[(9-oxo-9H-thioxanthen-2-yl)oxy]acetic acid

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:

30 Nov 2018 to 13 Dec 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:

Adopted April 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals

Version / remarks:

OECD series on testing and assessment number 23, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 97.75%
Test item storage: At room temperature protected from light desiccated

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept for possible analysis. The method of analysis is described in the appended Analytical Report (Appendix 2).

Frequency: at t=0 h and t=48 h
Volume: 2.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Additionally, reserve samples of 2.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

The batch of CMTX 2-carboxymethyloxy-thioxanthone tested was a yellow powder with a purity of 97.75% and was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item. Test solutions were prepared under dimmed light conditions and glassware was wrapped in aluminium foil to minimize exposure to light.

Preparation of test solutions started with a loading rate of 100 mg/L applying a 15-minute period of ultrasonic treatment followed by three days of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. At the end of the preparation procedure, all test solutions were clear with a gradient of coloration from colorless to yellow, increasing with concentration of the test item.

Any residual volumes were discarded.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.

Source: In-house laboratory culture with a known history.

Reason for selection: This system has been selected as an internationally accepted invertebrate species.

Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%(1), presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

Characteristics: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Breeding: Start of each batch Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all-glass culture vessel.

(1)Based on OECD Guideline 211. "Daphnia magna Reproduction Test", Adopted October 02, 2012.

Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation, half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Culture medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

The following salts and vitamins were added to freshly prepared test medium (see section 4.8.2) to reach the following concentrations:



Salts: H3BO3 0.71 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.090 mg/L
LiCl 0.076 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.038 mg/L
Na2MoO4.2H2O 0.015 mg/L
NaBr 0.0040 mg/L
CuCl2.2H2O 0.0042 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0032 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00057 mg/L
Na2EDTA.2H2O 0.62 mg/L
Na2SiO3.5H2O 7.5 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L

Vitamins: Thiamine hydrochloride 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

Hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

18-22 °C

pH:

Between 6 and 9

Dissolved oxygen:

8.7-9.4 mg/L

Salinity:

Not applicable

Conductivity:

Not measured

Nominal and measured concentrations:

Solutions containing 0.10, 1.0, 10, and 100% of the SS prepared at a loading rate of 100 mg/L.

Details on test conditions:

A combined limit/range-finding test was performed. Based on the obtained results, no further testing was required.

Test duration: 8 hours
Test type: Static
Test vessels: 60 mL, all-glass.
Test medium: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Number of daphnids: Control and limit concentration: 20 per test group
Lower concentrations: 10 per concentration
Loading: 5 per vessel containing 50 mL of test solution.
Light: The test was performed in the dark due to the light sensitivity of the test item.
Feeding: No feeding
Aeration: No aeration of the test solutions was applied.
Introduction of daphnids: Within 5 minutes after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Merck, Darmstadt, Germany) - See Appendix 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

See Appendix 2, "Reference Test", attached below.

Reported statistics and error estimates:

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Measured Test Item Concentrations

The results of analysis of the samples taken during the test are described in Table 2 of the appended Analytical Report.

Samples taken from the highest test concentration were analysed. Despite the filtration step, the measured concentrations were at the level of nominal (i.e. 86 – 88% relative to the loading rate) throughout the test.

Based on this result, effect parameters were expressed in terms of the analytically confirmed nominal concentrations.

Immobility

Table 1 shows the responses recorded during the test. No immobility was observed in the control and at any of the test concentrations throughout the exposure period. The results of this study allowed for reliable determination of the EC₅₀-values.

Table 1

Number of Introduced Daphnids and Incidence of Immobility

Time (h)	Replicate	CMTX 2-carboxymethyloxy-thioxanthone %SS prep. at a loading rate of 100 mg/L
		Control	0.10	1.0	10	100
0	A	5	5	5	5	5
	B	5	5	5	5	5
	C	5				5
	D	5				5
	Total introduced	20	10	10	10	20
24	A	0	0	0	0	0
	B	0	0	0	0	0
	C	0				0
	D	0				0
	Total immobilised	0	0	0	0	0
	Effect %	0	0	0	0	0
48	A	0	0	0	0	0
	B	0	0	0	0	0
	C	0				0
	D	0				0
	Total immobilised	0	0	0	0	0
	Effect %	0	0	0	0	0

Determination of Effect Concentrations

Table 2 shows the effect parameters based on the analytically confirmed nominal concentrations.

Table 2

Effect Parameters

Parameter	CMTX 2-carboxymethyloxy-thioxanthone Nominal conc. (mg/L)
24h, 48h-EC50	>100

Experimental Conditions

The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 3. These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: 3 mg/L at the end of the test).

The temperature continuously measured in a temperature control vessel varied between 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within ±1°C).

Table 3

pH and Oxygen Concentrations (mg/L)

CMTX 2-carboxymethyloxy-thioxanthone Nominal conc. (mg/L)	Start (t=0 h)		End (t=48 h)
	O2	pH	O2	pH
Control	9.6	7.9	9.3	7.9
100	8.7	7.9	9.4	7.9

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to CMTX 2-carboxymethyloxy-thioxanthone was beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L.

Executive summary:

The objective of the study was to evaluate CMTX 2-carboxymethyloxy-thioxanthone for itsability to generate acute toxic effects on the mobility of Daphnia magna during an exposureperiod of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202,2004. In addition, procedures were based on the test methods described in the OECD serieson testing and assessment number 23, 2000.

The batch of CMTX 2-carboxymethyloxy-thioxanthone tested was a yellow powder with a purity of 97.75% and was not completely soluble in test medium at the loading rate initiallyprepared. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used asthe highest test concentration. Lower concentrations were prepared by diluting the highestconcentration in test medium. Test solutions were prepared under dimmed light conditionsand glassware was wrapped in aluminium foil to minimize exposure to light.

A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to the SS prepared at aloading rate of 100 mg/L. In addition, ten daphnids per group (5 per replicate, duplicate) wereexposed to solutions containing 0.10, 1.0, and 10% of the SS. Since the test item was lightsensitive, exposure was performed in the dark. The total exposure period was 48 hours andsamples for analytical confirmation of exposure concentrations were taken at the start and atthe end of the test.

No immobility was observed in the control and at any of the test concentrations throughout the exposure period.

Samples taken from the highest test concentration were analysed. Despite the filtration step, the measured concentrations were at the level of nominal (i.e. 86 – 88% relative to the loading rate) throughout the test. Hence, effect parameters were expressed in terms of the analytically confirmed nominal concentration of 100 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EC₅₀ for Daphnia magna exposed to CMTX 2-carboxymethyloxy-thioxanthone was beyond the range of concentrations tested, i.e. exceeded the regulatory limitconcentration of 100 mg/L.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

EC₅₀ > 100 mg/L