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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from Givaudan study report.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Principles of method if other than guideline:
The method used is basically the one described under No. 301 F in the OECD Guidelines for testing of chemicals.
A measured volume of inoculated mineral medium, containing a known concentration of test substance (100 mg/l) as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (±1 degC) for upto 28 days. The consumption of oxygen is determined by measuring the quantity of oxygen required to maintain constant the gas volume in the respirometer flask. Evolved carbon dioxide is absorbed in soda lime pellets. The BOD, amount of oxygen taken up by the microbial population during biodegradation of the test chemical is expressed as a percentage of ThOD.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
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, Aire, Switzerland) was used.
- Laboratory culture: No data available
- Method of cultivation: No data available
- Storage conditions: No data available
- Storage length: No data available
- Preparation of inoculum for exposure: No data available
- Pretreatment: The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 mins, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.
- Concentration of sludge: 30 mg/l
- Initial cell/biomass concentration: No data available
- Water filtered: No data available
- Type and size of filter used, if any: No data available
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
other: nominal concentration
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Stock solutions of mineral components
The following stock solutions were prepared:
Solution A
KH2PO4 8.5 g
K2HPO4 21.75 g
Na2HPO4 . 2H2O 33.4 g
NH4Cl 0.5g
Dissolved in water and made up to 1 litre.

Solution B
CaCl2 27.5 g
Dissolved in water and made up to 1 litre.

Solution C
MgSO4 . 7H2O 22.5 g
Dissolved in water and made up to 1 litre.

Solution D
Dissolved in water and made up to 1 litre.
FeCl3 . 6H2O 0.25 g
HCl Conc. One drop
Dissolved in water and made up to 1 litre.

Mineral medium: Prepared by mixing 50 ml of solution A and 2000 ml deionised water, adding 5 ml of each of the solutions B, C and D and making upto 5 litres with deionised water. The pH is measured and if necessary adjusted to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide.
- Test temperature: 25 degC
- pH: 7.4 ± 0.2
- pH adjusted: yes
- Aeration of dilution water: No data available
- Determination of the dry weight of suspended solids: The dry weight of suspended solids is determined by taking two 50 ml samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105 - 110 degC for two hours and weighing the residue.
- Continuous darkness: no data available
- Other: Water: The water used during this study is deionised water containing less than 10 mg/l dissolved organic carbon.

TEST SYSTEM
- Culturing apparatus: No data available
- Number of culture flasks/concentration: No data available
- Method used to create aerobic conditions: No data available
- Method used to create anaerobic conditions: No data available
- Measuring equipment: The respirometer used during this study was a SAPROMAT D 12.
- Test performed in closed vessels due to significant volatility of test substance: No data available
- Test performed in open system: No data available
- Details of trap for CO2 and volatile organics if used: No data available
- Other: Test substance and/or reference samples (25 mg, corresponding to 100 mg/l in a 250 ml flask) are weighed in small aluminium boats and added directly to the test flasks of the SAPROMAT.
All flasks are filled with 250 ml of mineral medium. Samples of test or reference substance, or both, are added. Then, a volume of suspended sludge corresponding to 7.5 mg dry weight (as determined from 3.6, generally 1 to 3 ml) is added. The pH of each flask is measured. About 2g of soda lime is placed in an attachment of the stopper, the flasks are closed and placed in the water bath of the SAPROMAT. After tempe rature and pressure equilibration, the oxygen meters of the instrument are set to zero (time zero of the experiment).

SAMPLING
- Sampling frequency: No data available
- Sampling method: No data available
- Sterility check if applicable: No data available
- Sample storage before analysis: No data available
- Other: No data available

CONTROL AND BLANK SYSTEM
- Inoculum blank: No data available
- Abiotic sterile control: No data available
- Toxicity control: No data available
- Other: No data available

STATISTICAL METHODS: No data available
Reference substance:
aniline
Remarks:
The reference substance used to verify the activity of the inoculum was Aniline, analytical grade, purity: min. 99.0%. Reference substance concentration: 100 mg/l
Preliminary study:
The curves obtained with the reference substance alone and with Irisone pure + reference substance show no toxic effect of Irisone pure to the micro-organisms at the test concentration (100 mg/l).
Key result
Parameter:
% degradation (O2 consumption)
Value:
71
Sampling time:
28 d
Remarks on result:
other: readily biodegradable
Details on results:
The % biodegradation results are reported in Tables 1 and 2 below.
Parameter:
ThOD
Value:
2.91 other: mg O2/mg
Remarks on result:
other: For test substance
Parameter:
ThOD
Value:
2.41 other: mg O2/mg
Remarks on result:
other: For reference substance
Results with reference substance:
Aniline undergoes 75% biodegradation after 28 days in the test conditions.

