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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:
19 Feb. - 19 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern guideline conform study performed in GLP certified laboratory

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

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
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Batch number: RW 2013-10-22
Form: Powder
Color: Orange
Storage conditions: Room temperature
Expiring date: 22. 10. 2018

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Species:
Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 19 February 2014.
Preparation of Activated Sludge Inoculum:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with reconstituted water (see above) and then aerated until use (in this study 200 mL inoculum was prepared). After preparation the sludge was filtered through cotton wool. The pH of the activated sludge inoculum after preparation was 6.81. A pH adjustment before use was not performed.
The microbial inoculum was prepared on the day of the test and was not pre-adapted to the test chemical.
The microbial inoculum was continuously aerated (2 L/minute) at the test temperature of until use.
Duration of test (contact time):
>= 28 - <= 29 d
Initial test substance concentration
Initial conc.:
3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The Test Groups

1.) Test Item (flasks 1a and 1b):
Based on the measured chemical oxygen demand (COD) of the test item (2.05 mg O2/mg test item), at the start of the test a suitable volume (50 mL) of the test item solution was thoroughly mixed into the respective volume (5000 mL) of aqueous test medium corresponding to 3 mg/L test item, respectively a COD of about 6.15 mg O2/L.

2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)
Based on the theoretical oxygen demand (ThODNH3) of sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), the stock solution (360 mg/L) of sodium benzoate (50 mL) was thoroughly mixed into the respective volume of aqueous test medium (5000 mL), corresponding to 3.6 mg/L reference item concentration, respectively a ThODNH3 of about 3.6 x 1.67 = 6.012 mg O2/L.
More studies run in parallel and the corresponding procedure control was common.

3.) Inoculum Control (flasks 3a and 3b)
Only filtered inoculum (10 mL) was added to the aqueous test medium (5000 mL).
Microbial inoculum (2.0 mL per litre) was added to each preparation bottle.
More studies run in parallel and the corresponding procedure control was common.

4.) Toxicity Control (flasks 4a and 4b)]
Test (50 mL) and reference item (50 mL) stock solutions were mixed into the aqueous test medium (5000 mL) corresponding to the test item concentration of 3 mg/L [chosen based on the measured informative COD of the test item and the preliminary experiment] and to 3.6 mg/L concentration of the reference item.
In general: microbial inoculum (2.0 mL per litre) was added to each preparation bottle.

Course of the Test

Preparation of Test Flasks

A sufficient number of Winkler flasks were cleaned with 5 – 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing standing for about 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stopper.

Sulphuric acid: Supplier: CARLO ERBA; Batch Number: V2B680142B, Expiry date: February 2018
Potassium iodide: Supplier: REANAL (lach:ner); Batch Number: PP/2012/09667, Expiry date: 08 February 2015
Iodine: Supplier: VWR (PROLABO); Batch Number: 13B070011, Expiry date: February 2018

The Number of Test Flasks

The number of the test bottles was the following (according to the measurement days):
 10 (+2 reserve) bottles containing the test item and inoculum
 10 (+2 reserve) bottles containing the sodium benzoate and inoculum
 10 (+2 reserve) bottles containing only inoculum (inoculum control)
 10 (+2 reserve) bottles containing the test item and sodium benzoate (toxicity control)
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
Batch No.: A0300297; Expiry Date: 05 January 2016; Supplier: Reanal, Budapest Hungary, Manufacturer: ACRÕS

Results and discussion

Preliminary study:
In the preliminary experiment the test item biodegradability, toxicity was investigated for 14 days at the test item concentration of 3 mg/L. No toxic effect of the test item was found at the investigated concentration [in the toxicity control containing both, the test item and the reference item, a mean of 36.8 % biodegradation was noted within 14 days, thus, the test item was assumed not to inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days)].
Test performance:
Because of the N-containing test item, the oxidised nitrogen (nitrate and nitrite) concentrations were determined after each oxygen measurement with photometric method using nitrite and nitrate cell tests. The LOQ (Limit Of Quantification) of the measurements was 0.03 mg NO2 /L and 0.4 mg NO3 /L, respectively.
The measured quantities of nitrate were below the LOQ in the measured samples throughout the study.
The measured quantity of nitrite was in the measurable range in both parallel vessels of the test item and toxicity control groups on the 0, 7th, 14th, 21st and 28th days; furthermore in the inoculum control group on the 21st and 28th days. The measurable nitrite quantities were nearly the same throughout the test. Any correction of the BOD values with the observed nitrite values was considered as not necessary, because a correction with these low values would not have any influence on the results and conclusion of the study. The corrected BOD values would remain in the biological variability range of the applied test system.

% Degradation
Parameter:
% degradation (DOC removal)
Value:
>= 14.8 - <= 15.9
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of YELLOW LF 6881 reached a mean of 14.8 % after 28 days based on its COD (the highest value, 14.9 % was calculated on the 21st day of the test). The test item biodegradation reached its plateau already on the 7th day and from this the variations, slight changes were considered as being within the biological variability range of the applied test system. Based on the obtained values the test item can be considered to be not ready biodegradable.


