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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 May 1998 to 24 June 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (City of Geneva, Aïre) collected in the morning
- Storage length: Used on the same day
- Preparation of inoculum for exposure: Washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and re-suspending in mineral medium) and kept aerobic.
- Concentration of sludge: Dry weight of suspended solids 3.026 g/L. To obtain a concentration of 30 mg/L (dry weight) in a 250 mL flask, 2.48 mL of sludge is needed (inoculum).
Duration of test (contact time):
29 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 50 mL of solution A was mixed with 2000 mL deionised water and 5 mL of each of the solutions B, C and D were added; the solution was made up to 5 L with deionised water.
The water was deionised water containing less than 10 mg/L dissolved organic carbon and the 4 stock solutions were all prepared by dissolving the appropriate components and making up to one litre. The solutions contained the following substances: Solution A: 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4.2H2O and 0.5 g NH4CI; Solution B: 27.5 g CaCl2; Solution C: 22.5 g MgSO4.7H2O; and Solution D: 0.25 g FeCI3.6H2O and one drop conc. HCl.
- Additional substrate: No, the test material was the nominal sole source of organic carbon
- Test temperature: 22 ± 1 °C
- pH of mineral medium: 7.4 ± 0.2
- pH of test solutions: The pH of each flask was not measured but assumed to be the same as the mineral medium in order not to remove any floating undissolved test material from the test medium by dipping a glass electrode in it. At the end of the test period the pH of each flask is measured.
- pH of mineral medium adjusted: Yes (if required) with phosphoric acid or potassium hydroxide
- Aeration of dilution water: No
- Suspended solids concentration: 3.026 g dry material/L. The dry weight of suspended solids was determined by taking two 50 mL samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105 to 110 °C for two hours and weighing the residue.
- Continuous darkness: No data

TEST SYSTEM
- Culturing apparatus: 250 mL flask filled with 250 mL of medium
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The inoculum was kept aerobic prior to use
- Measuring equipment: The study was conducted using the flasks attached to the respirometer used to measure oxygen consumption, a SAPROMAT D 12 ( J.M. VOITH GmbH, D-7920 Heidenheim).
- Test performed in closed vessels: Yes in order to measure oxygen consumption
- Details of trap for CO2 and volatile organics if used: Evolved carbon dioxide is trapped in soda lime pellets (about 2 g is placed in an attachment of the stopper)

SAMPLING
- Sampling frequency: The oxygen consumption of each flask was recorded every day
- Sampling method: The oxygen concentrations were measured by the volumetric respirometer

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, two flasks containing neither the test nor reference material were prepared
- Toxicity control: Yes, two flasks containing both the test and reference materials were prepared

STATISTICAL METHODS:
The amount of oxygen taken up by the microbial population during biodegradation of the test material (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidised to carbon dioxide and hydrogen to water).
Reference substance:
benzoic acid, sodium salt
Remarks:
100 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
28 d
Details on results:
Oxygen uptakes, as read on the SAPROMAT meters, are corrected by deducting the basic oxygen uptake of sludge and proportionally to account for the small differences between actual and nominal concentrations of test and reference materials. Means of identical flasks were used.

Mean Theoretical Oxygen Demand (ThOD) of test material: 2.08 mg O2/mg.

The test material undergoes 66 % biodegradation after 29 days (65 % after 28 days) in the test conditions. Biodegradation really starts on day 14 and reaches only 56 % at the end of the 10-day window (days 14 to 24).
Thus, test material should be regarded as not readily but inherently biodegradable according to this test.
The curves obtained with the reference material alone and with the test material + reference material show no toxic effect of test material to the micro-organisms at the test concentration.
Results with reference substance:
Degradation of sodium benzoate exceeds 40 % after 7 days and 65 % after 14 days: the activity of the inoculum is thus verified and the test is considered as valid.

Table 1: Biological Oxygen Demand (BOD, mg O2/L, adjusted to nominal concentrations)

Parameter

Flask

Code

Day

5

14

21

24

28

29

BOD Sludge

1st flask

B1

17.0

23.0

27.0

29.0

33.0

33.0

2nd flask

B2

14.0

21.0

23.0

25.0

28.0

28.0

Mean

B

15.5

22.0

25.0

27.0

30.5

30.5

BOD Test Material

1st flask

C1

26.9

45.7

101.1

143.6

173.3

178.3

2nd flask

C2

22.8

44.3

128.9

143.6

156.3

158.2

1st flask corrected

C1 - B

11.4

23.7

76.1

116.6

142.8

147.8

2nd flask corrected

C2 - B

7.3

22.3

103.9

116.6

125.8

127.7

% Degradation

1st flask

D1

5

11

37

56

69

71

2nd flask

D2

4

11

50

56

60

61

Mean

D

4

11

43

56

65

66

B = (B1 + B2) / 2

D1 = 100 * (C1 - B) / ThOD * initial test material concentration

D2 = 100 * (C2 - B) / ThOD * initial test material concentration

D = (D1 + D2) / 2

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
Under the conditions of this study, the test material was determined not to be readily biodegradable but it is considered to be inherently biodegradable.
Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guideline OECD 301 F under GLP conditions using the manometric respirometry method.

