(Z)-α-[[2-(tert-butoxy)-1,1-dimethyl-2-oxoethoxy]imino]-2-(tritylamino)thiazol-4-acetic acid

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:

16 January 2018 to 14 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-α-[[2-(tert-butoxy)-1,1-dimethyl-2-oxoethoxy]imino]-2-(tritylamino)thiazol-4-acetic acid

EC Number:

272-056-6

EC Name:

(Z)-α-[[2-(tert-butoxy)-1,1-dimethyl-2-oxoethoxy]imino]-2-(tritylamino)thiazol-4-acetic acid

Cas Number:

68672-66-2

Molecular formula:

C32H33N3O5S

IUPAC Name:

(2Z)-2-({[1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl]oxy}imino)-2-{2-[(triphenylmethyl)amino]-1,3-thiazol-4-yl}acetic acid

Test material form:

solid: particulate/powder

Details on test material:

white powder

Specific details on test material used for the study:

Physical state/Appearance: white powder
Batch: 14TATT-S01025C
Purity: 100.1% w/w
Expiry Date: 24 December 2017
Storage Conditions:room temperature, over silica gel in darkness

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 15 January 2017 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 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. The suspended solids concentration was equal to 2.8 g/L prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

ca. 10 other: mg carbon/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 15 January 2017 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 preweighed 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 2.8 g/L prior to use.

Medium
The mineral medium used in this study (see Annex 3) 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).

Sampling
IC Analysis
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. All samples were analyzed for IC immediately. The remainder of all samples with the exception of all the Day 0 and Day 29 Absorber 2 samples were frozen for further analysis if required. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29. The samples were analyzed for IC using either a Shimadzu TOC-V CSH TOC analyzer or a Shimadzu TOC-L CSH TOC analyser. Samples (50 or 135 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 at least triplicate with 3 replicates being used in the calculation/reported.

IC/TC Ratio
Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item in order to calculate the IC content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to precondition the filter) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels
on Day 0 and filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. IC/TC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water. The samples were analyzed for IC and TC using a Shimadzu TOC-V CSH TOC analyzer and a Shimadzu TOC-V
CPH TOC Analyzer. Samples (50 µL) were injected into the TC and IC channels of the TOC analyzer. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in at least triplicate with 3 replicates being used in the calculation/reported.

Reference substance

Reference substance:

other: Sodium benzoate

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 1

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 74% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 101% biodegradation was attained. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 1% 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 was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. 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 and 24 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

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 1% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.