Ethyl 9-oxo-9H-thioxanthene-2-carboxylate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Jul - 23 Jul 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

2000

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: SANCO/3029/99 rev. 4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 9-oxo-9H-thioxanthene-2-carboxylate

EC Number:

280-960-7

EC Name:

Ethyl 9-oxo-9H-thioxanthene-2-carboxylate

Cas Number:

83817-60-1

Molecular formula:

C16H12O3S

IUPAC Name:

ethyl 9-oxo-9H-thioxanthene-2-carboxylate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations and method: duplicate samples from the freshly prepared test media filtrate (containing algae) and from the control were taken at the start of the test. Duplicate samples from the test media filtrates of the100 mg/L test concentrations and the control (containing algae) were taken also at the end of the test by pooling the replicates. Additional samples of the control blank and the dilution solvent were taken at test start and test end without any sample treatment.
- Sample storage conditions before analysis: all samples were diluted by a factor of 2 with acetonitrile and stored at ≤ - 20 °C, protected from light

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a stock suspension of 100 mg test item/L was prepared by suspending 100.3 mg test item in 1003 mL test water. The supersaturated stock suspension was mixed for 48 h at room temperature by overhead shaking. Then, medium was filtrated (0.45 μm cellulose acetate filter) and the filtrate was used as test medium.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source: laboratory culture (original source: Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen, Germany)
- Age of inoculum (at test initiation): 4 d old exponentially growing pre-culture
- Method of cultivation: under standardised conditions according to the test guidelines

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

21.8 - 22.7 °C

pH:

7.9 to 9.1

Dissolved oxygen:

-

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal test concentrations: (0) control and 100 mg/L (supersaturated solution)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (covered with glass dishes)
- Fill volume: 50 mL
- Initial cells density: 5000 algal cells per mL test medium
- Control end cells density: 65.515*10^4 cells /mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per blank control (replicates): 1 (blank without algae for the spectrophotometric measurements)
- Other: continuously stirred with magnetic stirrers

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: temperature was measured daily in a flask filled with water and incubated under the same conditions as the test flasks, pH was measured in all concentrations and the control at the start and the end of the test

OTHER TEST CONDITIONS
- Adjustment of pH in dilution water: yes with 1M HCl to pH 8.1
- Photoperiod: continuous
- Light intensity: 5617 lux (mean)

EFFECT PARAMETERS MEASURED: cell density was determined after 24, 48 and 72 h
- Determination of cell concentrations: spectrophotometrical measurement

TEST CONCENTRATIONS
- Range finding study: yes, non GLP (not included in final report)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes (131-fold increase within 72 h)
- Observation of abnormalities (for algal test): no
- Unusual cell shape: yes
- Effect concentrations exceeding solubility of substance in test medium: yes

Results with reference substance (positive control):

- Results with reference substance valid: yes
- ErC50: 1.50 mg potassium dichromate/L (95% CI: 1.43 - 1.58 mg/L)

Reported statistics and error estimates:

- The 72 h ErC10, ErC50 and EyC10, EyC50 could not be calculated by Probit analysis due to the lack of effects and were therefore determined directly from the raw data.
- The 72 h LOEC and NOEC, was tested for significant differences compared to the control values by Two sample t-test.
- The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Any other information on results incl. tables

Table 1: All biological results.

Parameter	Yield	Growth rate
	[mg test item/L]	[mg test item/L]
72-hour EC50	> 100	> 100
72-hour EC20	> 100	> 100
72-hour EC10	> 100	> 100
72-hour NOEC	≥100	≥100
72-hour LOEC	> 100	> 100

Table 2: Algal Cell Densities, Yield and Growth rate as well as the inhibition percentage after 72 h of exposure.

Mean measured concentration [mg/L]	Density of algal cells [1000/mL] after 72 h	Yields y [10000 cells/mL]	% inhibition of y	Growth rates μ [1/day]	% inhibition of μ
Control	65.515	65.015	-	1.625	-
100	72.256	71.756	-10.4	1.657	-2

The measured test concentration was both at the start and at the end of the test below the LOQ. Therefore, all biological results refer to the nominal value.

Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 12.1%

Coefficient of Variation of Average Growth between Control Replicates: 0.8%

Applicant's summary and conclusion

Validity criteria fulfilled:

yes