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Environmental fate & pathways

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:
Between 20 May 2008 and 3 September 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
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 (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
- Source of inoculum/activated sludge: Severn trent Water Plc
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture 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 21ºC 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 funnel three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC 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 3.0 g/l prior to use.
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper using Buchner funnel.

The activated sewage sludge sample was washed three times by settlement and resuspension in culture 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 21ºC 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 funnel three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC 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 3.0 g/l prior to use.
Duration of test (contact time):
28 d
Initial conc.:
5 other: mg C/l
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST SPECIES:
A mixed population of activated sewage sludge micro-organisms was obtained on 4 August 2008 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage

TEST SYSTEM
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium plus 100 mg silica gel.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 5 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium plus 100 mg silica gel to give a final concentration of 15 mg carbon/l to act as a toxicity control (one vessel only).
Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material 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 approximately 21C, in darkness.
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 30 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute 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.
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 de-gassed water.


SAMPLING
-CO2 analysis:
Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 14, 16, 22, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Days 12, 18 and 24 were stored at approximately 20C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed no significant change in degradation occurred during this time and therefore additional analyses were considered to be unnecessary.
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.
The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (300 or 50 µl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved Organic carbon (DOC) analysis:
Samples (20 ml) were removed from the test material and toxicity control vessels on Day 0 prior to the addition of the test material in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
DOC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
On Days 0 and 28 samples (20 ml) were removed from the control and standard material vessels and filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (27, 13 or 50 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate

pH Measurement:

The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
Evalaution of data: The theoretical amount of the carbon present in the test material was calculated using the equation: number of carbon atoms x molecular wt of carbon divided by molecular wt of the test material multiplied by 100%. Thus for a concentration of 5 mg C/l (a total of 31.2 mg of test material in 3 litres) the total organic carbon present was 15 mg C. The same formula was used to calculate the theoretical amount of the carbon content of the standard material, sodium benzoate.Thus for a 10 mg C/l test concentration (a total of 51.4 mg of sodium benzoate in 3 litres) the total organic carbon present for sodium benzoate was 30 mg C.

CONTROL AND BLANK SYSTEM
- Toxicity control:
For the purposes of the test, a toxicity control, 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 amount of test material (31.2 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh Sigma Lot No.101K3700) prior to dispersal in approximately 400 ml of culture medium with the aid of high shear mixing at approximately 7500 rpm for 15 minutes. The test material/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and an aliquot (51.4 ml) of the sodium benzoate stock solution added. The volume was adjusted to 3 litres to give a final concentration of 10.4 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 15 mg carbon/l.

Percentage degradation:
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, in the following equation:
The values of Replicates R1 and R2 are meaned for the control, test and standard materials before substitution in the equation.
%ThCo2(=%degradation)=mgICin test flask-mgICin control divided by mg TOC as test material multiplied by 100%. The percentage degradation from the results of the DOC analysis, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.

Equation: % degradation=[1-mg DOC in test flask on day 28/mg DOC in test flask on day 0] x 100%.

The total CO2 evolution in the 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 = mgIC in control x 100/%C of Co2 x 1/test volume.

Validation criteria:
The results of the degradation test are considered valid if in the same test the standard 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 end of the test is less than 20%.
The total CO2 evolution in the control vessels at the end of the test should not normally exceed 40 mg/l medium.
The IC content of the test material suspension in the mineral medium at the beginning of the test should be < 5% of the TC.




STATISTICAL METHODS:
Statistical analysis of the Day 29 IC values for the control and test material vessels was carried out using a Student’s t-test to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).
Reference substance:
other: sodium benzoate
Preliminary study:
In an initial experiment conducted at a concentration of 10 mg C/l, there was evidence that some inhibition of the activated sewage sludge micro-organisms had occured.
Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test material concentration was reduced to 5 mg C/l to overcome any possible inhibitory effects. It was not possible to test at concentrations below 5 mg C/l as below this concentration it is not possible to distinguish between background CO2 evolution from the inoculum and CO2 evolution due to biodegradation when using inorganic carbon analysis.
Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), 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.
Test performance:
Following the recommendations of the Test Guidelines, in the definitive test, the test material concentration was reduced to 5 mg C/l to overcome any possible inhibitory effects. It was not possible to test at concentrations below 5 mg C/l as below this concentration it is not possible to distinguish between background CO2 evolution from the inoculum and CO2 evolution due to biodegradation when using inorganic carbon analysis.
Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), 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.
Inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion were recorded. Percentage biodegradation values of the test and standard materials and the toxicity control were also documented. Total and Inorganic Carbon values in the culture vessels on Day 0 as well as the Dissolved Organic Carbon analyses performed on Days 0 and 28 a and the pH values of the test preparations on Day 28 were all recorded.
The total CO2 evolution in the control vessels on Day 28 was 31.66 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 was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
The total CO2 evolution in the control vessels on Day 28 was 31.66 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.
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 control Replicates R1 and R2, standard material Replicate R2 and test material Replicates R1 and R2. 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 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Statistical analysis of the Day 29 IC values for the control and test material vessels showed the test material IC values to be statistically lower than the control IC values (P< 0.05). However examination of the data generated from the study indicated that in terms of a biological response the IC values were similar. The test material was therefore considered not to have exhibited an inhibitory effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.
The toxicity control attained 57% degradation after 14 days and 56% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test. This slight decrease in degradation was considered to be due to sampling/analytical variation.
Sodium benzoate attained 82% degradation after 14 days and 102% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.
Results with reference substance:
Sodium benzoate attained 82% degradation after 14 days and 102% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), gave percentage degradation values of 100% for both Replicates R1 and R2.
Observations made throughout the test period showed the contents of the control vessels to be slightly cloudy light brown dispersions and the contents of the standard material vessels to be slightly cloudy light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were cloudy light brown dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a cloudy light brown dispersion with no undissolved standard material or test material visible.

Percentage Biodegradation value:

Day

% Degradation

Sodium Benzoate

% Degradation

Test Material

% Degradation

Test Material plus Sodium Benzoate Toxicity Control

0

0

0

0

1

21

0

13

2

40

0

29

3

43

0

37

6

54

0

47

8

52

0

53

10

73

0

51

14

82

0

57

16

82

0

53

22

92

0

54

27

87

0

46

28

94

0

49

29*

102

0

56


* Day 29 values corrected to include any carry-over of CO2detected in Absorber 2

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B
Executive summary:

Introduction. A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium.  The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC, and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

Methods. In an initial experiment conducted at a concentration of 10 mg C/l, there was evidence that some inhibition of the activated sewage sludge micro-organisms had occurred.

Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test material at a reduced concentration of 5 mg C/l was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), 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 degradation of the test material was assessed by the determination of carbon dioxide produced.  Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Results. The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Description of key information

Introduction:

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium.  The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC, and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

Methods:

In an initial experiment conducted at a concentration of 10 mg C/l, there was evidence that some inhibition of the activated sewage sludge micro-organisms had occurred.

Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test material at a reduced concentration of 5 mg C/l was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), 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 degradation of the test material was assessed by the determination of carbon dioxide produced.  Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Results:

The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

The toxicity control attained 57% degradation after 14 days and 56% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 82% degradation after 14 days and 102% degradation after 28 days therefore confirming the suitability of the inoculum and test conditions.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information