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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:
key study
Study period:
13 March 2018 - 10 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
A sample of activated sludge was taken from the aeration tank of Sewage Treatment Plant ”Czajka”, Warsaw, receiving predominantly domestic sewage. The sludge was aerated for 6 days, at the test temperature of about 22 °C, until application. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids. Before an application the sludge was washed in a mineral medium.
Duration of test (contact time):
28 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:
Prepare the following stock solutions, using analytical grade reagents.
(a) Monopotassium dihydrogen orthophosphate, KH2PO4 8.50 g
Dipotassium monohydrogen orthophosphate, K2HPO4 21.75 g
Disodium monohydrogen orthophosphate dihydrate Na2HPO4 · 2 H2O 33.40 g
Ammonium chloride, NH4Cl 0.50 g
Dissolve in water and make up to 1 litre The pH of the solution should be 7.4.
(b) Calcium chloride, anhydrous, CaCl2 27.50 g
Dissolve in water and make up to 1 litre
(c) Magnesium sulphate heptahydrate, MgSO4 · 7 H2O 22.50 g
Dissolve in water and make up to 1 litre.
(d) Iron(III) chloride hexahydrate, FeC13 · 6H2O 0.25 g
Dissolve in water and make up to 1 litre.
Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of solutions (b) to (d) and make up to 11 with dilution water.
- Test temperature: 22 ºC
- pH: 7.04-8.67
- pH adjusted: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Thermo-cabinet WTW TS 606 CZ-G/3-VAR
- Number of culture flasks/concentration: Triplicate, containing test item (100 mg/L) and inoculum (30 mg/L SS).
- Measuring equipment:
Closed WTW OxiTop OC 110 respirometer for BOD determination;
Spectrophotometer Hach DR 3900 for TOC concentration measurements;
Electronic temperature recorder EBI 310 –T;
pH-meter – multifunctional microcomputer meter ELMETRON CX-505;

SAMPLING
The oxygen uptake from the readings taken at regular and frequent intervals was calculated. The data were read out every 112 min during the 28 day test (40 320 min that is 360 readings) and were recoded and stored in the measuring heads of the sample bottles. These collected data, using the controller, were infrared read out from the heads and stored in the controller. At the end of incubation, normally 28 days, the pH of the contents of the flasks was measured.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (triplicate), only inoculum (30 mg/L SS)
- Toxicity control: Yes (triplicate), test item 100 mg/L, reference item (100 mg/L sodium acetate) and inoculum (30 mg/L SS)

Reference substance:
acetic acid, sodium salt
Remarks:
100 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
72.3
Sampling time:
28 d
Details on results:
At the 28th day of the test the measured aerobic biodegradation of the test item equals 72.3%. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 20 days and was not reached in a 10-day window in which biodegradation attained 53%. Lag time was 8 days and degradation time was 15 days. Thus, the test item cannot be classed as readily biodegradable.
Results with reference substance:
The reference item reached 91.7% of biodegradation and the level for ready biodegradability by 4 days.

The test item attained 72.3% of biodegradation. The pass level was attained after 20 days and was not reached in a 10-day window in which biodegradation attained 53%.

The reference item reached 91.7% of biodegradation and the level for ready biodegradability by 4 days.

In the toxicity test the biodegradation was equal to 56.0% in 14 days, not being inhibitory.

The oxygen uptake of the inoculum blank was equal to 37.9 mg/L in 28 days.

The pH values of all flasks were inside the range 7.04 -8.67.

 

