Z-109

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:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study was performed according to guideline. Method of sample preparation deviated from guideline. This deviation followed recommendations of ISO (1995) to improve bioavailability of poorly water soluble substance and did not affect integrity of data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Modified method of sample prep. according to recommendation of ISO 1995 & published literature of Handley et al, 2002. Substance was dissolved in auxiliary solvent & adsorpted onto granular silical get to aid dispersion and increase bioavailability

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

yes

Remarks:

Modified method of sample prep. according to recommendation of ISO 1995 & published literature of Handley et al, 2002. Substance was dissolved in auxiliary solvent & adsorpted onto granular silical get to aid dispersion and increase bioavailability

GLP compliance:

yes (incl. QA statement)

Remarks:

Issued by: The Department of Health of the government of the United Kingdom Inspection date: 19 August 2008

Test material

Test material information

Constituent 1

Details on test material:

- Lot/batch No.: CM71217681
- Storage condition of test material: room temperature in the dark
-water solubility: poorly soluble

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, 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 8 June 2009 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:
[1] The activated sewage sludge sample was washed three times by settlemnt and resuspension in culutre medium to remove excess amounts of dissolved organic carbon (DOC) that may have been present.

[2] The washed sample was then maintained on coninuous aeration in the laboratory at temperatures of approx. 21 deg. C and used on the day of collection.

[3] Determination of the suspended solids level (SSL) was carried out by filtering a 100 mL sample of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. The filter was rinsed three successive times with 10 mL deionized reverse osmosis water. The filter paper was dried in a 105 deg. C oven for 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.

- Concentration of sludge: The SSL (suspended solids level) was equal to 2.8 g/L prior to use

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

1 987 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: [A] potassium dihydrogen phosphate = 8.5 g/L + dipotassium hydrogen phosphate = 21.75 g/L + sodium phosphate dibasic dihydrate = 33.40 g/L + ammonium chloride = 0.50 g/L [B] calcium chloride = 27.50 g/L [C] magnesium sulfate heptahydrate = 22.50 g/L [D] iron chloride hexahydrate = 0.25 g/L.

The following volumes of solutions of A - D were mixed and diluted with reverse osmosis purified and deionized water to a final volume of 1 liter: [A] 10 mL + [B] 1 mL + [C] 1 mL + [D] 1 mL.


TEST SYSTEM
-Approx. 24 hours prior to addition of the test material and reference materials, 5 L glass culture vessels were filled with 2400 mL of culture medium and 32.1 mL of inoculum and aerated overnight. On Day 0, test and reference and control vessels were prepared. Each vessel contained inoculum at a final concentration of 30 mg suspended solids/L and was diluted with medium to a final volume of 3 L . The contents of the vessels were as follows:

-Control (duplicate): inoculated culture medium (30 ss/L) + 1987 mg silica gel adsorbant (final volume 3 L)

-Reference/sodium benzoate (duplicate): Inoculated culture medium(30 ss/L) + 1987 mg silica gel adsorbant + 51.4 mg sodium benzoate equivalent to 10 mg carbon/liter (final volume 3 L)

-Toxicity Control (single vessel): Inoculated culture medium (30 ss/L) + test material adsorbed onto silica gel (1987 mg) + sodium benzoate reference equivalent to 20 mg carbon/L (final volume 3 L)

-Test Material (duplicate): Inoculated culture medium (30 ss/L) + test material adsorbed onto silica gel (1987 mg) equivalent to 10 mg carbon/liter
(final volume 3 L)



CONDITIONS
- pH: Control = 7.5 - 7.6 / Reference = 7.6 / Test Material = 7.5 - 7.6 (measured on day 28)
- Test temperature: 21 deg. C
- Continuous darkness: YES
- Method used to create aerobic conditions: Culture vessels were sealed and CO2-free air bubbled through the solutions at a rate of approx. 40 mL/min with continuous stirring by magnetic stirrer. CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime granules.
-Details of trap for CO2 : CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05M NaOH prepared with purified de-gassed water.
-On day 28 (end of test) 1 mL of concentrated hydrochloric acid was added to each vessel to drive off inorganic carbonates. Vessels were re-sealed, aerated overnight and final samples taken on day 29 for analysis of inorganic carbon.


SAMPLING

-2 mL aliquots were taken for immediate CO2 analysis from the test material vessel, controls and reference vessels on days: 0, 2, 6, 8, 10, 14, 21, 28 and 29 to follow degradation.
-A 20 mL aliuout was taken and filtered through a Gelman 0.45 um Acrocap filter for DOC analysis from the test material vessel on day 0. DOC analysis was not possible on day 28 due to insoluble nature of test material in water.
-20 mL aliquots were taken and filtered through a Gelman 0.45 um Acrocap filters for DOC analysis from the control and reference vessels on days 0 and 28.


