Ethyl 3-[[bis(1-methylethoxy)phosphinothioyl]thio]propionate

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:

from 2014-04-16 to 2014-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to internationally accepted guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The evaluation of the concentrations of the test item was carried out from freshly prepared media after 0 hours and old test media after 72 hours. Samples at the start of the exposure were taken from an additional replicate without algae. At the end of the exposure, test samples were taken directly from the test replicates. Test concentrations were prepared by diluting the stock solution with dilution water.
- Control: Six replicates (without test item) were exposed under the same conditions as the test concentrations.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Stock solution: 15 mg/L, freshly prepared with dilution water
- Dispersion treatment: The stock solution was treated with ultrasound for 8 min at room temperature.
- The tested solutions were clear throughout the exposure period.
- Dilution water: According to the guidelines.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÂK, SAG 61.81
- Source: Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073, Göttingen, Germany
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE –m exp2 s exp -1 for 24 hours per day.
- Culture medium: Nutrient medium Z according to Liittge et al. (1994).
- Preculture: A three day old preculture, prepared in dilution water, was used as inoculum. For the start of the test, the preculture was directly pipetted into each test media and the control.

ACCLIMATION
- Culturing media and conditions: same as the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

22 – 23 °C

pH:

7.74 – 8.31

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

- Nominal concentrations: 0.938, 1.88, 3.75, 7.50 and 15.0 mg/L. Dilution Factor: 2
- Measured concentrations: 0.713, 1.50, 3.14, 7.25 and 13.5 mg/L. The measured concentrations of the test substance at the start of the exposure were in the range of 95 - 103 % of the nominal test item concentrations. At the end of the exposure, the measured concentrations were in the range of 59 - 91 % of the nominal test item concentrations. All effect values are based on the geometric mean measured concentrations of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs.
- Material, size, headspace, fill volume: 100 mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Initial cell density: aprox. 7085 cells/mL.
- Application: Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels.
- Incubation: The flasks were positioned randomly and repositioned daily.
- Temperature: 21 – 24 °C, controlled at +/- 2 °C
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline
- Intervals of water quality measurement: The pH-value at the start of the exposure was measured in one additional replicate of each test item concentration and the control. At the end of the exposure, it was measured in a pooled sample per test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of the test.

OTHER TEST CONDITIONS
- Light intensity: Approximately 60 – 120 µE/m2/s
- Light regime: 24 hours/day light
- Light source: Fluorescent tubes, Osram L 36 W/865, cool day light.

EFFECT PARAMETERS MEASURED: Alga, growth inhibition test for the determination of the EC10-EC20- and EC50-values of the growth rate after 0 and 72 hours.
- Frequency of Measurements of the biological parameter: Chlorophyll-a-fluorescence cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.
Microscopic evaluation: Microscopic evaluation of the cells was carried out at the start and the end of the exposure. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: The concentrations were based on the results of a preliminary range finding test (non GLP). The nominal test item concentrations in the range finding study were: 15, 1.5, 0.15, 0.015 mg/L. No self-fluorescence was found in the preliminary range finding test at the test concentration of 15 mg/L.
- Test concentrations: Based on a preliminary range finding test, five concentration levels were tested in a geometrical series with a dilution factor of 2 (nominal): 0.938, 1.88, 3.75, 7.50 and 15.0 mg/L corresponding to geometric mean measured test item concentrations of 0.713, 1.50, 3.14, 7.25 and 13.5 mg/L. The measured concentrations of the test substance at the start of the exposure were in the range of 95 - 103 % of the nominal test item concentrations. At the end of the exposure, the measured concentrations were in the range of 59 - 91 % of the nominal test item concentrations. All effect values are based on the geometric mean measured concentrations of the test item.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.93 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L. (3.48-4.38)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L. (7.98-8.64)

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

5.23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L. (4.86-5.55)

Details on results:

Microscopic evaluation of the cells at the start of the incubation period revealed no morphological abnormalities. At the end of the incubation period, the algae of the concentrations 7.50 and 15.0 mg/L were deformed. The other concentrations and the control showed no morphological abnormalities. All effect values are based on the geometric mean measured concentrations of the test item.
The EC-values and 95 % confidence intervals of the test substance (0-72 hours) based on geometric mean measured test item concentrations were determined to be as follows: ErC10 = 3.93 (95% C.L. = 3.48-4.38) mg/L, ErC20 = 5.23 (95% C.L. = 4.86 – 5.55) mg/L and the ErC50 = 8.28 (95% C.L.= 7.98-8.64) mg/L

Results with reference substance (positive control):

The ErC50-Values of the reference item (potassium dichromate) based on nominal concentrations (mg/L) (0-72 hours) were determined to be ErC50 = 0.661 mg/L with 95% confidence interval in the range from 0.638 - 0.685 mg/L for the growth rate inhibition and EyC50 = 0.341 mg/L with 95% confidence interval in the range of 0.286 and 0.377 mg/L

Reported statistics and error estimates:

EC10-, EC20 and EC50-values with confidence intervals of growth rate inhibition after 72 hours were calculated by sigmoidal dose response regression. The calculation of the confidence intervals was carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

Description of key information

Toxic effects were observed in an growth inhibition test with the freshwater green alga Pseudokirchneriella subcapitata according OECD 201. The EC-values based on geometric mean measured test item concentrations after 72 hours were determined to be as follows: ErC10 = 3.93 mg/L and the ErC50 = 8.28 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

8.28 mg/L

EC10 or NOEC for freshwater algae:

3.93 mg/L

Additional information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the OECD 201 and the (EC) No. 440/2008/ Method C.3. The aim of the study was to assess the effects on the growth rate over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 7085 cells/mL. Based on a preliminary range finding test, five concentration levels were tested in a geometrical series with a dilution factor of 2 (nominal): 0.938, 1.88, 3.75, 7.50 and 15.0 mg/L corresponding to geometric mean measured test item concentrations of 0.713, 1.50, 3.14, 7.25 and 13.5 mg/L.

All test concentrations of test item and the control were analytically verified by LC-MS/MS at the start and the end of the exposure. The measured concentrations of at the start of the exposure were in the range of 95 - 103 % of the nominal test item concentrations. At the end of the exposure, the measured concentrations were in the range of 59 - 91 % of the nominal test item concentrations.

All effect values are based on the geometric mean measured concentrations of the test item. The EC-values based on geometric mean measured test item concentrations after 72 hours were determined to be as follows: ErC10 = 3.93 mg/L and the ErC50 = 8.28 mg/L.