sewage sludge micro-organisms was obtained on 23 September 2014 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- The activated sewage sludge sample was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 °C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.3 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
13.4 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Test Item: Following the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was adsorbed onto silica gel prior to dispersion in mineral medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.
An amount of test item (40.2 mg) was adsorbed onto the surface of 500 mg of granular silica gel prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (7500 rpm, 15 minutes). The test item/silica gel/mineral mineral medium dispersion was then dispersed in inoculated mineral medium and the volume adjusted to 3 liters to give a final concentration of 13.4 mg/L, equivalent to 10 mg carbon/L.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

- Reference Item: A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

- Toxicity Control: A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An amount of test item (40.2 mg) was adsorbed onto the surface of 500 mg of granular silica gel prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (7500 rpm, 15 minutes). The test item/silica gel/mineral medium dispersion was then dispersed in inoculated mineral medium and an aliquot (51.4 mL) of the sodium benzoate stock solution added. The volume was adjusted to 3 liters to give a final concentration of 13.4 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

- Preparation of Test System: The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium plus 500 mg silica gel
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
Silica gel was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at 20 to 22 °C, in darkness. Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 39.1 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. The pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb ) granules. The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.
- Observations: The appearance of the test preparations was recorded on Days 0, 5, 12, 19 and 26
- pH Measurements: The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter or a Hach 160d handheld meter.
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
72
Sampling time:
28 d
Details on results:
Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an decrease in all replicate vessels. This decrease was considered to be due to sampling/analytical variation. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
The test item attained 72% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The toxicity control attained 70% biodegradation after 14 days and 83% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect an the sewage treatment micro-organisms used in the test.

Results with reference substance:
Sodium benzoate attained 86% biodegradation after 14 days and 96% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

 Table. Percentage Biodegradation Values

Day

% Biodegradation

Procedure Control

Test Item

Toxicity Control

0

0

0

0

1

41

1

27

5

72

33

53

8

90

55

76

10

97

63

79

14

86

72

70

21

98

76

86

28

99

75

87

29*

96

72

83

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

 

 Table. Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel

Total Carbon**
(mg/L)

Inorganic Carbon*
(mg/L)

IC Content (')/0 of TC)

Test Item

10 mg C/L R1

10.14

-0.02

0

Test Item

10 mg C/L R2

9.54

-0.02

0

R1 - R2 = Replicates 1 and 2

* Corrected for control values. Negative values are due to measured concentrations being less than control values

** Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

 

Table. pH Values of the Test Preparations an Days 0 and 28

Test Vessel

pH

Day 0
Pre-Adjustment

Day 0
Post-Adjustment

Day 28

Inoculum Control R1

7.8

7.6

7.5

Inoculum Control R2

7.8

7.6

7.5

Procedure Control R1

7.8

7.5

7.5

Procedure Control R2

7.8

7.5

7.5

Test Item R1

7.8

7.6

7.5

Test Item R2

7.8

7.6

7.5

Toxicity Control

7.8

7.5

7.5

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item attained 72% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

Readily biodegradable: 72% (CO2 evolution) in 28 days

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

One study is available assessing the ready biodegradability of Diisodecyl azelate (CAS 28472-97-1) in an aerobic aqueous medium (Harlan, 2014). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008.

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at 20 to 22 °C for 28 days. Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

The test item attained 72% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.