1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

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:

From 01 OCTOBER 2018 to 30 OCTOBER 2018

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:

yes

Remarks:

The carbon analyser was not calibrated monthly as stated in the study plan. Because quality control samples were measured daily, this can be considered as uncritical concerning the outcome of the study.

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

yes

Remarks:

The carbon analyser was not calibrated monthly as stated in the study plan. Because quality control samples were measured daily, this can be considered as uncritical concerning the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, 67435 NW-Lachen-Speyerdorf.
- Laboratory culture:
- Method of cultivation:
- Storage conditions:
- Storage length: From 28 SEPTEMBER 2018
- Preparation of inoculum for exposure: The sludge was filtrated, washed with test medium (2x) and re-suspended in test medium. It was then aerated until use. The dry matter was determined to contain 4940 mg of suspended solids/L.
- Pretreatment: The sludge was filtrated, washed with test medium (2x) and re-suspended in test medium. It was then aerated until use.
- Concentration of sludge: The dry matter was determined to contain 4940 mg of suspended solids/L.
- Initial cell/biomass concentration:
- Water filtered: yes/no
- Type and size of filter used, if any:

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 3.7 mg/L

Based on:

IC (inorganic carbon)

Initial conc.:

ca. 3.3 mg/L

Based on:

IC (inorganic carbon)

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
The medium was freshly prepared. 10 mL of solution a were mixed with 800 mL water, then
1 mL of solutions b, c and d were added and filled up to 1 L with H2O demin (volumes were
adapted to final volume needed in the test).
Composition:
Solution a 10 mL
Solution b 1 mL
Solution c 1 mL
Solution d 1 mL
H2O demin. ad 1000 mL

Where

6.6.1.1 Solution a
Potassium dihydrogen phosphate (KH2PO4) 8.5 g
Dipotassium hydrogen phosphate (K2HPO4) 21.75 g
Disodiumhydrogen phosphate dihydrate (Na2HPO4*2H2O) 33.4 g
Ammonium chloride (NH4Cl) 0.5 g
H2O demin. ad 1000 mL
The pH was 7.4.
6.6.1.2 Solution b
Calcium chloride (CaCl2) 27.5 g
H2O demin. ad 1000 mL
6.6.1.3 Solution c
Magnesium sulphate heptahydrate (MgSO4*7H2O) 22.5 g
H2O demin. ad 1000 mL
6.6.1.4 Solution d
Iron(III) chloride hexahydrate (FeCl3*6H2O) 0.25 g
Di-sodium-ethylene diaminetetraacetate dihydrate (Na2EDTA*2H2O) 0.4 g
H2O demin ad 1000 mL

- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: 20.1 – 22.8 °C without direct lighting
- pH: 7.6
- pH adjusted: no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes
- Other:

TEST SYSTEM
- Culturing apparatus: Heating chamber Heratherm OGS 60
- Number of culture flasks/concentration:
Apparatus blanks 2, containing mineral medium only
Blank Controls 2, containing mineral medium and inoculum
Positive control flasks 2, containing positive control, mineral medium and inoculum
Test flasks 2, containing test item, mineral medium and inoculum
Abiotic control 1, containing test item, mineral medium and HgCl2
Toxicity control 1, containing test item, positive control, mineral medium and inoculum
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment: Carbon analyser TOC multi N/C 2100S, Analytik Jena
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:

SAMPLING
- Sampling frequency: From each front scrubber flask, 9 samples were taken in order to determine the emitted CO2 (on day 0, 3, 7, 9, 11, 14, 17, 23 and 29)
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 60

Sampling time:

28 d

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

All validity criteria were met.
Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation was not observed. Both replicates of the test item showed good correspondence.
If degradation in the toxicity flask is below 25 % after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 46.1 % after 14
days, the test item can be stated as “not toxic towards the inoculum in a concentration of
34.7 mg/L”.
Ready biodegradability is defined in the guidelines as degradation surpassing 60% within
10 days after reaching a level of 10 %. The 10-day-window was not detected. Therefore, the
test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-
oxonicotinonitrile can be considered as “not readily biodegradable”. Degradation missed
60 % within 28 days, though. Therefore, the test item is considered as “not ultimately biodegradable within 28 days”, as well.
No observations were made which might cause doubts concerning the validity of the study
outcome.
The result of the test can be considered valid.

Description of key information

According to OECD 301B resp. EU C.4-C

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Therefore, when applying the 10-day-window, the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile is not readily biodegradable following OECD 301B and EU C.4-C respectively. As degradation missed 60% in the course of the test, 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile is
considered as not ultimately biodegradable, within 28 days as well.