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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:
From November 30, 2004 to December 28, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: An aqueous phase inoculum of non adapted activated sludge was obtained from the municipal sewage treatment plant, D-31137 Hildesheim, which treats predominantly municipal sewage.
- Pretreatment: The activated sludge was filtered with folded filter. The first 200 mL of the filtrate was not used. The second filtrate effluent from the domestic waste water was used to initiate inoculation.
- Colony forming units in the test vessels: 104-106 CFU/L
Duration of test (contact time):
ca. 28 d
Initial conc.:
3 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS:
- Composition of medium: The culture medium used in this study (mineral nutrient solution) was same as that recommended in the OECD Guidelines 301D
- Test temperature: 20-24 ± 1 °C
- pH: 7.4
- Continuous darkness: Yes, in an incubator

TEST SYSTEM:

- Culturing apparatus: 300 mL BOD bottles with glass stoppers
- Number of culture flasks: 5 x 2 each for test substance, reference substance and inoculum control and for toxicity control
- Test performed in closed vessels: Yes
- Method used to create aerobic conditions: 1 day before the test demineralised water for the test medium was aerated until oxygen saturation and thereafter left at room temperature for 20 hours.
- Other: All test solutions were made as stocks solutions.The referance substance was weighed directly. Test substance was prepared as a stock solution. 0.2 mL inoculum was added to each BOD bottle.

SAMPLING:
- Sampling frequency: Samples were taken from the BOD bottles on Days 0, 7, 14, 21 and 28 days for O2 determination, BOD determination and percentage degradation.

-Other: On Days 0 and 28, the O2 concentration and the pH-values of all the inoculum control, functional control, tetst substance and toxicity control was carried out.
- The temperature inside the incubator was checked every working day.

ANALYSIS:
- Measuring equipments: pH-meter, corning pH 240, Oximeter, WTW Oxi 530, Incubator, Rubarth.

CONTROL AND BLANK SYSTEM:
- Inoculum blank: Yes ( test flasks containing inoculum and nutrient solution)
- Toxicity control: Yes; (test flask containing 45 mL test substance solution/ 3L + 5 mg/L reference item + inoculum + nutrient solution)
- Reference control: Yes; (test flasks containing 10 mg/L of reference substance, i.e., sodium acetate)
Reference substance:
acetic acid, sodium salt
Remarks:
at 10 mg/L (ThOD = 0.78 mg02/mg)
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 70
Sampling time:
28 d
Details on results:
- The oxygen demand of the inoculum control was 0.97 mgO2/L after 28 days
- For the functional control adaptation phase transformed to a degradation phase with >10% degradation after 1 day, >60% after 5 days and 96% after 28 days. Hence clearing the validity criteria.
- The biodegradation of the toxicity control was 51% after 14 days and 81% after after 28 days. Hence, the biodegradation of the reference substance was not inhibited by the test substance.
- The test substance reached 10% degradation after 2 days, pass level of >60% after 26 days and a maximum degradation of 70% after 28 days
Results with reference substance:
Approximately 96 % degradation was observed after 28 d.




Refer attached background material for details.

Validity criteria fulfilment:

- The percentage degradation of the functional control reached the pass level of >60% after 14 d.

- The oxygen depletion in the inoculum control came to 0.97 mg dissolved oxygen/L after 28 d.

- The residual concentration of oxygen in the test bottles did not fall below 0.5 mg/L at any time.

- The difference of extremes of replicate values of removal of the test substance at the end of the test was less than 20 %.

- The percentage degradation of the toxicity control reached the pass level of 25 % after 14 d.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions, the test substance was considered to be readily biodegradable.

Executive summary:

A study was performed to assess the ready biodegradability of the test substance, C18-unsatd. DEA, according to OECD Guideline 301D (Closed bottle test), in compliance with GLP. Activated sewage sludge was exposed to the substance at a concentration of 3.0 mg/L in BOD bottles in the dark at 20–24°C for 28 d. Degradation was assessed by determination of oxygen consumption, expressed as percentage BOD. Biodegradation attained 70% after 28 d. The residual concentration of oxygen in the test vessels did not fall below 0.5 mg/L at any time. All other validity criteria were fulfilled. Under the study conditions, the test substance was considered to be readily biodegradable (Noack, 2005).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 8 April 2003 to 7 May 2003
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge from municipal sewage treatment plant, D-31137 Hildesheim
- Preparation of inoculum for exposure: The activated sludge was maintained in an aerobic condition by aeration for 4 h and then homogenized with a mixer. The sludge was filtered and the filtrate (30 mL) was subsequently used to initiate inoculation.
- Initial cell/biomass concentration: 10E7 - 10E8 CFU/mL
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
act. ingr.
Initial conc.:
67.9 other: %
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Test temperature: 20-24°C
- pH: The pH of media at the beginning of the test is not reported. However, the pH-value of all solutions was measured on the last day of the test (Day 28). The pH values for inoculum control, functional control and test flask at 15 mg/L were 7.14 (mean of 2 replicates), 7.44 and 7.13 (mean of 2 replicates) respectively.
- Aeration of dilution water: Before the start of the test, the mineral nutrient solution with inoculum was aerated for 24 h with CO2-free air to purge the system of CO2.
- Composition of medium: Mineral nutrient solution according to OECD 301 B/CO2 Evolution test

TEST SYSTEM
- Culturing apparatus: Brown glass bottles (5000 mL) as incubation vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The vessels were connected to the system for the production of CO2-free air and aerated for 24 h.
- Measuring equipment: The room temperature was measured continuously by a hygrothermograph. CO2 determination was carried out by titration subsequent to complete adsorption of the released CO2 in a basic solution. Back titration of the residual Ba(OH)2 with 0.05 N HCl 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.
- Details of trap for CO2 and volatile organics if used: The CO2-adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles.

