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Environmental fate & pathways

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:
August 8, 2012 - September 18, 2012
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
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Aeration tank of Sewage Treatment Plant ”Czajka” , Warsaw. The coarse particles were removed by filtration. Such prepared sludge was washed in the medium and was placed in laboratory-scale unit where the aerobic conditions were maintained by means of an intense aeration with compressor and aerators.- Preparation of inoculum for exposure: The concentrated sludge was suspended in mineral medium to yield a concentration of 3-5 g suspended solids/l and it was aerated until application. After complete re-suspension was achieved, a sample was withdrawn just before use for the determination of the dry weight of the suspended solids.- Pretreatment: Inocula was pre-conditioned to the experimental conditions. Pre-conditioning consisted of aerating activated sludge in mineral medium for 5 days at the test temperature of 22 ºC- Concentration of sludge: 30 mg/litre SS.
Duration of test (contact time):
28 d
Initial conc.:
138.6 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS- Composition of medium:(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. Dissolved in water and made up to 1 litre. The pH of the solution was ca 7,4.(b) Calcium chloride, anhydrous, CaCl2 27,50 g. Dissolved in water and made up to 1 litre(c) Magnesium sulphate heptahydrate, MgSO4. 7 H2O 22,50 g. Dissolved in water and made up to 1 litre.(d) Iron(III) chloride hexahydrate, FeCl3·6H2O 0,25 g. Dissolved in water and made up to 1 litre.10 ml of solution (a) was mixed with 800 ml dilution water, added 1 ml of solutions (b) to (d) and made up to 11 with dilution water.- Dilution water: Double-distilled water- Test temperature: 22 ± 2 ºC- pH: 7.4 ± 0.2.- pH adjustment: Yes, adjusted to 7.12 with 0.05 N NaOH.- Suspended solids concentration: 30 mg/L- Continuous darkness: yesTEST SYSTEM- Culturing apparatus: WTW OxiTop OC 110 respirometer flasks in WTW TS 606 CZ-G/3-VAR termo cabinets.- Number of culture flasks/concentration: 3 replicates, each containing test item (138 mg/l) and inoculum 30 mg/dm3 SS .- Measuring equipment: BOD was determined by closed WTW OxiTop OC 110 respirometer. The oxygen uptake from the readings taken at regular and frequent intervals was calculated, using the methods given by the manufacturer of the equipment. 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.COD was determined using LANGE Cuvette Test LCI 400 (potassium dichromate method). Nitrates were determined using LANGE Cuvette Test LCK339 and nitrites were determined using LANGE Cuvette Test LCK341. Spectrophotometer Hach DR 3900 and 45600 reactor COD was used for COD, nitrate and nitrite concentration measurements.- Test performed in closed vessels due to significant volatility of test substance: Closed respirometer flask.CONTROL AND BLANK SYSTEM- Inoculum blank: 3 replicates, each containing only inoculum 30 mg/dm3 SS.- Procedure control: 3 replicates, each containing reference item (sodium acetate 100 mg/L) and inoculum 30 mg/dm3 SS.- Toxicity control: 3 replicates, each containing test item (138.6 mg/L), reference item (sodium acetate 100 mg/L) and inoculum 30 mg/dm3 SS.
Reference substance:
acetic acid, sodium salt
Remarks:
(100 mg /l)
Key result
Parameter:
% degradation (O2 consumption)
Value:
3.8
Sampling time:
28 d
Details on results:
The biodegradation of the test item after 28 days was 3.5%.The biodegradation of the toxicity test after 28 days was 35.5%The oxygen uptake of the inoculum blank was equal to 46.0 mg/l in 28 days.The recorded temperature was 22 ± 0.9 ºC. - by 0.9ºC - the expected 22ºC.The pH values of all flasks were inside the range 6.3-8.4.The observed oxygen uptake by the reaction mixture was corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate and a nitrification process is not expected to impede a biodegradation process.
Results with reference substance:
At the 28th day of the test the aerobic biodegradation of the testing, the reference item reached 85.7% of biodegradability.

