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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Nov 2018 to 04 Jan 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

adopted 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25%
Test item storage: At room temperature protected from light desiccated

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Preparation of Test Solutions:
Test Item – Preparation and Test Concentrations:
The batch of CMTX 2-carboxymethyloxy-thioxanthone tested was a yellow powder with a purity of 98.25% (by area). No correction was made for the purity/composition of the test item. Preparation was as much as possible performed under low light conditions.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the amber coloured test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item – Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required concentrations (see Table 1, Table 2 and Table 3). Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.

Any residual volumes were discarded.

Reference Item – Preparation and Test Concentrations
The batch of activated sludge was checked for sensitivity by testing the reference item 3,5-dichlorophenol.

A 1.0 g/L stock solution was prepared by dissolving 250.5 mg 3,5-dichlorophenol in
Milli-RO water and making up to a total volume of 250 mL. The pH as used for the test was 7.8 in both full tests. The 3,5-dichlorophenol stock solution was stored in a freezer (≤-15°C) until use. The reference item solution was defrosted at room temperature and diluted to reach the test concentrations. Volumes of 0.5, 1.6, 5.0 and 16 mL from the stock solution were added to the test medium, resulting in the final test concentrations of 1.0, 3.2, 10 and 32 mg/L, respectively.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test system: Micro-organisms in activated sludge.

Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas','s -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Preparation of the sludge: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The concentration of the suspended solids was determined (3.0 g/L, as used for the test). The pH was 8.0 and 7.3 on the day of testing for the first and second full test, respectively. The batch of sludge was used one day after collection. Therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

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

Rationale Recognized by international guidelines as the recommended test system.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Approximately 10 minutes

Hardness:

Not reported

Test temperature:

20 ± 2°C

pH:

7.5 ± 0.5

Dissolved oxygen:

The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

First Full test: 4.8, 15, 49, 156 and 500 mg/L
Second Full Test: 3.2, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

Contact time: 3 hours, during which aeration and stirring took place.
Vessels: All glass open bottles/vessels.
Milli-RO / Milli-RO water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
Synthetic medium (=sewage feed): 16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

Air supply: Clean, oil-free air.

Performance of the test: The synthetic medium (16 mL) made up to 50 mL with Milli- RO and 200 mL test item solution were mixed (total volume 250 mL). The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test.
After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls.
The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.

Oxygen recording Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

254 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity.

Combined Limit/Range-Finding Test

Detailed study results are presented in Table 9 of Appendix 1.

The combined limit/range-finding showed 10%, 41% and 91% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC₅₀ was at a concentration between 100 and 1000 mg/L.

There was no oxygen uptake from abiotic processes.

The temperature continuously measured in the temperature control vessels ranged between 19.3 and 23.5°C during the test, and was slightly outside the range prescribed by the study plan (20 ± 2°C, see also Appendix 6).

Except for the temperature, all test conditions and acceptability criteria prescribed by the study plan were met.

First Full Test

Inhibition of the Respiration Rate

Detailed study results are presented in Table 10 of Appendix 1.

A summarized overview of the study results is presented in Table 6.

The effects observed were not in agreement with what was expected based on the results of the combined limit/range-finding test. There was no clear dose response observed in the first full test, therefore a second full test was performed to determine the NOEC and the EC₅₀.

Table 6

First Full Test – Overview of Results

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control		35.27	23.52
T1	4.8	28.23	18.82	19.96
T2	15	27	18	23.45
T3	49	28.95	19.3	17.93
T4	156	35.28	23.52	-0.02
T5	500	20.71	13.81	41.28

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

Experimental Conditions - First Full Test

The pH values in the individual vessels are presented in Table 10 of Appendix 1.

The pH in all test vessels, before addition of sludge, was between 5.9 and 7.3. After the 3 hour exposure period the pH was between 8.2 and 8.5.

The temperature continuously measured in the temperature control vessels ranged between 21.0 and 23.6°C during the test, and was outside the range prescribed by the study plan (20 ±2°C, see also Appendix 6).

Second Full Test

Inhibition of the Respiration Rate

Detailed study results are presented in Table 11 of Appendix 1.

