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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Nov 2018 to 15 Jan 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

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 the limit concentration 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=96 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.

Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already 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 visually not completely dissolved 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 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, the saturated solutions were clear and yellow, whereas the lower concentrations in the range-finding test were clear and ranged from colourless to light yellow with increasing test item concentration.

Any residual volumes were discarded.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
Mean length(1): Range-finding test: 3.5 ± 0.3 cm
Final test: 3.5 ± 0.3 cm
Mean weight(1): Range-finding test: 0.53 ± 0.16 g
Final test: 0.53 ± 0.14 g
Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 26

(1) Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.

Quarantine/Acclimatisation: At least 12 days after delivery
Medium: Adjusted ISO medium, formulated using RO-water (tap-water
purified by reverse osmosis; GEON Waterbehandeling, Berkel-
Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Measurements Conductivity, pH, nitrate, nitrite and ammonia concentration: Once a week
Temperature: Continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality parameters: Were kept within the optimum limits for the respective fish species.
Feeding: Daily with pelleted fish food (Essence 300-500 µm, Alltech® Coppens, Helmond, The Netherlands)
Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

180 mg/L (expressed as CaCO3).

Test temperature:

21°C

pH:

7.7 ± 0.3

Dissolved oxygen:

7.2-9.8 mg/L

Salinity:

Not applicable

Conductivity:

Not measured

Nominal and measured concentrations:

Saturated solution prepared at a loading rate of 100 mg/L.

Details on test conditions:

A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
-Three fish per concentration were exposed to solutions containing 0.10, 1.0, 10, and 100% of the SS prepared at a loading rate of 100 mg/L.
-Dissolved oxygen concentrations, pH and temperature were only measured in the lowest and the highest test concentration.
-Samples for possible analysis were taken from all test concentrations at the start, after 24 hours, and at the end of the test.

Test duration: 96 hours
Test type: Static
Test vessels: 10 L all-glass aquaria, containing 9 litres of test solution

Number of fish: 7 per concentration
Loading: 0.41 g fish/L, i.e. 7 fish in 9 litres of test medium
Illumination: The test was performed under dimmed light conditions due to light sensitivity of the test item
Aeration: The test media were not aerated during the test
Feeding: No feeding from 24 hours prior to the test and during the total test period
Introduction of fish: Within 30 minutes after preparation of the test solutions from a holding medium with comparable water quality parameters, i.e. pH and temperature between test solutions and the holding medium differed less than 1.0 unit and 1.0°C.

Measurements and Recordings
Mortality and other effects: At 3¼, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish. Dead fish were removed when observed.

Dissolved oxygen content, pH and temperature: Daily in all vessels with surviving fish, beginning at the start of the test (day 0).

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Results with reference substance (positive control):

See Appendix 1.

Reported statistics and error estimates:

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

Sublethal observations / clinical signs:

Range-Finding test

No mortality or clinical effects were observed at any of the test concentrations throughout the exposure period.

Based on this result, samples taken from the highest test concentration were analysed. Despite the filtration step, the actual exposure concentrations were at the level of nominal (i.e. 98–99% relative to the loading rate) throughout the test (see also Table 2 of the appended Analytical Report)

All test conditions were maintained within the limits prescribed by the study plan.

Limit Test

Measured Test Item Concentrations

The results of analysis of the samples taken during the final test are described in Table 3 of the appended Analytical Report.

Samples taken from the limit concentration were analysed. The measured concentration in the undiluted SS was 114 mg/L throughout the test. Since the actual exposure concentration was at the level of nominal throughout the test, it can be concluded that the undissolved material removed during preparation of test solutions was not test item related.

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

Mortality and Other Effects

Table 1 shows the mortality data recorded during the limit test. The obtained results were in agreement with the results of the range-finding test. No mortality or clinical effects were observed in the control and at the limit concentration throughout the test.

Table 1

Incidence of Mortality and Total Mortality During the Limit Test

Test item Nominal conc. (mg/L)	Initial Number of fish	Cumulative mortality					Total Mortality (%)
		3¼h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
100	7	0	0	0	0	0	0

Determination of Effect Concentrations

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

Table 2

Effect Parameters

Parameter	Concentration CMTX 2-carboxymethyloxy-thioxanthone Nominal conc. (mg/L)
24h, 48h, 72h, 96h-LC50	>100

Experimental Conditions

The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 3. The temperature of all test solutions was maintained at 21°C throughout the test (the temperature of the holding tank medium was 21°C upon test initiation). All test conditions remained within the ranges prescribed by the study plan (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation).

Table 3

pH and Oxygen Concentrations (mg/L) During the Limit Test

Test item Nominal conc. (mg/L)	Day 0		Day 1		Day 2		Day 3		Day 4
	pH	O2	pH	O2	pH	O2	pH	O2	pH	O2
Control	7.9	9.8	7.4	7.2	7.6	6.8	7.6	6.4	7.4	6.3
100	7.8	9.8	7.4	7.7	7.5	7	7.5	6.8	7.4	6.6

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 96h-LC50 for carp (Cyprinus carpio) exposed to CMTX 2-carboxymethyloxy-thioxanthone was beyond the maximum concentration 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 its ability to generate acute toxic effects in carp (Cyprinus carpio) during an exposure period of 96 hours and, if possible, to determine the LC50 at all observation times.

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, procedures were based on the test methods described in the OECD series on 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 visually not completely dissolved in test medium at the loading rate initially prepared. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest test concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. Test solutions were prepared under dimmed light conditions to minimize exposure to light.

A limit test was performed based on the results of a preceding range-finding test. Seven fish per group (single tank, non-replicated) were exposed to an untreated control and to the SS prepared at a loading rate of 100 mg/L. Since the test item was light sensitive, exposure was performed under dimmed light conditions. The total exposure period was 96 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

No mortality or clinical effects were observed in the control and at the limit concentration throughout the test.

Samples taken from the limit concentration were analysed. The measured concentration in the undiluted SS was 114 mg/L throughout the test. Based on this result, 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 96h-LC₅₀ for carp (Cyprinus carpio) exposed to CMTX 2-carboxymethyloxy-thioxanthone was beyond the maximum concentration tested, i.e. exceeded the regulatory limit concentration 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 fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

96h-LC₅₀ > 100 mg/L