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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:
07 February 2007 - 08 March 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP. Biodegradation test of DONA ammonium salt.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
Test temperature was 25.2 °C (>24 °C) for 4-hour period, negligible over 28-day test period.
Qualifier:
according to
Guideline:
other: European Commission (EC) directive 92/69/EEC, Part C, Pub No. L383, C.4-C; ISO International Standard 9439 (1999)
Deviations:
yes
Remarks:
Test temperature was 25.2 °C (>24 °C) for 4-hour period, negligible over 28-day test period.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 6675
- Substance type: single-constituent substance
- Physical state: clear colorless liquid
- Analytical purity: 29.9 ±0.1% in water
- Lot/batch no.: 140499-19/10
- Stability under test conditions: Stable in water at least 96 hours
- Storage condition of test material: At room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge from the 'Waterschap de Maaskant', 's-Hertogenbosch, The Netherlands, municipal sewage treatment plant.
- Storage conditions: Continuous aeration until use.
- Storage length: Not reported ("freshly obtained")
- Preparation of inoculum for exposure: Sludge allowed to settle 60 minutes and liquid decanted for use as inoculum.
- Pretreatment: None
- Concentration of sludge: 10 mL/L mineral medium
- Initial cell/biomass concentration: Not reported.
- Water filtered: Not reported
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
12 mg/L
Based on:
other: organic carbon
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Standard mineral media as per OECD 301B. Aerated with synthetic air overnight to purge the media of CO2.
- Solubilising agent (type and concentration if used): none
- Test temperature: 22.0 - 24.0 °C, with a short 4 hr period during which the temperature exceeded 24.0 °C with a maximum of 25.2 °C
- pH: 7.6 - 7.9
- pH adjusted: no
- Aeration of dilution water: Aerated each test system (without test or reference substance) with synthetic air (CO2 < 1 ppm) overnight prior to test initiation
- Suspended solids concentration: 4.4 g/L in concentrated sludge
- Continuous darkness: Not reported. Test vessels were brown-colored bottles
- Other: Test media aerated and stirred continuously during the test period

TEST SYSTEM
- Culturing apparatus: 2-liter all-glass brown colored bottles
- Number of culture flasks/concentration: 2 bottles test suspension; 2 bottles containing inoculum blank; 1 bottle positive control; 1 bottle toxicity control
- Method used to create aerobic conditions: Continuous aeration and stirring. Synthetic air (ca. 21% oxygen and ca. 79% nitrogen) was passed through 0.0125 M barium hydroxide at a rate of 1-2 bubbles/sec to remove any CO2 present prior to use to aerate the test system
- Measuring equipment: CO2 production determined by titrating remaining barium hydroxide with 0.05 M standardized HCl
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: 100 mL of 0.0125 M Ba(OH)2 in each of three bottles connected in series to the exit air line of each test bottle.

-SAMPLING
- Sampling frequency: Every second or third day during the first 10 days and thereafter at least every fifth day until the 28th day. Final titration was made on day 29.
- Sampling method: At each sample time, the CO2 absorber nearest to the test bottle was removed for analysis. Each of the remaining two absorbers was moved one position in the direction of the test bottle and a new absorber was placed at the far end of the series. Phenolphthalein was used as a pH indicator. On the 28th day, the pH of all test solutions was measured and 1 mL of 37% HCl was added to the inoculum blank and test suspension bottles. The bottles were aerated overnight to drive off any CO2 present. The final titrations were made on day 29.
- Sample storage before analysis: None
- Other: The theoretical CO2 production was calculated from the molecular formula.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Contained only mineral media and inoculum
- Abiotic sterile control: None
- Toxicity control: Contained test substance at 189 mg/L and sodium acetate at 40 mg/L plus mineral medium and inoculum
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Preliminary study:
N/A
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
6
Sampling time:
28 d
Details on results:
The ThCO2 of the active ingredient in ADONA was calculated to be 0.78 mg CO2/mg based on structure.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg based on structure.