A summarized overview of the study results is presented in Table 7.

No statistically significant inhibition of the respiration rate of the sludge was recorded at 100 mg CMTX 2-carboxymethyloxy-thioxanthone per liter (for more information on statistics see Appendix 5). At higher concentrations the inhibitory effect of CMTX 2-carboxymethyloxy-thioxanthone on aerobic waste water (activated sludge) bacteria increased with increasing concentration, with 59% inhibition at 320 mg/L and 97% at 1000 mg/L. The results of this test allowed for reliable determination of a NOEC and EC_x values.

Table 7

Second Full Test – Overview of Results

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control		36.98	24.65
T1	3.2	29.81	19.88	19.37
T2	10	32.9	21.93	11.04
T3	32	32.47	21.64	12.2
T4	100	32.73	21.82	11.48
T5	320	15.12	10.08	59.12*
T6	1000	1.15	0.77	96.88*

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Experimental Conditions – Second Full Test

The pH values in the individual vessels are presented in Table 11 of Appendix 1.

The pH in all test vessels, before addition of sludge, was between 6.1 and 7.4. After the 3 hour exposure period the pH was between 5.9 and 8.0.

The temperature continuously measured in the temperature control vessels ranged between 20.3 and 22.6°C during the test, and was slightly outside the range prescribed by the study plan (20 ± 2°C, see also Appendix 6).

Determination of Effect Parameters

Table 8 shows the effect parameters based on CMTX 2-carboxymethyloxy-thioxanthone concentrations; see also Appendix 4 and Appendix 5.

Table 8

Effect Parameters

Parameter	CMTX 2-carboxymethyloxy-thioxanthone Concentration (mg/L)
NOEC	100
EC10	81 (47 - 139)
EC20	120 (79 - 180)
EC50	254 (196 - 330)

( ) Between brackets the 95% confidence intervals are given.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present test CMTX 2-carboxymethyloxy-thioxanthone was not toxic to waste water bacteria (activated sludge) at or below a concentration of 100 mg/L (NOEC).

The EC10 was 81 mg/L (95% confidence interval: 47 - 139 mg/L).
The EC20 was 120 mg/L (95% confidence interval: 79 - 180 mg/L).
The EC50 was 254 mg/L (95% confidence interval: 196 - 330 mg/L).

Executive summary:

The objective of the study was to evaluate CMTX 2-carboxymethyloxy-thioxanthone for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC₅₀ and/or the no - observed effect concentration (NOEC).

The influence of CMTX 2-carboxymethyloxy-thioxanthone on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study procedures described in this report were based on the OECD guideline No. 209, 2010

The batch of CMTX 2-carboxymethyloxy-thioxanthone tested was a yellow powder with a purity of 98.25% (HPLC). No correction was made for the purity/composition of the test item. Preparation was as much as possible performed under low light conditions.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottlescontaining Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, synthetic medium, sludge and Milli - RO water were added, resulting in the required concentrations. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

The first full test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations were tested, ranging from 4.8 to 500 mg/L. Since there was no clear dose response at the first full test a second full test was performed. Six concentrations were tested, ranging from 3.2 to 1000 mg/L, and increasing with a factor 3.2. Five replicates per concentration and six replicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at 100 mg CMTX 2-carboxymethyloxy-thioxanthone per liter. At higher concentrations the inhibitory effect of CMTX 2-carboxymethyloxy-thioxanthone on aerobic waste water (activated sludge) bacteria increased with increasing concentration, with 59% inhibition at 320 mg/L and 97% at 1000 mg/L.

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.

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

CMTX 2-carboxymethyloxy-thioxanthone was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/L (NOEC).

The effect parameters for the inhibition of the respiration rate are presented below.

Parameter	CMTX 2-carboxymethyloxy-thioxanthone Concentration (mg/L)
NOEC	100
EC10	81 (47 - 139)
EC20	120 (79 - 180)
EC50	254 (196 - 330)

( ) Between brackets the 95% confidence intervals are given.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC50 for microorganisms:

254 mg/L