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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:
06 September 2016 to 05 October 2016
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)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO 10634 Water Quality – Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge: A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed twice by settlement and re-suspension 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 (Rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven) using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionised 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.
- Concentration of sludge: The suspended solids concentration was equal to 3.2 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
13.6 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
other: carbon/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: To 1 litre (final volume) of purified water (Reverse osmosis purified and deionised water, Elga Optima 15+ or Elga Purelab Option R-15 BP) was added the following volumes of solutions A to D: 10 mL of Solution A and 1 mL each of Solutions B, C and D.
Solution A: 8.50 g/L KH2PO4, 21.75 g/L K2HPO4, 33.40 g/L Na2HPO4.2H2O, 0.50 g/L NH4Cl; pH = 7.4
Solution B: 27.50 g/L CaCl2
Solution C: 22.50 g/L MgSO4.7H2O
Solution D: 0.25 g/L FeCl3.6H2O (one drop of concentrated HCl/L was added as a preservative)
- Solubilising agent (type and concentration if used): The test material was adsorbed onto silica gel prior to dispersion in mineral medium to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms. 40.8 mg of test material was adsorbed onto the surface of 500 mg of granular silica gel (230-400 mesh Sigma Lot No BCBM6195V) prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (7500 rpm, 5 minutes). The test item/silica gel/mineral medium dispersion was then dispersed in inoculated mineral medium.
- Test temperature: 21 to 24 °C
- Aeration of dilution water: Yes
- pH: 7.4 ± 0.2
- pH adjusted: Yes; diluted hydrochloric acid or sodium hydroxide solution were used prior to the volume in all the vessels being adjusted to 3 litres by the addition of mineral medium which had been purged overnight with CO2 free air.
- Suspended solids concentration: 30 mg (ss)/ L (final concentration per vessel)
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 5 litre test culture vessels containing 3 litres of solution.
- Number of culture flasks/concentration: Duplicate test material flasks (test material in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 10 mg carbon/L)
- Method used to create aerobic conditions: Approximately 24 hours prior to addition of the test and reference materials the vessels were filled with 2400 mL of mineral medium and 28.1 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added. The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Measuring equipment: CO2 absorbing solutions, pH meter and Shimadzu TOC-VCSH TOC analyser or a Shimadzu TOC-LCSH TOC analyser.
- Details of trap for CO2 and volatile organics if used: 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.

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
- Sampling method: Samples were analysed for IC using either a Shimadzu TOC-VCSH TOC analyser or a Shimadzu TOC-LCSH TOC analyser. Samples (135 or 50 μL) were injected into the IC channel of the TOC analyser. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2 M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.
- Sample storage before analysis: Samples were analysed for IC immediately.
- Other: To calculate the IC/TC ratio, samples (30 mL) were removed from the test material vessels on Day 0 prior to the addition of the test material in order to calculate the IC content in the test media. The samples were filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. IC/TC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 vessels consisting of inoculated mineral medium plus 500 mg silica gel
- Positive control (procedure control): 2 vessels containing the reference material in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 10 mg carbon/L
- Toxicity control: 1 vessel of the test material plus the reference material in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 20 mg carbon/L

STATISTICAL METHODS:
- Calculation of carbon content
The theoretical amount of carbon present in the test material was calculated as follows:
(No. of C atoms x mol wt of C / mol wt of test material) x 100 = (17 x 12.011 / 277.41) x 100 = 73.60 %
Thus for a 10 mg C/L test concentration the total organic carbon present for the test material was 30 mg C.
The theoretical amount of carbon present in the reference material was calculated as follows:
(No. of C atoms x mol wt of C / mol wt of sodium benzoate) x 100 = (7 x 12.011 / 144.11) x 100 = 58.34 %
Thus for a 10 mg C/L test concentration the total organic carbon present for sodium benzoate was 30 mg C.

