[No public or meaningful name is available]

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 May 2008 and 14 November 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Test guideline 2

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)

Remarks:

The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))

Test material

Test material information

Constituent 1

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 13 October 2008 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Laboratory culture: Not applicable
- Method of cultivation: Not applicable
- Storage conditions: The washed sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC
- Storage length: Used on day of collection.
- 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 filter 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.
- Pretreatment: As above.
- Concentration of sludge: Not stated
- Initial cell/biomass concentration: As above.
- Water filtered: yes
- Type and size of filter used, if any: Not applicable

Duration of test (contact time):

28 d

Details on study design:

TEST CONDITIONS
- Composition of medium:
Culture Medium
Solution a KH2PO4 8.50 g/l
K2HPO4 21.75 g/l
Na2HPO4.2H2O 33.40 g/l
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

To 1 litre (final volume) of purified water* was added the following volumes of solutions
a – d.

10 ml of Solution a
1 ml of Solution b
1 ml of Solution c
1 ml of Solution d

- Additional substrate: None
- Solubilising agent (type and concentration if used):None used
- Test temperature: 21°C
- pH: 7.4
- pH adjusted:no
- CEC (meq/100 g):Not available
- Aeration of dilution water: Not applicable
- Suspended solids concentration: 3.0mg/l
- Continuous darkness: yes
- Other:


TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
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.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of 5 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 15 mg carbon/l to act as a toxicity control (one vessel only).
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.

- Method used to create aerobic conditions: 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.
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: 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.
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: No
- 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 de-gassed water.
- Other:


SAMPLING
- Sampling frequency: Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
- Sampling method: The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 14, 16, 22, 28 and 29 were analysed for CO2 immediately. The samples taken on Days 12, 18, 20, 24 and 27 were stored at approximately 20°C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that these 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

On Days 0 and 28 samples (20 ml) were removed from all culture 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. Samples (27 or 13 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.

- Sterility check if applicable: Not available
- Sample storage before analysis: The samples taken on Days 12 and 18 were stored at approximately 20°C.
- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank: Not stated
- Abiotic sterile control: Not stated
- 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 aliquot (84.6 ml) of the test material stock solution was dispersed in inoculated culture 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 14.1 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 15 mg carbon/l.

- Other:


STATISTICAL METHODS: None

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test material did not absorb 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:

Observations made throughout the test period (see Table 6) showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels to be light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were light brown dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a light brown dispersion with no undissolved standard material or test material visible.

% Degradation

Parameter:

% degradation (test mat. analysis)

Value:

33

Sampling time:

28 d

Remarks on result:

other: Cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

BOD5 / COD results

Results with reference substance:

Analysis of the test media from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 12% and 15% respectively for the test material Replicates R1 and R2 and 76% for the toxicity control. Sodium benzoate attained 100% degradation for Replicates R1 and R2 calculated from the results of the DOC analyses. The degradation rates calculated from the results of the DOC analyses for sodium benzoate and the toxicity control vessels were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring. The degradation rates calculated from the results of the DOC analyses for the test material vessels were lower than those calculated from inorganic carbon analysis. This was considered to be due to sampling/analytical variation.

Observations made throughout the test period (see Table 6) showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels to be light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were light brown dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a light brown dispersion with no undissolved standard material or test material visible.

Any other information on results incl. tables

Table1              Inorganic Carbon Values on Each Analysis Occasion

Day	Control (mg IC)				Sodium Benzoate (mg IC)				Test Material (mg IC)				Test Material plus Sodium Benzoate Toxicity Control (mg IC)
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	1.05	1.63	1.28	1.40	1.05	1.40	1.05	1.63	1.05	1.40	1.05	1.40	1.05	1.75
1	3.48	-	2.20	-	4.87	-	4.06	-	3.59	-	2.78	-	4.87	-
2	5.19	-	2.77	-	15.92	-	12.92	-	4.50	-	3.81	-	17.53	-
3	7.57	-	3.33	-	21.67	-	15.48	-	8.26	-	4.93	-	24.77	-
6	9.81	-	4.79	-	23.49	-	18.70	-	8.78	-	6.84	-	28.39	-
8	11.90	-	6.12	-	28.67	-	21.87	-	11.56	-	8.73	-	30.71	-
10	13.18	-	10.14	-	37.18	-	27.15	-	11.49	-	8.68	-	33.46	-
14	12.92	-	16.70	-	38.74	-	36.18	-	15.59	-	9.80	-	38.41	-
16	13.94	-	15.71	-	39.84	-	40.50	-	18.26	-	11.07	-	39.18	-
22	17.28	-	19.67	-	42.60	-	42.60	-	22.60	-	17.71	-	41.73	-
28	19.41	-	21.12	-	43.73	-	44.69	-	27.31	-	23.25	-	43.31	-
29	18.55	1.85	20.25	2.09	41.34	2.09	40.60	1.85	25.97	2.09	22.68	1.97	48.76	1.85

 

R₁– R₂= Replicates 1 and 2

Abs= CO₂absorber vessels

Table2              Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
1	5	2	5
2	35	1	30
3	44	8	43
6	46	3	47
8	54	8	48
10	68	0	48
14	76	0	52
16	84	0	54
22	80	11	52
28	80	33	51
29*	72	33	65

 

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

Table3              Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Total Carbon* (mg/l)	Inorganic Carbon* (mg/l)	IC Content (% of TC)
Sodium Benzoate 10 mg C/lR1	9.22	0.27	3
Sodium Benzoate 10 mg C/l R2	9.53	-1.13	0
Test Material 5 mg C/l R1	4.50	-0.20	0
Test Material 5 mg C/l R2	5.10	-0.15	0
Test Material plus Sodium Benzoate Toxicity Control 15 mg C/l	13.68	-1.36	0

 

R₁– R₂= Replicates 1 and 2

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

Table4              Dissolved Organic Carbon (DOC) Values in the Culture Vessels on Days 0 and 28

Test Vessel	DOC*Concentration
	Day 0		Day 28
	mg C/l	% of Nominal Carbon Content	mg C/l	% of Initial Carbon Concentration	% Degradation
Sodium Benzoate 10 mg C/l R1	8.95	90	<control	0	100
Sodium Benzoate 10 mg C/l R2	10.66	107	<control	0	100
Test Material 10 mg C/l R1	4.70	94	4.15	88	12
Test Material 10 mg C/l R2	5.25	105	4.48	85	15
Test Material plus Sodium Benzoate Toxicity Control 20 mg C/l	15.02	100	3.66	24	76

 

Table5              pH Values of the Test Preparations on Day 28

Test Vessel	pH
ControlR1	8.2
Control R2	8.2
Sodium Benzoate 10 mg C/l R1	8.2
Sodium Benzoate 10 mg C/l R2	8.2
Test Material 5 mg C/l R1	8.1
Test Material 5 mg C/l R2	8.2
Test Material plus Sodium Benzoate Toxicity Control 15 mg C/l	8.2

 

Table6              Observations on the Test Preparations Throughout the Test Period

 

Test Vessel		Observations on Test Preparations
		Day 0	Day 6	Day 13	Day 20	Day 27
Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Standard Material	R1	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible
	R2	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible
Test Material	R1	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible
	R2	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible
Toxicity Control		Light brown dispersion, no undissolved test or standard material visible	Light brown dispersion, no undissolved test or standard material visible	Light brown dispersion, no undissolved test or standard material visible	Light brown dispersion, no undissolved test or standard material visible	Light brown dispersion, no undissolved test or standard material visible

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

The test material attained 33% 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; CO₂Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

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

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 33% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.