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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:
From Dec. 14, 2001 to July 1, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
Deviations, reliability, and validity evaluated against current OECD 301B guideline (1992).
Deviations:
no
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Taylor Creek Wastewater Treatment Plant (Harrison, OH) on Dec. 17, 2001. This STP receives predominantly domestic waste.
- Storage condition: The inoculum was aerated until use
- Storage length: 1 d
- Preparation of inoculum for exposure: The sludge solids were adjusted to 3,000 ±300 mg/L. Approx. 200 mL of sludge was gently centrifuged for 10 min. The supernatant was decanted, and replaced with media. The sludge was subsequently homogenized in a blender at low speed for approx. 2 min and used as inocula at a concentration of 1% (v/v).
- Pretreatment: None
- Concentration of sludge: Not reported
- Initial cell/biomass concentration: Not reported
Duration of test (contact time):
28 d
Initial conc.:
40 mg/L
Based on:
act. ingr.
Initial conc.:
16.3 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral media was prepared one day prior to test initiation according to the OECD 301B guideline.
- Solubilising agent: None used
- Test temperature: The test sample bottles were incubated at 22 ±1°C in a water bath throughout the test period
- pH: 7
- pH adjusted: Not reported
- Aeration of dilution water: Aerated overnight with CO2-free air.
- Suspended solids concentration: approx. 30 mg/L of TSS
- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 1 L bottles containing test chemical, activated sludge inoculum, and phosphate buffered media
- Number of culture flasks/concentration: Three for test material and blank treatment; two for reference substance
- Method used to create aerobic conditions: Continuous aeration with CO2-free air
- Measuring equipment: The carbon dioxide produced was measured using an electrolytic respirometer (Coordinated Environmental Services Ltd., UK). A conductivity probe immersed in 1% NaOH measured the production of carbon dioxide, and a pressure transducer monitored the decrease in pressure due to oxygen consumption. A sensor assembly charged with CuSO4 continually equalized the pressure as oxygen was consumed.
- Test performed in closed vessels: Yes
- Other: The study was terminated and aliquots of the test samples were centrifuged and analyzed to determine the remaining soluble organic carbon (SOC) using a Shimadzu Total Organic Carbon Analyzer, following Procter and Gamble Biological Safety Testing Standard, Volume II, Section IV(66).
- The details of ‘Electrolytic Respirometry Test set up’ is provided under ‘Any other information on materials and methods incl. tables’ section below

SAMPLING
- Sampling frequency: Sampling was performed via an automated data logging system which acquired data at 12 h time intervals, following a 1 h stabilization period.
- Sampling method: Automated sampling
- Sample storage before analysis: Not available

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, treatment flasks receiving no test material
- Reference control: Yes, containing reference substance (sodium benzoate) and inoculum
- Abiotic sterile control: No
- Toxicity control: No

STATISTICAL METHODS: Not reported
Reference substance:
benzoic acid, sodium salt
Remarks:
tested at 20 mg/L (13.69 mg Carbon/L )
Key result
Parameter:
% degradation (CO2 evolution)
Value:
14.3
St. dev.:
2.7
Sampling time:
29 d
Remarks on result:
other: Mean of replicates 1,2,3
Parameter:
% degradation (CO2 evolution)
Value:
12.3
Sampling time:
29 d
Remarks on result:
other: Replicate 1
Parameter:
% degradation (CO2 evolution)
Value:
13.2
Sampling time:
29 d
Remarks on result:
other: Replicate 2
Parameter:
% degradation (CO2 evolution)
Value:
17.4
Sampling time:
29 d
Remarks on result:
other: Replicate 3
Details on results:
- Final SOC (in mg/L) was 10.6, 10.8 and 11 for replicate 1, 2 and 3 respectively. The initial TOC was 16.3 mg/L.
- For details on biodegradation results, please refer to 'Table 1' in the ‘Any other information on results incl. tables’
Results with reference substance:
- The Reference material met the validity criteria for the test: >60% biodegradation within 14 d, and within the 10 d window. Here are details:

Test conducted with inoculum collected from Taylor Creek Wastewater Treatment Plant Harrison:
- Pass level of >60% reached by Day 4 in both replicates. (Mean value of replicates on Day 1 = 13%, Day 3 = 67.9% and Day 29 = 81.1% TCO2).
- Final mg Soluble Organic Carbon (SOC)/L were found to be 0.24 and 0.01 mg/L in replicate 1 and 2 respectively. The initial TOC was 13.69 mg/L.

Table 1. Ready biodegradation test (CO2 evolution) of N,N(bis)hydroxyethyl-p-phenylenediamine sulfate (inoculum source: Taylor Creek Wastewater Treatment Plant, Harrison, Ohio) (Study # 44407)

Time (days)

Replicate 1

Replicate 2

Replicate 3

Mean

Std. Dev.

