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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

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:

Frequency at t=0 h and t=72 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.

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.

In the combined limit/range-finding test, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

In the final test, 30 mL test solution was added per replicate since a different type of test vessel was used (see paragraph 4.8.2.2). Each replicate was inoculated with 0.6 mL algal suspension to obtain an algal cell density of 10^4 cells/mL.

Any residual volumes were discarded.

Control: Test medium without test item or other additives.
Replicates: 3 replicates of each test concentration,
6 replicates of the control,
1 extra replicate of each test group for sampling purposes after
24 hours of exposure,
1 or 2 replicates of each test concentration without algae.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh Water Algae Culture: Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

(Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 to 23°C

pH:

pH 8.1 ± 0.2

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

2-carboxymethyloxy-thioxanthone %SS prep. at a loading rate of 100 mg/L
Nominal concentrations: 0.24, 1.1, 4.9, 4.9*, 22, 100

Measured concentration (mg/L):

t=0h: 0.24, 1.1, 4.8, 4.9*, 22, 99
t=72h: 0.15, 0.70, 3.4, 3.5*, 19, 93

Average exposure conc. (mg/L): 0.19, 0.86, 4.0, 4.1*, 21, 96

* solution incubated without algae.

Reference substance (positive control):

yes

Remarks:

Potassium DIchromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Statistical significance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Biological relevance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Statistical significance and biological relevance

Results with reference substance (positive control):

See Appendix 5

Combined Limit/Range-Finding Test

The mean cell densities measured during the combined limit/range-finding test are presented in Table 1. Table 2 and Table 3 present the percentages growth rate inhibition and yield inhibition per concentration, respectively.

Inhibition of growth rate and yield increased with increasing concentration of CMTX 2-carboxymethyloxy-thioxanthone, resulting in 65% growth rate and 97% yield inhibition at the highest test concentration.

Based on these results, samples taken from solutions containing 0.10 and 100% of the SS were analysed. The concentration measured in the undiluted filtrate was between 95 and 97 mg/L throughout the test. The concentration measured in the lowest test concentration was 0.091 mg/L at the start of the test and 0.063 mg/L at the end of the exposure (i.e. 68% relative to nominal, see also Table 2 of the appended Analytical Report).

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

Table 1

Mean Cell Densities (x10⁴ Cells/mL) during the Combined Limit/Range-Finding Test

Time (h)	CMTX 2-carboxymethyloxy-thioxanthone %SS prep. at a loading rate of 100 mg/L
	Control	0.1	1	10	100
0	1.0	1.0	1.0	1.0	1.0
24	4.4	n.d.	n.d.	n.d.	2.9
48	37	n.d.	n.d.	n.d.	4.1
72	195	177	107	22	6.4

Table 2

Percentage Inhibition of Growth Rate during the Combined Limit/Range-Finding Test

CMTX 2 -carboxymethyloxy-thioxanthone %SS prep. at a loading rate of 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.757	0.0237	6
0.1	1.726	0.0174	3	1.8
1.0	1.556	0.0111	3	11
10	1.036	0.0187	3	41
100	0.615	0.0684	6	65

Table 3

Percentage Inhibition of Yield during the Combined Limit/Range-Finding Test

CMTX 2-carboxymethyloxy-thioxanthone %SS prep. at a loading rate of 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	193.9	14.1	6
0.1	176.3	9.38	3	9.1
1.0	105.6	3.58	3	46
10	21.4	1.27	3	89
100	5.4	1.3	6	97

Final 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 all test concentrations and the control were analysed. The measured concentrations at the start of the test were at the level of the nominal loading rate and dilutions thereof (i.e. 97-101% relative to nominal loading rate). Measured concentrations were at the level of 64 to 94% of the initial concentrations at the end of the test.

It should be noted that a test item response was observed in the control sample taken at the end of the test. Since no test item was detected in the control at the start of the test, the test solutions in the exposure vessels contained most likely no test item. Introduction of test item to the control sample rather occurred during sampling or sample pretreatment.

Based on these results, the average exposure concentrations were calculated and used to express effect parameters (see Table 4).

The concentrations measured in the solution incubated without algae were comparable to its counterpart incubated with algae. Hence, it can be stated that the presence of the algae had no effect on the test item concentrations over time.

Table 4

Determination of Average Exposure Concentrations

Test item^1 %SS prep. at a loading rate of 100 mg/L	Measured concentration (mg/L)		Average exposure conc. (mg/L)
	t=0h	t=72h
0.24	0.24	0.15	0.19
1.1	1.1	0.7	0.86
4.9	4.8	3.4	4.0
4.9*	4.9*	3.5*	4.1*
22	22	19	21
100	99	93	96

¹CMTX 2-carboxymethyloxy-thioxanthone, *solution incubated without algae.

Mean Cell Densities

Figure 1 shows growth curves at different concentrations of CMTX 2-carboxymethyloxy-thioxanthone. The individual and group mean cell densities measured at 24h intervals are given in Table 10.

Inhibition of Growth Rate and Inhibition of Yield

Table 5 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 6 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 7 (see Appendix 1 for the individual values). Statistical analysis of the data is shown in Appendix 2 and Appendix 3.

Inhibition of growth rate and yield increased with increasing concentration of CMTX 2-carboxymethyloxy-thioxanthone from average exposure concentrations of 0.86 mg/L upwards, resulting in 84% growth rate and 99% yield inhibition at 21 mg/L. However, growth inhibition and yield were inhibited by 56 and 93%, respectively, at the highest concentration of 96 mg/L.