time, days

1

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2uptake, mg/L

a1

5.8

7.5

11.1

16.0

31.7

62.8

77.6

88.3

99.0

113.5

118.7

125.6

132.8

a2

6.8

8.5

12.7

18.0

25.9

43.6

63.0

81.3

97.1

118.6

128.2

135.9

146.5

a3

5.4

9.2

13.9

27.4

63.7

90.9

103.9

115.2

122.8

132.4

135.8

140.3

146.8

am. avg

6.0

8.4

12.6

20.5

40.5

65.8

81.5

94.9

106.3

121.5

127.6

133.9

142.0

Blank test O2uptake mg/L

b1

6.3

11.8

14.6

21.5

24.1

29.0

29.5

32.5

33.5

36.3

40.0

40.2

41.6

b2

5.6

10.4

13.7

16.7

18.2

23.9

24.2

26.9

27.9

29.3

34.3

35.9

37.1

b3

7.2

10.2

13.6

18.1

20.6

24.1

25.7

27.3

28.1

28.9

33.0

32.3

35.1

bm. avg

6.4

10.8

13.9

18.8

21.0

25.6

26.5

28.9

29.8

31.5

35.8

36.1

37.9

Reference item O2uptake. mg/L

w1

24.0

53.5

67.3

77.7

84.2

89.6

95.0

97.0

98.5

101.2

103.9

104.2

107.0

w2

23.3

55.3

68.6

78.3

85.7

92.1

95.8

98.6

100.1

105.1

107.4

108.8

111.9

w3

24.6

55.4

69.1

83.2

90.2

98.9

104.5

108.2

112.2

115.1

118.4

119.1

122.0

wm. avg

24.0

54.7

68.3

79.7

86.7

93.6

98.5

101.3

103.6

107.1

109.9

110.7

113.6

Toxicity control O2uptake. mg/L

tox1

13.1

63.3

70.4

80.0

97.1

125.2

140.9

151.5

159.8

170.6

174.3

179.6

186.8

tox2

11.4

60.3

70.2

77.7

85.3

133.1

153.4

167.0

177.6

191.2

197.4

204.4

211.4

tox3

11.9

61.2

72.8

84.1

98.7

142.2

158.2

170.0

178.2

190.3

194.9

197.6

205.3

toxm.avg

12.1

61.6

71.1

80.6

93.7

133.5

150.8

162.8

171.9

184.0

188.9

193.9

201.2

Corrected

test item O2uptake, mg/L

(a1-bm)

-0.6

-3.3

-2.8

-2.8

10.8

37.1

51.1

59.4

69.2

82.0

82.9

89.4

94.9

(a2-bm)

0.5

-2.3

-1.3

-0.7

4.9

18.0

36.5

52.4

67.3

87.1

92.4

99.8

108.6

(a3-bm)

-1.0

-1.6

0.03

8.6

42.8

65.3

77.5

86.3

93.0

100.9

100.0

104.2

108.9

Reference item % degradation

ThOD = 0.78

mgO2/mg

C = 100 mg/L

R1(w1)

22.6

54.8

68.5

75.6

81.1

82.0

87.9

87.3

88.1

89.3

87.3

87.3

88.5

R2(w2)

21.8

57.0

70.1

76.3

83.1

85.2

88.9

89.4

90.0

94.3

91.8

93.1

94.8

R3(w3)

23.3

57.2

70.7

82.6

88.8

94.0

100.0

101.7

105.6

107.1

105.9

106.4

107.8

Rwavg

22.2

55.9

69.3

76.0

82.1

83.6

88.4

88.4

89.1

91.8

89.5

90.2

91.7

Test item% degradation

ThOD = 2.60

mgO2/mg

C = 100 mg/L

R1(a1)

0.0

0.0

0.0

0.0

7.5

25.8

35.5

41.2

48.0

56.9

57.6

62.1

65.9

R2(a2)

0.3

0.0

0.0

0.0

3.4

12.5

25.4

36.4

46.7

60.5

64.2

69.3

75.4

R3(a3)

0.0

0.0

0.0

6.0

29.7

45.3

53.8

59.9

64.6

70.0

69.4

72.3

75.6

Raavg

0.1

0.0

0.0

2.0

13.5

27.9

38.2

45.8

53.1

62.5

63.7

67.9

72.3

Toxicity test

% degradation

 

R1(tox1)

3.0

23.6

25.4

27.6

34.3

44.9

51.5

55.2

58.5

62.6

62.4

64.6

67.0

R2(tox2)

2.3

22.3

25.4

26.6

29.0

48.4

57.2

62.2

66.6

71.9

72.8

75.8

78.2

R3(tox3)

2.5

22.7

26.5

29.4

35.0

52.5

59.4

63.6

66.8

71.5

71.7

72.7

75.4

Rtoxavg

2.6

22.9

25.8

27.9

32.8

48.6

56.0

60.3

64.0

68.7

69.0

71.0

73.5

Validity criteria:

- The difference of extremes of replicate values of the BOD of the test item at the plateau and at the end of the test is less than the limit of 20% or slightly exceeds this limit.

- The reference item has reached the pass level (60%) on day 4 (the limit is by day 14).

- In a toxicity test, containing both the test item and a reference item, on the 14th  test day the biodegradation (based on ThOD) attained 56.0%.  

