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Environmental fate & pathways

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:
5 July 2010 and 3 August 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
other: Sewage sludge microorganism from the secondary treatment stage.
Details on inoculum:
Test Species
A mixed population of sewage sludge micro-organisms was obtained on 6 July 2010 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.


Preparation of inoculum
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 1 hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.5 using 7 M sodium hydroxide and the inoculum allowed to settle for 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.

* CO2-free air produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
METHODS
Preliminary Work
The test item 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 item was added directly to the test vessels using a high precision volumetric syringe. Using this method enabled relatively small amounts of test item to be accurately added to each test vessel.
Preliminary work conducted showed that a volume of 4.2 µl of test item injected into a test vessel using a gas tight micro-syringe gave a measured weight of 2.86 mg (mean of 15 separate weighings).

Experimental Preparation
For the purpose of the test, the test item was dispersed directly in culture medium.
Inoculum (350 ml) was dispersed in culture medium to a final volume of 3.5 litres and 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 (4.2 µl) of test item was injected through the septum of each vessel to give the required test concentration of 26.7 mg/l, equivalent to 20 mg carbon/l.

A test concentration of 20 mg carbon/l was employed in the study following the recommendations of the test guidelines.
Control vessels (33 replicates) were prepared as above without the addition of test item.
Data from the control vessels was shared with similar concurrent studies.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Reference Item
For the purposes of the test, a reference item, sodium benzoate (C6H5COONa) (Sigma Lot No. 096K1231), was used. An initial stock solution of 1000 mg/l was prepared by dissolving the reference item directly in culture medium. An aliquot (137.2 ml) of this stock solution was dispersed with inoculum (400 ml) and culture medium in a final volume of 4 litres, to give a test concentration of 34.3 mg/l, equivalent to 20 mg carbon/l. Aliquots (107 ml) of the 34.3 mg/l test concentration were dispensed to each of 33 replicate test vessels (125 ml glass Wheaton bottles) and the vessels sealed using Teflon lined silicon septa and aluminium crimp caps.

Toxicity Control
For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the study.
An aliquot (34.3 ml) of the reference item stock solution was dispersed with inoculum (100 ml) and culture medium in a final volume of 1 litre, to give a reference item concentration of 34.3 mg/l, equivalent to 20 mg carbon/l. Aliquots (107 ml) of the 34.3 mg/l reference item 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 (4.2 µl) of test item was injected through the septa of each vessel to give the required test item concentration of 26.7 mg/l, equivalent to 20 mg carbon/l.
The final concentration in the toxicity control vessels was 26.7 mg test item/l plus 34.3 mg reference item/l, equivalent to 40 mg carbon/l.

Culture medium
The culture medium used in this study (see Appendix 2) was that recommended in the OECD and ISO Guideline.

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 reference item (sodium benzoate) in inoculated culture medium, to give a final concentration of 20 mg carbon/l, 33 replicate vessels.
c) The test item in inoculated culture medium, to give a final concentration of 20 mg carbon/l, 29 replicate vessels.
d) The test item plus the reference item 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, reference item, test item and toxicity control at each sampling occasion (five replicates for analysis on Day 28). Additional control and reference item 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±1°C with constant shaking in darkness at approximately 125 rpm (INFORS Version 2 Multitron® Incubator).



Evaluation of data
For calculation of carbon content and percentage degradation see in attached section.