Fresh activated sludge collected from a biological waste water plant for domestic sewage was washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and re-suspending in mineral medium) and kept aerobic until being used on the same day. The sludge was exposed to the test material in duplicate at a nominal concentration of 100 mg/L for a period of 28 days at 22 ± 1 °C. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure.

As reference, sodium benzoate was tested simultaneously under the same conditions and functioned as a procedure control. Additionally, flasks containing both the test and reference materials were run in parallel and acted as toxicity control.

The test material undergoes 66 % biodegradation after 29 days (65 % after 28 days) in the test conditions. Biodegradation really starts on day 14 and reaches only 56 % at the end of the 10-day window (days 14 to 24). Thus, test material should be regarded as not readily but inherently biodegradable according to this test.

No toxic effect of test material to the micro-organisms at the test concentration was observed. Degradation of sodium benzoate exceeds 40 % after 7 days and 65 % after 14 days: the activity of the inoculum is thus verified and the test is considered as valid.

Under the conditions of this study, the test material was determined not to be readily biodegradable but it is considered to be inherently biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 July 2002 to 24 October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge freshly obtained from a municipal sewage treatment plant; 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands.
- Storage conditions: The sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (30 to 90 minutes) and the liquid decanted for use as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: The concentration of suspended solids was 3.4 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant).
Duration of test (contact time):
28 d
Initial conc.:
15.5 - 16.5 mg/L
Based on:
test mat.
Initial conc.:
11 other: mg TOC/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 L of mineral medium contains 10 mL of solution A, 1 mL each of solutions B to D and Milli-RO water. The stock solutions contained the following components dissolved in 1 L Milli-Q water.
Solution A: 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O and 0.50 g NH4Cl (pH 7.4 ± 0.2); Solution B: 22.50 g MgSO4.7H2O; Solution C: 36.40 g CaCI2.2H2O; and Solution D 0.25 g FeCI3.6H2O.
Tap-water was purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion­exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).
- Test temperature: 22 ± 1 °C
- pH: 7.4 to 7.8
- pH adjusted: No
- Aeration of dilution water: Yes, with CO2-free air. A mixture of oxygen (21 %) and nitrogen (79 %) was led through a bottle, containing 0.5- 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The CO2-free air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min). The test was started by bubbling CO2-free air through the solution at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min). A temporary breakdown in the aeration (< 1 day) was noted on day 12. However, this breakdown was considered to have no effect on the outcome of this study. The solutions were continuously stirred throughout the test period.
- Suspended solids concentration: 3.4 g/L
- Continuous darkness: No data

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles. Mineral components, Milli-RO water (ca. 80 % total volume) and inoculum (1 % final volume) were added to each bottle. This mixture was aerated with CO2-free air overnight to purge the system of CO2. The test material and positive control were added to the bottles. The volumes of suspensions were made up to 2 litres with Milli-RO water.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Aeration with CO2-free air
- Measuring equipment: The CO2 evolved was measured by titration
- Test performed in closed vessels: Yes to trap evolved CO2
- Details of trap for CO2 and volatile organics if used: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle. The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardised HCl.

SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day.
- Sampling method: Each time the CO2 absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2- absorber was placed at the far end of the series. Phenolphthalein was used as pH-indicator.
On the 28th day, the pH of the test suspensions was measured and 1 mL of concentrated HCl was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes; 2 bottles
- Abiotic sterile control: No
- Toxicity control: Yes; 1 bottle
- Positive control: Yes; 2 bottles
Reference substance:
acetic acid, sodium salt
Remarks:
40 mg/L
Key result
Parameter:
% degradation (CO2 evolution)
Value:
24
Sampling time:
28 d
Remarks on result:
other: The average value from two replicates
Details on results:
THEORETICAL CO2 PRODUCTION
The Theoretical CO2 production (ThCO2) of the test material was calculated to be 2.52 mg CO2/mg.
The concentration was 33.2 (A) and 30.9 mg (B) in 2 litres of test medium. Hence, the theoretical CO2 production following complete degradation was 83.7 mg per 2 litres for A and 77.9 mg per 2 litres for B.
The toxicity control contained 80.0 mg sodium acetate and 33.2 mg test material in 2 litres of test medium. Hence, the theoretical CO2 production following complete degradation of the test material plus sodium acetate was 169.3 mg per 2 litres.

