5-[4-Carboxy-4-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-butyrylamino]-2-nitro-benzoic acid, L-

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:

17 April - 24 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with OECD guideline no. 301F and Good Laboratory Practice.

Data source

Reference

Reference 1

Reference Type:

other: study amendment

Title:

Unnamed

Year:

2021

Report date:

2021

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1992)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-[(S)-4-Carboxy-4-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-butyrylamino]-2-nitro-benzoic acid

EC Number:

926-197-1

Cas Number:

2561176-48-3

Molecular formula:

C20 H15 N3 O9

IUPAC Name:

5-[(S)-4-Carboxy-4-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-butyrylamino]-2-nitro-benzoic acid

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): P-Glupa-C
- Molecular formula: C20H15N3O9
- Molecular weight: 441.35 g/mol
- CAS number: 2561176-48-3
- Appearance: Yellow solid
- Theoretical Oxygen Demand: ThOD(NH4): 1.23 mg oxygen per mg test item, ThOD(NO3): 1.69 mg oxygen per mg test item

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (sewage plant Bensheim, Germany) was used.
The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

103 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

127 mg/L

Based on:

other: THOD(NH4) of P-Glupa-C (the purity of the test material did not affect the results as other constituents had similar ThOD, see confidential details in test materials for more details)

Initial test substance concentration 3

Initial conc.:

172 mg/L

Based on:

other: ThOD(NO3) of P-Glupa-C (the purity of the test material did not affect the results as other constituents had similar ThOD, see confidential details in test materials for more details)

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD301F
- Test temperature: 22 ± 1 °C
- pH: 7.3 - 7.6
- pH adjusted: no
- Suspended solids concentration: 29 mg/L

TEST SYSTEM
- Culturing apparatus: Manometric Test System with 500 mL flasks
- Number of culture flasks/treatment: 2
- Measuring equipment: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany
- Test performed in closed vessels: Yes, the test flasks were closed gas-tight by a measuring head
- Details of trap for CO2: 45% Potassium hydroxide solution (this trap was only for adsorbing, it was not analysed)

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 flasks
- Abiotic sterile control: Yes
- Toxicity control: Yes
- Procedural control (with reference substance): Yes

PREPARATION OF THE FLASKS
Test substance and/or reference substance samples (~25 mg) were weighed and added directly to the test flasks.
In the abiotic sterile control flask 5 mL of a 1 g/L HgCl solution was added
Then all test flasks, except for the abiotic sterile control flask were filled with test medium inoculated with 2 mL of a 3.5 g/L suspended sludge solution to a total volume of 244 mL. The abiotic sterile contriol flask was was filled with test medium to a total volume of 244 mL.

The closed test flasks were incubated in a climatised room under continuous stirring.

MEASUREMENTS

Oxygen:
The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel, which was measured daily with the BSB/BOD sensor.

Temperature:
The temperature was recorded by means of the automated software AMR Wincontrol©.

pH-Value:
pH-values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at the end of the test, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

ThOD(NH4): 1.67 mg oxygen per mg sodium benzoate (calculated)

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Value:

40

Sampling time:

28 d

Remarks on result:

other: based on ThOD(NH4)

% Degradation 2

Parameter:

% degradation (O2 consumption)

Value:

29

Sampling time:

28 d

Remarks on result:

other: based on ThOD(NO3)

Details on results:

The test item P-Gluca-C contains nitrogen; therefore the evaluation of biodegradation has to be based on ThOD(NH4) and ThOD(NO3).
The mean biodegradation at test end after 28 days was 40% (ThOD(NH4)) and 29% (ThOD(NO3)).
The nitrate concentration was not determined and therefore it is not known whether nitrification is relevant. As the results based on ThOD(NH4) and THOD(NO3) were in the same range order and substance is not ready biodegradable, it can be assumed to be not relevant for the conclusion.

BOD5 / COD results

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 72% after 14 days and to 81% after 28 days of incubation.: the activity of the inoculum was thus verified (validity criterion).

Any other information on results incl. tables

The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no use to correct the degradation of the test item and toxicity control.

The validity criteria were fulfilled as follows:

Inoculum Control:

The oxygen demand of the inoculum control (medium and inoculum) was 25 mg O2/L and thus not greater than 60 mg

O2/L within 28 days as required by the test guideline.

pH-Value:

The pH-value of the test item flasks at the end of the test was 7.5 and therefore within the range of pH 6.0 to 8.5 as

required by the test guideline.

Reference Item:

The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14

days as required by the test guideline. The reference item sodium benzoate was degraded to more than 60% after 3 days of incubation.

Test Item:

The difference of duplicate values for the degradation of the test item at the plateau, and at the end of the 10-day window

was less than 20%. The difference of duplicate values at the end of the 10-day window and at day 28 was 14%

Toxicity Control:

If in a toxicity test, containing both the test substance and a reference compound less than 25% biodegradation (based

on total ThOD) occurred within 14 days, the test substance can be assumed to be inhibitory. The biodegradation was

45% (ThOD(NH4)) at day 14; the test item was not toxic.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See above

Interpretation of results:

other: The test substance is not ready biodegradable.

Conclusions:

Although the test substance biodegraded (40% and 29% after 28 d based on ThOD(NH4) and ThOD(NH3), respectively), it is not ready biodegradable.

Executive summary:

The ready biodegradability of the test substance was investigated according to OECD guideline no. 301F and Good Laboratory Practice.

The test item P-Gluca-C contains nitrogen; therefore the evaluation of biodegradation has to be based on ThOD(NH4) and ThOD(NO3).

The mean biodegradation at test end after 28 days was 40% (ThOD(NH4)) and 29% (ThOD(NO3)).

Based on these results it can be concluded that the test substance is not ready biodegradable.

The test substance was not inhibitory to the inoculum.