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Environmental fate & pathways

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:
02 SEP - 01 OCT 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge from the municipal wastewater treatment plant Breisgauer Bucht was used as inoculum.
- Preparation of inoculum for exposure: the activated sludge was washed twice by settling the sludge, decanting the supernatant and resuspending the sludge in aerated tap water.
- Concentration of sludge: to 1484.8 mL mineral medium 15.2 mL activated sludge was added, corresponding to 30 mg/L dry solids.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Solution A: KH2PO4: 8.50 g, K2HPO4: 21.75 g, Na2HPO4 x 2H2O: 33.40 g, NH4Cl: 0.50 g; Solution B: CaCl2 x 2H2O: 36.4 g; Solution C: MgSO4 x 7H2O: 22.50 g; Solution D: FeCl3 x 6H2O: 0.25 g. For preparation of the mineral medium 10 mL of solution A is mixed with 800 mL demineralised water, 1 mL of solutions B, C and D are added and made up to 1 litre each.
- Test temperature: 21.0 °C - 22.1 °C
- Aeration of dilution water: aeration with CO2-free air at a rate of 50 - 100 mL/min
- Suspended solids concentration: dry solids of the acitvated sludge was determined as 2.96 g/L by weight measurements after 2 h drying at 105 °C (mean of triplicate measurements)


TEST SYSTEM
- Culturing apparatus: gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septums were used as reactors.
- Number of culture flasks/concentration: 3
- Measuring equipment:
- Details of trap for CO2 and volatile organics if used: CO2-absorption medium: 72 g NaOH were dissolved in 9000 mL deionised water in closed recipients (0.2 M NaOH).
- Other: the CO2-free air production system consists of an air compressor, two 1000 mL gas wash bottles filled with dry soda lime, followed by one bottle filled with 0.1 M NaOH. At the end of the system was one gas wash bottle filled with demineralised water, followed by an empty one to catch any drops of condensation water. A colour range of the soda lime from white to blue indicates, that CO2 absorption capacity is depeted. The CO2-free air is passed on to an air distributer with two imput and 22 output channels and through PE-tubes.


SAMPLING
- Sampling frequency: 4, 7, 11, 14, 21 and 28 days
- Sampling method: sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 mL PE syrings.


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes - 3 replicates
- Toxicity control: yes - 1 replicate containing test item and reference compound (concentration: 40 mg/L organic carbon)

Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
93.433
Sampling time:
28 d
Remarks on result:
other: mean of 3 replicates
Results with reference substance:
The reference compound reached the pass levels for ready biodegradability within 7 days. The reference substance was degraded by 90.267% after 14 days.

Table 1: Biodegradation rates

 

0 d

4 d

7 d

11 d

14 d

21 d

28 d

28 d(after acidification)

Test flask

0

29.7

55.7

70.3

74.8

81.5

75.2

78.4

Test flask

0

42.6

60.8

76.1

75.0

86.2

83.3

91.0

Test flask

0

42.3

57.6

74.7

68.9

78.4

83.3

100.9

Reference

0

54.5

72.7

84.4

84.9

91.1

91.2

92.2

Reference

0

32.4

72.2

83.8

86.7

93.2

94.8

101.5

Reference

0

67.2

87.7

98.0

99.2

97.2

98.7

96.3

Toxicity control

0

59.1

74.8

89.8

99.4

98.5

95.6

100.7

blank

0

8.8

18.1

20.1

24.9

29.2

29.1

30.1

 

Table 2: Validity criteria for OECD 301 B.

Criterion from the guideline

Outcome

Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.

<10%

yes

Percentage degradation of the reference compound reached the pass level by day 14 (≥ 60%).

90.26%

 

The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.

99.4%

yes

The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.

<5%

 

The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.

< 40%

 

 

Validity criteria fulfilled:
yes
Remarks:
Please refer to table 2 at "Any other information on results incl. tables"
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
07 SEP - 05 OCT 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No abiotic control performed
Qualifier:
according to guideline
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge from the Marl-Ost municipal sewage treatment plant
- Preparation of inoculum for exposure: the sewage sludge was centrifugated for 15 min at 3000 rpm. The supernatant was rejected and the sludge resuspended with mineral medium (washing). After another centrifugation step 15 min at 3000 rpm, the sludge was resuspended.
- Concentration of sludge: content of dry matter in the inoculum used was 3.45 g/L.
- Water filtered: yes
Duration of test (contact time):
28 d
Initial conc.:
11.4 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: solution A: 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g (NH4)Cl; solution B: 22.5 g MgSO4 x 7 H2O; solution C: 27.5 g CaCl2; solution D: 0.25 g FeCL3 x 6 H2O. Composition of mineral medium: solution A: 10 mL/L deionized water, solution B-D: 1 mL/L deionised water
- Test temperature: 21.9 - 22.2 °C
- Suspended solids concentration: 27.6 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2000 mL concial flasks (Erlenmeyer) filled with 1000 mL with loose aluminium foil closure
- Number of culture flasks/concentration: 2

SAMPLING
- Sampling frequency: 0 (3 hours), 7, 14, 21, 27 and 28 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
- Reference control: 2 replicates
- Abiotic sterile control: no

Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (DOC removal)
Value:
97
Sampling time:
28 d
Results with reference substance:
Sodium benzoate was degraded by 98.5% in the course of 10 days after first reaching the 10%-degradation level. It can be concluded therefrom that the inoculum used had a sufficient biological activity.

Table 1 Degradation rates

 

% DOC degradation after n days

Day

0

7

14

21

27

28

Test substance

0

97

99

98

100

97

 Control substance  0  98  99  100  99  99
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 FEB - 13 MAR 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No abiotic control
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-B (Determination of the "Ready" Biodegradability - Modified OECD Screening Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): for the inoculation of the inorganic medium, the secondary effluent from the Marl-Ost municipal sewage treatment plant was used
- Preparation of inoculum for exposure: the inoculum sample was treated in accordance with specifications of the laboratory. It was filtered through a paper filter, the first 200 mL were rejected. The filtrate of the inoculation suspension was kept aerobic until it was used for the test at the same day.
- Initial cell/biomass concentration: 0.5 mL bacterial suspension / L medium was used as inoculum
Duration of test (contact time):
19 d
Initial conc.:
ca. 5 mg/L
Based on:
other: a-WAS
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Test temperature: 25±1°C
- Suspended solids concentration:
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
2 vessels containing test substance ,
2 vessels containaing contol substance 1 (readily biodegradable),
2 vessels containaing contol substance 2 (poorly biodegradable)
- Measuring equipment: a-WAS analysis

SAMPLING
- Sampling frequency: at day 0, 5, 8, 10, 12, 14, 16, 18 and 19
Parameter:
% degradation (DOC removal)
Value:
99
Sampling time:
19 d
Details on results:
Control substance 1 reached a degradation of 97% in 19 days
Control substance 2 reached a degradation of 25% in 19 days
Results with reference substance:
97% degradation after 19 days

 

% a-WAS degradation after n days

Day

0

5

8

10

12

14

16

18

19

Test substance

0

96

97

96

97

98

99

99

99

 Control substance 1  0 7  70  100  97  96  96  97  97
 Control substance 2  0  4  21  24  25  23  22  21  25
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable

Description of key information

Readily biodegradable: 78-100% (ThCO2) in 28 days (OECD 301B)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Three studies investigating the ready biodegradability of Alcohols, C12-14 (even numbered), ethoxylated (<=2.5 moles EO), sulfated, monoisopropanolamine salt are available.

The key study was conducted according to OECD guideline 301B. A total of 20 mg/L tests substance (based on DOC) was incubated with activated sludge from the municipal wastewater treatment plant. After a test period of 28 days a biodegradation rate range of 78- 100% was determined based on CO2 evolution.

This result is supported by two GLP-studies performed according to OECD guidelines 301E and OECD 301 A, respectively. In the screening test according to 301E a biodegradation rate of 99% (DOC-removal) after 19 days was determined. In the study conducted according to OECD 301 A, a biodegradation rate of 97% (DOC removal) after 28 days was reported. 

Based on the available results, the test substance is considered to be readily biodegradable.  

 

The ready biodegradability of the counter ion was demonstrated in a study performed according to OECD guideline 301F. The substance reached a degradation rate of >= 78 % after 28 days based on O2 consumption.