- The oxygen uptake of the inoculum blank is 37.9 mg O2/l in 28 days (should not be greater than 60 mg/L).

- The pH values of all flasks were inside the range 6-8.5 and the oxygen consumption by the test item was greater than 60%.

Validity criteria fulfilled:
yes
Remarks:
(BOD differences between replicates < 20%, reference test item reached the pass level on day 4, in the toxicity test the biodegradation attained 56.0%, the oxigen update of inoculum blank was 37.9 mgO2/L in 28 days, the pH values were inside 6-8.5)
Interpretation of results:
inherently biodegradable
Conclusions:
At the 28th day of the test the measured aerobic biodegradation of the test item equals 72.3%. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 20 days and was not reached in a 10-day window in which biodegradation attained 53%. Lag time was 8 days and degradation time was 15 days. Thus, the test item cannot be classed as readily biodegradable.
Executive summary:

A ready biodegradation test was performed according to OECD Guideline 301F and EU Method C.4 -D (GLP study). A measured, stirred volume of a mineral solution containing 100 mg/L of test item was inoculated with microorganisms (activated sludge, 30 mg/L SS) and incubated under aerobic conditions (oxygen presence) in a closed respirometer flask at temperature of 22 ± 0.3 °C for 28 days. The blank tests were run in parallel with only inoculum but without test item. A reference item (sodium acetate) was run in parallel to check the operation of the procedures. The degradation was followed by the determination of oxygen uptake and measurements were taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation. To check the possible inhibitory effect of the test item the toxicity test was run in parallel. The solutions containing 100 mg/L, of both the test and a reference item, in the mineral medium, were inoculated. The consumption of oxygen was determined from the change in pressure in the apparatus. The carbon dioxide, evolved during test item degradation, was absorbed in a solution of potassium hydroxide. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of calculated ThOD of the test item. All the validity criteria were fullfilled. The reference item reached 91.7% of biodegradation and the level for ready biodegradability by 4 days. In the toxicity test the biodegradation was equal to 56.0% in 14 days, not being inhibitory. The oxygen uptake of the inoculum blank was equal to 37.9 mg/L in 28 days. The pH values of all flasks were inside the range 7.04 -8.67. At the 28th  day of the test the measured aerobic biodegradation of the  test item equals 72.3%. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 20 days and was not reached in a 10-day window in which biodegradation attained 53%. Lag time was 8 days and degradation time was 15 days. Thus, the test item cannot be classed as readily biodegradable.

Endpoint:
biodegradation in water: screening tests
Type of information:
calculation (if not (Q)SAR)
Remarks:
Migrated phrase: estimated by calculation
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Internationally accepted method, EPI-Suite EPA (USA)
Principles of method if other than guideline:
Calculation by EPI-Suite, EPA (USA) / BIOWIN v4.10
GLP compliance:
no
Key result
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

The substance is predicted to be not readily biodegradable by EPI-Suite, EPA (USA) / BIOWIN v4.10.

Validity criteria fulfilled:
not applicable
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The substance is predicted to be not readily biodegradable by EPI-Suite, EPA (USA) / BIOWIN v4.10.
Executive summary:

The substance is predicted to be not readily biodegradable by EPI-Suite, EPA (USA) / BIOWIN v4.10.

Description of key information

Key study: OECD Guideline 301F and EU Method C.4 -D. GLP study. The test item reached 72.3% biodegradation after 28 days, but only reaching a 53% biodegradation within the 10-day window. Thus, it cannot be classed as readily biodegradable. However, it can be deemed rapidly degradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window
Type of water:
freshwater

Additional information

Key study: OECD Guideline 301F and EU Method C.4 -D. GLP study. At the 28th  day of the test the measured aerobic biodegradation of the  test item equals 72.3%. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 20 days and was not reached in a 10-day window in which biodegradation attained 53%. Lag time was 8 days and degradation time was 15 days. Thus, the test item cannot be classed as readily biodegradable.

Supporting study: The substance predicted to be not readily biodegradable according to EPI-Suite, EPA (USA) v4.1 (BIOWINN v4.10).

However, since the substance can be degraded (biotically and/or abiotically) in the aquatic environment to a level > 70 % within a 28-day period, it can be considered as a rapidly degradable substance, according to 4.1.2.9.5. (c) of Annex I to CLP.