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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:
Experimental Starting Date:21 February 2012 Experimental Completion Date:11 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Principles of method if other than guideline:
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) is to dissolve the test item in an auxiliary solvent prior to adsorption onto filter paper. High shear mixing was also applied to break up the filter paper containing the test item. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 12 March 2012 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

The activated sewage sludge sample was washed once by settlement and resuspension in mineral 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 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage 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 deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC 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 3.0 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
5.1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparation of Test System
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:

a) An inoculated control, in duplicate, consisting of inoculated mineral medium plus a filter paper*.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus a filter paper* to give a final concentration of 10 mg carbon/L.
c) The test item on a filter paper*, in duplicate, in inoculated mineral medium to give a final concentration of 5.1 mg carbon/L.
d) The test item on a filter paper* plus the reference item in inoculated mineral medium to give a final concentration of 15.1 mg carbon/L to act as a toxicity control (one vessel only).

A filter paper with chloroform evaporated to dryness was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item 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 21ºC, in darkness.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 30 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. If necessary the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.

The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to
100 mL/min per vessel 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 (Carbosorb®) 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 and Analysis
CO2 Analysis
Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were all sampled on Days 0 and 29.

The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 were analyzed 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 analyzed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyzer. Samples (300 µL) were injected into the IC (Inorganic Carbon) channel of the TOC analyzer. 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 reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.


Inorganic Carbon/Total Carbon Analysis
Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis.

IC/TC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.

Samples (30 mL) were removed from the inoculum control vessels on Day 0 after dosing and filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis.

The samples were analyzed for IC and TC using a Shimadzu TOC-VCPH TOC Analyzer. Samples (50 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyzer. Total carbon analysis is carried out at 680ºC using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in triplicate.

pH Measurements
The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Reference substance:
other: Sodium benzoate
Preliminary study:
An initial experiment was conducted at a concentration of 10 mg C/L. The toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25% biodegradation after 14 days. These results indicated that, under the strict terms and conditions of the OECD Guidelines, the test item would be classed as exhibiting inhibitory effects.
Following the recommendations of the OECD Guidelines it was considered appropriate to reduce the test concentration employed in the study to 5.1 mg C/L in order to minimize the toxic effect that the test item exhibited on the sewage treatment micro-organisms used in the study. It was not possible to test at concentrations below 5.1 mg C/L as below this concentration it was not possible to distinguish between background CO2 evolution from the inoculum and CO2 evolution due to biodegradation using inorganic carbon analysis.
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Remarks on result:
other: cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Details on results:
Inorganic carbon values for the test item, reference item, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3. The pH values of the test preparations on Days 0 and 28 are given in Table 4. Observations made on the contents of the test vessels are given in Table 5.

The total CO2 evolution in the inoculum control vessels on Day 28 was 30.10 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3) 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 hence satisfied the validation criterion given in the OECD Test Guidelines.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

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 the exception of control replicate R1, test item replicate R2 and the toxicity control. 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 item attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

The toxicity control attained 34% degradation after 14 days and 30% degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. The slight decrease in degradation between days 14 and 28 was considered to be due to sampling / analysis variation.
Results with reference substance:
Sodium benzoate attained 65% degradation after 14 days and 79% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table1     Inorganic Carbon Values on Each Analysis Occasion

Day

Inoculum Control (mg IC)

Sodium Benzoate
(mg IC)

Test Item (mg IC)

Test Item
plus Sodium Benzoate Toxicity Control
(mg IC)