It should be noted that the cell densities recorded at 21 mg/L varied considerably between replicates (CV was 88% for growth rate and 107% for yield inhibition) and therefore, are slightly less reliable. As during the combined limit/range-finding test comparable inhibition of growth rate (i.e. 65%) was observed at the undiluted SS at which similar concentrations were measured (i.e. 95 – 97 mg/L). For this reason, the values obtained in the undiluted SS seem to be more reliable. In order to prevent a bias, all values were included in thecalculations of EC_x-values.

Statistically significant growth rate and yield inhibition was found at average exposure concentrations of 0.86 mg/L and higher. Growth rate inhibition found at 0.86 mg/L was however below 10%, and therefore, considered to be biologically not relevant. The NOEC for growth rate inhibition was thus 0.86 mg/L based on biological relevance.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to an average exposure concentration of 4.0 mg/L when compared to the control.

Table 5

Growth Rate and Percentage Inhibition for the Total Test Period

CMTX 2-carboxymethyloxy-thioxanthone Average exposure conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.484	0.0324	6
0.19	1.488	0.0185	3	-0.24
0.86	1.373	0.0204	3	7.5#
4	0.971	0.0245	3	35*
21	0.231	0.2024	3	84*
96	0.656	0.0895	3	56*

* Effect was statistically significant, # effect was statistically significant but biologically not relevant (<10%).

Table 6

Growth Rate and Percentage Inhibition at Different Time Intervals

Test item^1 Average exposure conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.247	1.54	1.665
0.19	3	1.047	16	1.748	-13	1.667	-0.13
0.86	3	0.000	100	2.625	-70	1.492	10
4.0	3	1.000	20	1.136	26	0.776	53
21	3	0.753	40	-0.287	119	0.227	86
96	3	0.496	60	1.636	-6.2	-0.164	110

¹CMTX 2-carboxymethyloxy-thioxanthone

Table 7

Yield and Percentage Inhibition for the Total Test Period

CMTX 2-carboxymethyloxy-thioxanthone Average exposure conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	85.1	8.46	6
0.19	85.8	4.74	3	-0.77
0.86	60.5	3.71	3	29*
4.0	17.4	1.36	3	80*
21	1.3	1.34	3	99*
96	6.3	1.81	3	93*

* Effect was statistically significant.

Determination of Effect Concentrations

Table 8 shows the effect parameters based on average exposure concentrations, see also Appendix 4.

Table 8

Effect Parameters

Parameter (mg/L)		NOEC*	NOEC#	EC10	EC20	EC50
Growth rate	Value	0.19	0.86	0.39	1.24
	lower 95%-cl			0.21	0.8	8.8
	upper 95%-cl			0.62	1.7	14
Yield	Value	0.19	0.19	0.39	0.63	1.6
	lower 95%-cl			0.34	0.57	1.5
	upper 95%-cl			0.44	0.69	1.7

cl – confidence limit, * based on statistical significance, # based on biological relevance.

Experimental Conditions

Table 9 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within ± 1°C).

Table 9

pH Levels Recorded during the Final Test

CMTX 2-carboxymethyloxy-thioxanthone Average exposure conc. (mg/L)	pH
	t=0h	t=72h
Control	7.9	7.9
0.19	7.9	7.8
0.86	7.9	7.8
4.0	7.8	7.7
21	7.8	7.6
96	7.5	7.5

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), CMTX 2-carboxymethyloxy-thioxanthone inhibited growth rate and yield of this fresh water algae species significantly at average exposure concentrations of 0.86 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was 11 mg/L with a 95% confidence interval ranging from 8.8 to 14 mg/L.

The 72h-EC50 for yield inhibition (EYC50) was 1.6 mg/L with a 95% confidence interval ranging from 1.5 to 1.7 mg/L.

The 72h-NOEC for growth rate inhibition was 0.19 mg/L based on statistical significance and 0.86 mg/L based on biological relevance.

The 72h-NOEC for yield inhibition was 0.19 mg/L based on both statistical significance and biological relevance.

Executive summary:

The objective of the study was to evaluate CMTX 2-carboxymethyloxy-thioxanthone for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀, EC₂₀ and EC₅₀ for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. 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 not completely soluble 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 and glassware was wrapped in aluminium foil to minimize exposure to light.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing 0.24, 1.1, 4.9, 22, and 100% of the SS prepared at a loading rate of 100 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start and end of the test.

Samples taken from all test concentrations and the control were analysed. Based on these results, average exposure concentrations were 0.19, 0.86, 4.0, 21 and 96 mg/L in solutions containing 0.24, 1.1, 4.9, 22, and 100% of the SS, respectively.

Inhibition of growth rate and yield increased with increasing concentration of CMTX 2-carboxymethyloxy-thioxanthone from average exposure concentrations of 0.86 mg/L upwards, resulting in 84% growth rate and 99% yield inhibition at 21 mg/L. However, growth inhibition and yield were inhibited by 56 and 93%, respectively, at the highest concentration of 96 mg/L. Statistically significant growth rate and yield inhibition was found at average exposure concentrations of 0.86 mg/L and higher. Growth rate inhibition found at 0.86 mg/L was below 10%, and therefore, considered biologically not relevant.

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

The effect parameters (based on average exposure concentrations) obtained in this study are summarized in the table below.

Parameter (mg/L)		NOEC^*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	0.19	0.86	0.39	1.24	11
	lower 95%-cl			0.21	0.8	8.8
	upper 95%-cl			0.62	1.7	14
Yield	Value	0.19	0.19	0.39	0.63	1.6
	lower 95%-cl			0.34	0.57	1.5
	upper 95%-cl			0.44	0.69	1.7

cl – confidence limit, ^*based on statistical significance, ^#based on biological relevance.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC50 for freshwater algae:

11 mg/L