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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:
14 November 2008 to 15 December 2008
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)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial (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, Peney-Dessous) was used.
- The sludge was collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.
- Concentration of sludge: 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 – 110 °C for two hours and weighing the residue. 2.00 mL of suspended sludge, diluted to a concentration of 1.53 g/L dry matter, corresponding to 3.06 mg dry weight was added to the flasks.
Duration of test (contact time):
31 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: The following stock solutions were prepared:
Solution A: KH2PO4 8.5 g, K2HPO4 21.75 g, Na2HPO4·2 H2O 33.4 g and NH4CI 0.5 g 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: FeCl3.6H2O 0.25 g and HCI conc. one drop, dissolved in water and made up to 1 litre.
Mineral medium was 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 up to 5 litres with deionised water. The pH was measured and if necessary adjusted to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide.
- Test material samples (10.2 mg, corresponding to 100 mg/L in 102 mL of mineral medium) were weighed in small aluminium boats and added directly to the test flasks of the Oxitop, whereas reference the substance (sodium benzoate) was added as 1.00 mL of a 10.2 g/L solution in mineral medium, to give a total volume of 103 mL.
- Test temperature: 22 °C
- pH: 7.56 – 8.05
- pH adjusted: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes, placed in the cupboard of the Oxitop system.

TEST SYSTEM
- Culturing apparatus: test flasks of the Oxitop
- Number of culture flasks/concentration: 2
- Measuring equipment: measuring unit of the Oxitop test flasks
- Details of trap for CO2: Two sodium hydroxide pellets are placed in the quivers on top of the bottle, and the flasks are closed tightly with the measuring heads.

SAMPLING
- Everyday the oxygen consumption of each flask was recorded and correct temperature and stirring were checked. At the end of the test period (normally 28 days), the pH of each flask was measured again.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Two flasks
- Toxicity control: As suggested in the OECD 301 F method, the toxicity of the test material for the inoculum was checked. Therefore, a pair of flasks of the volumetric respirometer were filled with mineral medium + test material (100 mg/L) + reference substance (100 mg/L) + inoculum and their respirations were recorded as for the other flasks. If they were lower than those of the flasks containing mineral medium + reference substance (100 mg/L) + inoculum, the test material was assumed to be inhibitory to the inoculum used.
Reference substance:
benzoic acid, sodium salt
Test performance:
The repeatability validity criterion (not more than 20% difference between replicates) was fulfilled. Therefore, the test was considered valid.
Key result
Parameter:
% degradation (O2 consumption)
Value:
80
Sampling time:
28 d
Details on results:
- The test material underwent 80% biodegradation after 28 days (82% after 31 days) under the test conditions. The 10-day window criterion was also fulfilled (15% biodegradation on day 7 and 63% on day 17).
- The curves obtained with the reference substance alone and with the test material + reference substance showed no toxic effect of the test material to the micro-organisms at the test concentration (100 mg/L).
- The test material should be regarded as readily biodegradable according to this test.
Results with reference substance:
Degradation of sodium benzoate exceeded 40% after 7 days and 65% after 14 days: the activity of the inoculum was thus verified (validity criterion).

Table 1: Biodegradability of the Test Material

 

 

Days

5

7

17

21

28

31

O2 uptake of sludge (inoculum blank)

1

B1

10.5

15.8

31.5

36.8

36.8

36.8

2

B2

10.5

21.0

26.3

31.5

42.1

42.1

Mean

B

10.5

18.4

28.9

34.2

39.5

39.5

O2 uptake of test material + sludge

9

C1

36.7

57.7

204.7

225.6

257.6

262.6

10

C2

26.3

57.7

188.7

215.6

246.6

251.6

O2 uptake of test material

 

C1-B

26.2

39.3

175.8

191.5

218.1

223.1

 

C2-B

15.8

39.3

159.8

181.5

207.1

212.1

% biodegradation of test material

 

D1

10

15

66

72

82

84

 

D2

6

15

60

68

78

80

Mean

D

8

15

63

70

80

82

% degradation of test material in presence of reference substance (co-metabolism)

 

Dco1

8

14

39

47

63

67

 

Dco2

12

14

44

53

69

75

 

Dco

10

14

42

50

66

71

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)/Th0D*[S]

D2 = 100 * (C2 - B) / ThOD * [S]

Dco2 = 100 * (E2 - A2) / Th OD * [S]

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

[S] : Initial test material concentration (mg/L)

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of this study, the test material is considered to be readily biodegradable.
Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301F, EU Method C.4 -D and EPA OPPTS 835.3110, under GLP conditions, following the Manometric Respirometry Test method.

Under the conditions of the study the test material underwent 80% biodegradation after 28 days (82% after 31 days). The 10-day window criterion was also fulfilled (15% biodegradation on day 7 and 63% on day 17). The curves obtained with the reference substance alone and with the test material + reference substance showed no toxic effect of the test material to the micro-organisms at the test concentration (100 mg/L).

The test material was therefore considered to be readily biodegradable.

Description of key information

Under the conditions of the study, the test material was considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301F, EU Method C.4 -D and EPA OPPTS 835.3110, under GLP conditions, following the Manometric Respirometry Test method. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Under the conditions of the study the test material underwent 80 % biodegradation after 28 days (82 % after 31 days). The 10-day window criterion was also fulfilled (15% biodegradation on day 7 and 63 % on day 17). The curves obtained with the reference substance alone and with the test material + reference substance showed no toxic effect of the test material to the micro-organisms at the test concentration (100 mg/L).

The test material was therefore considered to be readily biodegradable.