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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:
30 April 2015 to 15 June 2015
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- A nominal amount of test material (1000 mg) was dissolved in mineral medium with the aid of ultrasonication (5 minutes) and the volume adjusted to 1 litre to give a 1000 mg/L stock solution. An aliquot (65.1 mL) of this stock solution was dispersed in inoculated mineral medium and the volume adjusted to 3 litres to give a final concentration of 21.7 mg/L, equivalent to 9.3 mg carbon/L. The volumetric flask containing the test material was inverted several times to ensure homogeneity of the solution.
- At the time the study was performed it was calculated that a test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines. However this was calculated using the incorrect empirical formula, therefore the test concentration actually employed was 9.3 mg carbon/L.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source: 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 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 ºC and used on the day of collection.
- Concentration of sludge: 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 ºC 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 3.3 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
21.7 mg/L
Based on:
test mat.
Initial conc.:
9.3 mg/L
Based on:
other: Carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral 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 – d: 10 mL of Solution a, 1 mL of Solution b, 1 mL of Solution c and 1 mL of Solution d.
Solution a: KH2PO4 8.50 g/L, K2HPO4 21.75 g/L, Na2HPO4.2H2O 33.40 g/L and NH4Cl 0.50 g/L, pH = 7.4.
Solution b: CaCl2 27.50 g/L.
Solution c: MgSO4.7H2O 22.50 g/L.
Solution d: FeCl3.6H2O 0.25 g/L.
- Test temperature: 22-24 °C
- pH: 7.4 ± 0.2
- pH adjusted: yes, using diluted hydrochloric acid or sodium hydroxide solution 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.
- Aeration of dilution water: Approximately 24 hours prior to addition of the test and reference materials the vessels were filled with 2400 mL of mineral medium and 27.3 mL of inoculum and aerated overnight.
- Suspended solids concentration: 30 mg (ss)/L
- Continuous darkness: yes/no

TEST SYSTEM
- Culturing apparatus: 5 litre test culture vessels each containing 3 litres of solution
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: 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.
- 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
- IC Analysis: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 24, 28 and 29. The second absorber vessels were sampled on Days 0 and 29. All samples were analysed for IC immediately. 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.
- IC/TC Ratio: Samples (30 mL) were removed from the inoculum control and test material 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. The samples were analysed for IC and TC using a Shimadzu TOC-VCPH TOC Analyser.
- Observations: The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.

CONTROL AND BLANK SYSTEM
- Inoculum blank: An inoculated control, in duplicate, consisting of inoculated mineral medium was used in the study.
- Reference material: A reference material, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels in duplicate. An initial stock solution of 1000 mg/L was prepared by dissolving the reference material directly in mineral medium with the aid of ultrasonication for approximately 10 minutes. 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 litres to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference material was inverted several times to ensure homogeneity of the solution.
- Toxicity control: A toxicity control (one vessel only), containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test. An aliquot (65.1 mL) of the test material stock solution was dispersed in inoculated mineral medium along with an aliquot (51.4 mL) of the sodium benzoate stock solution. The volume was adjusted to 3 litres to give a final concentration of 21.7 mg test material/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 19.3 mg carbon/L.

DATA EVALUATION
- Calculation of Carbon Content:
The theoretical amount of carbon present in the test material (C12H17N4OSClHCl) was calculated as follows:
[(No. of C atoms x mol wt of C) / mol wt of test material] x 100
[(12 x 12.011) / 337.27] x 100 = 42.73 %
Thus for a concentration of 9.3 mg C/L the total organic carbon present was 27.9 mg C.

The theoretical amount of carbon present in the reference material, sodium benzoate (C6H5COONa) 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 = 58.24 %
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 meaned 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 material] x 100

- 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 inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned 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)