Correction for oxygen uptake for interference by nitrification:

Test ítem:

Days

0

28

Difference

Flasks

#1

#2

#3

#1

#2

#3

1) Concentration of nitrate (mg N/l)

0.4

0.6

0.3

7.8

8.3

8.3

7.7

0.4 ± 0.2

8.1 ± 0.3

2) Oxygen equivalent (4.57× N) (mg/l)

 

35.2

3) concentration of nitrite (mg N/l)

0.030

0.037

-

16.6

-

-

16.57

0.034 ± 0.005

16.6

4) Oxygen equivalent (3.43×N) (mg/l)

 

26.8

5) total oxygen equivalent 2) + 4)

 

92.0

 

Toxicity test:

Days

0

28

Difference

Flasks

#10

#11

#12

#10

#11

#12

1) Concentration of nitrate (mg N/l)

0.4

0.5

0.4

-

8.2

8.3

7.9

0.4 ± 0.1

8.3 ± 0.1

2) Oxygen equivalent (4.57× N) (mg/l)

 

36.1

3) concentration of nitrite (mg N/l)

-

0.034

0.045

-

15.7

-

15.66

0.04 ± 0.01

15.7

4) Oxygen equivalent (3.43×N) (mg/l)

 

53.7

5) total oxygen equivalent 2) + 4)

 

89.8

 

Sample oxygen uptake (mean value 3 flasks): biodegradability

 

Days

 

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2 uptake, mg/l

9.4

18.8

52.7

62.1

70.6

75.3

78.0

82.1

91.3

115.5

120.1

141.5

Blank test O2 uptake. mg/l

18.5

25.6

29.2

31.0

34.3

38.5

39.4

40.1

42.8

43.7

44.7

46.0

Reference ítem O2 uptake. mg/l

70.0

86.0

94.9

98.6

103.7

106.2

108.0

109.9

112.5

113.9

115.4

116.3

Toxicity control O2 uptake. mg/l

74.9

92.9

118.1

137.0

148.3

152.3

155.0

157.6

166.3

177.2

185.4

211.7

Reference ítem %:

BOD/ThODx100

ThOD = 82.0 mgO2/l

63.6

73.7

80.2

82.5

84.7

82.5

83.7

85.1

84.9

85.5

86.2

85.7

After correction for oxygen uptake for interference by nitrification the final biodegradation of test item onn 28th test day was: 3.8%.

After correction for oxygen uptake for interference by nitrification and by the blank inoculum control the final biodegradation in toxicity test on the 28th test day was: 35.5%.

The biodegradability was calculated assuming the measured COD value of a dry substance as a substitute of the ThODNO3 of the substance. It could be assumed that the measured COD value of 1 mg of dry substance is a measure of the amount of oxidisable matter present in the dry substance and is an evaluation of unknown ThODNO3 value.

Validity criteria fulfilled:
yes
Remarks:
(difference of replicates < 20%, reference item reached pass level (60%) on day 2, biodegradation of toxicity test on day 28 was 35.5%, oxygen update of blank was 46.0 mgO2/l on day 28, pH was inside 6-8.5 range, oxygen consumption by test item was <60%).
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance cannot be classed (according to OECD principles) as readily biodegradable under aerobic aqueous conditions.
Executive summary:

The study of ready biodegradability of test substance in an aerobic aqueous medium with manometric respirometry method was carried out according to OECD 301F and EU C.4-D Method (GLP study). 30 mg/L SS of activated sludge were exposed to 138.6 mg/L of test item during 28 days under aerobic conditions at 22 ºC. The observed oxygen uptake by the reaction mixture was corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate. All the acceptability criteria were met and the study was considered valid. The test item (46.3% dry substance) attained 3.8% of biodegradation after 28 days of incubation. Taking into account the ThOD value of the test item was unknown, the biodegradation % could be lower than the real value. Based on these results, the test item cannot be classed (according to OECD principles) as readily biodegradable under aerobic aqueous conditions. Moreover, the final biodegradation in the toxicity test on the 28th test day was determined to be 35.5 % (after correction), and therefore it was determined that the test item did not inhibit the respiration of inoculum.

Description of key information

Key study: OECD Guideline 301F. GLP Study. Test item was considered not to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Key study: A ready biodegradability test with a manometric respirometry method was performed according to OECD 301F and EU C.4-D Method (GLP study). 30 mg/L SS of activated sludge were exposed to 138.6 mg/L of test item during 28 days under aerobic conditions at 22 ºC. The test item attained 3.8% of biodegradation after 28 days of incubation, and therefore it was considered not to be readily biodegradable.