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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 study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Qualifier:
according to guideline
Guideline:
other: ECHA guidance for implementation of REACH,Guidance on information requirements and chemical safety assessment,chapter R7b:Endpoint specific guidance,May 2008(Version 1.1)
Principles of method if other than guideline:
The ready biodegradability of test chemical was determined by the manometric Respirometry Test.
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used.

The sludge is 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.

- Laboratory culture:
- Method of cultivation:
- Storage conditions:
- Storage length:
- Preparation of inoculum for exposure:
- Pretreatment:
- Concentration of sludge:
- Initial cell/biomass concentration:
- Water filtered: yes/no
- Type and size of filter used, if any:
Duration of test (contact time):
62 d
Initial conc.:
30 mg/L
Based on:
test mat.
Initial conc.:
100 mg/L
Based on:
other: Reference substance
Parameter followed for biodegradation estimation:
other: ThoD
Parameter followed for biodegradation estimation:
other: BOD
Details on study design:
TEST CONDITIONS
- Composition of medium:50 ml of solution A (KH2P04 ,K2HP04,Na2HP04 · 2 H20,NH4CI) and 2000 ml deionised water, adding 5 ml of each of the solutions B(CaCl2 dissolved in water), C(MgSO4.7H2O dissolved in water) and D( FeCl3.6H2Oand making up to 5 litres with deionised water.
- Test temperature:21.9-22.5 deg C
- pH:7.4
- pH adjusted: yes- The pH is measured and if necessary adjusted to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide.
- Suspended solids concentration:Dry weight of suspended solids: 4.05 g/l, diluted to 1.53 g/l
- Other:A measured volume of inoculated mineral medium, containing a known concentration of test substance as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (± 1 °C) for up to 28 days.

TEST SYSTEM
- Culturing apparatus:The respirometer used during this study is an Oxitop Control System, made by
Wissenschaftlich-Technische Werkstatten (WTW), Weilheim, Germany.
- Number of culture flasks/concentration:Flasks are filled with 250 ml of mineral medium (flasks containing reference substance: 100 ml). Samples of test or reference substance are added. Then suspended sludge diluted to a concentration of 1.53 g/l dry matter is added
- Measuring equipment:respirometer
- Other:Test substance samples (7.65 mg, corresponding to 30.0 mg/I in 255 ml of test medium) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop, whereas reference the substance (sodium benzoate) is added as 1.00 ml of a 10.2 g/l solution in mineral medium, to give a total volume of 103 ml.
Flasks are filled with 250 ml of mineral medium (flasks containing reference substance: 100 ml). Samples of test or reference substance are added. Then suspended sludge diluted to a concentration of 1.53 g/l dry matter is added. Except when the test substance has an acid or alkaline character, the pH of each flask is not measured but assumed to be the same as the mineral medium (see 3.5.), in order not to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances, even sodium benzoate, were shown not to affect the pH of the medium by more than 0.1 pH unit. Two sodium hydroxide pellets are placed in the quivers on top of the bottle, and the flasks are closed tightly with the measuring heads. The flasks are allowed to equilibrate to the test temperature. The measurement is started by programming the measuring unit of the Oxitop test flasks, and the test flasks are placed in the temperature controlled cupboard of the Oxitop system. After temperature equilibration, the controller of the instrument starts the data acquisition (time zero of the experiment).

CONTROL AND BLANK SYSTEM
- Inoculum blank: No data available
- Abiotic sterile control: No data available
- Toxicity control: An optional toxicity control as suggested by OECD guideline 301F was not performed
- Other: Everyday the oxygen consumption of each flask is recorded and correct temperature and stirring are checked. At the end of the test period (normally 28 days), the pH of each flask is measured again.
Reference substance:
other: Sodium benzoate , purity: min. 99.0 %.
Test performance:
Oxygen uptakes, as read on the Oxitop controller, are corrected to account for the small differences between actual and nominal concentrations of test and reference substances.
Parameter:
% degradation (O2 consumption)
Value:
88
Sampling time:
28 d
Remarks on result:
other: The 10-day window criterion is also fulfilled (20% biodegradation on day 1 and 77% on day 11 ).
Parameter:
% degradation (O2 consumption)
Value:
87
Sampling time:
62 d
Remarks on result:
other: The 10-day window criterion is also fulfilled (20% biodegradation on day 1 and 77% on day 11 ).
Details on results:
Degradation of sodium benzoate exceeded 40 % after 7 days and 65 % after 14 days: the activity of the inoculum was thus verified (validity criterion).
The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test substance. Therefore, the test is considered valid.
Results with reference substance:
Degradation of sodium benzoate exceeded 40 % after 7 days and 65 % after 14 days