SAMPLING
- Sampling frequency: On Day 1, 3, 6, 7, 9, 14, 17, 21, 24, 28 and 29
- Other: On the 28th day 1 mL concentrated HCl 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.

CONTROL AND BLANK SYSTEM
- Inoculum control: Yes
- Toxicity control: Yes
Reference substance:
acetic acid, sodium salt
Remarks:
Batch # 420773/153701, CAS # 127-09-3, Purity >99%, Replicate: Single, Test concentration: 35 mg/L, ThCO2: 1.07 mg CO2/mg, ThTOC: 0.29 mg C/mg, Carbon content in the vessel: 10.2 mg C/L
Parameter:
% degradation (CO2 evolution)
Value:
86
Sampling time:
28 d
Remarks on result:
other: Mean of 2 replicates
Details on results:
Carbon content of the test substance: Based on the carbon content a ThCO2 of 2.49 mg CO2/mg test substance was calculated. On this basis the test concentration of 15 mg/L, corresponding to a carbon content of 10.2 mgC/L in the test vessels was selected.

CO2-production and biodegradation: The total amount of CO2 produced in 28 d was analysed by titration in 12 measurements.
- In the control group a maximum of 43.2 mg CO2/L was formed after 28 d (validity criterion: <70 mg CO2/L after 28 d.
- In the test group gross CO2 production after 28 d was 69.9 mg/L (replicate 1) and 89.4 mg/L (replicate 2). The 10% biodegradation level was reached after 5 days. In the 10-d-window the pass level of a biodegradation of more than 60% was reached. The mean biodegradation came to 79% after 14 d and to a maximum of 86% after 28 d.
- In the toxicity control 62% biodegradation occurred within 14 d and came to a maximum of 78% after 28 d. The test substance did not inhibit the biodegradation of the reference substance.
Results with reference substance:
The adaptation phase of the functional control group changed after 2 d into the degradation phase (degradation ≥ 10%). The course of the degradation phase was rapid and reached a degradation rate of 60% on Day 7. The validity criterion of a degradation of 60% after 14 d was fulfilled.

Criteria met for the validity of the study:

- The total CO2 evolution in the inoculum blank at the end of the test did not exceed 70 mg/L.

- The biodegradation of the reference compound reached the pass level of ≥60 % by Day 14.

- The difference of extremes of replicate values of the test substance at the end of the 10-d window was less than 20%.

Table 1: Biodegradation of the test substance

 

Biodegradation [%]

Study day

6

14

21

28

Test substance (15 mg/L)

37

79

86

86

Reference substance (35 mg/L)

56

100

100

100

Toxicity control (15 mg/L test substance + 35 mg/L reference substance)

31

62

67

78

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

A study was conducted to determine the ready biodegradability of the test substance, C18-unsatd. DEA, according to OECD Guideline 301B (modified Sturm test), in compliance with GLP. A solution of the test substance (15 mg/L) in nutrient solution, corresponding to 10.2 mg TOC/L, was inoculated with activated sludge. The test vessels were aerated by the passage of CO2-free air and incubated under aerobic conditions for 28 d. The biodegradation of the test substance was followed by titrimetric analyses of the quantity of CO2 produced on Days 1, 3, 6, 7, 9, 14, 17, 21, 24, 28 and 29 of the study. The reference substance used was sodium acetate at a concentration of 35 mg/L. The reference substance reached the pass levels for ready biodegradability after 7 d. The mean biodegradation of the test substance was 79% after 14 d and reached a maximum of 86% after 28 d (after acidification). Under the conditions of the study, the test substance was considered to be readily biodegradable (Noack, 2003).

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

A study was conducted to determine the ready biodegradability of the test substance, C18-unsatd. DEA, according to OECD Guideline 301B (modified Sturm test), in compliance with GLP. A solution of the test substance (15 mg/L) in nutrient solution, corresponding to 10.2 mg TOC/L, was inoculated with activated sludge. The test vessels were aerated by the passage of CO2-free air and incubated under aerobic conditions for 28 d. The biodegradation of the test substance was followed by titrimetric analyses of the quantity of CO2 produced on Days 1, 3, 6, 7, 9, 14, 17, 21, 24, 28 and 29 of the study. The reference substance used was sodium acetate at a concentration of 35 mg/L. The reference substance reached the pass levels for ready biodegradability after 7 d. The mean biodegradation of the test substance was 79% after 14 d and reached a maximum of 86% after 28 d (after acidification). Under the conditions of the study, the test substance was considered to be readily biodegradable (Noack, 2003).

A study was performed to assess the ready biodegradability of the test substance, C18-unsatd. DEA, according to OECD Guideline 301D (Closed bottle test), in compliance with GLP. Activated sewage sludge was exposed to the substance at a concentration of 3.0 mg/L in BOD bottles in the dark at 20–24°C for 28 d. Degradation was assessed by determination of oxygen consumption, expressed as percentage BOD. Biodegradation attained 70% after 28 d. The residual concentration of oxygen in the test vessels did not fall below 0.5 mg/L at any time. All other validity criteria were fulfilled. Under the study conditions, the test substance was considered to be readily biodegradable (Noack, 2005).