Validation criteria
Test items giving a result of = 60% yield of ThIC within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation exceeding 10%.
The test is considered valid if the reference item degradation rate is = 60% by day 14.
The toxicity control should attain = 25% degradation by day 14 for the test item to be considered as non-inhibitory.
The TIC produced from the control bottles at the end of the test should be = 15% of the TOC added initially as test item
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
Not applicable.
Test performance:
The mean TIC in the control vessels on Day 28 was 0.86 mg/l; equivalent to 4% of the organic carbon added initially as test item to the test vessels andtherefore satisfied the validation criterion given in the Test Guideline.
The test item attained 12% degradation after 28 days and hence cannot be considered to be readily biodegradable.
The toxicity control attained 49% degradation after 14 days and thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the study.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
12
Sampling time:
28 d
Remarks on result:
other: degradation obtained
Details on results:
Total inorganic carbon values for the test item, reference item, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values for the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1 (please see attached Figure 1). The results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 3.
The mean TIC in the control vessels on Day 28 was 0.86 mg/l; equivalent to 4% of the organic carbon added initially as test item to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

The test item attained 12% degradation after 28 days and hence cannot be considered to be readily biodegradable.
The toxicity control attained 49% degradation after 14 days and thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the study.




BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 74% degradation after 14 and 28 days thereby confirming the suitability of the inoculum and test conditions.

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.

DOC analyses conducted on samples taken from the reference item vessels on Days 0 and 28 (see Table 3) showed that the reference item attained 100% degradation for each replicate vessel. The degradation rates for the reference item 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.

Any other information on results incl. tables

Table1              Inorganic Carbon Values on Each Analysis Occasion

Day

Control (mg TIC)

Sodium Benzoate (mg TIC)

Test Item (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

0.04

0.07

0.07

-

-

0.07

0.07

0.07

-

-

0.05

0.05

0.05

-

-

0.05

0.07

0.05

2

0.05

0.05

0.07

-

-

1.24

1.42

1.50

-

-

0.11

0.09

0.07

-

-

-

-

-

6

0.08

0.08

0.07

-

-

1.46

1.67

1.62

-

-

0.12

0.17

0.20

-

-

-

-

-

8

0.10

0.08

0.08

-

-

1.55

1.51

1.49

-

-

0.26

0.23

0.27

-

-

1.69

1.84

1.99

10

0.08

0.08

0.08

-

-

1.68

1.62

1.72

-

-

0.29

0.29

0.32

-

-

-

-

-

14

0.10

0.07

0.07

-

-

1.67

1.60

1.71

-

-

0.33

0.31

0.27

-

-

2.24

2.10

2.16

16

0.13

0.11

0.11

-

-

1.96

1.91

1.85

-

-

0.28

0.22

0.36

-

-

-

-

-

21

0.11

0.12

0.10

-

-

1.95

1.66

1.60

-

-

0.29

0.31

0.24

-

-

-

-

-

28

0.12

0.08

0.08

0.08

0.10

1.56

1.60

1.86

1.26

1.59

0.37

0.33

0.31

0.23

0.32

-

-

-

R1– R5= Replicates 1 to 5- = No value determined

Table 2              Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

Test Item

Toxicity Control

0

0

0

0

2

62

1

-

6

70

4

-

8

67

7

41

10

74

10

-

14

74

10

49

16

84

8

-

21

76

8

-

28

74

12

-

 

- = No value determined

Table3              Dissolved Organic Carbon (DOC) Values in the Control and Reference Item 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

1.83

-

-

1.51

-

-

 

R2

1.79

-

-

1.48

-

-

Reference Item

R1

20.36

18.55

93

1.26

<control

100

 

R2

19.83

18.02

90

1.41

<control

100

 


R1– R2= Replicates 1 and 2

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 12% degradation after 28 days and therefore cannot be considered to be readily biodegradable.
Executive summary:

Introduction.

A study was performed to assess the ready biodegradability of the test item 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 (CO2headspace test)”.

Methods.

The test item, at a concentration of 20 mg C/l, was exposed to sewage treatment micro-organisms with culture medium in sealed culture vessels in the dark at 20 ± 1°C for 28 days. This test was conducted in sealed vessels and assessed the biodegradation of the test item by measuring the inorganic carbon present in the headspace of the vessels.

The degradation of the test item 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 reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results.

The test item attained 12% degradation after 28 days and therefore cannot be considered to be readily biodegradable.

Conclusion.

The test item attained 12% degradation after 28 days and therefore cannot be considered to be readily biodegradable.