bis(2,2,6,6-tetramethyl-1-(phenylthio)piperidin-4-yl)carbonate

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:

2019-09-27 - 2019-10-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted 23rd March, 2006, Annex 5 corrected: 28th July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) No 266/2016, Annex Part C, C.3: „Freshwater Alga and Cyanobacteria, Growth Inhibition Test“ (published in the Official Journal of the European Union L 54 of 01 March 2016)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, No. 23 (Second Edition), ENV/JM/MONO(2000)6/REV1,

Version / remarks:

8 February 2019

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L nominal, control
- Sampling method: For determination of the test item concentration six replicate samples (2 mL per replicate) from the test concentration were taken on each day of the study and six replicate samples (2 mL per replicate) from the control group were taken at the start and at the end of the test.
- Sample storage conditions before analysis: not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The preparation of test solution was performed by direct weighing and stirring for 24 h with subsequent filtration (according to OECD Series on Testing and Assessment No. 23). 0.0800 g of the test item was suspended in 800 mL dilution water (OECD Medium; see 4.4) in order to give the loading rate of nominal 100 mg test item/L. The solution was handled by ultrasonic bath for approx. 15 minutes thereafter stirred for a period of approximately 24 hours to achieve equilibrated concentration. The solution was then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material.
- Controls: The dilution water (OECD Medium) without addition of test item was used as untreated control solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Raphidocelis subcapitata and Selenastrum capricornutum)
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for four days before this test.)
- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells observed: The algal cultures used in this study did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

Culture temperature was checked at the beginning of the study and every 24 hours thereafter in a flask filled with water, in the climatic chamber. In addition temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.5 and 22.8 °C measured in the flask and in the range of 21.4 – 23.5 °C measured within the climate chamber.

pH:

The pH was measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.79 – 8.48 during the experiment. The pH of the test solutions was not adjusted during the study.

Nominal and measured concentrations:

nominal: 100 mg/L nominal loading rate
geometric mean measured: 13.0 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Material, size, headspace, fill volume: Volumes of 100 mL algal suspension per replicate were continuously shaken by a laboratory orbital shaker in 250 mL Erlenmeyer flasks. The test flasks were covered with air-permeable stoppers.
- Initial cells density: 10E4 cells/mL in each test flask.
- Control end cells density: 52.83 * 10E4/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Other: The test containers were kept during the test in a climate chamber with controlled environmental conditions.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD Medium, according to OECD 201) was used as dilution water in the experiment. Separate stock solutions were first prepared in deionised water (prepared in the laboratory of TOXI-COOP ZRT. by Millipore Elix 3 water purification system). The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations in the prepared algal growth medium. For further information on the detailed composition of the OECD Medium please see "Any other information on materials and methods incl. tables".
- Culture medium different from test medium: No. The pre-culture was prepared with OECD Medium and incubated for four days before this test.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: The light intensity was checked and recorded at the start of the test. The average light intensity measured at the position occupied by algal culture flasks was 5261 lux (SD: 108 lux), which was ensured with fluorescent lamps (with a spectral range of 400 - 700 nm). The differences in light intensity between the test vessels did not exceed ±15 % and, therefore, provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. The cell morphology was examined in parallel.
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Range finding study: In order to select appropriate test concentrations for the definitive test, a non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item. Algal cells were exposed to one test concentration (100 mg/L nominal) of the test item plus a control, for 72 hours. The test was performed with two replicates in the test item treatment group and six replicates in the control. For preparation of the test solution an amount of 0.0803 g test item was dispersed in 803 mL OECD Medium to obtain the 100 mg/L nominal concentration. Thereafter the test solution was handled in ultrasonic bath for approx. 15 minutes, stirred rigorously for 24 hours and then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. Based on the results of the non-GLP Preliminary Range-Finding Test and the lack of effects, the main test was a limit test performed by direct weighing and stirring for 24 h with subsequent filtration (according to OECD Series on Testing and Assessment No. 23) including a nominal loading rate of 100 mg/L and a concurrent control group.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the reliability of the applied test system and the experimental conditions Potassium dichromate is tested at least twice a year. The date of the latest study performed in the laboratory of TOXI-COOP ZRT. was: 2019-03-11 to 14.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: A very slight stimulation of algal growth was observed in the only treatment group of nominal 100 mg/L compared to the control. As this stimulation is not significant, it can be considered to be an effect of natural biological variation.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- EC50: 72h ErC50: 0.83 mg/L, (95 % confidence limits: could not be calculated), 72h EyC50: 0.57 mg/L, (95 % confidence limits: 0.45 – 0.72 mg/L)