MEASURING EQUIPMENT

-Samples were analyzed for CO2 (carbon dioxide) using a Tekmar-Dohrmann Apollo 9000 TOC analyzer and a Shimadzu TOC-Vcsh TOC analyzer.
calibrated with standard solutions of sodium carbonate ( in tripicate).

-Samples were analyzed for DOC using a Shimadzu TOC-5050A analyzer and a Shimadzu TOC-Vchs analyzer calibrated with standard solutions of potassium hydrogen phthalate and sodium carbonate in deionized water (in triplicate).


CALCULATIONS/STATISTICAL METHODS:

- The % degradation (%ThCO2) of the test material was calculated from the inorganic carbon in the test substance corrected for the inorganic carbon in the control using the equation:

%ThCO2 = ( mg IC test - mg IC control)/ mg TOC test material

where: IC = inorganic carbon and TOC = total organic carbon


-Statistical analysis of the Day 29 IC values for the control and test material was determined using a Student's t-test to determine statistically significant differences between the test and control groups. Statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001)

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

No unusual occurrences were noted during performance of the test that would affect the interpretation of the results.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Details on results:

The test material attained 0% degradation after 28 days.
The toxicity control attained 30% degradation after 14 days .
The sodium benzoate reference attained 91 degradation after 14 days and 98% after 28 days.

Statistical analysis of the Day 29 inorganic carbon values for the control and test material vessels showed no statistically significant differences (P ≥ 0.05) between the control and the test material.

BOD5 / COD results

Results with reference substance:

Sodium benzoate showed 91% degradation after 14 days and 98% degradation after 28 days.

Any other information on results incl. tables

Table 1: Inorganic Carbon (IC) Values on Each analysis Occasion

Day	Control (mg IC)		Sodium Benzoate (mg IC)		Toxicity Control (mg IC)	Test Material (mg IC)
	Rep 1	Rep 2	Rep 1	Rep 2	Rep 1	Rep 1	Rep 2
0	1.63 /1.87	1.87 /2.10	1.75 / 1.75	1.87 / 1.63	1.63 / 1.63	1.63 / 1.63	1.63 / 1.63
2	6.38	5.33	7.54	15.43	8.24	12.41	5.57
6	12.8	12.69	9.92	46.36	26.37	13.81	13.73
8	17.77	17.89	42.2	53.89	33.48	17.31	18.81
10	19.38	17.44	44.23	46.17	37.17	18.47	17.9
14	22.89	21.87	19.98	49.19	40.46	22.67	22.55
21	21.63	25.01	54.42	54.42	-	23.32	23.32
28	24.75	25.31	54.32	54.99	-	25.43	25.31

Table 2: % Biodegradation

Day	Sodium Benzoate	Toxicity Control	Test Material
0	0	0	0
2	19	4	10
6	51	26	3
8	101	26	1
10	89	31	0
14	91	30	1
21	104	terminated	0
28	99	terminated	1
29	98	terminated	0

Table 3: Total carbon (TC) and Inorganic Carbon (IC) Values on Day 0

Test Vessel	Replicate	Total Carbon (mg/L)	Inorganic Carbon (mg/L)	IC as % of TC
Sodium Benzoate    (10 mg Carbon / L )	1	10.07	0.17	2
	2	9.99	0	0
Toxicity Control - Test Material + Reference (20 mg Carbon / L)	1	19.19	-0.96	0
Test Material               (10 mg Carbon / L )	1	10.26	0.32	3
	2	10.11	0.23	2

Table 4: Dissolved Organic Carbon (DOC) on Day 0 and Day 28

Vessel	Replicate	Day 0		Day 28
		mg Carbon/L	% of Nominal	mg Carbon/L	% of Nominal	% Degradation
Sodium Benzoate    (10 mg Carbon/L )	1	9.91	99	< control	0	100
	2	10	100	< control	0	100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material is not readily biodegradable.

Executive summary:

The ready biodegradability of the test substance in an aerobic aqueous medium was evaluated according to OECD 301B guidelines.

Following the recommendations of the International Standards Organization (ISO 1995) and published literature (Handley JW, Mead C, Rausina GA, Waid L J, Gee JC and Herron SJ (2002) - "The Use of Inert Carriers in Regulatory Biodegradation Tests of Low Density Poorly Water-soluble Substances", Chemosphere 48 529 -34), the test material was dissolved in aceton solvent and adsorbed onto granular silica gel to aid dispersion and increase the surface area of the test material exposed to the micro-organisms.

The test substance, at a final concentration of 10 mg carbon/L was exposed to activated sewage sludge for 28 days at 21 deg. C. Degradation was assessed by the determination of carbon dioxide produced. A sodium benzoate reference substance, a control solution with inoculum only and a toxicity control containing test material + reference were included to validate the study.

Results of the study showed 0% degradation of the test substance after 28 days indicating that the test substance is not readily biodegradable under the conditions of the OECD 301B guideline.

The guideline criteria for a vaild test were met.