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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:
2015-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
yes
Remarks:
The deviation did not adversely affect the test results and quality.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
A fresh sample of activated sludge was collected from the aeration tank of Shenyang North Sewage Treatment Plant 2 days before the test was started. It is a well functioning treatment plant receiving predominantly domestic sewage.
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 29.2 mg/L
Based on:
DOC
Initial conc.:
ca. 29 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
Test Conditions
Concentration of test item: 29.20 and 29.00 mg/L as DOC (determined values, duplicate)
Concentration of microbial inoculums: 29.8 mg/L as suspended solids (SS)
Test temperature: 22°C±2°C (monitored temperature range: 20.7-23.5°C)
Test period: 28 days
The test was conducted in diffuse light.

The suspended sludge was prepared with mineral medium to yield a concentration of 4 g/L as suspended solids (SS). The suspended sludge was keep aerobic at 22.3-23.8°C for two days until used for the test. sludge was dried at 105°C for 1 hour.
The final concentration of suspended sludge was 29.8 mg/L as suspended solids (SS).

Test Item Test Vessel: The nominal concentration of test solution was equivalent to 29.6 mg/L as DOC (average). The pH of the solution was checked (for sample TS-1 &TS-2): it was 7.56 and 7.55
Reference Item Test Vessel: The nominal concentration of test solution was equivalent to 28.4 mg/L as DOC. The pH of the solution was checked to be 7.53.Inoculum Blank Control Test Vessel: the suspended solid concentration of 29.8 mg/L in the final volume. Inoculum blank control test vessel was prepared in duplicate (identified as IC-1 and IC-2).Toxicity Control Test Vessel: the suspended solid concentration of 29.8 mg/L in the final volume. One replicate (toxicity control identified as TC) sufficed.

Bioassay Conditions
The openings of the vessels were capped with air-permeable membrane to allow free exchange of air between the flask and the surrounding atmosphere. The test vessels were inserted into the shaker and incubated in the diffused light. The room temperature was kept at 20.7-23.5°C during the test. DO (dissolved oxygen) was checked on each day of sampling, the DO was in the range of 7.30-9.32 mg/L, which met the criterion of DO>1 mg/L.
The DO results are presented in Section 15 Table 1.

Sampling
Approx 25 ml samples are taken :
- Immediately after addition of inoculums into test vessels and mixing;
- on the 1st, 4th, 6th, 8th, 11th, 14th, 19th and 23rd day;
- on the 28th days.
The concentration of DOC in samples was analysised by TOC analyzer.
see table 1: Dissolved Oxygen Values at Every Sampling Occasion during 28 Day Test
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (DOC removal)
Value:
97.9
Sampling time:
6 d
Remarks on result:
readily biodegradable based on QSAR/QSPR prediction
Parameter:
% degradation (DOC removal)
Value:
45.1
Sampling time:
4 d
Details on results:
The starting concentration of DOC in the test suspension measured was 29.20 and 29.00 mg/L (corrected for inoculums control). Samples of suspension were taken on 1st, 4th, 6th, 8th, 11th, 14th, 19th, 23rd and 28th day to determine DOC concentration. After 4 days, the percentage removal of DOC in the test suspension attained 45.1% (average), so 10% biodegradation may be obtained on 2nd or 3rd day and when the 10-day window began. After 6 days, the percentage removal of DOC in the test suspension attained 97.9% (average), it can be assumed that the percentage biodegradation in activated sludge exceeded 70% pass level (based on removal of DOC) within 10-day window.
The percentage biodegradation of reference item (sodium benzoate) attained 96.5% after 1 day and 99.4% at the 14th day, which exceeded 70% pass level. In the toxicity control containing the test item, reference item and inoculums, the percentage degradation based on total DOC attained 46.1% after 1 day and 99.4% at the 14th day, which exceeded 35%, which indicated that the test item can be assumed to be not inhibitory under the conditions of the test. As mentioned above, the test was considered to be valid.
The DOC measurement and biodegradation results are presented in Section 15 Table 4 and Table 5, biodegradation curve is presented in Section 15 Figure 1.
Table 4. Measured DOC Concentration in Filtrates during 28 Days Test

Table 4. Measured DOC Concentration in Filtrates during 28 Days Test

Sampling time

(day)

Injection

replicate

Concentration of DOC measured (mg/L)

