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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:
2011-10-20 to 2011-11-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
adopted 2008-05-31
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
adopted 1992-07-17
Deviations:
yes
Remarks:
The pH values of the procedural control, the abiotic control, and the toxicity control were outside the range of 6.0 - 8.5 (8.3 till 9.0). However, degradation in the procedural control and the toxicity control was above 60 %.
GLP compliance:
yes (incl. QA statement)
Remarks:
signed on 2011-02-07
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum: fresh samples of activated sludge are withdrawn on 2011-10-20 from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany, which is mainly fed with municipal wastewater.
- Since it was not necessary, the samples were not washed with mineral medium after the arrival in the laboratory but kept aerobic until use.
- The concentration used in the test was 29.5 mg dry mass/litre (7.38 mg dry mass/250 mL).
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Initial conc.:
25 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST PRINCIPLE/CONDITIONS
- Inoculated mineral medium (measured volume) with a known concentration of test item (100 mg test item/L giving at least 50-100 mg ThOD/L) as the nominal sole source of organic carbon
- Stirred in a closed flask
- Test temperature: 22 °C ± 1 °C (constant)
- Test duration: 28 days
- Consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas volume in the respirometer flask
- Evolved carbon dioxide is absorbed in a suitable absorbent.
- The amount of oxygen taken up by the microbial population during biodegradation of the test item (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD.
- Water: deionized water, free from inhibitory concentrations of toxic substances
- following reagents (analytical grade) were used for the preparation of stock solutions for mineral medium.

The mineral medium applied containes 10 mL/L and 1 mL/L of the mineral stock solution a and b-d, respectively:
(a) KH2PO4: 8.50 g/L
K2HPO4: 21.75 g/L
Na2HPO4 x 2 H2O: 33.40 g/L
NH4Cl: 0.50 g/L
(b) CaCl2 x 2 H2O: 36.40 g/L
(c) MgSO4 x 2 H2O: 22.50 g/L
(d) FeCl3 x 6 H2O: 0.25 g/L

- pH: determined and adjusted to 7.4 ± 0.2 before test start, determined at the end of the test.
- Measurement/recording of oxygen demand: continuously (SAPROMAT respirometer (VOITH Inc.)
- Reference item: Sodium benzoate. Concentration in test vessels with reference item(procedual control, toxicity control): 100 mg per litre mineral test medium (25 mg/250 mL).
- Concentration of test item (SymSitive 1609 pur):
25 mg per litre mineral test medium (6.25 mg/250 mL, test suspension A) and
100 mg per litre mineral test medium (25 mg/250 mL, test suspension B, abiotic control, toxicity control).
- additional assay: 25 mg test item (74 mg ThOD, meeting the required concenrtation of 50-100 mg ThOD/L) was conducted

TEST SYSTEM/VESSELS
- Test suspension A:
2 vessels containing test item (25 mg/L / ThOD 74 g) and inoculum
- Test suspension B:
2 vessels containing test item (100 mg/L / ThOD 296 g) and inoculum

CONTROL AND BLANK SYSTEM
- Inoculum blank:
2 vessels containing only inoculum
- Abiotic control:
2 vessels containing test item (100 mg/L) and a sterilising agent
- Procedual control:
2 vessels containing reference item (100 mg/L) and inoculum
- Toxicity control:
2 vessels sontaining test item (100 mg/L), reference item (100 mg/L) and inoculum

TEST PROCEDURE
- SymSitive 1609 pur (in concentrations of about 25 and 100 mg per litre mineral medium, respectively) and
sodium benzoate (in concentrations of about 100 mg per litre mineral medium) were incubated with 29.5 mg dry mass inoculum per litre mineral medium in 500 mL glass vessels at a medium volume of 250 mL.
- Test was run for 28 days, in darkness at 22 °C ± 1 °C.
- The suspension was aerated during the whole test.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
not applicable
Test performance:
The guideline OECD 301 (1992) stated that "if the pH value is outside the range 6 -8.5 AND the oxygen consumption by the test substance is less than 60 %, the test should be repeated with a lower concentration of test substance".
The pH values of the procedural control, the abiotic control, and the toxicity control were outside the range of 6.0 - 8.5 (8.3 till 9.0). However, degradation in the procedural control and the toxicity control was above 60 %. Therefore, the results are plausible and according to the guideline the test does not have to be repeated.
Key result
Parameter:
% degradation (O2 consumption)
Value:
66
St. dev.:
1
Sampling time:
28 d
Remarks on result:
other: 25 mg test substance
Parameter:
% degradation (O2 consumption)
Value:
57
St. dev.:
9.6
Sampling time:
28 d
Remarks on result:
other: 100 mg test substance
Details on results:
The biodegradation of the test item after 28 days of incubation in the static test was found to be 66 % (SD = 1.0 %) and 57 % (SD = 9.6 %) in the assay with 25 mg/L and 100 mg/L, respectively. The biodegradation within the 10-day-window was 66 % and 57 % in the assay with 25 mg/L and 100 mg/L, respectively.
For further informations on the results, please refer to "Any other information on results incl. tables" below.
Results with reference substance:
The reference item sodium benzoate was degraded to 85 % within the first 14 days.

