2,4-diethyl-9H-thioxanthen-9-one

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:

12 September 2017 to 11 OCtober 2017

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)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test material was dissolved in an auxiliary solvent prior to adsorption onto filter paper. High shear mixing was also applied to break up the filter paper containing the test material. Using this method the test material is evenly distributed throughout the test medium and the surface area of test material exposed to the test organisms is increased thereby increasing the potential for biodegradation.
- A nominal amount of test material (1 000 mg) was dissolved in 10 mL of acetone to give a 1 000 mg/10 mL solvent stock solution. An aliquot (396 μL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to inoculated mineral medium. The volume was then adjusted to 3 liters to give a final concentration of 13.2 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
- A filter paper was added to each control vessel in order to maintain consistency between the test and procedure control vessels. Acetone (396 μL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to each vessel.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: A mixed population of activated sewage sludge micro-organisms was obtained on 11 September 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 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 °C 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 (rinsed 3 times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionised 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 2.4 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

13.2 mg/L

Based on:

test mat.

Initial conc.:

10 mg/L

Based on:

other: mg Carbon/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: To 1 litre (final volume) of purified water (Reverse osmosis purified and deionised water, Elga Optima 15+ or Elga Purelab Option R-15 BP) was added 10 mL of Solution A and 1 mL each of Solutions B, C and D.
Solution A: 8.50 g/L KH2PO4, 21.75 g/L K2HPO4, 33.40 g/L Na2HPO4.2H2O and 0.50 g/L NH4Cl; pH = 7.4
Solution B: 27.50 g/L CaCl2
Solution C: 22.50 g/L MgSO4.7H2O
Solution D: 0.25 g/L FeCl3.6H2O (one drop of concentrated HCl/L was added as a preservative)
- A nominal amount of test material (1000 mg) was dissolved in 10 mL of acetone to give a 1000 mg/10 mL solvent stock solution. An aliquot (396 μL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to inoculated mineral medium. The volume was then adjusted to 3 liters to give a final concentration of 13.2 mg/L, equivalent to 10 mg carbon/L.
- The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.
- Test temperature: 22 to 24 °C
- pH: 7.4 ± 0.2
- pH adjusted: Yes using diluted HCl or NaOH prior to the volume in all the vessels being adjusted to 3 litres by the addition of mineral medium which had been purged overnight with CO2 free air. The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.
- Suspended solids concentration: 30 mg (ss)/ L (final concentration per vessel)
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 5 litre test culture vessels each containing 3 litres of solution.
- Number of culture flasks/concentration: 2; the test material was added to inoculated mineral medium to give a final concentration of 10 mg carbon/L.
- Method used to create aerobic conditions: Approximately 24 hours prior to addition of the test and reference materials the vessels were filled with 2400 mL of mineral medium and 34.6 mL of inoculum and aerated overnight. On Day 0 the test and reference materials were added and the volume was then adjusted to 3 L by the addition of mineral medium that had been purged overnight with CO2-free air. 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. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Measuring equipment: Hach HQ40d Flexi handheld pH meter, Shimadzu TOC-VCSH TOC analyser or a Shimadzu TOC-LCSH TOC analyser.
- 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. All samples were analysed for IC immediately.
- 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.
- Sampling method: The samples were analysed for IC using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyser. Samples (50 or 135 μL) were injected into the IC channel of the TOC analyser. 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.
- Sample storage before analysis: All samples were analysed for IC immediately. The remainder of all samples were frozen for further analysis if required.

CONTROL AND BLANK SYSTEM
- Inoculum blank: In duplicate, consisting of inoculated mineral medium plus a filter paper.
- Positive (procedure) control: In duplicate, containing the reference material in inoculated mineral medium plus a filter paper, to give a final concentration of 10 mg carbon/L.
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume 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.
A filter paper was added to each vessel in order to maintain consistency between the test and procedure control vessels. Acetone (396 μL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to each vessel.
- Toxicity control: The test material on a filter paper, plus the reference material in inoculated mineral medium to give a final concentration of 20 mg carbon/L (one vessel only).
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 aliquot (396 μL) of the test item solvent stock solution was dispensed onto a filter paper* and the solvent allowed to evaporate for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to the test vessel containing inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume was adjusted to 3 liters to give a final concentration of 13.2 mg test item/L plus 17.1 mg sodium benzoate/L equivalent to a total of 20 mg carbon/L.
- A filter paper with acetone evaporated to dryness was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.

