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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 2019-06-19 to 2019-07-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: A03-18-0081
Purity: 99.9%
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: collected from the aeration tank of Shenyang North Sewage Treatment Plant 2 days before the test exposure was started. It is a well-functioning treatment plant receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The inoculum was pre-treated as follows: 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. 4 replicates of 10 mL suspended sludge were weighed and dried at 105 °C for 1 hour and 33 minutes and reweighed to calculate initial sludge concentration. The measured concentration of suspended sludge was 3.82 g/L as suspended solids (SS). The suspended sludge was kept aerobic at 21.9 ~ 23.3 °C until used for the test.
- Concentration of sludge: On the day of the test beginning, 5 replicates of 10 mL sludge were weighed and dried at 105 °C for 1 hour and 50 minutes and reweighed. The measured concentration was 3854 mg/L as SS.
- Type and size of filter used, if any: a fine sieve
Duration of test (contact time):
28 d
Initial conc.:
36.38 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
The stock solutions were prepared as follows using analytical grade reagents, except that Na2HPO4•2H2O was HPLC grade.
(A) Dissolve 4.2517 g potassium dihydrogen orthophosphate (KH2PO4), 10.8758 g dipotassium hydrogen orthophosphate (K2HPO4), 16.7002 g disodium hydrogen orthophosphate dihydrate (Na2HPO4•2H2O), and 0.2505 g ammonium chloride (NH4Cl) in distilled water and make up to 500 mL. The pH of the solution was 7.59.
(B) Dissolve 3.6419 g Calcium chloride dihydrate, CaCl2•2H2O in distilled water and make up to 100 mL.
(C) Dissolve 2.2507 g magnesium sulfate heptahydrate (MgSO4•7H2O) in distilled water and make up to 100 mL.
(D) Dissolve 0.0252 g iron (III) chloride hexahydrate (FeCl3•6H2O) in distilled water and make up to 100 mL.
There were no precipitates formed in above stock solutions, and the solutions were used for preparation of mineral medium.
For 1 liter of 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. 5 L mineral medium was prepared. The pH was checked to be 7.59.
- Test temperature: 21.3 °C - 23.7 °C
- pH: 7.75 - 7.93
- Suspended solids concentration: The measured concentration was 3854 mg/L as SS.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Biochemical incubator
- Number of culture flasks/concentration: duplicate
- Measuring equipment: Dissolved oxygen meter, BOD analyzer, pH meter

CONTROL AND BLANK SYSTEM
- Inoculum blank: Test vessels containing only 250 mL inoculated mineral medium were prepared in duplicate and served as inoculum controls.
- Abiotic sterile control: 0.00950 g of test item was weighed into test flask and 237.5 mL of mineral medium was added. 12.5 mL of 3,5-dichlorophenol solution in mineral medium at the concentration of 500 mg/L was added as a sterilizing agent, the concentration of 3,5-dichlorophenol in final volume was 25 mg/L. The concentration of test item was 38.00 mg/L, equivalent to 53.20 mg/L ThODNH3. No inoculum was added to test flask which served as an abiotic sterile control identified as ASC. One replicate sufficed.
- Toxicity control: Toxicity control test vessel contained both test item and reference item. Both a weighed quantity (0.00905 g) of test item and a weighed quantity (0.00799 g) of reference item were weighed and added into test vessel. The concentration of test item was 36.20 mg/L (equivalent to 50.68 mg/L as ThODNH3) and the concentration of reference substance was 31.96 mg/L (equivalent to 53.37 mg/L as ThOD). The concentration expressed as total ThOD (based on ThODNH3) was 104.05 mg/L. One replicate (toxicity control identified as TC) sufficed.

Measuring of Oxygen Uptake
After the test flasks were prepared, the following operation was performed as soon as possible: Insert the magnetic stirring rod, place 1 pill of NaOH into the seal gasket. Then insert the gasket in the neck of the bottle. Screw the BOD measuring head to the BOD sample bottles. Place the sample bottles in the inductive stirring system. Keep the BOD system in the biochemical incubator in constant temperature (22 °C ± 1 °C) and start the stirring and the measurement of BOD values. The BOD values in all test vessels were automatically recorded for the 28 days period by the system. At the end of the period of incubation, call up and read the BOD values. Incubation temperature was measured by automated temperature monitoring system, the measured range was 21.3 - 23.7 °C. Stirrer was checked on every working day to determine proper operation.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
82.9
Sampling time:
28 d
Details on results:
The biodegradation of test item after 28 days averaged 82.9% (2 replicates). The percentage degradation of the test item reaches the pass level of 60% in a 10-day window within the 28 days period of the test.
In the Toxicity Control (TC) test mixture, 75.2% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test item can be assumed to be not inhibitory under the conditions of this test.
In the Abiotic Sterile Control (ASC) Test Vessel, 21.2% degradation occurred after 28 days.
Results with reference substance:
The biodegradation of the reference substance (Sodium benzoate) attained 60.2% after 2 days 13 hours, 83.6% after 14 days and 89.0% after 28 days.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The percentage biodegradation within 28 days exceeds the pass level of 60% ThOD. The test item meets the criteria for “ready biodegradability” under the described test conditions.
Executive summary:

The ready biodegradability test of the test item was conducted with unacclimatised sewage micro-organisms by measuring oxygen consumption over the 28-day test period according to OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test).

The test item was added directly to test vessels at a level of 36.38 mg/L (equivalent to 50.93 mg/L as Theoretical Oxygen Demand or “ThODNH3”) (average, 2 replicates). The test concentration of sodium benzoate, used as reference substance in the Procedure Control, was 30.80 mg/L (equivalent to 51.44 mg/L as ThOD). In the Toxicity Control (TC) containing both test item and reference substance, the test item was added to the test vessel at a level of 36.20 mg/L (equivalent to 50.68 mg/L as ThODNH3) and the reference substance was added at a level of 31.96 mg/L of sodium benzoate (equivalent to 53.37 mg/L as ThOD), the concentration expressed as total ThOD was 104.05 mg/L. In the Abiotic Sterile Control (ASC) Test Vessel without inoculum addition and containing sterilizing agent, the test item was added to the test vessel at a level of 38.00 mg/L (equivalent to 53.20 mg/L as ThODNH3).

The biodegradation of test item after 28 days averaged 82.9% (2 replicates). The percentage degradation of the test item reaches the pass level of 60% in a 10-day window within the 28 days period of the test.

The biodegradation of the reference substance (Sodium benzoate) attained 60.2% after 2 days 13 hours, 83.6% after 14 days and 89.0% after 28 days.

In the Toxicity Control (TC) test mixture, 75.2% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test item can be assumed to be not inhibitory under the conditions of this test.

In the Abiotic Sterile Control (ASC) Test Vessel, 21.2% degradation occurred after 28 days.

 

Based on the results, the percentage biodegradation within 28 days exceeds the pass level of 60% ThOD. The test item meets the criteria for “ready biodegradability” under the described test conditions.

Description of key information

The percentage biodegradation within 28 days exceeds the pass level of 60% ThOD. The test item meets the criteria for “ready biodegradability” under the described test conditions.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The ready biodegradability test of the test item was conducted with unacclimatised sewage micro-organisms by measuring oxygen consumption over the 28-day test period according to OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test).

The test item was added directly to test vessels at a level of 36.38 mg/L (equivalent to 50.93 mg/L as Theoretical Oxygen Demand or “ThODNH3”) (average, 2 replicates). The test concentration of sodium benzoate, used as reference substance in the Procedure Control, was 30.80 mg/L (equivalent to 51.44 mg/L as ThOD). In the Toxicity Control (TC) containing both test item and reference substance, the test item was added to the test vessel at a level of 36.20 mg/L (equivalent to 50.68 mg/L as ThODNH3) and the reference substance was added at a level of 31.96 mg/L of sodium benzoate (equivalent to 53.37 mg/L as ThOD), the concentration expressed as total ThOD was 104.05 mg/L. In the Abiotic Sterile Control (ASC) Test Vessel without inoculum addition and containing sterilizing agent, the test item was added to the test vessel at a level of 38.00 mg/L (equivalent to 53.20 mg/L as ThODNH3).

The biodegradation of test item after 28 days averaged 82.9% (2 replicates). The percentage degradation of the test item reaches the pass level of 60% in a 10-day window within the 28 days period of the test.

The biodegradation of the reference substance (Sodium benzoate) attained 60.2% after 2 days 13 hours, 83.6% after 14 days and 89.0% after 28 days.

In the Toxicity Control (TC) test mixture, 75.2% degradation occurred within 14 days and the value exceeded 25% based on total ThOD, which indicated that the test item can be assumed to be not inhibitory under the conditions of this test.

In the Abiotic Sterile Control (ASC) Test Vessel, 21.2% degradation occurred after 28 days.

 

Based on the results, the percentage biodegradation within 28 days exceeds the pass level of 60% ThOD. The test item meets the criteria for “ready biodegradability” under the described test conditions.