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

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Nov 2018 to 06 Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

adopted July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 97.75% (assumed 100% for testing)
Test item storage: At room temperature, protected from light, desiccated

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage. Freshly obtained sludge was kept under continuous aeration until further treatment. Before use, sludge was coarsely sieved (1 mm). After treatment concentration of suspended solids (SS) was determined to be 4.8 g/L in concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 3 mL per litre of mineral medium, leading to a SS concentration of 14.4 mg/L.

Duration of test (contact time):

28 d

Initial conc.:

19 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

CMTX 2-carboxymethyloxy-thioxanthone was a yellow powder with a purity of 97.75% (HPLC). Test item was tested in duplicate at a target concentration of 19 mg/L, corresponding to 12 mg TOC/L. Organic carbon content was based on the molecular formula. Preparation was as much as possible performed under low light conditions.

Since CMTX 2-carboxymethyloxy-thioxanthone was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to 2-litre test bottles containing medium with microbial organisms and mineral components (test item bottle A: 38.06 mg; test item bottle B: 38.02 mg and toxicity control bottle: 38.07 mg). To this end, 10 mL of Milli- RO water was added to each weighing bottle containing test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. Test solutions were continuously stirred during the test, to ensure optimal contact between test item and test organisms.

Any residual volumes were discarded.

Testing Strategy and Experimental Design:
Test Procedure and Conditions:
Test duration 28 days for inoculum blank and test item (last CO2 measurement on day 29).
14 days for procedure and toxicity control (last CO2 measurement on day 15).
During the test period, test media were aerated and stirred continuously.
Test vessels 2 litre brown coloured glass bottles.
Milli- RO water Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.
 
Stock solutions of mineral components
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2

B) 22.50 g MgSO4.7H2O dissolved in Milli- RO water and
made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water and
made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water and
made up to 1 litre.

Mineral medium 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.

Barium hydroxide 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from air.

Synthetic air (CO2 < 1 ppm) A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. Synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

Illumination Test media were excluded from light.

Preparation of Bottles:

Pre-incubation medium: The day before start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Type and number of bottles: Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Procedure control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).
Preparation: At the start of the test (day 0), test and reference item were added to bottles containing microbial organisms and mineral components.
Volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before.
Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

Determination of CO2:
Experimental CO2 production CO2 produced in each test bottle reacted with barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).
Measurements Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for inoculum blank and test item. Titrations for procedure and toxicity control were made over a period of at least 14 days.
Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On the penultimate day, pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the inoculum blank and test suspension. Bottles were aerated overnight to drive off CO2 present in the test suspension. Final titration was made on day 15 (procedure and toxicity control) and on day 29 (remaining vessels).
Theoretical CO2 production Theoretical CO2 production was calculated from the molecular formula.

Measurements and Recordings:
pH: At the start of the test (day 0) and on the penultimate day (day 14 for procedure and toxicity control and day 28 for inoculum blanks and test item), before addition of concentrated HCl.
Temperature of medium: Continuously in a vessel with Milli- RO water in the same room.

Reference substance:

acetic acid, sodium salt

Test performance:

In the toxicity control, more than 25% biodegradation occurred within 14 days (45%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

Parameter:

% degradation (CO2 evolution)

Value:

6

Sampling time:

28 d

Details on results:

See below ("Any other information on results incl. tables").

Theoretical CO₂ Production

ThCO₂ of CMTX 2-carboxymethyloxy-thioxanthone was calculated to be 2.31 mg CO₂/mg.

ThCO₂ of sodium acetate was calculated to be 1.07 mg CO₂/mg.

If applicable ThCO₂ per test bottle is given in the subscript of the tables (see Appendix 1).

Biodegradation

All data are presented in Appendix 1. Results of CO₂ production and biodegradation in blank bottles, background bottles and each test bottle are listed in Table 2 to 8. Table 9 compares biodegradation of CMTX 2-carboxymethyloxy-thioxanthone in bottles A and B.

Figure 1 shows curves for biodegradation of the bottles with CMTX 2-carboxymethyloxy-thioxanthone, procedure control and toxicity control. (attached below)

 

Monitoring of Temperature and pH

Temperature recorded in a vessel with water in the same room varied between 21 and 23°C, and complied with the requirements as laid down in the study plan. The pH values of different test vessels are presented in Table 1.

Table 1       

pH Values of Different Test Media

Test medium:	At the start of the test:	On day 14:	On day 28:
Blank control (A)	7.7 → 7.6 1	-	7.7
Blank control (B)	7.5	-	7.6
Procedure control	7.5	7.8	-
CMTX 2-carboxymethyloxy-thioxanthone (A)	7.5	-	7.6
CMTX 2-carboxymethyloxy-thioxanthone (B)	7.5	-	7.6
Toxicity control	7.5	7.7	-

1: Adjusted using 1 M HCl (Merck, Darmstadt, Germany)

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

The objective of the study was to evaluate test item CMTX 2-carboxymethyloxy-thioxanthone for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge.

Study procedures described in this report were in compliance with OECD guideline No. 301 B, 1992. In addition, procedures were designed to meet test methods of ISO standard 10634, 1995.

CMTX 2-carboxymethyloxy-thioxanthone was a yellow powder with a purity of 97.75% (HPLC). The test item was tested in duplicate at a target concentration of 19 mg/L, corresponding to 12 mg TOC/L. Organic carbon content was based on the molecular formula. Theoretical CO₂ production (ThCO2) of CMTX 2-carboxymethyloxy-thioxanthone was calculated to be 2.31 mg CO₂/mg.

The study consisted of six bottles:

•       2 inoculum blanks (no test item),

•       2 test bottles (CMTX 2-carboxymethyloxy-thioxanthone),

•       1 procedure control (sodium acetate) and

•       1 toxicity control (CMTX 2-carboxymethyloxy-thioxanthone plus sodium acetate).

Since CMTX 2-carboxymethyloxy-thioxanthone was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to 2 litre test bottles containing medium with microbial organisms and mineral components. To this end, 10 mL of Milli- RO water was added to each weighing bottle containing test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. Test solutions were continuously stirred during the test to ensure optimal contact between test item and test organisms. Test duration was 28 days for inoculum blank and test item (last CO2 measurement on day 29) and 14 days for procedure and toxicity control (last CO2 measurement on day 15). Handling of test item was performed under low light conditions.

Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of CMTX 2-carboxymethyloxy-thioxanthone (8% and 3%, based on ThCO2).

In the toxicity control, CMTX 2-carboxymethyloxy-thioxanthone was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone was designated as not readily biodegradable.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of CMTX 2-carboxymethyloxy-thioxanthone (8% and 3%, based on ThCO2).

In the toxicity control, CMTX 2-carboxymethyloxy-thioxanthone was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone was designated as not readily biodegradable.