INORGANIC CARBON (IC)/TOTAL CARBON (TC) RATIO
Samples (30 mL) were removed from the test material vessels on Day 0 prior to the addition of the test material 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 pre-condition 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 material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
The samples were analysed for IC and TC using a Shimadzu TOC-VCPH TOC Analyser. Samples (50 μL) were injected into the TC and IC channels of the TOC analyser. 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 deionised water. Each analysis was carried out in triplicate.

STATISTICAL METHODS
-Calculation of Carbon Content
The theoretical amount of carbon present in the test material was calculated as follows:
[(No. of C atoms x mol wt of C) / mol wt of test material] x 100
[(16 x 12.011) / 254.35] x 100 = 75.56 %
Thus for a concentration of 10 mg C/L the total organic carbon present was 30 mg C.

The theoretical amount of carbon present in the reference material was calculated as follows:
[(No. of C atoms x mol wt of C) / mol wt of sodium benzoate] x 100
[(7 x 12.0110)/144.11] x100 = 58.34 %
Thus for a 10 mg C/L test concentration the total organic carbon present for sodium benzoate was 30 mg C.

-Percentage Biodegradation
The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and reference materials before substitution into the following equation:
%ThCO2 (= % biodegradation*) = [(mg IC in test flask – mg IC in control flask)/ mg TOC added as test chemical] x100
*The conversion factor for carbon to carbon dioxide is 3.67

-Total CO2 Evolution
The total CO2 evolution in the inoculum control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation:
Total CO2 evolution (mg C/L) = mg IC in control x (100 / %C of CO2) x (1 / test volume)
= mg IC in control x (100 / 27.29) x (1 / 3)

Reference substance:

benzoic acid, sodium salt

Test performance:

- The total CO2 evolution in the inoculum control vessels on Day 28 was 25.04 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
- The IC content of the test material 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.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

5

Sampling time:

28 d

Remarks on result:

other: St. dev. not reported.

Details on results:

- 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 material.
- 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 (<3%) into the second absorber vessels occurred.
- The test material attained 5 % 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 material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.
- Percentage biodegradation values of the test and reference materials and the toxicity control are given in Table 1.

Results with reference substance:

Sodium benzoate attained 87 % biodegradation after 14 days and 90 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table 1: Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Material	Toxicity Control
0	0	0	0
2	49	4	25
6	57	0	28
8	84	0	29
10	83	0	39
14	87	0	40
21	87	6	39
28	87	1	39
29*	90	5	41

*Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) cannot be considered to be readily biodegradable.

Executive summary:

The ready biodegradability of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was determined in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and US EPA OCSPP 835.3110 under GLP conditions using the CO2 evolution test.

The study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The test material, 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.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)) and the published literature (Handley et al, 2002), the test material 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 material was evenly distributed throughout the test medium and the surface area of test material exposed to the test organisms is increased thereby increasing the potential for biodegradation.

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

The test material attained 5 % 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 material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 87 % biodegradation after 14 days and 90 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Under the conditions of the study the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) attained 5 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable.

Description of key information

- 5 % biodegradation after 28 days [using the CO2 evolution test of OECD 301B, EU Method C.4-C and US EPA OCSPP 835.3110; GLP; test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9)];

- Based on the results for 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9), the structurally related registration substance 2,4-diethyl-9H-thioxanthen-9-one (CAS 82799-44-8, EC 280-041-0) cannot be considered to be readily biodegradable as well.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

No biodegradation studies are available for the registration substance 2,4-diethyl-9H-thioxanthen-9-one ("DETX", CAS 82799-44-8, EC 280-041-0) itself.

However, reliable data of the structurally related substance 2-Isopropylthioxanthone ("ITX", CAS 5495-84-1, EC 226-827-9) can be used to adress the endpoint, which is entirely appropriate to draw conclusions on the ready biodegradability of DETX.

The only difference between the source substance ITX and the target substance DETX can be reduced to slightly differing substituents: ITX possesses one isopropyl group, whereas DETX two ethyl groups.

The ready biodegradability of the test material ITX was determined in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and US EPA OCSPP 835.3110 under GLP conditions using the CO2 evolution test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The test material, 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.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)) and the published literature (Handley et al, 2002), the test material 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 material was evenly distributed throughout the test medium and the surface area of test material exposed to the test organisms is increased thereby increasing the potential for biodegradation.

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

The toxicity control attained 40 % biodegradation after 14 days and 41 % biodegradation after 28 days thereby confirming that the test material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 87 % biodegradation after 14 days and 90 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Under the conditions of the study the test material 2-Isopropylthioxanthone ("ITX", CAS 5495-84-1, EC 226-827-9) attained 5 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

[Type of water: freshwater]