- Percentage Biodegradation
The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values into the following equation. The values of Replicates R1 and R2 are averaged for the inoculum control, test and reference materials before substitution into the following equation:
% ThCO2 (%biodegradation*) = [(mg IC in test flask – mg IC in control flask) / mg TOC added as test chemical] x 100
*the conversion factor for carbon to carbon dioxide is 3.67

- Total CO2 Evolution
The total CO2 evolution in the inoculum control vessels at the end of the test is calculated from the equation below. The mean inorganic carbon values for Replicates R1 and R2 on Day 28 are calculated before substitution into the equation:
Total CO2 evolution (mg C/L) = mg IC in control x (100 / %C of CO2) x (1 / test volume) = mg IC in control x (100 / 27.29) x (1/3)
Reference substance:
benzoic acid, sodium salt
Test performance:
All validation criteria for this test were met so the study is considered to be valid.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
85
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 material. The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels.
The test material attained 85 % biodegradation after 28 days and satisfied the 10-Day window validation criterion.
The toxicity control attained 52 % biodegradation after 14 days and 78 % biodegradation after 28 days thereby confirming that the test material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

VALIDATION CRITERIA
-The results of the degradation test are considered valid if in the same test the reference material yields ≥ 60 % degradation (in a 10-Day window) by Day 14.
-The test material may be considered to be readily biodegradable if ≥ 60 % degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10 %.
-The toxicity control (test material and sodium benzoate) should attain ≥ 25 % degradation by Day 14 for the test material to be considered as non-inhibitory.
-The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the time the plateau is reached, at the end of the test or at the end of the 10-Day window as appropriate, is less than 20 %.
- The total CO2 evolution in the inoculum control vessels at the end of the test should not normally exceed 40 mg/L medium (= 120 mg/3 litres, corresponding to 33 mg C per flask), however values up to 70 mg/L are acceptable.
-The IC content of the test material suspension in the mineral medium at the beginning of the test should be <5 % of the TC.
Results with reference substance:
Sodium benzoate attained 82 % biodegradation after 14 days and 96 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table 1: Percentage Biodegradation Values

Day

% Biodegradation

Procedure Control

Test Material

Toxicity control

0

0

0

0

2

58

4

28

6

69

7

35

8

78

23

48

10

86

52

62

14

82

51

52

21

99

67

65

28

74

94

76

29*

96

85

78

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

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of this study the test material was considered to be readily biodegradable.
Executive summary:

The ready biodegradability of the test material was determined in accordance with the standardised guidelines OECD 301B, EU Method C.4-C, US EPA OCSPP 835.3110 and ISO 10634 under GLP conditions using the CO2 evolution test.

The test material, 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 21 to 24 °C for 28 days. Preliminary solubility work conducted showed the test material was insoluble in water at a concentration of 100 mg/L, therefore the test material was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

The biodegradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test material attained 85 % biodegradation after 28 days and satisfied the 10-Day window validation criterion. The toxicity control attained 52 % biodegradation after 14 days and 78 % biodegradation after 28 days thereby confirming that the test material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. Sodium benzoate attained 82 % biodegradation after 14 days and 96 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Under the conditions of this study the test material was considered to be readily biodegradable.

Description of key information

Under the conditions of this study the test material was considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The ready biodegradability of the test material was determined in accordance with the standardised guidelines OECD 301B, EU Method C.4-C, US EPA OCSPP 835.3110 and ISO 10634 under GLP conditions using the CO2 evolution test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material, 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 21 to 24 °C for 28 days. Preliminary solubility work conducted showed the test material was insoluble in water at a concentration of 100 mg/L, therefore the test material was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

The biodegradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test material attained 85 % biodegradation after 28 days and satisfied the 10-Day window validation criterion. The toxicity control attained 52 % biodegradation after 14 days and 78 % biodegradation after 28 days thereby confirming that the test material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. Sodium benzoate attained 82 % biodegradation after 14 days and 96 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Under the conditions of this study the test material was considered to be readily biodegradable.

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