- Effect concentrations exceeding solubility of substance in test medium: No

% biodegradation vs time plot - see Figure 1

BOD5 / COD results

Results with reference substance:
The reference substance sodium acetate was degraded at least 60% (74%) within 14 days. It had a calculated ratio of 85.6 mg CO2/2L for CO2 produced (cumulative) and the ThCO2 of sodium acetate.

Any other information on results incl. tables

Table 1:  pH values during the test

Test Medium:

At the start of the test:

On Day 28

Blank Control (A)

7.6

7.5

Blank Control (B)

7.6

7.5

Sodium Acetate

7.6

7.9

ADONA (A)

7.6

7.4

ADONA (B)

7.6

7.3

Toxicity Control

7.6

7.7

Table 2: CO2production in the blank

Day

HCl (0.05 N) titrated (mL)

CO2Produced

(mL HCl)

CO2Produced

(mg)

Cumulative CO2(mg)

Ba(OH)2A

Blank (mean)

0

---

---

---

---

0

2

47.63

47.87

0.00

0.0

0.0

5

50.71

48.51

2.21

2.4

2.4

7

50.43

46.97

3.46

3.8

6.2

9

50.86

47.94

2.92

3.2

9.4

14

50.30

46.29

4.02

4.4

13.9

19

49.85

45.12

4.73

5.2

19.1

23

48.46

42.87

5.59

6.1

25.2

27

49.71

44.53

5.18

5.7

30.9

29

47.51

44.53

2.98

3.3

34.2

29

49.83

47.39

2.44

2.7

36.9

29

48.47

48.30

0.17

0.2

37.0

A) “Strength” of untreated 0.0125 M Ba(OH)2solution

Table 3: CO2 production and % biodegradation of the reference substance

Day

HCl (0.05 N) titrated (mL)

CO2Produced

(mL HCl)

CO2Produced

(mg)

Cumulative CO2(mg)

DegradationA

(%)

Blank (mean)

Sodium acetate

0

---

---

---

---

---

0

2

47.87

40.08

7.79

8.6

8.6

10

5

48.51

26.40

22.11

24.3

32.9

38

7

46.97

35.60

11.37

12.5

45.4

53

9

47.94

39.76

8.18

9.0

54.4

64

14

46.29

37.81

8.47

9.3

63.7

74

19

45.12

38.03

7.08

7.8

71.5

84

23

42.87

39.01

3.86

4.2

75.8

89

27

44.53

42.55

1.98

2.2

77.9

91

29

44.53

39.55

4.98

5.5

83.4

97

29

47.39

45.99

1.40

1.5

84.9

99

29

48.30

48.50

0.00

0.0

84.9

99

A) Calculated as ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.6 CO2/2L

Table 4: CO2 production and % biodegradation of ADONA (bottle A)

Day

HCl (0.05 N) titrated (mL)

CO2Produced

(mL HCl)

CO2Produced

(mg)

Cumulative CO2(mg)

DegradationA

(%)

Blank (mean)

Bottle A

0

---

---

---

---

---

0

2

47.87

48.66

0.00

0.0

0.0

0

5

48.51

47.45

1.05

1.2

1.2

1

7

46.97

47.13

0.00

0.0

1.2

1

9

47.94

46.37

1.57

1.7

2.9

3

14

46.29

45.54

0.74

0.8

3.7

4

19

45.12

44.76

0.35

0.4

4.1

5

23

42.87

42.81

0.06

0.1

4.2

5

27

44.53

44.15

0.38

0.4

4.6

5

29

44.53

43.71

0.82

0.9

5.5

6

29

47.39

45.14

2.25

2.5

7.9

9

29

48.30

48.93

0.00

0.0

7.9

9

A) Calculated as ratio between CO2 produced (cumulative) and the ThCO2 of ADONA: 88.2 mg CO2/2L

Table 5: CO2 production and % biodegradation of ADONA (bottle B)

Day

HCl (0.05 N) titrated (mL)

CO2Produced

(mL HCl)

