4-methylbenzothiazol-2-ylamine

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:

2016-11-15 - 2016-12-14 (Experimental)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

4-methylbenzothiazol-2-ylamine

EC Number:

216-028-3

EC Name:

4-methylbenzothiazol-2-ylamine

Cas Number:

1477-42-5

Molecular formula:

C8H8N2S

IUPAC Name:

4-methyl-1,3-benzothiazol-2-amine

Test material form:

solid: particulate/powder

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 14 November 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: 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 pre-weighed 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.
- Concentration of sludge: The suspended solids concentration was equal to 3.0 g/L prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 mg/L

Based on:

IC (inorganic carbon)

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium used in this study was that recommended in the OECD Guidelines.
- Solubilising agent (type and concentration if used): From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) it was concluded that the best testable dispersion was found to be obtained when using the high shear mixing method of preparation.
- Test temperature: 22-24°C
- pH: 7.4 ± 0.2
- pH adjusted: yes (acidification with hydrochloric acid)
- Aeration of dilution water: yes, CO2-free air
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 liter test culture vessels each containing 3 liters of solution
- Number of culture flasks/concentration: duplicates
- Method used to create aerobic conditions: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer.
- Measuring equipment: The samples were analyzed for IC using a Shimadzu TOC-VCSH or TOC-LCSH TOC analyzer. Samples (135 or 50 μL) were injected into the IC channel of the TOC analyzer. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.

SAMPLING
- 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.
- Sample storage before analysis: All samples were analyzed for IC immediately.

CONTROL AND BLANK SYSTEM
- Inoculum blank: An inoculated control, in duplicate, consisting of inoculated mineral medium plus a filter paper.
- Toxicity control: A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An amount of test item (51.3 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 17.1 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

22

Sampling time:

28 d

Details on results:

Validation Criteria
The total CO2 evolution in the inoculum control vessels on Day 28 was 34.07 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.
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 22% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301B.
The toxicity control attained 29% biodegradation after 14 days and 40% 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.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 70% biodegradation after 14 days and 72% biodegradation after 28 days

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The study was conducted under GLP according to OECD guideline 301B on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the ready biodegradability of the test substance.
The test item attained 22% 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:

A study according to OECD 301B and EU method C.4-C (GLP) was performed 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 22 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.

The test item attained 22% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301B.