R1

R2

R1

R2

R1

R2

R1

Abs1

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

1.17

1.40

1.40

1.28

1.40

1.40

1.40

1.40

1.40

1.75

1.87

1.75

2.33

1.87

2

10.79

-

11.37

-

29.12

-

29.47

-

12.76

-

11.48

-

24.71

-

6

13.03

-

12.92

-

33.68

-

34.14

-

12.22

-

11.88

-

29.52

-

8

14.33

-

13.87

-

32.22

-

34.86

-

13.42

-

13.19

-

27.98

-

10

17.78

-

16.53

-

40.59

-

42.52

-

16.53

-

16.42

-

35.34

-

14

18.93

-

16.77

-

36.83

-

37.97

-

16.77

-

17.00

-

33.21

-

21

26.70

-

22.20

-

47.66

-

50.59

-

22.76

-

26.36

-

43.04

-

28

26.43

-

22.85

-

45.47

-

49.17

-

22.96

-

25.65

-

42.78

-

29

26.28

2.78

23.60

2.67

48.21

2.90

49.32

2.44

23.71

2.44

24.83

2.90

38.75

4.29


R1– R2          =   Replicates 1 and 2

Abs              =  CO2absorber vessels

-                =  No value determined

Table2     Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Item

% Degradation

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

61

7

30

6

70

0

37

8

65

0

31

10

81

0

40

14

65

0

34

21

82

1

41

28

76

0

40

29*

79

0

30


*Day 29 values corrected to include any carry-over of CO2detected in Absorber 2

Table3     Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel

Total Carbon*

(mg/L)

Inorganic Carbon*

(mg/L)

IC Content (% of TC)

Test Item

5.1 mg C/L R1

6.52**

0.67

0

Test Item

5.1 mg C/L R2

6.32**

0.66

0


R1– R2     =  Replicates 1 and 2

*       Corrected for control values.

**    Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

Table4     pH Values of the Test Preparations on Days 0 and 28

Test Vessel

pH

prior to adjustment on Day 0

pH

after adjustment on Day 0

pH on Day 28

Inoculum ControlR1

8.0

7.6

7.2

Inoculum Control R2

8.0

7.6

7.4

Sodium Benzoate

10 mg C/L R1

8.0

7.5

7.3

Sodium Benzoate

10 mg C/L R2

8.0

7.5

7.2

Test Item

5.1 mg C/L R1

8.0

7.6

7.1

Test Item

5.1 mg C/L R2

8.0

7.5

7.1

Toxicity Control

15.1 mg C/L

8.0

7.5

7.2

 

 

R1– R2    =      Replicates 1 and 2

Table5     Observations on the Test Preparations Throughout the Test Period

Test Vessel

Observations on Test Preparations

Day 0

Day6

Day 13

Day 20

Day 27

Control

R1

Light brown cloudy dispersion containing broken up pieces of filter paper

Light brown cloudy dispersion containing broken up pieces of filter paper

Light brown cloudy dispersion containing broken up pieces of filter paper

Light brown cloudy dispersion containing broken up pieces of filter paper

Light brown cloudy dispersion containing broken up pieces of filter paper

 

R2

Light brown cloudy dispersion containing broken up pieces of filter paper

Light brown cloudy dispersion containing broken up pieces of filter paper

Light brown cloudy dispersion containing broken up pieces of filter paper

Light brown cloudy dispersion containing broken up pieces of filter paper

Light brown cloudy dispersion containing broken up pieces of filter paper

Sodium Benzoate

R1

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved reference item visible

 

R2

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved reference item visible

Test Item

R1

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test item visible

 

R2

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test item visible

Toxicity Control

 

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test or reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test or reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test or reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test or reference item visible

Light brown cloudy dispersion containing broken up pieces of filter paper. No undissolved test or reference item visible

 

R1– R2= Replicates 1 and 2

Appendix2   Mineral 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

27.50 g/L

Solution c

MgSO4.7H2O

22.50 g/L

Solution d

FeCl3.6H2O

0.25 g/L

 

 

 

To 1 liter (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 deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP)

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item attained 0% 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:

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

 

 Methods

An initial experiment was conducted at a concentration of 10 mg C/L. The toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25% biodegradation after 14 days. These results indicated that, under the strict terms and conditions of the OECD Guidelines, the test item would be classed as exhibiting inhibitory effects.

 

Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test item at a reduced concentration of 5.1 mg C/L was exposed to activated sewage sludge micro-organisms with mineral 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), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

 

The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes

 

 Results

The test item attained0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

 

Description of key information

The test item attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Key value for chemical safety assessment

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

Additional information