Results

The results are presented as single values (table 3, table 4, table 5, table 6, and table 7) and mean values (table 4, table 5, table 6, and table 7).

Table1: pH values at test end. Single values of the parallel test vessels

vessel

Test suspension A

Test suspension B

Inoculum blank

Abiotic control

Procedural control

Toxicity control

1

8.2

8.2

8.3

8.8

8.5

9.0

2

8.2

8.3

8.7

8.3

8.6

8.9

 

Table2: Oxygen consumption. Cumulated consumption (mg O2/L) after 14 days of incubation. Single and mean values of the parallel test vessels and standard deviation. Test suspension A: 25 mg/L; Test suspension and procedural control B: 100 mg/L; Toxicity control: 200 mg/L.

vessel

Test suspension A

Test suspension B

Inoculum blank

Abiotic control

Procedural control

Toxicity control

1

31

21

26

3

169

164

2

30

27

29

2

168

177

Mean

31

24

28

3

169

171

SD

1

4

2

1

1

9

 

Table3: Percent degradation. Degradation (%) after 14 days of incubation. Single and mean values of the parallel test vessels and standard deviation.

vessel

Test suspension A

Test suspension B

 

Abiotic control

Procedural control

Toxicity control

1

4.7

-2.2

 

1.0

85.0

29.5

2

3.4

0.2

 

0.7

84.4

32.4

Mean

4.1

-1.2

 

0.8

84.7

30.9

SD

1.0

1.4

 

0.2

0.4

2.0

 

Table4: Oxygen consumption. Cumulated consumption (mg O2/L) after 28 days of incubation. Single and mean values of the parallel test vessels and standard deviation. Test suspension A: 25 mg/L; Test suspension and procedural control B: 100 mg/L; Toxicity control: 200 mg/L.

vessel

Test suspension A

Test suspension B

Inoculum blank

Abiotic control

Procedural control

Toxicity control

1

82

182

33

6

179

310

2

83

222

35

7

177

339

Mean

83

202

34

7

178

325

SD

1

28

1

1

1

21

 

Table5: Percent degradation. Degradation (%) after 28 days of incubation. Single and mean values of the parallel test vessels and standard deviation.

vessel

Test suspension A

Test suspension B

 

Abiotic control

Procedural control

Toxicity control

1

65.0

50.1

 

2.0

87.1

59.7

2

66.3

63.6

 

2.4

85.9

66.0

Mean

65.7

56.9

 

2.2

86.5

62.9

SD

1.0

9.6

 

0.2

0.8

4.4

 

The biogradation within the 10-day-window was 66 % and 57 % in the assays with 25 mg/L and 100 mg/L, respectively.

The 10-day-window starts at day 18-22 (25 mg/L) and day 22-25 (100 mg/L), respectively. A distinct lag phase / adaption phase was noticeable.

No significant abiotic degradation of the test item was noticeable after 28 days of incubation. The biodegradation of the item mixture in the toxicity control was found to be 31 % after 14 days of incubation. Thus, the demanded threshold value of 25 % is exceeded and the test item can be identified as non inhibitory to microorganisms at a tested concentration of 100 mg/L in a ready biodegradability test.

The test item can be identified as ready biodegradable under the thosen test conditions.

Even when the pH value of the procedural control, the abiotic control, and the tocxicity control was outside the range of 6.0 - 8.5 at test end, degradation in the procedural control and toxicity control was above 60 %. Therefore, the results are plausible and according to the guideline the test does not have to be repeated.

Validity and interpretation:

The test considered valid, as

- the difference of extremes of replicate values of the removal of the test item at the end of the test was less than 20 %,

- the percentage degradation of the reference item has exceeded the pass level of 60 % by day 14,

- the oxygen uptake of the inoculum blank is < 60 mg/L in 28 days,

- the pH value for almost all assays was inside the range of 6.0 - 8.5 and the degradation rate in the only assay outside the demanded range was > 60 %.

Validity criteria fulfilled:
yes
Remarks:
please refer for any information on the validity criteria to "Any other information on results incl tables" above
Interpretation of results:
readily biodegradable
Conclusions:
In conclusion, the test item can be considered to be ready biodegradable under the chosen conditions. The test item can be identified as non inhibitory to microrganisms at a tested concentration of 100 mg/L in a ready biodegradability test.

Description of key information

Biodegradation in water: readily biodegradable (OECD 301F; EC method C.4-D; GLP compliant)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information