Table 5: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days
Treatment Concentration Flask Percent of biodegradation after n days of exposure
[mg/L] No. 7 14 21 28
Test item 1a 12.6 10.9 15.9 14.8
3 1b 12.3 11.5 13.8 14.8
mean 12.4 11.2 14.9 14.8
Reference item 2a 55.1 59.2 62.5 71.0
3.6 2b 58.8 69.8 68.0 75.0
mean 56.9 64.5 65.3 73.0
Toxicity control Test item: 3
Reference item: 3.6 4a 27.4 29.2 30.2 31.1
4b 25.2 29.4 34.4 35.6
mean 26.3 29.3 32.3 33.3
Biodegradation % =
where:
T.i. = test item
R.i. = reference item
i.control = inoculum control

BOD5 / COD results

BOD5 / CODopen allclose all
Parameter:
COD
Value:
>= 2.05 - <= 2.051 mg O2/g test mat.
Parameter:
BOD5
Value:
>= 0.3 - <= 0.33 mg O2/g test mat.
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 64.5 % after 14 days, and to a mean of 73.0 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum. In the experiment multiple test items were tested with the reference values from the common parallel reference control.

Any other information on results incl. tables

Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

mg O2/L after n days of exposure

[mg/L]

No.

0

7

14

21

28

Test item

 

1a

8.67

6.99

6.58

6.31

6.30

3

1b

8.81

7.15

6.68

6.58

6.44

 

mean

8.74

7.07

6.63

6.45

6.37

Reference item

 

2a

8.87

4.66

3.90

3.74

3.15

3.6

2b

8.89

4.46

3.28

3.43

2.93

 

mean

8.88

4.56

3.59

3.59

3.04

Inoculum control

3a

8.78

7.91

7.38

7.50

7.30

3b

8.84

7.90

7.40

7.36

7.40

mean

8.81

7.91

7.39

7.43

7.35

Toxicity control

Test item: 3
Reference item: 3.6

4a

8.78

4.51

3.78

3.70

3.51

4b

8.87

4.88

3.85

3.27

3.04

mean

8.83

4.70

3.82

3.49

3.28

 

 

Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

mg O2/L after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

3

1a

0.78

0.67

0.98

0.91

1b

0.76

0.71

0.85

0.91

Reference item

3.6

2a

3.31

3.55

3.75

4.26

2b

3.53

4.19

4.08

4.50

Toxicity control

Test item: 3
Reference item: 3.6

4a

3.37

3.58

3.70

3.81

4b

3.09

3.60

4.22

4.37

 

oxygen depletion : (mt0- mtx) - (mbo- mbx), where:

        mt0: oxygen concentration (mg/L) of test group on day 0 (1a, 2a, 4a and 1b, 2b, 4b from Table 2)

mtx: oxygen concentration (mg/L) of test group on day x (1a, 2a, 4a and 1b, 2b, 4b from Table 2)

mb0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b from Table 2)

mbx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b from Table 2)


BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

BOD after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

3

1a

0.26

0.22

0.33

0.30

1b

0.25

0.24

0.28

0.30

Reference item

3.6

2a

0.92

0.99

1.04

1.18

2b

0.98

1.16

1.13

1.25

Toxicity control

Test item: 3
Reference item: 3.6

4a

0.51

0.54

0.56

0.58

4b

0.47

0.55

0.64

0.66

 

BOD = = mg O2/mg T.i and/or R.i.

where:

T.i.        =test item

R.i.       =reference item

i.control=inoculum control

 

 

Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

Flask

Percent of biodegradation after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

 

1a

12.6

10.9

15.9

14.8

3

1b

12.3

11.5

13.8

14.8

 

mean

12.4

11.2

14.9

14.8

Reference item

 

2a

55.1

59.2

62.5

71.0

3.6

2b

58.8

69.8

68.0

75.0

 

mean

56.9

64.5

65.3

73.0

Toxicity control

Test item: 3
Reference item: 3.6

4a

27.4

29.2

30.2

31.1

4b

25.2

29.4

34.4

35.6

mean

26.3

29.3

32.3

33.3

Biodegradation % =

where:

T.i.        =test item

R.i.        =reference item

i.control=inoculum control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item is considered to be not ready biodegradable.
According to the test guidelines the pass level for ready biodegradability is removal of 60 % COD in a 10-day window.
The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.
According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.
Executive summary:

The purpose of this study was to determine the ready biodegradability of the test item
YELLOW LF 6881. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

Under the test conditions the percentage biodegradation ofYELLOW LF 6881reached a mean of 14.8 % after 28 days based on its COD (the highest value, 14.9 % was calculated on the 21stday of the test). The test item biodegradation reached its plateau already on the 7thday and from this the variations, slight changes were considered as being within the biological variability range of the applied test system. Based on the obtained values the test item can be considered to be not ready biodegradable.

The reference item Sodium benzoate was sufficiently degraded to a mean of 64.5 % after 14 days, and to a mean of 73.0 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

In the toxicity control containing both, the test item and the reference item, a mean of 29.3 % biodegradation was noted within 14 days and 33.3 % biodegradation after 28 days of incubation. Thus, the test item can be assumed not to inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).