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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 July to 04 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 301F with GLP statement. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

The Netherlands GLP Compliance Programme (inspected on 28 February - 7 March 2014 / signed on 30 April 2014)

Specific details on test material used for the study:

Phys-Chem Properties:
Water Solubility: Well soluble in water (481 mglL in 2 days experiment; 875 mglL in 3 days experiment)
Partition Coefficient: log Pow=3.8
Vapour Pressure: 5.8 Pa at 20 °C, 11 Pa at 25 °C

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Inoculum was activated sludge derived from a conventional treatment plant for domestic waste in Shenyang. A fresh sample of activated sludge was collected from the aeration tank of Shenyang Northern Sewage Treatment Plant, one day before the test item exposure was started.
- Preparation of inoculum for exposure: On the day of sludge collection, coarse particles were removed by filtration through a fine sieve and the sample was settled. After the supernatant was discarded, the sludge was washed twice with mineral medium and the concentrated sludge was suspended in mineral medium. 3 replicates of 10 mL suspended sludge was weighed and dried at 105 °C for about 1.5 hours and reweighed to calculate initial sludge concentration. The measured concentration of suspended sludge was 5.36 g/L as suspended solids (SS). The suspended sludge was prepared with mineral medium to give a concentration of 4.17 g/L and then kept aerobic at 21.7 -23.9 °C until used for the test. On the day of the beginning of the test, 5 replicate of 10 mL sludge were weighed and dried at 105 °C for 1.5 hours and reweighed. The mean concentration of suspended sludge measured was 3728 mg/L as SS. From this result, the added amount of wet sludge was calculated.
- Concentration of microbial inoculum: 30 mg/L (as suspended solids (SS))

Duration of test (contact time):

28 d

Initial conc.:

31.5 mg/L

Based on:

test mat.

Remarks:

equivalent to 93.0 mg/L as ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium*: For mineral medium, 10 mL stock solution A was added to approximately 800 mL distilled water and the mixture stirred before adding, sequentially, 1 mL each of stock solutions B, C and D. After mixing the mixture was made up to 1 liter with distilled water to prepare 1 L mineral medium. 2 L mineral medium was prepared and mixed to check the pH on the day the study started. The pH was checked to be 7.78, and adjusted to 7.56 with HCL.
- Test temperature: 19.68-20.48 °C
- pH: 7.37-7.56
- Suspended solids concentration: 30 mg/L (as suspended solids (SS))
- Continuous darkness: Yes; test was conducted in darkness

TEST SYSTEM
- Culturing apparatus: BOD (Biochemical Oxygen Demand) bottles of 500 mL filled with 244 mL test solution with sealed BOD sensor
- Preparation of inoculated mineral medium: An unspecified amount mineral medium was added to a 2 L flask, followed by the addition of 16 mL of the prepared activated sludge and the mixture was made up to volume to give a concentration of 30 mg/L as SS. 2 L inoculated mineral medium were prepared. The inoculated mineral medium was brought into an overflow measuring flask (244 mL) to measure the volume of the sample precisely and then transferred into each BOD bottle respectively.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: No data
- After the test vessels were prepared, KOH (45% w/w) solution was added into the seal gasket to absorb carbon dioxide produced metabolically by bacteria. Then BOD bottles were kept in an incubator for 28 days.
- Measuring equipment: BOD values in all test vessels were recorded on a daily basis for the 28 day period using an automated respirometer.
- Incubation temperature was measured and recorded to be in the range of 19.68-20.48 °C at 1 hour interval by automated temperature monitoring system.
- pH of the various test vessels was measured at the end of the incubation period (on Day 28).

CONTROL AND BLANK SYSTEM
- Inoculum blank control: BOD bottles containing only inoculated mineral medium were prepared in duplicate and served as inoculum controls
- Toxicity control: BOD bottle contains test substance (32.4 mg/L; equivalent to 95.5 mg/L as ThOD) and reference substance (56.8 mg/L; equivalent to 94.5 mg/L as ThOD). The concentration expressed as total ThOD was 193.5 mg/L; one replicate was used.

