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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:
The study was conducted between 15 November 2013 and 13 December 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-3-[(3-{[3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)(dimethyl)ammonio]propane-1-sulfonate
EC Number:
695-243-8
Cas Number:
500731-87-3
Molecular formula:
C18H28N2O6S
IUPAC Name:
2-hydroxy-3-[(3-{[3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)(dimethyl)ammonio]propane-1-sulfonate
Test material form:
other: liquid
Details on test material:
- Name of test material: Galaxy SunBeat
- Physical state: extremely pale yellow liquid
- Analytical purity: 51.38%
- Lot/batch No.: 07
- Expiration date of the lot/batch: 01 January 2015
- Storage condition of test material: room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
A mixed population of sewage treatment micro-organisms was obtained on 15 November 2013 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and the filtrate maintained on continuous aeration in a temperature controlled room at approximately 21 °C prior to use.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
4.5 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Experimental Preparation:
The test item was dissolved directly in mineral medium.

An amount of test item (100 mg) was dissolved in mineral medium and the volume adjusted to 100 mL to give a 1000 mg/L stock solution. An aliquot (27 mL) of this stock solution was dispersed in a final volume of 6 liters of inoculated mineral medium to give a test concentration of 4.5 mg/L.

The volumetric flask containing the stock solution was inverted several times to ensure homogeneity of the solution.

A test concentration of 4.5 mg/L was employed in the study as the Theoretical Oxygen Demand (ThOD) of the test item was calculated to be 1.07 mg O2/mg. Hence, if complete degradation of the test item occurred, the oxygen depletion in the test vessels would be 4.82 mg O2/L and as such de-oxygenation of the test media would not occur.

As it was not a requirement of the Test Guidelines, no analysis was carried out 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.

Toxicity Control:
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 treatment micro-organisms used in the study.

An aliquot (13.5 mL) of the 1000 mg/L test item stock solution plus an aliquot (4.5 mL) of the sodium benzoate stock solution were dispersed in a final volume of 3 liters of mineral medium to give a final concentration of 4.5 mg test item/L plus 1.5 mg sodium benzoate/L.

The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the solutions.

Preparation of Test System:
The following test preparations were prepared and inoculated in 250 mL Biological Oxygen Demand (BOD) bottles (darkened glass) with ground glass stoppers:

a) An inoculum control, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in inoculated mineral medium to give a concentration of 3.0 mg/L.
c) The test item, in inoculated mineral medium to give a concentration of 4.5 mg/L.
d) The test item (4.5 mg/L) plus the reference item (1.5 mg/L), in inoculated mineral medium to act as a toxicity control.

The reference item concentration was reduced to 1.5 mg/L in the toxicity control to prevent complete deoxygenation of the test media occurring.

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.

Test media a to d were inoculated with sewage treatment micro-organisms at a rate of 1 drop of inoculum per liter.

The pH of all of the above test preparations were 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 being transferred by siphon to BOD bottles, which were firmly stoppered to exclude all air bubbles. Sufficient bottles were prepared to allow a single oxygen determination per bottle with duplicate bottles for each test medium at each sampling occasion.

The BOD bottles were incubated in darkness at approximately 21 °C.

Data Evaluation:
Percentage Biodegradation:
The dissolved oxygen depletion for each replicate flask for the test item, reference item and toxicity control for each time period is calculated as follows:

Oxygen depletion = (Mto - Mtx) - (Mbo - Mbx) mg O2/L

where:

Mto = oxygen concentration in test flask at time 0
Mtx = oxygen concentration in test flask at time x
Mbo = mean oxygen concentration of inoculated control flasks at time 0
Mbx = mean oxygen concentration of inoculated control flasks at time x

The oxygen depletion for the inoculated control (blank) flasks is calculated as follows:
Blank oxygen depletion = Mbo - Mbx

The percentage biodegradation for each replicate test item, reference item and toxicity control flask was calculated as follows:

% biodegradation = dissolved oxygen depletion / concentration of test substance x ThOD (or COD) x 100

Mean % biodegradation = % biodegradation of replicate 1 + % biodegradation of replicate 2 / 2

Validation Criteria:
The results of the degradation test are considered valid if in the same test the reference item yields ≥ 60% biodegradation (in a 10-Day window) by Day 14.