0

0

0

0

0

0

0.5

-0.86

-0.86

-0.93

-0.88

0.04

1

-0.92

-0.86

-0.73

-0.84

0.1

1.5

-0.48

-0.15

0.11

-0.18

0.3

2

-0.26

-0.13

0.33

-0.02

0.31

2.5

0.07

0.33

0.91

0.44

0.43

3

0.35

0.28

1.06

0.56

0.43

3.5

0

0.2

1.29

0.5

0.7

4

0.11

0.88

1.65

0.88

0.77

4.5

0.2

0.65

1.8

0.88

0.83

5

0.32

1.22

2.56

1.37

1.13

5.5

1.18

1.69

3.65

2.17

1.31

6

1.81

2.44

4.02

2.76

1.14

6.5

2.55

2.92

5.12

3.53

1.39

7

3.19

3.38

5.63

4.07

1.36

7.5

3.46

4.03

6.39

4.63

1.56

8

3.81

4.37

6.85

5.01

1.62

8.5

3.97

4.65

7.07

5.23

1.63

9

4.44

5.56

7.96

5.99

1.8

9.5

5.04

5.84

8.42

6.43

1.77

10

5.4

6.26

8.77

6.81

1.75

10.5

7.91

8.94

11.52

9.46

1.86

11

7.93

9.2

11.66

9.6

1.9

11.5

8.44

9.29

12.1

9.94

1.91

12

8.39

9.54

12.23

10.05

1.97

12.5

8.71

9.73

12.41

10.29

1.91

13

8.84

9.8

12.54

10.4

1.92

13.5

8.92

10.35

12.79

10.69

1.96

14

8.73

10.23

12.78

10.58

2.05

14.5

9.09

10.35

13.13

10.86

2.07

15

9.08

10.45

13

10.84

1.99

15.5

9.24

10.67

13.51

11.14

2.17

16

9.37

10.56

13.52

11.15

2.13

16.5

9.23

9.83

13.21

10.76

2.14

17

9.18

10.26

13.04

10.82

1.99

17.5

9.53

10.49

13.5

11.17

2.07

18

8.64

9.95

13.09

10.56

2.29

18.5

9.28

10.41

13.65

11.12

2.27

19

9.72

10.67

14.03

11.47

2.26

19.5

9.67

10.8

13.8

11.42

2.13

20

9.66

10.78

14.13

11.52

2.33

20.5

9.95

11.2

14.36

11.84

2.27

21

10.02

11.38

14.31

11.91

2.19

21.5

10.48

11.49

14.76

12.25

2.24

22

9.99

10.88

14.45

11.77

2.36

22.5

8.82

9.76

13.33

10.64

2.38

23

10.24

11.42

14.81

12.15

2.37

23.5

10.76

11.82

15.27

12.62

2.36

24

11.1

11.98

15.71

12.93

2.45

24.5

10.97

11.92

15.76

12.89

2.54

25

11.16

12.05

15.66

12.96

2.38

25.5

11.34

11.81

15.95

13.03

2.53

26

11.69

12.4

16

13.36

2.31

26.5

11.67

12.61

16.32

13.53

2.46

27

11.84

12.61

16.66

13.7

2.59

27.5

11.42

12.65

16.76

13.61

2.8

28

12.29

12.93

16.98

14.07

2.54

28.5

12.39

13.03

17.31

14.24

2.68

29

12.25

13.24

17.4

14.3

2.73

Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily biodegradable
Conclusions:
N,N(bis)hydroxyethyl-p-phenylenediamine sulfate (A050) is not readily biodegradable.
Executive summary:

A ready biodegradation test, following the OECD 301B guideline, was conducted on N,N(bis)hydroxyethyl-p-phenylenediamine sulfate (A050). The test material was tested at 40 mg active/L (16.32 mg TOC/L). The inoculum was activated sludge collected from Taylor Creek Wastewater Treatment Plant (Harrison, OH) which receives predominantly domestic sewage. The test treatment and inoculum blank were measured in triplicate, while the reference was measured in duplicate. The deviation between replicate treatments was <20% (met the validity criteria of the guideline). 

The test material, N,N(bis)hydroxyethyl-p-phenylenediamine sulfate (A050), did not meet the criteria for ready biodegradation.

The final percentage of biodegradation (CO2 evolution) on Day 29 was 14.3 % (mean of 3 flasks with 12.3, 13.2 and 17.4%, respectively).

The reference material, sodium benzoate, reached >10% biodegradation at Day 1 and >60% at Day 4 in both replicates. Hence, it met the validity criteria established in the guideline for a reference material.

This ready biodegradation test is classified as acceptable, and satisfies the guideline requirements for the OECD 301B.

Description of key information

N,N(bis)hydroxyethyl-p-phenylenediamine sulphate is not readily biodegradable.

Key value for chemical safety assessment

Additional information

A ready biodegradation test, following the OECD 301B guideline, was conducted on N,N(bis)hydroxyethyl-p-phenylenediamine sulphate. The test material was tested at 40 mg active/L (16.32 mg TOC/L). The inoculum was activated sludge collected from Taylor Creek Wastewater Treatment Plant which receives predominantly domestic sewage. The test treatment and inoculum blank were measured in triplicate, while the reference was measured in duplicate. The deviation between replicate treatments was <20% (met the validity criteria of the guideline). 

The percentage of biodegradation of the tested substance on Day 29 was 14.3 % and it is considered as not readily biodegradable.

The reference material, sodium benzoate, reached >60% at Day 4 in conformity with the validity criteria for the reference material.