VALIDATION CRITERIA
- The results of the degradation test are considered valid if in the same test the reference material yields ≥ 60 % degradation 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. Data from studies where values in excess of 70 mg/L are obtained should be critically examined.
- The IC content of the test material suspension in the mineral medium at the beginning of the test should be <5 % of the TC.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test material did not adsorb to filter matrices or to activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test material.
Test performance:
- The total CO2 evolution in the inoculum control vessels on Day 28 was 35.70 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
- The IC content of the test material suspension in the mineral medium at the start of the test was below 5 % of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
- The difference between the values for CO2 production at the end of the test for the replicate vessels was <20 % and hence satisfied the validation criterion given in the OECD Test Guidelines
Key result
Parameter:
% degradation (CO2 evolution)
Value:
115
Sampling time:
28 d
Details on results:
BIODEGRADATION
- 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 with the exception of test material replicate 1 and the toxicity control. 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 material attained 115 % 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 material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
- Biodegradation values in excess of 100 % were considered to be due to an increase in the numbers of viable micro-organisms in the test material vessels as a result of the readily biodegradable nature of the test material. This effect occurs due to the micro-organisms utilising the test material as a carbon source for cellular growth resulting in a greater number of viable micro-organisms in these vessels when compared to the control vessels. The increased number of micro-organisms in these vessels gave rise to increased respiration rates and hence background CO2 evolution was greater than in the control vessels. This increase in background CO2 evolution resulted in biodegradation rates in excess of 100 %.
- The toxicity control attained 51 % 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.
Results with reference substance:
Sodium benzoate attained 88 % biodegradation after 14 days and 98 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions

Table 1: Dissolved Organic Carbon (DOC) Values from the Preliminary Investigational Work

Sample

DOC Concentration

% of Nominal Carbon Content

mg C/L

mg C/L Correct for Appropriate Control

Mineral medium

<LOQ

 

-

-

Control, inoculated at 30 mg ss/L, Filtered

<LOQ

 

-

-

Control, inoculated at 30 mg ss/L, Centrifuged

<LOQ

 

-

-

100 mg/L Untreated

42.98

42.98

93

100 mg/L Filtered

42.85

42.85

93

100 mg/L, inoculated at 30 mg ss/L, Filtered

42.53

42.53

92

100 mg/L, inoculated at 30 mg ss/L, Centrifuged

42.53

42.53

92

Table 2: Percentage Biodegradation Values

Day

% Biodegradation

Procedure Control

Test Material

Toxicity Control

0

0

0

0

2

33

0

25

6

34

0

32

8

87

14

37

10

88

9

34

14

88

52

51

21

93

108

68

24

94

111

79

28

96

116

79

29*

98

115

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 attained 115 % biodegradation after 28 days and satisfied the 10-Day window validation criterion, the test material can therefore be considered to be readily biodegradable.
Executive summary:

The biodegradation potential of the test material was investigated in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and EPA OCSPP 835.3110, under GLP conditions.

The study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The test material, at a concentration of 9.3 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 22 to 24 °C for 28 days. 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 115 % 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 material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Biodegradation values in excess of 100 % were considered to be due to an increase in the numbers of viable micro-organisms in the test material vessels as a result of the readily biodegradable nature of the test material. This effect occurs due to the micro-organisms utilising the test material as a carbon source for cellular growth resulting in a greater number of viable micro-organisms in these vessels when compared to the control vessels. The increased number of micro-organisms in these vessels gave rise to increased respiration rates and hence background CO2 evolution was greater than in the control vessels. This increase in background CO2 evolution resulted in biodegradation rates in excess of 100 %.

All validity criteria of the test were met.

Under the conditions of this study the test material attained 115 % biodegradation after 28 days and satisfied the 10-Day window validation criterion, the test material can therefore be considered to be readily biodegradable.

Description of key information

Under the conditions of this study the test material attained 115 % biodegradation after 28 days and satisfied the 10-Day window validation criterion, the test material can therefore be considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The biodegradation potential of the test material was investigated in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and EPA OCSPP 835.3110, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The test material, at a concentration of 9.3 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 22 to 24 °C for 28 days. 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 115 % 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 material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Biodegradation values in excess of 100 % were considered to be due to an increase in the numbers of viable micro-organisms in the test material vessels as a result of the readily biodegradable nature of the test material. This effect occurs due to the micro-organisms utilising the test material as a carbon source for cellular growth resulting in a greater number of viable micro-organisms in these vessels when compared to the control vessels. The increased number of micro-organisms in these vessels gave rise to increased respiration rates and hence background CO2 evolution was greater than in the control vessels. This increase in background CO2 evolution resulted in biodegradation rates in excess of 100 %.

All validity criteria of the test were met.

Under the conditions of this study the test material attained 115 % biodegradation after 28 days and satisfied the 10-Day window validation criterion, the test material can therefore be considered to be readily biodegradable.

[Type of water: freshwater]