BIODEGRADATION
The relative degradation values calculated from the measurements performed during the test period revealed 29 and 19 % degradation of the test material for replicates A and B respectively. Thus, the criterion for ready biodegradability (at least 60 % biodegradation was not reached within ten days of biodegradation exceeding 10 %) was not met.
In the toxicity control more than 25 % degradation occurred within 14 days (based on ThCO2). Therefore, the test material was assumed to be not inhibitory on microbial activity.
Results with reference substance:
The positive control contained 80.0 mg sodium acetate (ThCO2 = 1.07 mg CO2/mg) resulting in a theoretical CO2 production following complete degradation of 85.6 mg per 2 litres.
The positive control had achieved 64 % degradation after 7 days and 96 % degradation by the end of the test period.

Table 1: CO2 Production in the Blank

Day

HCl (0.05 N) Titrated (mL)

Produced CO2 (mL HCl)

Produced CO2 (mg)

Cumulative CO2 (mg)

Ba(OH)2*

Blank (mean)

0

-

-

-

-

0.0

2

5.0.0

46.06

3.94

4.3

4.3

5

50.22

45.06

5.16

5.7

10.0

7

49.19

45.64

3.56

3.9

13.9

9

51.81

48.01

3.80

4.2

18.1

14

49.44

46.75

2.69

3.0

21.1

19

49.89

46.39

3.50

3.8

24.9

23

49.86

44.70

5.16

5.7

30.6

27

50.03

46.17

3.86

4.2

34.8

29

50.75

46.72

4.04

4.4

39.3

29

48.96

45.52

3.45

3.8

43.1

29

48.59

47.58

1.01

1.1

44.2

*”Strength” of untreated 0.0125 M Ba(OH)2 solution (mean value)

 

Table 2: CO2 Production and Percentage Degradation of the Test Material

Day

HCl (0.05 N) Titrated (mL)

Produced CO2 (mL HCl)

Produced CO2 (mg)

Cumulative CO2 (mg)

Degradation (%)

Blank (mean)

Test Material Replicate

Replicate A

0

-

-

-

-

-

0

2

46.06

44.19

1.87

2.1

2.1

2

5

45.06

42.87

2.19

2.4

4.5

5

7

45.64

44.81

0.82

0.9

5.4

6

9

48.01

48.13

0.00

0.0

5.4

6

14

46.75

47.49

0.00

0.0

5.4

6

19

46.39

45.48

0.91

1.0

6.4

8

23

44.70

43.14

1.56

1.7

8.1

10

27

46.17

42.40

3.77

4.1

12.2

15

29

46.72

39.81

6.91

7.6

19.8

24

29

45.52

42.40

3.12

3.4

23.3

28

29

47.58

46.99

0.59

0.6

23.9

29

Replicate B

0

-

-

-

-

-

0

2

46.06

43.74

2.32

2.6

2.6

3

5

45.06

42.95

2.11

2.3

4.9

6

7

45.64

45.52

0.11

0.1

5.0

6

9

48.01

47.00

1.01

1.1

6.1

8

14

46.75

46.94

0.00

0.0

6.1

8

19

46.39

45.70

0.69

0.8

6.9

9

23

44.70

44.21

0.49

0.5

7.4

10

27

46.17

43.76

2.41

2.6

10.1

13

29

46.72

44.35

2.37

2.6

12.7

16

29

45.52

44.33

1.19

1.3

14.0

18

29

47.58

46.82

0.76

0.8

14.8

19

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of this study, the test material was determined not to be readily biodegradable.
Executive summary:

The ready biodegradability of the test material was investigated in a CO2-evolution test conducted in accordance with the standardised guidelines OECD 301 B and EU Method C.4-C under GLP conditions.

The test system was a microbial inoculum of activated sludge obtained from a municipal sewage treatment plant. The sludge (suspended solids concentration 3.4 g/L) was exposed to the test material in duplicate for a period of 28 days at 22 ± 1 °C. The biodegradation was followed by the carbon dioxide evolution of the microorganisms during exposure. As reference, sodium acetate was tested simultaneously under the same conditions and functioned as a positive control. Blank and toxicity controls were run concurrently.

