N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide)

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:

Experimental Phase: 16 September 2020 to 14 October 2020.

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m))

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Source of Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 14 September 2019 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
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 a pre-weighed Whatman 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 0.9 g/L prior to use.

Duration of test (contact time):

ca. 28 d

Initial conc.:

14.7 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

TEST CONDITIONS
- Composition of medium: Activated Sludge
- Test temperature: 22 °C to 24 °C
- pH: 7.5 to 7.6 throughout the study
- pH adjusted: no
- Aeration of dilution water: Yes (continuous aeration)
- Suspended solids concentration: 30 mg suspended solids (ss)/L
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: 5L test vessels (containing 3L of preparation)
- Number of culture flasks/concentration: Two replicates for test item, inoculum control, Procedure control nad one vessel for Toxicity control.
- Method used to create aerobic conditions: CO2 free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer.
- Measuring equipment: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO
2 absorbing solutions were prepared using deionised reverse osmosis water.
- Test performed in open system: No

SAMPLING (Inorganic Carbon Analysis)
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
- Sampling method: The samples were analyzed for inorganic carbon using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer.


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

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Results with reference substance:

Sodium benzoate attained 61% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Validation Criteria and Biodegradation

The total CO2 evolution in the inoculum control vessels on Day 28 was 45.69 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines. Although the CO2 evolution was in excess of 40 mg/L at the end of the test, this was considered not to affect the integrity of the study given that the upper level of 70 mg CO2/L given in the OECD Test Guidelines was not exceeded, and that all other validation criteria were satisfied.

 

The IC content of the test item suspension in the mineral medium at the start of the test, before dosing, 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 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.

 

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore, any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

 

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. The inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

 

The toxicity control attained 41% biodegradation after 14 days and 50% 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.  

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

Introduction

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium when exposed to sewage sludge micro-organisms under defined conditions over a 28-day period. The method followed was designed to be compatible with the following guidelines:

OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008

US EPA Fate, Transport and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

Methods

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 22 °C and 24 °C for 28 days.

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B. Sodium benzoate attained 61% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Conclusion

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

Biodegradation in water data summary.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.