sodium 4-[(4-chlorobenzoyl)amino]benzoate

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

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 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

yes

Remarks:

see "Principles of method if other than guideline" for further information

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see "Principles of method if other than guideline" for further information

Principles of method if other than guideline:

On Day 19 of the study the temperature in a vessel containing water which was incubated under the same conditions as the test vessels was recorded as being 24.7 °C which is outside the temperature range of 22 ± 2 °C. This slight deviation was considered to have had no adverse effect on the study given that all validation criterion were met.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

sodium 4-[(4-chlorobenzoyl)amino]benzoate

EC Number:

807-461-4

Cas Number:

1489170-67-3

Molecular formula:

C14H9ClNNaO3

IUPAC Name:

sodium 4-[(4-chlorobenzoyl)amino]benzoate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Storage condition of test material: room temperature in the dark
- Description: white powder

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: A mixed population of activated sewage sludge micro-organisms was obtained on 13 April 2015 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

- Pretreatment: The activated sewage sludge sample was washed twice by settlement and resuspension 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 °C and used on the day of collection.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

17.7 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
Mineral Medium
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl (pH = 7.4)
b) 27.5 g CaCl2
c) 22.5 g MgSO4 x 7H2O
d) 0.25 g FeCl3 x 6H2O
To 1 liter (final volume) of purified water* was added the following volumes of solutions a-d.
10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d

- Test temperature: 20 - 25 °C
- pH: 7.4 ± 0.2
- pH adjusted: yes

TEST SYSTEM
- Number of culture flasks/concentration:
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 plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
- Other:
Volume of test vessels: 5 L
Volume test solution: 3 L (toxicity control flask 125 mL)

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did adsorb to filter matrices and activated sewage sludge. Therefore, for the purpose of the study, in order to ensure that the test vessels were dosed accurately the required amount of test item was dispersed into the test system with the aid of ultrasonication. Samples were taken for DOC analysis prior to dosing and filtered in order to remove the suspended solids present.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

104

Sampling time:

28 d

Remarks on result:

other: Biodegradation value s in excess of 100% were considered to be due to sampling/analytical variation.

Details on results:

Validation Criteria
The total CO2 evolution in the inoculum control vessels on Day 28 was 30.30 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.

Biodegradation
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 with the exception of test item replicate 1 and the toxicity control.
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 test item attained 104% 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. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation.
The toxicity control attained 101% biodegradation after 14 days and 100% 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. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation. The slight decrease in biodegradation between days 14 and 28 was considered to be due to sampling/analytical variation.
Sodium benzoate attained 97% biodegradation after 14 days and 102% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 97% biodegradation after 14 days and 102% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

A reliability of Klimisch 1 has been assigned as the study was performed according to OECD Guideline 301B ("Ready Biodegradability; CO2 Evolution Test") and EU Method C.4-C to assess the ready biodegradability of the test item in an aerobic aqueous medium. 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 20 to 25 °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.
The test item attained 104% 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.
The toxicity control attained 101% biodegradation after 14 days and 100% 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. The slight decrease in biodegradation between days 14 and 28 was considered to be due to sampling/analytical variation.
Sodium benzoate attained 97% biodegradation after 14 days and 102% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation.