Benzene, ethylenated, residues, distn. lights

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:

December 8, 2008 - January 5, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted under GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

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 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO Guideline No 14593 "Water quality - Evaluation of ultimate aerobic biodegradability of organic compunds in aqueous medium-Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)

Deviations:

no

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethylbenzene

EC Number:

700-371-5

Cas Number:

178535-25-6

IUPAC Name:

ethylbenzene

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Study design

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 Loghborough, Leicester, UK, which treats predominantly domestic sewage.

Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 ml discarded).
In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air* for approximately 1 hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.3 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2 litres) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2-free air until use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

20 mg/L

Based on:

other: carbon

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

inorg. C analysis

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 CaCl2.2H2O 36.40 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 incubated in 125 ml glass Wheaton bottles (total volume when full 160 ml) each containing 107 ml of solution.
a) A control consisting of inoculated mineral salts medium, 33 replicate vessels.
b) The standard material (sodium benzoate) in inoculated mineral salts medium, to give a final concentration of 20 mg carbon/l, 33 replicate vessels.
c) The test material in inoculated mineral salts medium, to give a final concentration of 20 mg carbon/l, 29 replicate vessels.
d) The test material plus the standard material in inoculated mineral salts medium, to give a final concentration of 40 mg carbon/l to act as a toxicity control, 9 replicate vessels.

Test media a-d were inoculated with the prepared inoculum at a final concentration of 100 ml/l. Aliquots (107 ml) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel with triplicate vessels for the control, standard material, test material and toxicity control at each sampling occasion (five replicates for analysis on Day 28). Additional control and standard material vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on days 0 and 28 (duplicate vessels per sampling occasion).

TEST SYSTEM
CO2 production in the vessels was determined by measuring the increase in the concentration of Inorganic Carbon (IC) in the headspace. Triplicate control, standard material and test material vessels were sacrificed on days 0, 2, 4, 8, 10, 14, 16 and 21 for IC analysis. On day 28, five replicate vessels were sacrificed for IC analysis. Triplicate toxicity control vessels were sacrificed on days 0, 8 and 14 for IC analysis.

Prior to sampling, concentrated orthophosphoric acid (1 mL) was injected through the septum of each vessel taken for analysis in order to lower the pH of the medium to < 3*. The vessels were then continued to be shaken at approximately 150 rpm (INFORS TR-225 orbital platform shaker) for 1 hour at 20±1 degC before a sample was withdrawn from the headspace and analysed for IC.

SAMPLING
On Days 0 and 28 samples (20 ml) were removed from the control and standard material vessels prepared for DOC analysis 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. See analytical methods for further details.

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.

Reference substance

Reference substance:

other: sodium benzoate

Results and discussion

Preliminary study:

Preliminary work showed that the test material was volatile and hence the conditions employed for a Modified CO2 Evolution Test (OECD Guideline No 301B) would not be suitable for the determination of the biodegradability of the test material. The test method was therefore changed to a CO2 Headspace Test, which is recommended for volatile materials.

The test material was added directly to the test vessels using a high precision volumetric syringe. Preliminary work conducted showed that a volume of 2.7 µl of test material injected into a test vessel using a gas tight micro-syringe gave a measured weight of 2.4 mg, mean of 15 separate weighings.

Test performance:

No further information.

% Degradation

Parameter:

% degradation (inorg. C analysis)

Value:

15

Sampling time:

28 d

Details on results:

The biodegradation was assessed by measuring the inorganic carbon present in the headspace of the vessels. Total inorganic carbon values for the test material, standard material, toxicity control and control vessels was measured and are reported in the table below.

The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 5% of the organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline. The test material attained 15% degradation after 28 days and hence cannot be considered to be readily biodegradable.

The toxicity control attained 41% degradation after 8 days and 44% degradation after 14 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

DOC analysis was also conducted and the results tabulated below. Samples taken from the standard material vessels on Days 0 and 28 (see Table 3) gave percentage degradation values of 100% and 99% respectively for Replicates R1 and R2. The degradation rates for the standard material were higher than those determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO2 evolution occurring.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 81% degradation after 14 days and 87% degradation 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

The results from the biodegradation tests are below:

Table1            Inorganic Carbon Values on Each Analysis Occasion

Day	Control (mg TIC)					Sodium Benzoate (mg TIC)					Test Material (mg TIC)					Toxicity Control (mg TIC)
	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5
0	0.09	0.13	0.11	-	-	0.09	0.09	0.09	-	-	0.10	0.09	0.09	-	-	0.09	0.08	0.09	-	-
2	0.09	0.12	0.09	-	-	1.36	1.32	1.08	-	-	0.09	0.09	0.09	-	-	-	-	-	-	-
4	0.09	0.14	0.13	-	-	1.70	1.73	1.74	-	-	0.13	0.10	0.11	-	-	-	-	-	-	-
8	0.18	0.13	0.13	-	-	2.06	2.00	1.94	-	-	0.25	0.18	0.17	-	-	1.90	1.84	2.00	-	-
10	0.08	0.12	0.13	-	-	1.88	1.93	1.82	-	-	0.23	0.20	0.21	-	-	-	-	-	-	-
14	0.14	0.14	0.14	-	-	1.87	1.80	1.95	-	-	0.49	0.55	0.48	-	-	1.87	2.09	2.13	-	-
16	0.15	0.17	0.13	-	-	1.76	1.86	1.46	-	-	0.48	0.41	0.47	-	-	-	-	-	-	-
21	0.07	0.18	0.16	-	-	1.78	1.74	1.84	-	-	0.48	0.59	0.51	-	-	-	-	-	-	-
28	0.07	0.10	0.11	0.12	0.11	1.94	2.06	1.86	2.06	1.80	0.48	0.26	0.52	0.52	0.43	-	-	-	-	-

Table2              Percentage Biodegradation Values

Day	% Degradation
	Sodium Benzoate	Test Material	Toxicity Control
0	0	0	0
2	54	0	-
4	75	0	-
8	86	2	41
10	83	5	-
14	81	17	44
16	72	14	-
21	77	18	-
28	87	15	-

Table 3              Dissolved Organic Carbon (DOC) Values in the Control and Standard Material Vessels on Days 0 and 28

DOC Concentration
Test Vessel		Day 0			Day 28
		mg C/l	mg C/l Corrected for Mean Control Value	% of Nominal Carbon Content	mg C/l	mg C/l Corrected for Mean Control Value	% Degradation
Control	R1	2.18	-	-	<LOQ	-	-
	R2	2.11	-	-	1.43	-	-
Standard Material	R1	23.05	20.90	105	1.25	<control	100
	R2	23.17	21.02	105	1.65	0.22	99

R₁– R₂= Replicates 1 and 2

LOQ = Limit of quantitation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material attained 15% degradation after 28 days and therefore cannot be considered to be readily biodegradable.

Executive summary:

Preliminary work showed that the test material was volatile and hence the conditions employed for a Modified CO2 Evolution Test (OECD Guideline No. 301B) would not be suitable for the determination of the biodegradability of the test material. The test method was therefore changed to a CO2 Headspace test, which is recommended for volatile materials. This test was conducted in sealed vessels and assessed the biodegradation of a test material by measuring the inorganic carbon present in the headspace of the vessels.

The test material, at a concentration of 20 mg C/l, was exposed to activated sewage sludge micro-organisms with mineral salts medium in sealed culture vessels in the dark at 20±1.0°C for 28 days.

The degeneration of the test material was assessed by the determination of carbon dioxide produced on Days 0, 2, 4, 8, 10, 14, 16, 21 and 28. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

The mean TIC in the control vessels on Day 28 was 0.10 mg/l; equivalent to 5% of the organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline. The test material attained 15% degradation after 28 days and hence cannot be considered to be readily biodegradable.

The toxicity control attained 41% degradation after 8 days and 44% degradation after 14 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study. Sodium benzoate attained 81% degradation after 14 days and 87% degradation 28 days thereby confirming the suitability of the inoculum and test conditions. All validity criteria was therefore met.