CO2Produced

(mg)

Cumulative CO2(mg)

DegradationA

(%)

Blank (mean)

Bottle B

0

---

---

---

---

---

0

2

47.87

48.00

0.00

0.0

0.0

0

5

48.51

47.60

0.90

1.0

1.0

1

7

46.97

47.08

0.00

0.0

1.0

1

9

47.94

47.34

0.60

0.7

1.7

2

14

46.29

45.80

0.48

0.5

2.2

2

19

45.12

45.02

0.09

0.1

2.3

3

23

42.87

43.36

0.00

0.0

2.3

3

27

44.53

44.90

0.00

0.0

2.3

3

29

44.53

44.93

0.00

0.0

2.3

3

29

47.39

47.86

0.00

0.0

2.3

3

29

48.30

49.25

0.00

0.0

2.3

3

A) Calculated as ratio between CO2 produced (cumulative) and the ThCO2 of ADONA: 88.2 mg CO2/2L

 

Table 6: CO2 production and % biodegradation of toxicity control

Day

HCl (0.05 N) titrated (mL)

CO2Produced

(mL HCl)

CO2Produced

(mg)

Cumulative CO2(mg)

DegradationA

(%)

Blank (mean)

Toxicity Control

0

---

---

---

---

---

0

2

47.87

38.84

9.03

9.9

9.9

6

5

48.51

27.76

20.75

22.8

32.8

19

7

46.97

36.08

10.89

12.0

44.7

26

9

47.94

41.30

6.64

7.3

52.0

30

14

46.29

36.29

10.00

11.0

63.0

36

19

45.12

39.86

5.26

5.8

68.8

40

23

42.87

40.10

2.77

3.0

71.9

41

27

44.53

40.78

3.75

4.1

76.0

44

29

44.53

43.19

1.34

1.5

77.5

45

29

47.39

47.42

0.00

0.0

77.5

45

29

48.30

48.25

0.05

0.0

77.5

45

A) Calculated as ratio between CO2 produced (cumulative) and the ThCO2 of ADONA and sodium acetate : 173.8 mg CO2/2L

ThCO2 ADONA: 88.2 mg CO2/2L

ThCO2 Sodium acetate: 85.6 mg CO2/2L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
At day 14 the reference substance showed > 60% biodegradation; duplicate flasks had less than 20% difference in % biodegradation; total CO2 release in blank at end of test < 40 mg/L
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
ADONA was not readily biodegradable under test conditions
Executive summary:

'DONA is an acid that will be entirely deprotonated at pH>4. ADONA is the ammonium salt of DONA.

The ready biodegradability of ADONA was studied in a 28-day CO2 evolution test according to OECD method 301B. Activated sludge from a domestic sewage treatment plant was used as the source of inoculum. Brown glass bottles were filled with oxygen-saturated, CO2-free medium and spiked with one of the following: ADONA at 186 mg/L (12 mg TOC/L), sodium acetate (reference substance) at 40 mg/L (12 mg TOC/L), ADONA plus sodium acetate (toxicity control), or inoculum alone (inoculum blank). Evolved CO2 was titrated every second or third day during the first 10 days and thereafter at least every fifth day until the 28th day. Final titration was made on day 29. The 28-day biodegradation of ADONA was found to be 6% by CO2 evolution. In the toxicity control more than 25% biodegradation occurred within 14 days (36%) therefore the test substance did not inhibit microbial activity. The reference substance sodium acetate showed 74% biodegradation at 14 days demonstrating the viability of the inoculum. Based on these data, ADONA was found to not be readily biodegradable under the conditions of the CO2 evolution test.Because ammonia metabolism is not detected in CO2-evolution tests and because the acid will be deprotonated at pH >4, the results of the ADONA test are directly applicable to DONA biodegradability.

This study is classified as acceptable and satisfies the guideline requirements for test method OECD301B, ready biodegradability: CO2 evolution test.

Results Synopsis

Percent degradability: 6% 95% C.I.: not applicable