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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 December 2017 to 17 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Samples were taken from the control and the 100 % v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis.
- All samples were stored frozen prior to analysis.
- Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
- Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document, therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
- As a result of the trial the test material was prepared using a saturated solution method of preparation.
- A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre condition the filter) to give a 100 % v/v saturated solution.
- The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
- For the ranfe-finding study a series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.
- The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 48 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: In house
- Age: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Feeding during test: No

ACCLIMATION
- Acclimation conditions: Adult daphnids were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a cycle of 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods. Culture conditions ensured that reproduction was by parthenogenesis.
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. - Feeding frequency: Daily

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

20 - 22 °C

pH:

7.9 - 8.0

Dissolved oxygen:

8.7 - 9.0 mg O2/L

Nominal and measured concentrations:

Nominal: 100 % v/v Saturated Solution
0-Hour Measured test concentration: 0.028 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers.
- Type: The test vessels were covered to reduce evaporation.
- Material, size, headspace, fill volume: 100 mL of test preparation.
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water, Elendt M7, contained the following substances in mg/L: H3BO3 0.715, MnCl2.4H2O 0.090, LiCl 0.077, RbCl 0.018, SrCl2.6H2O 0.038, NaBr 0.004, Na2MoO4.2H2O 0.016, CuCl2.2H2O 0.004, ZnCl2 0.013, CoCl2.6H2O 0.010, KI 0.0033, Na2SeO3 0.0022, NH4VO3 0.00058, Na2EDTA.2H2O 0.625, FeSO4.7H2O 0.249, CaCl2.2H2O 293.8, NaHCO3 64.8, MgSO4.7H2O 123.3, Na2SiO3.9H2O 10, KCl 5.8, NaNO3 0.274, K2HPO4 0.184, KH2PO4 0.143, Thiamine hydrochloride 0.075, Cyanocobalamine (vitamin B12) 0.0010 and D(+) biotin (vitamin H) 0.00075.
- Water quality assessments: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods
- Light intensity: 200 to 1200 Lux

VEHICLE CONTROL PERFORMED: Yes. The control group was maintained under identical conditions but not exposed to the test item.

EFFECT PARAMETERS MEASURED:
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 % v/v saturated solution.
- Results used to determine the conditions for the definitive study: Yes. Based on the results of the range finding test a "limit test" was conducted at a concentration of 100 % v/v saturated solution to confirm that at highest attainable test concentration no immobilisation or adverse reactions to exposure were observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.028 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 0.028 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST
- No immobilisation was observed in any test concentrations.
- No sub-lethal effects of exposure were observed throughout the test.
- Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the LOQ of the analytical method employed were obtained which was determined to be 0.0068 mg/L. This does not infer that no test material was in solution, just that any dissolved test material present was at a concentration of less than the LOQ.

DEFINITIVE TEST
- Verification of Test Concentrations: Analysis of the test preparations at 0 hours showed a measured test concentration of 0.028 mg/L. There was no significant change in the measured concentration at 48 hours and so the results are based on the 0 hour measured test concentration only.
- Immobilisation Data: There was no significant immobilisation in 20 daphnids exposed to a test concentration of 0.028 mg/L for a period of 48 hours. Inspection of the immobilisation data gave the following results: 24 and 48 hour EC50 > 0.028 mg/L. The No Observed Effect Concentration after 24 and 48 hours exposure was 0.028 mg/L.
- No sub lethal effects of exposure were observed throughout the test.
- Validation Criteria: The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.
- Water Quality Criteria: Temperature was maintained at 20 to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 910 to 1130 Lux.
- Observations on Test Material Solubility: At the start and throughout the test the control and test solutions were observed to be clear colourless solutions.