Reported statistics and error estimates:

Mean values and standard deviations were calculated at each observation period using Microsoft Excel for Windows.
A limit test was performed and no toxic effects were observed, therefore, statistical comparisons were not necessary. The ECx values, NOEC and LOEC of the test item related to growth rates and yield were determined from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD Guideline 201 and EU Method C.3 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the registered substance towards algae.
Accordingly, the 72-hour EC50 and the 72-hour EC10 for the parameter growth rate and yield were determined to be > 100 mg test item/L, corresponding to a geometric mean measured concentration of 13.0 µg/L and representing the saturation concentration or the limit of solubility. The 72-hour NOEC was determined to be ≥ 100 mg test item/L and the associated 72-hour LOEC is > 100 mg test item/L, both values also based on the nominal concentration for the parameter growth rate and yield. Based on the geometric mean measured concentration the 72-hour NOEC was determined to be > 13.0 µg/L and the associated 72-hour LOEC is > 13.0 µg/L for growth rate and yield, respectively.

Executive summary:

The effect of the test item on the growth of a unicellular green algal species was assessed using exponentially-growing cultures of Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). Algal cells were exposed to the test item under defined conditions according to OECD 201 under GLP. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. Based on the results of a non-GLP preliminary test the main test was performed as a limit test. The test concentration of nominal 100 mg/L and a control were tested in order to demonstrate that the test item has no influence on algal growth up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration).

Due to the poor water solubility of the test substance the test concentration of nominal 100 mg/L was stirred for 24h and filtrated through a membrane filter to remove any non-dissolved test material.

The test design included six replicates at the test concentration and six replicates for the untreated control. OECD Medium was used as dilution water. The alga cell concentration was approximately 10⁴ cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. Environmental conditions were recorded. All measured values remained within acceptable ranges.

The analytically measured test item concentration was below 80 % of the nominal at the end of the test, however due to the test item characteristic keeping the concentration within ± 20 % of the nominal was not possible (semi-static or flow through system is not applicable in alga test). Therefore, in this limit test biological endpoints are based on the nominal and the geometric mean measured concentration of the test item.

Geometric mean exposure concentration was determined as 13.00 µg/L.

No inhibition of algal growth and no morphological changes of algal cells were observed in the control group and the limit concentration of nominal 100 mg/L after 72 hours of exposure. Accordingly, the 72-hour EC₅₀ and the 72 -hour EC₁₀ for the parameter growth rate and yield were determined to be > 100 mg test item/L, corresponding to a geometric mean measured concentration of 13.0 µg/L and representing the saturation concentration or the limit of solubility. The 72-hour NOEC was determined to be ≥ 100 mg test item/L and the associated 72-hour LOEC is > 100 mg test item/L, both values also based on the nominal concentration for the parameter growth rate and yield. Based on the geometric mean measured concentration the 72 -hour NOEC was determined to be > 13.0 µg/L and the associated 72 -hour LOEC is > 13.0 µg/L for growth rate and yield, respectively.

The results were determined directly from the raw data.

All validity criteria were met and, therefore, the study can be considered as valid.

Description of key information

- Key, toxicity to aquatic algae and cyanobacteria: EC₅₀ (72h) > 100 mg/L nominal, corresponding to > 13.0 µg/L based on geometric mean measured concentrations of the test item for Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) (OECD 201, limit-test, GLP)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The study was conducted under GLP according to OECD Guideline 201 and EU Method C.3 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable.

The toxic effect of the test item to the unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was assessed in a static limit test at the saturation concentration (equivalent to 100 mg/L nominal loading rate). Due to the poor water solubility of the test substance the test concentration of nominal 100 mg/L was stirred for 24h and filtrated through a membrane filter to remove any non-dissolved test material. No inhibition of algal growth and no morphological changes of algal cells were observed in the control group and the limit concentration of nominal 100 mg/L after 72h of exposure. Accordingly, the 72-hour EC₅₀ and the 72 -hour EC₁₀ for the parameter growth rate and yield were determined to be > 100 mg test item/L, corresponding to a geometric mean measured concentration of 13.0 µg/L and representing the saturation concentration or the limit of solubility. The 72-hour NOEC was determined to be ≥ 100 mg test item/L and the associated 72-hour LOEC is > 100 mg test item/L, both values also based on nominal concentration for the parameter growth rate and yield. Based on the geometric mean measured concentration the 72-hour NOEC was determined to be > 13.0 µg/L and the associated 72-hour LOEC is > 13.0 µg/L for growth rate and yield, respectively.