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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 November 2017 - 12 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-N-1H-1,2,4-triazol-3-ylbenzamide
EC Number:
253-021-4
EC Name:
2-hydroxy-N-1H-1,2,4-triazol-3-ylbenzamide
Cas Number:
36411-52-6
Molecular formula:
C9H8N4O2
IUPAC Name:
2-hydroxy-N-1H-1,2,4-triazol-3-ylbenzamide
Test material form:
solid: particulate/powder
Details on test material:
Batch:102Z5
Purity: 99.9%
white powder

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.0 g/L prior to use.
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 18.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: (Mineral medium) KH2PO4 -8.50 g/L, K2HPO4 -21.75 g/L, Na2HPO4.2H2O - 33.40 g/L, NH4Cl - 0.50 g/L, CaCl2 - 27.50 g/L, MgSO4.7H2O - 22.50 g/L, FeCl3.6H2 - 0.25 g/L
- Solubilising agent (type and concentration if used):
- Test temperature: 20 - 24 °C
- pH: 7.4 ±0.2
- pH adjusted: yes using diluted hydrochloric acid or sodium hydroxide solution
- Aeration of dilution water: overnight
- Suspended solids concentration: 4.0 g/L

TEST SYSTEM
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 20 and 24 °C for 28 days.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 22.5 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. If necessary the pH was adjusted to pH 7.4 ±0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2-free air. The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules. The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.
Reference substance
Reference substance:
other: Sodium benzoate

Results and discussion

Preliminary study:
not specified
Test performance:
The total CO2 evolution in the inoculum control vessels on Day 28 was 33.52 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.
% Degradationopen allclose all
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 10
Sampling time:
6 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 30
Sampling time:
8 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 57
Sampling time:
10 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 44
Sampling time:
14 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 68
Sampling time:
21 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 62
Sampling time:
28 d
Details on results:
The test item attained 62% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
The toxicity control attained 47% biodegradation after 14 days and 56% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 80% biodegradation after 14 days and 100% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item attained 62% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

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