Results with reference substance (positive control):

- A positive control used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
- 24h: EC50: 0.79 mg/L (0.73 - 0.86 mg/L), NOEC: 0.56 mg/L and LOEC: 1.0 mg/L.
- 48h: EC50: 0.75 mg/L (0.56 - 1.0 mg/L), NOEC: 0.56 mg/L and LOEC: 1.0 mg/L.
- The NOEC is based upon equal to or less than 10% immobilization at this concentration.
- The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:

An estimate of the EC50 values was given by inspection of the immobilisation data.

For the positive control, analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations.

Table 1: Cumulative Immobilisation Data and Observations in the Definitive Test After 24 Hours

Nominal Concentration (% v/v Saturated Solution)	0-Hour Measured test concentration (mg/L)	24 Hours
		Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	-	0	0	0	0	0	0	5N	5 N	5 N	5 N
100	0.028	0	1	0	0	1	5	5 N	4 N	5 N	5 N

R = Replicate

N = Normal

Table 2: Cumulative Immobilisation Data and Observations in the Definitive Test After 48 Hours

Nominal Concentration (% v/v Saturated Solution)	0-Hour Measured test concentration (mg/L)	48 Hours
		Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	-	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0.028	0	1	0	0	1	5	5 N	4 N	5 N	5 N

R = Replicate

N = Normal

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the acute toxicity of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) to the freshwater invertebrate Daphnia magna has been investigated and based on the 0 Hour measured test concentration gave a 48 hour EC50 value of greater than 0.028 mg/L. The NOEC was 0.028 mg/L.

Executive summary:

The acute toxicity of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions using an acute daphnia immobilisation study.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.025 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100 % v/v saturated solution for 48 hours at a temperature of 20 to 22 °C under static test conditions. The test material solution was prepared by stirring an excess (50 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution of the test material. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparation at 0 hours showed a measured test concentration of 0.028 mg/L. There was no significant change in the measured concentration at 48 hours and so the results are based on the 0 Hour measured test concentration only.

This study showed that there were no toxic effects at saturation.

Under the conditions of this study the acute toxicity of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) to the freshwater invertebrate Daphnia magna has been investigated and based on the 0 Hour measured test concentration gave a 48 hour EC50 value of greater than 0.028 mg/L. The NOEC was 0.028 mg/L.

Description of key information

- (48h) EC50 for freshwater invertebrate D. magna: > 0.028 mg/L "100 % v/v saturated solution" (measured, based on mobility) [OECD 202; GLP; test material: 2-Isopropylthioxanthone (CAS 5495-84 -1, EC 226-827-9)];

- (48h) NOEC for freshwater invertebrate D. magna: > 0.028 mg/L "100 % v/v saturated solution" (measured, based on mobility) [OECD 202; 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 acute toxicity of the registration substance 2,4-diethyl-9H-thioxanthen-9-one (CAS 82799-44-8, EC 280-041-0) to aquatic invertebrates is considered to be no effects at the water solubility limit (saturation).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.028 mg/L

Additional information

No studies for aquatic invertebrates 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 closely related substance 2-Isopropylthioxanthone ("ITX", CAS 5495-84-1, EC 226-827-9) can be used to address the endpoint, which is entirely appropriate to draw conclusions on the toxicity of DETX to aquatic invertebrates.

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 acute toxicity of the structurally related test material ITX to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions using an acute daphnia immobilisation study. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.025 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100 % v/v saturated solution for 48 hours at a temperature of 20 to 22 °C under static test conditions. The test material solution was prepared by stirring an excess (50 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution of the test material. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparation at 0 hours showed a measured test concentration of 0.028 mg/L. There was no significant change in the measured concentration at 48 hours and so the results are based on the 0 Hour measured test concentration only.

This study showed that there were no toxic effects at saturation.

Under the conditions of this study the acute toxicity of the test material 2-Isopropylthioxanthone ("ITX", CAS 5495-84-1, EC 226-827-9) to the freshwater invertebrate Daphnia magna has been investigated and based on the 0 Hour measured test concentration gave a 48 hour EC50 value of greater than 0.028 mg/L. The NOEC was 0.028 mg/L.