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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:
2018-09-19 to 2018-11-14
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
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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): Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Method of cultivation: The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present.
- Storage conditions: The washed sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection.
- Pretreatment: none
- Concentration of sludge: 30 mg dry solids per litre
- Initial cell/biomass concentration: The suspended solids concentration was 2.1 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
A: Potassium dihydrogenphosphate KH2PO4 8.50 g
Dipotassium hydrogenphosphate K2HPO4 21.75 g
Disodium hydrogenphosphate dihydrate Na2HPO4 * 2 H2O 33.40 g
Ammonium chloride NH4Cl 0.50 g
are dissolved in demineralised water and made up to 1 litre.

B: Calcium chloride CaCl2 27.50 g
is dissolved in demineralised water and made up to 1 litre.

C: Magnesium sulfate heptahydrate MgSO4 * 7H2O 22.5 g
is dissolved in demineralised water and made up to 1 litre.

D: Iron (III) chloride hexahydrate FeCl3 * 6H2O 0.25 g
is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 1 litre.

For preparation of the mineral medium 10 mL of solution (A) is mixed with 900 mL demineralised water, 1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre.

- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature: 22 °C - 24 °C
- pH: 7.4 (day 0 post-adjustment) to 7.5 (day 28)
- pH adjusted: yes
- Suspended solids concentration: 2.1 g/L
- Continuous darkness: yes
- Other: dry solid concentration: 180 mL activated sludge was filled up to 3000 mL with 2820 mL mineral medium corresponding to 30 mg/L dry solids.

TEST SYSTEM
- Culturing apparatus: not stated
- Number of culture flasks/concentration: 2 vessels in the inoculated control, the procedure control plus the reference item and the test item. 1 vessel only for the test item plus the reference item
- Method used to create aerobic conditions: The test vessels are aerated by the passage of carbon dioxide-free air.
- Measuring equipment: IC measurement was performed with a total carbon analyser (either TOC-V or TOC-L Shimadzu) by conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas.
- Test performed in closed vessels due to significant volatility of test substance:
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was absorbed in two 500 mL Dreschel bottles each filled with 350 mL 0.05 M NaOH.

SAMPLING
- Sampling frequency: IC analysis samples on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 from the first CO2 absorver and days 0 and 29 for the second CO2 absorver.
- Sampling method: IC analysis: 2 mL samples from the first of two CO2-absorber flasks was sampled and the IC's were determined. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. IC/TC: 30 mL samples from the inoculum control and test item vessels and filtered through 0.45 µm Gelman AcroCap fillters and the IC/TC were determined. Samples (50 µL) were injected into the TC and IC channels of the TOC analyzer.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The results obtained from the samples taken for organic carbon analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis could be filtered to remove the suspended solids present without the loss of any test item.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
- The degradation extent of the test substance at the end of the test was 0%.
- The degradation extent in the toxicity control was 30% within 14 days and 38% biodegradation after 28 days. The test substance had no inhibitory effect on the inoculum.
- The mean CO2-evolution of the blank flasks was 34.27 mg/L on day 28.
- Test control Sodium benzoate attained 70% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 87% biodegradation was attained.
Results with reference substance:
The reference compound sodium benzoate reached the pass levels for ready biodegradability (61% ThCO2 by day 8) within 2 days.

Table 1 Inorganic Carbon Values on Each Analysis Occasion


 Day           Inorganic Carbon (mg IC)                                       
 Inoculum Control           Procedure Control            Test Item           Toxicity Control   
 R1     R2     R1    R2    R1    R2    R1   
 Abs1  Abs2   Abs1   Abs2   Abs1   Abs2   Abs1   Abs2   Abs1   Abs2   Abs1   Abs2   Abs1   Abs2
 0 1.75   1.75   1.75  1.75   1.75  1.05   1.75   1.17   1.75  1.75   1.75   1.05  1.75   1.75 
 2 5.45   -  6.61

-

19.72

 15.08

 7.89

 -

6.85

20.53

 6

 14.65

 14.07

 -

 31.03

27.91

14.07 

 13.73

37.71 

 8

12.84 

 -

 14.91

 -

 35.09

 -

29.01

 14.68

 -

15.02

 33.83

 10

16.42 

 -

 16.65

 -

 37.62

35.34 

17.67

11.29 

 33.29

 14

 19.72

20.63

 -

44.88 

 -

37.29

19.04

 -

18.47

38.31

 21

 23.77

28.28 

 -

51.26

 -

45.40 

26.03 

 -

26.59

43.15

 28

37.47/26.32*

 -

37.30/29.79*

 

61.93/53.76* 

 -

58.91/50.51* 

 -

41.22/27.55*

 -

34.27/28.00*

 63.28/49.73*

 29

 29.62

 2.09

 31.95

 2.09

 57.00

2.09

56.89

 2.09

 32.06

 2.09

 28.06

 2.09

 53.76

2.09

R = Replicate Abs = CO2 absorber vessel - = No value determined * Results from re-analysis of sample as original result was deemed to be erroneous

Table 2: Percentage Biodegradation Values


 Day   

 Biodegradation (%)      

 Procedure Control

 Test Item

 Toxicity Control

 0

 0

 0

 0

 2

38 

4

 24

 6

 50

0

 39

 8

 61

 3

 33

 10

 66

 0

 28

 14

 70

 0

 30

 21

 74

 1

 29

 28  80  0  36
 29*  87  0  38

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2


Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The biodegradation of the test item was determined according to OECD 301 B under GLP conditions. The test item attained 0% biodegradation 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:

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

Description of key information

The test item attained 0% biodegradation 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