(E)-N'-(2-cyano-4-(3-(1-hydroxy-2-methylpropan-2-yl)thioureido)phenyl)-N,N-dimethylformimidamide

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:

Experimental start date 14 January 2020 Experimental completion date 08 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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

Constituent 1

Reference substance name:

E-N'-{2-cyano-4-[3-(2-hydroxy-1,1-dimethylethyl)-thioureido]-phenyl}-N,N-dimethylformamidine

Cas Number:

1429755-57-6

Molecular formula:

C15H12N5OS

IUPAC Name:

E-N'-{2-cyano-4-[3-(2-hydroxy-1,1-dimethylethyl)-thioureido]-phenyl}-N,N-dimethylformamidine

Specific details on test material used for the study:

Identification: SPS 5290 (Stage 3)
Batch: 80034242B
Purity: 99.5%
Physical state / Appearance: Yellow crystalline powder
Expiry date: 05 July 2020
Storage conditions: Room temperature in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 09 March 2020 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
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
4.3 g/L prior to use.

Medium
The mineral medium used in this study was that recommended in the OECD Guidelines.
The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).
Solution a KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H2O 33.40 g/L
NH4Cl 0.50 g/L
pH = 7.4
Solution b CaCl2.2H2O 36.40 g/L
Solution c MgSO4.7H2O 22.50 g/L
Solution d FeCl3.6H2O 0.25 g/L
(In order to avoid having to prepare solution (d) immediately before use, one drop of concentrated HCl per liter was added as a preservative).
To 1 liter (final volume) of purified water* was added the following volumes of solutions a to d
10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d
* Reverse osmosis purified and deionized water (Elga Purelab Option R-15 or Elga Purelab Option R-15 BP)

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:

Preliminary Solubility Work
The following preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation:
i) Ultrasonication and High Shear Mixing: A nominal amount of test item (100 mg) was dispersed in 1 liter of deionized reverse osmosis purified water with the aid of shaking by hand for approximately 30 seconds, this formed a slightly hazy dispersion with many particles of test item visible dispersed throughout, prior to ultrasonication for 45 minutes. This formed a hazy dispersion with many particles
of test item visible dispersed throughout. This was then subjected to high shear mixing (approximately 7500 rpm, 30 minutes) and formed a very hazy dispersion with a few particles of test item visible dispersed throughout and a thin layer of foam on the surface.
This work confirmed that the test item was insoluble in water. Therefore the following additional solubility work was conducted to ascertain the best method to employ in the biodegradation test.
i) Ultrasonication: A nominal amount of test item (50 mg) was dispersed in approximately 400 mL of mineral media with the aid of ultrasonication for 15 minutes. The volume was then adjusted to a final volume of 3 liters with mineral media. This formed a very slightly hazy dispersion with a few particles of test item visible dispersed throughout and at the surface, many particles of test item were
visible settled on the bottom of the vessel. After 50 hours of magnetic stirring this formed a very slightly hazy dispersion with a few specks of test item visible at the surface.
ii) High Shear Mixing: A nominal amount of test item (50 mg) was dispersed in approximately 400 mL of mineral media with the aid of high shear mixing (approximately 7500 rpm, 15 minutes). The volume was then adjusted to a final volume of 3 liters with mineral media. This formed a hazy dispersion with a few particles of test item visible dispersed throughout and at the surface with small particles of test item visible settled on the bottom of the vessel. After 50 hours of magnetic stirring this formed a very slightly hazy dispersion with a few specks of test item visible at the surface.
iii) Preliminary Solution in a Volatile Solvent: The addition of a test item solvent stock to glass fibre filter paper was attempted.
A nominal amount of test item (1000 mg) was dissolved in THF (10 mL), and with the aid of ultrasonication (15 minutes) formed a yellow solution. An aliquot (450 μL) of this solvent stock solution was dispensed to filter paper. The solvent was allowed to evaporate to dryness for approximately 15 minutes. The filter paper was then added to approximately 400 mL mineral medium and subjected to high
shear mixing (approximately 7500 rpm, 15 minutes). The volume was then adjusted to 3 liters with mineral medium. This formed a very cloudy dispersion containing tiny broken up pieces of filter paper throughout with some small particles of test item visible at the surface. After 50 hours of magnetic stirring this formed a very cloudy dispersion containing tiny broken up pieces of filter paper dispersed
throughout and many specks of test item visible at the surface.
iv) Preliminary Solution in a Non-Volatile, Non-Degradable Solvent: A nominal amount of test item (100 mg) was dispersed in silicone oil (10 mL) with the aid of shaking by hand for approximately 30 seconds and ultrasonication (15 minutes). This formed a very cloudy pale yellow dispersion with many particles of test item visible dispersed throughout with test item visible settled on the bottom of the
vessel.
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 ultrasonication method of preparation.

Test Item Preparation
From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, 1995) and Handley et al (2002) it was concluded that the best testable dispersion was found to be obtained when using the ultrasonication method of preparation.
The test item was dispersed directly in mineral medium.
In an initial experiment conducted at a concentration of 10 mg C/L, the test failed as the replicate test vessels at day 28 failed the validation criteria. A further test was therefore conducted at a concentration of 10 mg C/L,
An amount of test item (53.1 mg) was dispersed directly in approximately 400 mL of mineral medium, in duplicate, with the aid of ultrasonication for approximately 15 minutes. It was then allowed to cool to a temperature of 24 °C prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 17.7 mg/L, equivalent to 10 mg carbon/L.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Reference Item Preparation
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution was prepared by dissolving a nominal amount of the reference item (1000 mg) directly in mineral medium and the volume adjusted to 1 liter to give a 1000 mg/L stock solution. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium prior to the volume being adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure
homogeneity of the solution.

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 (53.1 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication prior to allowing to cool to a temperature of 24°C 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 17.7 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Preparation of Test System
The following were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of preparation:
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).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 20 °C and 24 °C for 28 days, in darkness. The temperature was recorded daily from a vessel incubated alongside the test containing 3 liters of reverse osmosis water.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 20.9 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. The pH values of the medium were adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid solution prior to the volume in all of the vessels being adjusted to 3 liters by the addition of mineral medium, which had been purged overnight with CO2-free air. The inoculum control vessels were prepared in a similar manner without the addition of test item or reference item.
In order to confirm that the sodium benzoate solution was prepared correctly, a diluted 100 mg/L stock solution (in reverse osmosis water) was sampled for TOC analysis.
The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a moisture trap prior to being passed through a glass column containing self-indicating soda lime (Carbosorb®) granules.
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 deionized reverse osmosis water.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

6

Sampling time:

28 d

Details on results:

Validation Criteria and Biodegradation
The total CO2 evolution in the inoculum control vessels on Day 28 was 37.01 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, 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 <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 a decrease in all replicate vessels with the exception of Inoculum Control R1 and R2. This decrease was considered to be due to sampling/analytical variation.
The IC 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 6% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
The toxicity control attained 40% biodegradation after 14 days and 41% 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.
These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines. After 28 days 71% biodegradation was attained.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 68% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window.
Total organic carbon of the diluted sodium benzoate stock solution confirmed that it had been prepared correctly.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 6% 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

A 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. Carbon dioxide (CO2) evolution from the test item was followed by means of Inorganic Carbon (IC) analysis over a period of 28 days when exposed to sewage sludge micro-organisms under defined conditions. The method followed was designed to be compatible with the 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 and 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 20 °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 6% 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 68% 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.