Theoretical oxygen demand:                                                                                                                                                              

Formula:      C11H14NO2                                          

Molecular weight:    178 g/mol                                                                                                                                                         

Theoretical oxygen demand (ThOD): 2.42 mg O2/mg                                                                                                                                                                                                                                                        

Biodegradability:                                                                                                                                                                               

 

Days

1

7

11

14

21

28

62

02 uptake of sludge

(inoculum blank)

1a

B1

6.7

22.9

28.3

31.0

35.0

37.7

55.2

2a

B2

8.1

24.2

29.6

32.3

36.3

40.4

57.9

mean

B

7.4

23.6

29.0

31.7

35.7

39.1

56.6

02 uptake of Test

Subst.+ sludae

Sa

C1

21.5

75.4

86.2

91.5

98.3

104.0

121.0

6a

C2

22.9

72.6

83.4

87.4

96.7

101.8

117.8

02 uptake of Test

Substance

 

C1-B

14.1

51.9

57.3

59.9

62.7

65.0

64.5

 

C2-B

15.5

49.0

54.4

55.7

61.1

62.8

61.3

%biodegradation

of test

substance

 

D1

19

71

79

82

86

89

89

 

02

21

68

75

77

84

86

84

mean

D

20

69

77

80

85

88

87

Calculations:                                                                                                                                                                                 

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

B=(B1 +B2)/2                                                                                                                                                                       

D1=100*(C1 -B)/ThOD*(S)                                                                                                                                                          

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

D=(D1 +D2)/2                                                                                                                                                                               

S=Initial test substance concentration(mg/L)                                                                                                                                                                                                                                                                                                                      

Biodegradability of reference substance:                                                                                                                                             

Theoretical oxygen demand:

Formula:   C7H5O2Na                                                                                                                                                       

Molecular weight:    144.1 g/mol                                                                                                                                                 

ThOD: 1.67 mg O2/ mg

                                                                                                                                                                  

 

Days

5

7

14

21

28

02 uptakeofsludge

(inoculumblank)

1a

B1

18.8

22.9

31.0

35

37.7

2a

B2

20.2

24.2

32.3

36.3

40.4

mean

B

19.5

23.6

31.7

35.7

39.1

02 uptakeofTest

Subst.+sludae

17a

A1

146.2

162.3

193.6

204.6

214.7

18a

A2

146.2

162.3

183.5

193.5

204.6

02 uptakeofTest

Substance

 

A1-B

126.7

138.8

161.9

169.0

175.5

 

A2-B

126.7

138.8

151.8

157.9

165.6

99

 

D1

76

83

97

101

105

 

02

76

83

91

95

99

mean

D

76

83

94

98

102

     

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

B=(B1 +B2)/2                                                                                                                                                                   

D1=100*(A1 -B)/ThOD*(S)                                                                                                                                                     

D2=100*(A2 -B)/ThOD*(S)                                                                                                                                                        

D=(D1 +D2)/2                                                                                                                                                                        

S=Initial test substance concentration(mg/L)

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Test chemical undergoes 88 % biodegradation after 28 days (87% after 62 days) in the test conditions. The 10-day window criterion was also fulfilled (20% biodegradation on day 1 and 77% on day 11 ). Thus, based on the % degradation of test chemical, test chemical consider to be readily biodegradable.
Executive summary:

The Ready Biodegradability of test chemical was determined by the Manometric respirometry. Test conducted in accordance with OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test). A measured volume of inoculated mineral medium containing a known concentration 30 mg/l of test substance as the nominal sole source of organic carbon was stirred in a closed flask at a constant  temperature (± 1 °C) for up to 28 days. Evolved carbon dioxide was absorbed in sodium hydroxide pellets. Fresh activated sludge from a biological  waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland)  was used. The consumption  of oxygen was determined  by measuring  the pressure drop in the respirometer  flask. The Biological Oxygen Demand (BOD), amount of oxygen taken up by the microbial population during biodegradation of the test chemical (corrected for uptake by blank inoculum, run in parallel)  was expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidized to carbon dioxide,  hydrogen to water and nitrogen to ammonium, nitrite or nitrate). Test chemical undergoes 88 % biodegradation after 28 days (87% after 62 days) in the test conditions. The 10-day window criterion was also fulfilled (20% biodegradation on day 1 and 77% on day 11 ). Thus, based on the % degradation of test chemical, test chemical consider to be readily biodegradable. Test chemical did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

Description of key information

The Ready Biodegradability of test chemical was determined by the Manometric respirometry. Test conducted in accordance with OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test). A measured volume of inoculated mineral medium containing a known concentration 30 mg/l of test substance as the nominal sole source of organic carbon was stirred in a closed flask at a constant  temperature (± 1 °C) for up to 28 days. Evolved carbon dioxide was absorbed in sodium hydroxide pellets. Fresh activated sludge from a biological wastewater treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used. The consumption of oxygen was determined by measuring the pressure drop in the respirometer flask. The Biological Oxygen Demand (BOD), amount of oxygen taken up by the microbial population during biodegradation of the test chemical (corrected for uptake by blank inoculum, run in parallel)  was expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidized to carbon dioxide,  hydrogen to water and nitrogen to ammonium, nitrite or nitrate). Test chemical undergoes 88 % biodegradation after 28 days (87% after 62 days) in the test conditions. The 10-day window criterion was also fulfilled (20% biodegradation on day 1 and 77% on day 11). Thus, based on the % degradation of test chemical, test chemical considers to be readily biodegradable. Test chemical did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Two different experimental studies from different sources have been reviewed to determine biodegradability of test chemical and their results are summarized below.

The Ready Biodegradability of test chemical was determined by the Manometric respirometry. Test conducted in accordance with OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test). A measured volume of inoculated mineral medium containing a known concentration 30 mg/l of test substance as the nominal sole source of organic carbon was stirred in a closed flask at a constant  temperature (± 1 °C) for up to 28 days. Evolved carbon dioxide was absorbed in sodium hydroxide pellets. Fresh activated sludge from a biological wastewater treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used. The consumption of oxygen was determined by measuring the pressure drop in the respirometer flask. The Biological Oxygen Demand (BOD), amount of oxygen taken up by the microbial population during biodegradation of the test chemical (corrected for uptake by blank inoculum, run in parallel)  was expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidized to carbon dioxide,  hydrogen to water and nitrogen to ammonium, nitrite or nitrate). Test chemical undergoes 88 % biodegradation after 28 days (87% after 62 days) in the test conditions. The 10-day window criterion was also fulfilled (20% biodegradation on day 1 and 77% on day 11). Thus, based on the % degradation of test chemical, test chemical considers to be readily biodegradable. Test chemical did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

 

Above study further supported by the second study from experimental source. 28-days Manometric respirometry test was conducted following the OECD guideline 301F to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 20± 1°C. Mixture of domestic wastewater, surface soil and soil samples was used as a test inoculum for the study. This inoculum was collected and were mixed to get diluted suspension. The inoculum was kept aerobic until being used for experiment by supplying organic and inorganic sources required by micro flora to sustain at controlled laboratory conditions.  This gave the bacterial count as 10e7 to 10e8 CFU/ml. At the regular interval microbial plating was also performed on agar to confirm the vitality and CFU count of microorganism. OECD mineral medium was used for the study. The test system included control, test item and reference item. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 100 mg/L, while that of inoculum was 10 ml/l. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % Degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (Sodium Benzoate) was also calculated using BOD & ThOD and was determined to be 75.80 %. The mean BOD values (mg O2/l) in control on 28th day was 37.3 mg O2/l, thereby fulfilling the control validity criteria (i.e., The oxygen consumption of the inoculum blank is normally 20-30 mg O2/l and should not be greater than 60 mg/l in 28 days). Degradation of Sodium Benzoate exceeds 60 % after 14 days. The activity of the inoculums is thus verified, and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.967 mgO2/mg. ThOD was calculated as 2.424 mgO2/mg. Accordingly, the % degradation of the test item after 28 days of incubation at 20 ± 1°C according to manometric respirometry test was determined to be 81.15%.Based on the results, the test item, under the test conditions, was determined to be readily-Biodegradable at 20 ± 1°C over a period of 28 days.

Thus, based on the above both studies, test chemical consider to be readily biodegradation in water.