* Note:
Stock solution A (in water): 100 mL contains 0.8507 g KH2PO4, 2.1757 g K2HPO4, 3.3406 g Na2HPO4.2H2O and 0.0505 g NH4CI
Stock solution B (in water): 100 mL contains 3.6405 g CaCl2.2H2O
Stock solution C (in water): 100 mL contains 2.2507 g MgSO4.7H2O
Stock solution D (in water): 100 mL contains 0.0258 g FeCl3.6H2O

Reference substance:

benzoic acid, sodium salt

Remarks:

56.8 mg/L (equivalent to 94.5 mg/L as ThOD)

Preliminary study:

None

Key result

Parameter:

% degradation (O2 consumption)

Value:

78.6

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Details on results:

The percentage biodegradation of test substance attained 12.9% (2 replicates) by Day 3, 73.2% by Day 13, 78.6% by Day 28. The test substance showed biodegradation exceeding the pass level of 60% within 10-day window within the 28 day test duration.
In the Toxicity Control (TC) test mixture, 73.7% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test substance can be assumed to be not inhibitory under the conditions of this test.

Results with reference substance:

Biodegradation of the reference substance (Sodium benzoate) attained 61.9% by Day 4, 78.8 % by Day 14 and 82.5% by Day 28.

See attached background material section for results - table

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the test conditions, test substance is considered as readily biodegradable.

Executive summary:

The ready biodegradability of the test substance has been determined by the Manometric Respirometry Test according to the OECD Guideline No. 301F with GLP compliance. The ready biodegradability test of test substance was conducted with unacclimatised sewage micro-organism by measuring oxygen consumption over the 28-day test period.

The test substance was added directly to test vessels at a level of 31.5 mg/L (equivalent to 93.0 mg/L as Theoretical Oxygen Demand or "ThOD") (2 replicates). The test concentration of sodium benzoate, used as reference substance in the Procedure Control, was 56.8 mg/L (equivalent to 94.5 mg/L as ThOD). In the Toxicity Control (TC) containing both test substance and reference substance, the test substance was added directly to the test vessel at a level of 32.4 mg/L (equivalent to 95.5 mg/L as ThOD ) and the reference substance was added at a level of 58.8 mg/L of sodium benzoate (equivalent to 98.0 mg/L as ThOD). The concentration expressed as total ThOD was 193.5 mg/L. After the test vessels were prepared, BOD bottles were incubated at 19.68-20.48 °C for 28 days.

 

The percentage biodegradation of test substance attained 12.9% (2 replicates) by Day 3, 73.2% by Day 13, 78.6% by Day 28. The test substance showed biodegradation exceeding the pass level of 60% within 10-day window within the 28 day test duration. In the Toxicity Control (TC) test mixture, 73.7% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test substance can be assumed to be not inhibitory under the conditions of this test. Biodegradation of the reference substance (Sodium benzoate) attained 61.9% by Day 4, 78.8 % by Day 14 and 82.5% by Day 28.

 

Under the test conditions, test substance is considered as readily biodegradable.

Description of key information

OECD Guideline 301F, GLP, key study, validity 1:

78.6% biodegradation was observed after 28 days fulfilling the 10-day window criterion, so the substance can be considered rapidly biodegradable, according to CLP Regulation No 1272/2008

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

One valid key study is available (SYRICI, 2015) to assess the readily biodegradation of the registered substance.

The ready biodegradability of the test substance has been determined by the Manometric Respirometry Test according to the OECD Guideline No. 301F with GLP compliance. The ready biodegradability test on the test substance was conducted with unacclimated sewage micro-organisms by measuring oxygen consumption over the 28-day test period.

The test substance was added directly to test vessels at a level of 31.5 mg/L (equivalent to 93.0 mg/L as Theoretical Oxygen Demand or "ThOD") (2 replicates). The test concentration of sodium benzoate, used as reference substance in the Procedure Control, was 56.8 mg/L (equivalent to 94.5 mg/L as ThOD). In the Toxicity Control (TC) containing both test substance and reference substance, the test substance was added directly to the test vessel at a level of 32.4 mg/L (equivalent to 95.5 mg/L as ThOD ) and the reference substance was added at a level of 58.8 mg/L of sodium benzoate (equivalent to 98.0 mg/L as ThOD). The concentration expressed as total ThOD was 193.5 mg/L. After the test vessels were prepared, BOD bottles were incubated at 19.7 -20.5 °C for 28 days.

 The percentage biodegradation of test substance attained 12.9% (2 replicates) by Day 3, 73.2% by Day 13, 78.6% by Day 28. The test substance showed biodegradation exceeding the pass level of 60% within 10-day window within the 28 day test duration. In the Toxicity Control (TC) test mixture, 73.7% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test substance can be assumed to be not inhibitory under the conditions of this test. Biodegradation of the reference substance (Sodium benzoate) attained 61.9% by Day 4, 78.8 % by Day 14 and 82.5% by Day 28.

Under the test conditions, the substance is considered as readily biodegradable.