IC-1

IC-2

PC

TS-1

TS-2

TC

1

2

1

2

1

2

1

2

1

2

1

2

0hour

1

1.44

0.1459

0.2104

0.3246

27.26

27.62

29.79

29.85

29.35

29.68

55.93

55.95

2

1.38

0.1361

0.2175

0.3373

27.75

27.77

29.53

29.72

29.50

29.56

56.55

56.16

1

1

0.1432

0.1189

0.1605

0.1538

1.54

0.6913

29.15

29.79

29.70

29.54

30.27

30.10

2

0.1737

0.1273

0.1563

0.2023

1.47

0.6546

29.57

29.78

29.37

29.23

30.04

30.21

4

1

0.2725

0.1687

0.1502

0.1963

0.8674

0.6914

16.03

16.48

15.80

16.00

27.04

27.40

2

0.1995

0.1458

0.1758

0.1774

0.9284

0.6776

16.52

16.57

15.99

15.94

27.17

27.30

6

1

0.2061

0.3883

0.3238

0.3531

0.7143

0.7031

0.9548

1.06

0.7680

0.8931

15.66

15.04

2

0.2517

0.3280

0.3255

0.3657

0.7354

0.6779

0.9468

1.06

0.8095

0.9313

16.02

15.16

8

1

0.1963

0.3659

0.2943

0.1954

0.6235

0.5899

0.8741

1.13

0.6761

0.6128

1.14

1.72

0.3064

0.3500

0.2523

0.1992

0.5779

0.5829

0.8773

1.07

0.6939

0.5615

1.17

1.71

11

1

*3.58

0.3743

0.3823

0.2231

0.5956

0.8539

0.7530

1.02

0.5063

0.6002

0.7355

0.9475

2

*3.72

0.2601

0.4015

0.2287

0.6757

0.8523

0.7060

0.982

0.5009

0.6548

0.7870

1.0169

14

1

0.6771

0.3305

0.5562

0.4323

0.6569

0.6543

0.7858

1.00

0.7226

0.6834

0.8004

0.9243

2

0.6704

0.2743

0.6064

0.4213

0.6363

0.6974

0.8271

1.05

0.6515

0.6353

0.7977

0.8794

19

1

0.5287

1.23

0.6746

1.40

1.37

1.01

1.70

2.13

2.11

2.31

1.67

1.34

2

0.4807

1.23

0.6396

1.33

1.36

1.04

1.66

2.15

2.10

2.27

1.63

1.30

23

1

0.5939

0.3657

0.9600

0.3845

1.09

0.5986

0.8757

1.22

0.8813

0.9576

1.35

1.37

2

0.5878

0.3649

0.9633

0.3817

1.21

0.6550

1.008

1.32

0.9485

1.023

1.34

1.21

28

1

0.7395

0.5908

0.5077

0.4237

0.8100

0.7983

1.15

1.005

1.18

1.00

1.55

1.36

2

0.8871

0.6224

0.4543

0.3952

0.7956

0.8788

1.12

0.9670

1.17

1.03

1.66

1.36

Note: IC = Inoculums Control; TS = Test Suspension; PC =Procedure Control; TC= Toxicity Control.

Two samples were taken from each test vessel and every sample was injected for two times.

*The determined values were exceptionally higher than other IC values, so the data were not used for calculation.

Table 5. Biodegradation Results during 28 Days Test

Time (Day)

Biodegradation

PC

TS-1

TS-2

Average of TS

TC

1

96.5%

0.0%

0.0%

0.0%

46.1%

4

97.8%

44.5%

45.7%

45.1%

51.4%

6

98.6%

97.6%

98.2%

97.9%

72.8%

8

98.8%

97.5%

98.7%

98.1%

97.9%

11

98.4%

98.1%

99.1%

98.6%

99.0%

14

99.4%

98.6%

99.4%

99.0%

99.4%

19

99.1%

96.7%

95.7%

96.2%

99.0%

23

98.8%

98.2%

98.7%

98.5%

98.7%

28

99.1%

98.3%

98.2%

98.3%

98.4%

Note: PC =Procedure Control;TS = Test Suspension;TC= Toxicity Control.


Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Based on these results, the test item can be assumed to be readily biodegradable under the test conditions.
Executive summary:

The test assessed theready biodegradabilityoftest itemby determination of Dissolved Organic Carbon (DOC) removal in the filtered samples of suspension.

The test item was dissolved with 1.5 L mineral medium at a nominal concentration of29.6 mg/L as DOC, and the prepared activated sludge was added to flasks to give a concentration of inoculums at29.8mg/L as suspended solids (SS) in the final inoculated mixture.After mixing, samples were taken from each test vessel to determine starting DOC concentration. The starting concentration of DOC in the test suspension measured was29.20 and 29.00 mg/L (corrected for inoculums control).Samples of suspension were takenon 1st, 4th, 6th, 8th, 11th, 14th, 19th, 23rdand 28thday to determine DOC concentration. After 4 days, thepercentageremoval of DOC in the test suspension attained45.1%(average), so10% biodegradationmay be obtained on 2ndor 3rdday andwhen the 10-day window began. After 6 days, thepercentageremoval of DOC in the test suspension attained97.9%(average), it can be assumed that the percentage biodegradation in activated sludge exceeded 70% pass level (based on removal of DOC) within 10-day window.

The percentagebiodegradation ofreference item(sodium benzoate)attained96.5%after 1 day and 99.4% at the14thday, whichexceeded70%pass level. In the toxicity control containing the test item, reference item and inoculums, the percentage degradationbased on total DOC attained46.1%after 1 day and99.4%at the14thday, whichexceeded35%,which indicated that the test item can be assumed to be not inhibitory under the conditions of the test.As mentioned above, the test was considered to be valid.

Based on these results,the test itemcan be assumed to be readily biodegradable under the test conditions.

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information