5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile

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:

1996-04-23 to 1996-05-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-300-1

EC Name:

-

Cas Number:

138564-59-7

Molecular formula:

C12H9N3O2S

IUPAC Name:

5-methyl-2-[(2-nitrophenyl)amino]thiophene-3-carbonitrile

Test material form:

solid: particulate/powder

Details on test material:

97.7%
Item code QA405K
Lilly lot no: 356MO1

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor and 356M01
- Purity test date: 1995-11-15

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
- Solubility in the test solution: Up to 1.5 mg/L

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: An appropiate amount of the test substance was dissolved in methanol
- Final preparation of a solid: Using a methanol solution, the insides of the test flasks were coated with the test substance by drying the solution.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Coating the walls of the selected volumetric flask.

Study design

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): Greenfield sewage plant
- Storage conditions: Aerated continously and NANOpure water was added if the volume decreased significantly.
- Storage length: 92.5 hours
- Preparation of inoculum for exposure: 30 mL was filtered through a glass fiber filter, and this was used as the source of inoculum.
- Concentration of sludge: 1.6 mL iinoculant/7.9984 L of test solution
- Water filtered: no

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial conc.:

ca. 1.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: 80 mL of BOD dilution water, 78.2 L of NANOpure water, 1.6 mL
- Test temperature: 20 +-1
- pH adjusted: no
- Aeration of dilution water: Yes
- Continuous darkness: yes
- Other: BOD Dilution water composition:
Solution Chemical Formula of Componets Mass (g) of Chemical/L
a. Phosphate buffer
potassium dihydrogen phosphate KH2PO4 8.5
dipotassium hydrogen phosphate K2HPO4 21.75
disodium hydrogen phosphate-heptahydrate Na2HPO4·7H2O 33.4
ammonium chloride NH4Cl 0.5
b. Magnesium sulfate MgSO4·7H2O 22.5
c. Calcium chloride CaCl2·6H2O 54.28
d. Ferric chloride(one drop of concentrated HCI was added to stabilize solution) FeCl3·6H20 0.25

TEST SYSTEM
- Number of culture flasks/concentration: 20
- Method used to create anaerobic conditions: Closed bottle
- Measuring equipment: Oxygen electrode attached to a pH meter for the DO measurements, or the stated HPLC method for test concentrations.
- Test performed in closed vessels due to significant volatility of test substance: Test is closed bottle but non-volatile.

SAMPLING
- Sampling frequency: Once every 3-4 days, including at test intiation.
- Sampling method: Oxygen electrode and suitable pH meter for dissolved oxygen concentrations, and the stated HPLC method for test concentrations.
- Sample storage before analysis: Samples were analyzed in situ or analyzed immediately.

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

STATISTICAL METHODS:
Oxygen Consumption/Depletion
The theoretical oxygen demand of an organic compound in mg O2/mg of the test chemical for an organic compound with molecular formula CcHhClclNnNanaOoPpSs was calculated by the following equation:
= 16*(2c+1/2(h-cl-3n)+3s+5/2p+1/2na-o)/(molecular weight) mg/mg

This calculation is based on the following assumptions: C is mineralized to CO2, H to H2O, P to P2O5, and Na to Na2O. Halogen is eliminated as a hydrogen halide and nitrogen as ammonia. The average change in oxygen concentration in mg/L is calculated as the average O2 concentration at Day 0 minus the average O2 concentration at time t. This calculation normalizes the changes in DO to the initial DO levels at the beginning of the study.

The experimental O2 demand is determined by substracting the average change in oxygen concentration in mg/L of inoculum blank-A at time t from the average change in oxygen in mg/Lat time t of the sample.

A calculation of the percentage biodegradability after n days can be made by dividing the oxygen demand by the total theoretical oxygen demand in the flask as follows:
% Biodegradability = (Experimental O2 Demand/Theoretical Oxygen Demand in Flask)*100

For the toxicity control samples, the theoretical oxygen demand was clalculated as the sum of the contributions to the thoeretical oxygen demand in the flask from the referennce compound andn the test article.

A determination of the culmulative O2 demand and plot versus time was not warranted for this study given the test results.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

No preliminary study was conducted.

Test performance:

The controls met the criteria necessary for the test to be determined to be valid.

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 0

Sampling time:

0 d

% Degradation 2

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. -10.68

Sampling time:

1 d

% Degradation 3

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. -12.5

Sampling time:

3 d

% Degradation 4

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. -5.21

Sampling time:

7 d

% Degradation 5

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. -7.55

Sampling time:

10 d

% Degradation 6

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. -1.82

Sampling time:

14 d

% Degradation 7

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. -13.28

Sampling time:

17 d

% Degradation 8

Key result

Parameter:

% degradation (O2 consumption)

Value:

-4.17

Sampling time:

21 d

% Degradation 9

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. -5.47

Sampling time:

24 d

% Degradation 10

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. -1.56

Sampling time:

28 d

Details on results:

The dissolved oxygen level in the reference compound blank samples (D) yielded a 48.35% biodegradability level at Day 1 and a 51.35% level at Day 28. This corresponded well with the analytical data which indicates that sodium benzoate is degraded to other chemical species by Day 1 such that it is not detected or quantifiable at a level of 0.030 µg/mL. (This figure corresponded to >98% degradation of an initial level of 2.0 µg/mL.) Similarly, the reference compound samples (E) were 48.31 % and 60.89% biodegraded on Day 1 and Day 28, respectively, based on dissolved oxygen levels. Although the percent biodegradability of the toxicity control samples (F) indicates that only 30.74% and 35.67% at Day 1 and Day 28, the figures reflect the recalcitrant intermediate 290437 as part of the theoretical oxygen demand in the flask. Observation of the absolute O2 demand shows the same approximate level of oxygen utilization in the toxicity control samples
and other samples which only contained sodium benzoate.

After 28 days of incubation the test substance was stable as indicated by the oxygen demand and percent biodegradability in test systems B and C. After 28 days, the O2 demand for the test vesselswith inoculum and the test article was 0.03 mg O2/L

BOD5 / COD results

Results with reference substance:

The concentrations of sodium benzoate declined rapidly within the first day of incubation. The 0 Day values are all at levels near the theoretical target level of 2.0µg/mL. On Day 1,the sodium benzoate levels had decreased below the limit of quantitation of 0.030µg/mL and in many cases were not even detectable. These results are confirmed by the rapid reduction in dissolved oxygen in these same sample vessels reference compound One sample (reference compound E-Replicated 8, Day 7) provided unusual data in that a small peak was found in the chromatogram where sodium benzoate occurs. The peak was equivalent to a level of 0.032 ug/mL to 0.033 ug/mL as sodium benzoate. This peak may be contributed to the autosampler vial contamination.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

This study validly found that the test substance does not readily biodegrade in the test system according to GHS criteria.

Executive summary:

The study was carried out in accordance with EU Method C4 -E.

The results of this study indicate that over a 28-day period, the test substance did not degrade in the test systems.