Test items giving a measured BOD value which is ≥ 60% of the ThOD value (within 28 days) are regarded as being ready biodegradable. This level must be reached within 10 days of biodegradation exceeding 10%.

The toxicity control (test item and sodium benzoate) should attain ≥ 25% biodegradation by Day 14 for the test item to be considered as non-inhibitory.

The oxygen depletion for the inoculated control should not exceed 1.5 mg O2/L after 28 days.

The test is considered valid if the difference between the extremes of replicate oxygen consumption values at the time the plateau is reached, at the end of the test or at the end of the 10-day window, as appropriate is less than 20%.

The residual oxygen concentration in the test bottles should not fall below 0.5 mg O2/L at any time.























Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
The oxygen depletion of the inoculated control did not exceed 1.5 mg O2/L after 28 days, the residual oxygen concentration in the test bottles remained at 4.6 mg O2/L or greater in all test vessels and the difference between the extremes of replicate oxygen depletion values at the end of the test was less than 20% in all vessels thereby satisfying the validation criteria.
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
The test item attained 0% biodegradation after 28 days and, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301D.

Variation in biodegradation rates on different sampling days was considered to be due to normal biological variation in respiration rates between control and test vessels.

The toxicity control attained 25% biodegradation after 14 days and 29% biodegradation after 28 days therefore confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.



BOD5 / COD results

Results with reference substance:
The reference item, sodium benzoate, attained 65% biodegradation after 14 days and 65% biodegradation after 28 days thereby confirming the suitability of the test method and culture conditions.

Any other information on results incl. tables

Table1     Dissolved Oxygen Measurements

Test Series

Dissolved Oxygen (mg O2/L)

Day

0

3

5

7

11

14

18

21

28

a)      Inoculum Control

R1

8.60

8.40

8.35

8.35

8.30

8.30

8.15

8.10

8.00

R2

8.60

8.40

8.40

8.35

8.30

8.25

8.15

8.00

8.00

b)      Procedure Control

R1

8.50

5.85

5.80

5.00

4.85

4.80

4.85

4.75

4.65

R2

8.40

5.60

5.65

5.00

5.10

4.90

4.65

4.60

4.55

c)      Test Item

R1

8.40

8.45

8.40

8.30

8.05

8.00

8.10

7.95

7.90

R2

8.40

8.50

8.40

8.30

8.00

7.90

8.00

7.80

7.75

d)      Toxicity Control

R1

8.40

-

-

-

-

6.10

-

-

5.60

R2

8.40

-

-

-

-

6.40

-

-

5.80


R1- R2= Replicates 1 and 2

Table2     Oxygen Depletion and Mean Percentage Biodegradation Values

Test Series

 

3

5

7

11

14

18

21

28

a)      Inoculum Control

             Mean O2Depletion (mg O2/L)*

 

0.200

0.225

0.250

0.300

0.325

0.450

0.550

0.600

b)      Procedure Control

             O2Depletion (mg O2/L)*

R1

2.450

2.475

3.250

3.350

3.375

3.200

3.200

3.250

R2

2.600

2.525

3.150

3.000

3.175

3.300

3.250

3.250

             % Biodegradation (mean)

 

51

50

64

64

65

65

65

65

c)      Test Item

             O2Depletion (mg O2/L)*

R1

-0.250

-0.225

-0.150

0.050

0.075

-0.150

-0.100

-0.100

R2

-0.300

-0.225

-0.150

0.100

0.175

-0.050

0.050

0.050

             % Biodegradation (mean)

 

-6

-5

-3

2

3

-2

-1

-1

d)      Toxicity Control

             O2Depletion (mg O2/L)*

R1

-

-

-

-

1.975

-

-

2.200

R2

-

-

-

-

1.675

-

-

2.000

             % Biodegradation (mean)

 

-

-

-

-

25

-

-

29


*Corrected for oxygen depletion due to nitrification

R1- R2= Replicates 1 and 2

Table3     pH Values of the Test Preparations on Day 0

Test Preparation

pH

Inoculum Control

7.6

Procedure Control

7.6

Test Item

7.6

Toxicity Control

7.5

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item attained 0% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301D.


Executive summary:

Introduction:

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

Methods:

The test item, at a concentration of 4.5 mg/L, was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 21°C for 28 days. The degradation of the test item was assessed by the determination of the amount of oxygen consumed. 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 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301D.