BIODEGRADABILITY - RESPIROMETRIC METHOD

Table 1: O2 uptake (mg O2/l, adjusted to nominal concentrations):

 

Days

3

4

14

21

28

O2 uptake of sludge (inoculum blank)

6

B1

18.0

21.0

37.0

43.0

47.0

12

B2

15.0

18.0

29.0

29.0

29.0

mean

B

16.5

19.5

33.0

36.0

38.0

O2 uptake of Test Subs. + sludge

1

C1

19.1

108.2

229.1

240.4

248.6

2

C2

13.1

22.9

213.1

230.9

241.7

O2 uptake of test substance

 

C1-B

2.6

88.7

196.1

204.4

210.6

 

C2-B

-3.4

3.4

180.1

194.9

203.7

% biodegradation of test substance

 

D1

1

30

67

70

72

 

D2

-1

1

62

67

70

mean

D

0

16

65

69

71

% degrade. Of test substance in presence of reference subst. (co-metabolism)

 

Dco1

9

5

70

74

75

 

Dco2

32

1

64

70

72

 

Dco

21

3

67

72

74

 

Calculations:

B1, B2, C1, C2, A1, A2, E1, E2: experimental O2 uptake values

B = (B1 + B2) / 2

D1 = 100* (C1-B) / ThOD* [S]       Dco1 = 100* (E1 – A1)/ ThOD* [S]

D2 = 100* (C2-B) / ThOD* [S]       Dco2 = 100* (E2 – A2)/ ThOD* [S]

D = (D1 + D2) / 2      Dco = (Dco1 + Dco2)/2

[S] : Initial test substance concentration (mg/l)

 

BIODEGRADABILITY (AND TEST SUBSTANCE TOXICITY CONTROL)

Table 2: O2 uptake (mg O2/l, adjusted to nominal concentrations):

 

Days

5

7

14

21

28

O2 uptake of sludge (inoculum blank)

6

B1

24.0

29.0

37.0

43.0

47.0

12

B2

20.0

24.0

29.0

29.0

29.0

mean

B

22.0

26.5

33.0

36.0

38.0

O2 uptake of Refer. Subs. + sludge

3

A1

178.1

186.2

196.3

196.2

196.2

9

A2

206.1

222.9

236.7

236.8

240.8

O2 uptake of Refer. Substance

 

A1-B

156.1

159.7

163.3

160.2

158.2

 

A2-B

184.1

196.4

203.7

200.8

202.8

O2 uptake of Refer. Substance + Test subs. + sludge

4

E1

203.3

290.1

399.4

410.7

414.7

5

E2

207.1

326.5

422.7

440.2

450.0

mean

E

205.2

308.3

411.1

425.4

432.3

%biodegradation of reference substance

 

D1

65

66

68

66

66

 

D2

76

81

85

83

84

mean

D

71

74

76

75

75

 

Calculations:

B1, B2, C1, C2, A1, A2, E1, E2: experimental O2 uptake values

B = (B1 + B2) / 2

D1 = 100* (A1-B) / ThOD* [S]       

D2 = 100* (A2-B) / ThOD* [S]       

D = (D1 + D2) / 2                    E = (E1 + E2)/2

[S] : Initial reference substance concentration (mg/l)

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test chemical undergoes 71% biodegradation after 28 days in the test conditions. The 10 -day window criterion is also fulfilled (16% biodegradation on day 4 and 65% on day 14). The curves obtained with the reference substance alone and with test chemical + reference substance show no toxic effect of to the micro-organisms at the test concentration (100 mg/l). Thus, the test chemical should be regarded as readily biodegradable according to this test.
Executive summary:

The Ready Biodegradability of test chemical was determined by the Manometric Respirometry Test according to the OECD Guidelines for Testing of Chemicals, Method No. 301 F. The test chemical undergoes 71% biodegradation after 28 days in the test conditions. The 10 -day window criterion is also fulfilled (16% biodegradation on day 4 and 65% on day 14). The curves obtained with the reference substance alone and with test chemical + reference substance show no toxic effect to the micro-organisms at the test concentration (100 mg/l). Thus, the test chemical should be regarded as readily biodegradable according to this test.

Description of key information

The Ready Biodegradability of test chemical was determined by the Manometric Respirometry Test according to the OECD Guidelines for Testing of Chemicals, Method No. 301 F. The test chemical undergoes 71% biodegradation after 28 days in the test conditions. The 10 -day window criterion is also fulfilled (16% biodegradation on day 4 and 65% on day 14). The curves obtained with the reference substance alone and with test chemical + reference substance show no toxic effect to the micro-organisms at the test concentration (100 mg/l). Thus, the test chemical should be regarded as readily biodegradable according to this test.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Various experimental studies has been investigated for the test chemical for reviewing the biodegradation endpoint which have been summarized as below;

 

The Ready Biodegradability of test chemical was determined by the Manometric Respirometry Test according to the OECD Guidelines for Testing of Chemicals, Method No. 301 F. The test chemical undergoes 71% biodegradation after 28 days in the test conditions. The 10 -day window criterion is also fulfilled (16% biodegradation on day 4 and 65% on day 14). The curves obtained with the reference substance alone and with test chemical + reference substance show no toxic effect to the micro-organisms at the test concentration (100 mg/l). Thus, the test chemical should be regarded as readily biodegradable according to this test.

 

The another Manometric Respirometry Test was conducted for test chemical to assess its percent degradation as per the OECD Guidelines for Testing of Chemicals, Method No. 301 F. The Test chemical undergoes 60% biodegradation after 28 days (65% after 36 days) in the test conditions. Biodegradation exceeds 10% on day 8 and reaches only 52% at the end of the 10-day window (days 8 to 18).The curves obtained with the reference substance alone and with Test chemical + reference substance show no toxic effect of Test chemical to the micro-organisms at the test concentration (100 mg/I).Thus, Test chemical should be regarded as readily biodegradable according to this test.

 

The above results were supported by Biodegradation study conducted according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) for 28 days for evaluating the percentage biodegradability of test substance. The percentage degradation of the test substance was determined to be 75.4% by using CO2 evolution parameter. Thus, based on percentage degradation of test chemical, the chemical was considered to be readily biodegradable in nature.

By considering results of all the studies mentioned above, it can be concluded that the test chemical can be expected to be readily biodegradable in nature.