Since the solubility of the test material was below 1 g/L, weighed amounts of the test material were added to test bottles. Approximately 10 mL of Milli-RO water was added to each weighing bottle and after thoroughly mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test was started by bubbling CO2-free air through the solution at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min). The solutions were continuously stirred throughout the test period. The CO2 evolved was trapped by Ba(OH)2 and measured by titration with HCl. Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. On the 28th day, 1 mL of concentrated HCl was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

The relative degradation values calculated from the measurements performed during the test period revealed 29 and 19 % degradation of the test material for replicates A and B respectively. Thus, the criterion for ready biodegradability (at least 60 % biodegradation was not reached within ten days of biodegradation exceeding 10 %) was not met.

In the toxicity control more than 25 % degradation occurred within 14 days (based on ThCO2). Therefore, the test material was assumed to be not inhibitory on microbial activity. The positive control had achieved 64 % degradation after 7 days and 96 % degradation by the end of the test period.

Under the conditions of this study, the test material was determined not to be readily biodegradable.

Description of key information

Weight of Evidence Approach

There are 2 studies available, both are GLP and guideline compliant.

Due to the fact that both studies are assigned with the reliablility score of 1, a weight of evidence approach was conducted.

The test material was determined not to be readily biodegradable in both a CO2 evolution test and in a manometric respirometry test but was considered to be inherently biodegradable in the manometric respirometry test.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

Weight of Evidence Approach

Two studies were conducted to assess the ready biodegradability of the test material in water. Both studies were conducted in accordance with standardised guidelines and under GLP conditions. As both studies were assigned with a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997), a weight of evidence approach was conducted.

Study 1: Manometric respirometry test

The ready biodegradability of the test material was investigated in accordance with the standardised guideline OECD 301 F under GLP conditions using the manometric respirometry method.

Fresh activated sludge collected from a biological waste water plant for domestic sewage was washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and re-suspending in mineral medium) and kept aerobic until being used on the same day. The sludge was exposed to the test material in duplicate at a nominal concentration of 100 mg/L for a period of 28 days at 22 ± 1 °C. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure.

As reference, sodium benzoate was tested simultaneously under the same conditions and functioned as a procedure control. Additionally, flasks containing both the test and reference materials were run in parallel and acted as toxicity control.

The test material undergoes 66 % biodegradation after 29 days (65 % after 28 days) in the test conditions. Biodegradation really starts on day 14 and reaches only 56 % at the end of the 10-day window (days 14 to 24). Thus, test material should be regarded as not readily but inherently biodegradable according to this test.

No toxic effect of test material to the micro-organisms at the test concentration was observed. Degradation of sodium benzoate exceeds 40 % after 7 days and 65 % after 14 days: the activity of the inoculum is thus verified and the test is considered as valid.

Under the conditions of this study, the test material was determined not to be readily biodegradable but it is considered to be inherently biodegradable.

Study 2: CO2 -evolution test

The ready biodegradability of the test material was investigated in a CO2-evolution test conducted in accordance with the standardised guidelines OECD 301 B and EU Method C.4-C under GLP conditions.

The test system was a microbial inoculum of activated sludge obtained from a municipal sewage treatment plant. The sludge (suspended solids concentration 3.4 g/L) was exposed to the test material in duplicate for a period of 28 days at 22 ± 1 °C. The biodegradation was followed by the carbon dioxide evolution of the microorganisms during exposure. As reference, sodium acetate was tested simultaneously under the same conditions and functioned as a positive control. Blank and toxicity controls were run concurrently.

Since the solubility of the test material was below 1 g/L, weighed amounts of the test material were added to test bottles. Approximately 10 mL of Milli-RO water was added to each weighing bottle and after thoroughly mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test was started by bubbling CO2-free air through the solution at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min). The solutions were continuously stirred throughout the test period. The CO2 evolved was trapped by Ba(OH)2 and measured by titration with HCl. Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. On the 28th day, 1 mL of concentrated HCl was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

The relative degradation values calculated from the measurements performed during the test period revealed 29 and 19 % degradation of the test material for replicates A and B respectively. Thus, the criterion for ready biodegradability (at least 60 % biodegradation was not reached within ten days of biodegradation exceeding 10 %) was not met.

In the toxicity control more than 25 % degradation occurred within 14 days (based on ThCO2). Therefore, the test material was assumed to be not inhibitory on microbial activity. The positive control had achieved 64 % degradation after 7 days and 96 % degradation by the end of the test period.

Under the conditions of this study, the test material was determined not to be readily biodegradable.

Conclusion

The test material was determined not to be readily biodegradable in both a CO2 evolution test and in a manometric respirometry test but was considered to be inherently biodegradable in the manometric respirometry test.

Therefore, the test material is considered to be inherently biodegradable.