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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:
From 06 JUL 2002 to 05 JUL 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
e.g. missing information on the suspended matter content of the inoculum and the inorganic carbon (IC) content of the test of the test item suspension in the mineral medium at the beginning of the test (validity criteria)
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted 1992-07-17
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
TOC: 29.8% (TOC determined at test facility (NON-GLP-State))
ThCO2: 1.10 mg CO2/mg test item
pH value in water: 13-14
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Source of inoculum
Test inoculum consisted of the aqueous phase of non-adapted activated sludge. Activated sludge originated from the sewage plant at Hildesheim (Municipal sewage treatment plant, Hildesheim) consisting of mainly municipal sewage and hardly industrial chemical waste.

Pre-treatment
The activated sludge was maintained in an aerobic condition by aeration for four hours and then homogenized with a mixer. The sludge was filtered and the filtrate (30 mL) was subsequently used to initiate inoculation.

Colony forming units of the inoculum: 107- 108 CFU/L
Colony forming units in the test vessels: 105- 106 CFU/L

The following test solutions were prepared in 5 L brown glass bottles as incubation vessels:
• two incubation vessels for the test item concentration (R1, R2)
• one incubation vessel for the reference item
• two incubation vessels for the inoculum control (C1, C2)
• one incubation vessel for the toxicity control (T1)
Duration of test (contact time):
28 d
Initial conc.:
10.44 mg/L
Based on:
TOC
Initial conc.:
35 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral nutrient solution acc. to OECD 301 B/CO2 Evolution Test
- Additional substrate: no
- Test temperature: 22 ± 2 °C
- pH: 7.62, measured on day 28 before acidification
- Aeration of dilution water: yes, for 24 hours
- Suspended solids concentration: not reported
- Continuous darkness: not reported

TEST SYSTEM
- Culturing apparatus: 5000 mL, brown glass
- Volume of the test medium: 3000 mL
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration for 24 hours
- Measuring equipment:
pH-Meter, CORNING pH 240
Thermohygrograph, LUFFT FELLBACH
Flow meter, KROHNE DUISBURG TYP DK 800 PV
- Type and frequency of measurements:
Temperature - The room temperature was measured continuously by a hygrothermograph.
pH - On day 28, the pH-value of all solutions was measured prior to acidification.

SAMPLING
- Sampling frequency: The total amount of CO2 produced in 28 days was analysed by titration in 12 measurements.
- Sampling method: Back titration of the residual Ba(OH)2 with 0.05 N HCI was carried out three times a week during the first ten days and thereafter twice weekly. On the 28th day the pH of all solutions were measured prior to acidification.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Abiotic sterile control: n/a
- Toxicity control: 1 (35 mg/L test item + 35 mg/L reference substance)
- Positive/Functional control (reference substance: sodium acetate): 1

RESULTS
- Calculation of results were performed according to the formulas indicated in any other information on materials and methods.
Reference substance:
acetic acid, sodium salt
Test performance:
The necessary amounts of aqua bidest., nutrient media and inoculum were placed in each of the incubation vessel. The vessels were then connected to the system for the production of CO2 free air and aerated for 24 h.
After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles. The test and reference item concentrations were placed into the incubation vessels, the vessels made up to 3 L with CO2 free aqua bidest. and connected to the system for the production of CO2 free air.
Incubation took place in a temperature range of 22 ± 2 °C. All vessels were stirred continuously throughout the test. On the 28th day 1 mL concentrated HCI was added to each of the vessels. Aeration was continued for a further 24 h and on the 29th day the quantity of CO2 released in the last two gas wash bottles was determined.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
39
Sampling time:
28 d
Details on results:
Biodegradation of the test item
The 10% level (beginning of biodegradation) was reached after 7 days. The pass level of a biodegradation > 60 % was not reached in the 10-d-window. The mean percentage of biodegradation came up to 39 % after 28 days. Therefore, the test item must be regarded to be not readily biodegradable in the 10-d-window and after 28 days.

Course of degradation of the test item:
0 % degradation after 1 d
13 % degradation after 8 d
26 % degradation after 14 d
34 % degradation after 21 d
38 % degradation after 28 d

In the inoculum blank a maximum of 36.8 mg CO2/L was formed after 28 days (validity criterion: <40 mg CO2/L after 28 days).
In the toxicity control 36% biodegradation occurred within 14 days and came to a maximum of 49 % after 28 days. The test item did not inhibit the biodegradation of the reference item.

Validity criteria
The validity criteria were fulfilled according to the guideline:
• The total CO2 evolution in the blank at the end of the test was lower than 40 mg/L and did not exceed 70 mg/L, i.e. 36.8 mg CO2/L was formed after 28 days.
• The degradation of the functional control reached the pass level of 60% by day 14, i.e. 74% on day 14 (see field 'results with reference substance').
Results with reference substance:
The adaptation phase of the reference substance (sodium acetate) changed after 2 days into the degradation phase (degradation 10 %). The course of the degradation phase was rapid and reached a degradation rate of 74% on day 14. The validity criterion of 60% degradation after 14 days was fulfilled.

