Alkenes, C15-18-branched and linear, maleated, hydrolyzed

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 27, 2009 - March 4, 2009

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)

Deviations:

yes

Remarks:

Please see below

Qualifier:

according to guideline

Guideline:

other: US EPA Fate, Transport and Transformation test Guidelines OPPTS 835.3110 (Paragraph (m))

Deviations:

no

Principles of method if other than guideline:

The test method, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21 degC 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 mediumand to increase the surface area of the test material exposed to 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.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

A mixed population of activated sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, U.K., which treats predominantly domestic sewage.

The activated 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 21 °C and used on the day of collection. Determination of the suspended solids level of the activated 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 105 °C 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 2.9 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

other: Carbon

Initial conc.:

13.9 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
Culture Medium:
Solution a KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H20 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

The following volumes of the above solutions were added to 1 litre (final volume) of purified water.
10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d

The following test preparations were prepared and inoculated in 5 L glass culture vessels each containing 3 L 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 slica gel to give a final concentration of 10 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 20 mg carbon/L to act as a toxicity control (one vessel only).

In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organisation (ISO 1995) and the published literature (Handley et al., 2002) is to adsorb the test material onto an inert support prior to dispersion in the test vessels. Using this method the test material is evenly distributed throughout the test medium and the surface area of test material exposed to the test organisms is increased thereby increasing the potential for biodegradation.

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 31 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 L by the addition of culture medium.

TEST SYSTEM
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 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
Samples (2mL) were taken from the first CO2 absorber vessel on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.

The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 were analysed for CO2 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.

The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 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.


CONTROL AND BLANK SYSTEM
Control vessels were prepared containing 100 mg silica gel per 3 litres of inoculated culture medium in order to maintain consistency between the control and test material vessels.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Pre-study solubility work using ultrasonication and high shear mixing confirmed the test material had limited solubility in water, and following additional solubility work to ascertain which method would provide the best testable dispersion, it was concluded appropriate to adsorb the test material onto silica gel and disperse with the aid of high shear mixing as this method appeared to increase the dispersibility of the test material over the other methods.

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 39.75 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines. In addition, 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 therefore also satisfied the OECD criteria.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

22

Sampling time:

28 d

Details on results:

The test material attained 22% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Results with reference substance:

The toxicity control attained 47% degradation after 14 days and 49% degradation after 28 days thereby confirming the test material was not toxic to the sewage treatment micro-organisms used in the test. Sodium benzoate attained 70% degradation after 14 days and 89% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

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 exception of the toxicity control vessel. 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 22% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B. Please see Table 1 for further values.

Table 1: Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
2	47	3	30
6	62	8	42
8	65	11	42
10	66	8	48
14	70	13	47
21	88	25	54
28	88	21	52
29*	89	22	49

 

Table 2. 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
	Abs 1	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	0.47	0.35	0.35	0.35	0.35	0.35	0.35	0.70	0.47	0.70	0.35	0.70	0.35	0.70
2	7.31	-	6.61	-	20.53	-	21.46	-	6.85	-	11.25	-	26.80	-
6	16.49	-	17.30	-	33.56	-	37.60	-	17.19	-	21.80	-	39.21	-
8	16.86	-	20.07	-	36.23	-	39.79	-	19.72	-	23.85	-	38.87	-
10	22.34	-	24.28	-	40.59	-	45.72	-	23.83	-	31.46	-	46.17	-
14	24.37	-	26.18	-	44.99	-	47.60	-	28.22	-	30.26	-	49.30	-
21	29.41	-	30.42	-	53.52	-	59.04	-	32.45	-	35.94	-	56.33	-
28	30.80	-	34.27	-	57.46	-	60.14	-	33.94	-	36.51	-	55.44	-
29	33.29	1.39	35.51	1.51	60.56	1.51	61.46	1.74	32.62	1.51	40.30	2.09	58.34	1.28

R1 - R2 = Replicates 1 and 2

Abs = CO2 adsorber vessels

Table 3. Total and Inorganic Carbon Values in the Culteure Vessels on Day 0.

Test vessel	Total Carbon* (mg/l)	Inorganic Carbon* (mg/l)	IC Content (% of TC)
Sodium Benzoate 10 mg C/l R1	10.01	0.45	4
Sodium Benzoate 10 mg C/l R2	9.47	-1.49	0
Test material 10 mg C/l R1	8.97**	-1.37	0
Test material 10 mg C/l R2	9.00**	-1.21	0
Test material plus Sodium Benzoate Toxicity Control 20 mg C/l	20.26**	0.28	1

R1 - R2 = Replicates 1 and 2

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

** Total Carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test material and sodium benzoate where applicable.

Table 4. 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 Content	% Degradation
Sodium Benzoate 10 mg C/l R1	9.56	96	0.12	1	99
Sodium Benzoate 10 mg C/l R2	10.96	110	-0.03	0	100

* Corrected values. Negative values are dur to measured concentrations being less than control values.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test material attained 22% 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:

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The test material, at a concentration of 10 mg carbon/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 (Handleyet 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.

The test material attained 22 % degradation after 28 days and therefore cannot be considered readily biodegradable under the strict terms and conditions of OECD Guideline No. 301 B.

Description of key information

1 - 10 tonnes of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes is imported into Europe per annum. The data requirements for this tonnage band require that a Ready Biodegradability test is carried out on the substance.

Therefore, a study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The test material, at a concentration of 10 mg carbon/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 (Handleyet 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.

The test material attained 22 % degradation after 28 days and therefore cannot be considered readily biodegradableunder the strict terms and conditions of OECD Guideline No. 301 B.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater