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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:
The study was conducted between 7 October 2008 and 4 November 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO Guideline No 14593 “Water quality – Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium – Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of Inspection 21/08/07 Date of Signature 15/10/07
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
A mixed population of sewage sludge micro-organisms was obtained on 7 October 2008 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK

- Laboratory culture:
Not applicable

- Method of cultivation:
Aliquots (107 ml) of inoculated culture medium were dispensed to each of 29 replicate test vessels and the test vessels sealed using Teflon lined silicon septa and aluminium crimp caps. After sealing, an aliquot (3.7 µl) of test material was injected through the septum of each vessel to give the required test concentration of 23.8 mg/l, equivalent to 20 mg carbon/l.

- Storage conditions:
Not Stated

- Storage length:
Not Stated

- Preparation of inoculum for exposure:
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 (3 litres) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2-free air until use.

* CO2-free air produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules

- Pretreatment:
The test material was a poorly water soluble, volatile, non-viscous liquid and hence following the recommendations of the International Standards Organisation (ISO 1995) for dealing with such compounds, for the purpose of the biodegradation test the test material was added directly to the test vessels using a high precision volumetric syringe. Using this method enabled relatively small amounts of test material to be accurately added to each test vessel.
Preliminary work conducted showed that a volume of 3.7 µl of test material injected into a test vessel using a gas tight micro-syringe (SGE P/N 001100 5FX) gave a measured weight of 2.5 mg (mean of 15 separate weighings).

- Concentration of sludge:
A test concentration of 20 mg carbon/l was employed in the study following the recommendations of the test guidelines.

- Initial cell/biomass concentration:
Not recorded

- Water filtered: yes

- Type and size of filter used, if any:
Coarse filter paper
Duration of test (contact time):
28 d
Initial conc.:
20 other: mg C/l
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of 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 CaCl2.2H2O 36.40 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
* Reverse osmosis purified and deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)

- Additional substrate:
Not applicable

- Solubilising agent (type and concentration if used):
Not applicable

- Test temperature:
incubated at 20±1 deg C

- pH:
An aliquot (1.0 ml) of concentrated orthophosphoric acid was injected through the septum of each vessel taken for analysis in order to lower the pH of the medium to < 3

- pH adjusted:
yes using concentrated orthophosphoric acid

- CEC (meq/100 g):
Not recorded

- Aeration of dilution water:
Not recorded

- Suspended solids concentration:
Not recorded

- Continuous darkness:
no


TEST SYSTEM
Preparation of test system
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 culture medium, 33 replicate vessels.

b) The standard material (sodium benzoate) in inoculated culture medium, to give a final concentration of 20 mg carbon/l, 33 replicate vessels.

c) The test material in inoculated culture medium, to give a final concentration of 20 mg carbon/l, 29 replicate vessels.

d) The test material plus the standard material in inoculated culture 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).
All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps and incubated at 20±1oC with constant shaking at approximately 150 rpm (INFORS Version 2 Multitron® Incubator or a Gallenkamp INR-401-010W).

CONTROL AND BLANK SYSTEM
- Inoculum blank:
Not recorded

- Abiotic sterile control:
Not recorded

- 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 study.
An aliquot (34.3 ml) of the standard material stock solution was dispersed with inoculum (100 ml) and culture medium, final volume 1 litre, to give a standard material concentration of 34.3 mg/l, equivalent to 20 mg carbon/l. Aliquots (107 ml) of the 34.3 mg/l standard material concentration were dispensed to 9 replicate test vessels and each test vessel sealed using Teflon lined silicon septa and aluminium crimp caps. After sealing, an aliquot (3.7 µl) of test material was injected through the septa of each vessel to give the required test material concentration of 23.8 mg/l, equivalent to 20 mg carbon/l.
The final concentration in the toxicity control vessels was 23.8 mg test material/l plus 34.3 mg standard material/l, equivalent to 40 mg carbon/l.

Reference substance:
benzoic acid, sodium salt
Preliminary study:
The Sponsor requested that a Modified CO2 Evolution Test (OECD 301B) was conducted on the test material, however preliminary work showed that the test material was volatile and hence the conditions employed for a Modified CO2 Evolution Test (OECD Guideline 310) 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.
Test performance:
The mean TIC in the control vessels on Day 28 was 0.07 mg/l; equivalent to 3% 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 toxicity control attained 36% degradation after 14 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.
Variation in the degradation rates obtained on different sampling days (see Table 2) was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the sacrificial nature of the study design, the degradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative degradation values determined from a single vessel sampled on numerous occasions and as such variation in degradation rates on different sampling days was to be expected
Parameter:
% degradation (CO2 evolution)
Value:
6
Sampling time:
28 d
Remarks on result:
other: t½ =315 days
Details on results:
Total inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values for the test and standard materials and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1. The results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 3.

The test material attained 6% degradation after 28 days and hence cannot be considered to be readily biodegradable.
Results with reference substance:
DOC analyses conducted on samples taken from the standard material vessels on Days 0 and 28 (see Table 3) showed that the replicate standard material vessels attained 100% degradation for each replicate vessel. 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.

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

0

0.03

0.08

0.04

-

-

0.04

0.03

0.04

-

-

0.04

0.05

0.03

-

-

0.05

0.05

0.05

2

0.07

0.07

0.05

-

-

1.28

1.35

1.40

-

-

0.06

0.05

0.05

-

-

-

-

-

6

0.06

0.06

0.06

-

-

1.54

1.48

1.44

-

-

0.14

0.09

0.13

-

-

-

-

-

8

0.07

0.05

0.06

-

-

1.75

1.75

1.62

-

-

0.15

0.16

0.13

-

-

1.66

1.68

1.57

10

0.10

0.06

0.06

-

-

1.62

1.69

1.70

-

-

0.12

0.13

0.10

-

-

-

-

-

14

0.05

0.08

0.07

-

-

1.61

1.63

1.73

-

-

0.16

0.16

0.17

-

-

1.68

1.62

1.55

16

0.02

0.06

0.07

-

-

1.75

1.43

1.50

-

-

0.16

0.14

0.14

-

-

-

-

-

21

0.05

0.09

0.09

-

-

1.66

1.89

1.75

-

-

0.22

0.16

0.16

-

-

-

-

-

28

0.07

0.08

0.06

0.07

0.07

1.68

1.64

1.71

1.77

1.82

0.21

0.19

0.16

0.19

0.18

-

-

-


R1– R5= Replicates 1 to 5

- = value not determined

Table2              Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

Test Material

Toxicity Control

0

0

0

0

2

60

0

-

6

67

3

-

8

77

4

37

10

75

2

-

14

74

4

36

16

71

5

-

21

79

5

-

28

75

6

-

 

Table3              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

3.69

-

-

1.35

-

-

 

R2

2.37

-

-

1.28

-

-

Standard Material

R1

22.74

19.71

99

1.88

<LOQ

100

 

R2

22.60

19.57

98

2.19

<LOQ

100

 

R1– R2= Replicates 1 and 2

LOQ = Limit of quantitation


Validity criteria fulfilled:
yes
Interpretation of results:
other: Not readily Biodegradable
Conclusions:
The test material attained 6% degradation after 28 days and hence cannot be considered to be readily biodegradable.
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 (2006) No. 310 and the ISO Guideline No 14593 “Water quality – Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium – Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)”.

Methods.

The Sponsor requested that a Modified CO2Evolution Test (OECD 301B) was conducted on the test material, however preliminary work showed that the test material was volatile and hence the conditions employed for a Modified CO2 Evolution Test (OECD Guideline 310) would not be suitable for the determination of the biodegradability of the test material. The test method was therefore changed to a CO2Headspace 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 culture medium in sealed culture vessels in the dark at 20 ± 1.0°C for 28 days.

The degradation of the test material was assessed by the determination of carbon dioxide produced on Days 0, 2, 6, 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.

Results. The test material attained 6% degradation after 28 days and therefore cannot be considered to be readily biodegradable.

Description of key information

Oxooil LS9 reached a biodegradation rate of 6% in 28 days in a  CO2 Headspace Test (OECD 310).  Therefore, readily biodegradation could not be shown for the test substance.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

Oxooil LS9 was tested for its biodegradation potential in a CO2 Headspace test according to OECD test guideline 310 (CO2 evolution test suitable for volatile substances). The activated sludge inoculum obtained from a domestic sewage treatment plant (Loughborough, UK) proved its suitability, because the reference substance sodium benzoate reached biodegradation value of 75 % after 28 days. With a biodegradation value of 6 % (ThIC) after 28 days, Oxooil LS 9 is not readily biodegradable.