Course of degradation:
2 % degradation after 1 d
63 % degradation after 8 d
74 % degradation after 14 d
78 % degradation after 21 d
75 % degradation after 28 d

CO2-Production and Biodegradation for all Determination Points in the Control, Functional Control and Toxicity Control Samples

study day date control 1

functional control

(reference substance)

toxicity control
[mg CO2/3L] [mg CO2/3L] degr. [mg CO2/3L] degr.
mean gross net [%] gross net [%]
1 07.06. 3.8 6.1 2.3 2 4 0.2 0
4 10.06. 13.5 50.1 36.6 33 43.8 30.3 13
6 12.06. 20.2 80.1 59.9 53 68.7 48.5 21
8 14.06. 28.3 99.6 71.3 63 85.5 57.2 25
11 17.06. 37.7 115.2 77.5 69 107.1 69.4 30
14 20.06. 50.2 133 82.8 74 131.9 81.7 36
18 24.06. 64.1 151 86.9 77 155.7 91.6 40
21 27.06. 76 163.7 87.7 78 171.8 95.8 42
25 01.07. 90 177.6 87.6 78 190.2 100.2 44
28 04.07. 102.9 187.4 84.5 75 203.1 100.2 44
29* 05.07. 110.4 194.9 84.5 75 221.4 111 49

* results of the last two gas wash bottles; degr. = degradation, mv = mean value

CO2-Production and Biodegradation for all Determination Points in the Control and Test Item Samples

study day date control 2 R1 - test item R2 - test item
[mg CO2/3L] [mg CO2/3L] degr. [mg CO2/3L] degr.
mean gross net [%] gross net [%]
1 07.06. 3.8 3.5 -0.3 0 2.4 -1.4 0
4 10.06. 13.5 19.3 5.8 5 10.2 -3.3 0
6 12.06. 20.2 31.1 10.9 9 25.8 5.6 5
8 14.06. 28.3 44.9 16.6 14 41.9 13.6 12
11 17.06. 37.7 62.5 24.8 21 57.9 20.2 17
14 20.06. 50.2 83.6 33.4 29 75.1 24.9 22
18 24.06. 64.1 106 41.9 36 92.6 28.5 25
21 27.06. 76 122.3 46.3 40 108.3 32.3 28
25 01.07. 90 142.7 52.7 46 125.4 35.4 31
28 04.07. 102.9 158.2 55.3 48 135.4 32.5 28
29* 05.07. 110.4 168.5 58.1 50 142.3 31.9 28

* results of the last two gas wash bottles; degr. = degradation, mv = mean value

On day 28, the pH-value of all solutions was measured prior to acidification. Results are summarised below.

control functional control
(reference substance)
test item toxicity control
C1 C2   R1 R2  T1
7.55 7.55 7.89 7.62 7.62 7.98
Validity criteria fulfilled:
yes
Remarks:
(not reported: The inorganice carbon content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.)
Interpretation of results:
not readily biodegradable
Conclusions:
The submission substance is not readily biodegradable in the 10d-window and after 28 days. Percentage biodegradation of the test substance was 39% after 28 days.
Executive summary:

The ready biodegradability of the submission substance was investigated acc. to OECD 301B / CO2 Evolution (Modified Sturm Test) (adopted 1992) and in compliance with GLP.

The test item was tested in a concentration of 35 mg/L in duplicates, corresponding to a carbon content (TOC) of 10.44 mg C/L. The percentage biodegradation of the test item was quantified by titrimetric analyses of CO2 that was produced by the respiration activity of inoculum bacteria. The degradation period was completed on day 28 by acidification. The last titration was made on day 29, after soluble CO2 was turned out over a period of 24 h. The CO2 production was calculated as percentage of ThCO2.

In order to check the activity of the test system, the reference substance sodium acetate was used as functional control. The percentage degradation of the reference substance reached the pass level of > 60 % after 7 days. After 21 days a maximum degradation rate of 78% was reached. The toxicity control – containing both test and reference item – showed that the submission substance did not inhibit the respiration activity of the inoculum. The test item reached the 10%-level (beginning of biodegradation) after 7 days. The pass level of a biodegradation > 60 % could not be reached in the 10d-window. The mean biodegradation of the test item came up to 39% after 28 days. Consequently, the test item must be regarded not readily biodegradable in the 10d-window and after 28 days.

The study was considered adequate and reliable for the evaluation of the ready biodegradability of the submission substance. Despite deficiencies in reporting (e.g., missing information on the suspended matter content of the inoculum, pH was only reported after 28 days exposure period and missing information of the inorganic carbon (IC) content of the test of the test item suspension in the mineral medium at the beginning of the test (validity criteria)). All other reported validity criteria of the guideline were fulfilled.

Description of key information

The submission substance is not readily biodegradable in the 10d-window and after 28 days. Percentage biodegradation of the test substance was 39% after 28 days (OECD 301B, 2002).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A reliable study is available on the ready biodegradability of the submission substance (RL2). The study was performed acc. to OECD 301B / CO2 Evolution (Modified Sturm Test) and in compliance with GLP.

The test item was tested in a concentration of 35 mg/L in duplicates, corresponding to a carbon content (TOC) of 10.44 mgC/L. The percentage biodegradation of the test item was quantified by titrimetric analyses of CO2 that was produced by the respiration activity of inoculum bacteria. The pass level of a biodegradation > 60 % could not be reached in the 10d-window. The mean biodegradation of the test item came up to 39% after 28 days. Consequently, the test substance must be regarded not readily biodegradable in the 10d-window and after 28 days. The study was considered adequate and reliable for the evaluation of the ready biodegradability of the submission substance.