ethyl N,S-bis(4-oxo-4-(2,6,6-trimethylcyclohex-3-en-1-yl)butan-2-yl)cysteinate

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:

March - April, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

GLP compliance:

no

Remarks:

internal study performed in the spirit of GLP

Analytical monitoring:

yes

Remarks:

HPLC-UV/VIS and LC-MS

Details on sampling:

To confirm the concentration of GR-87-0307 in samples of test media during this test, at the given time points, the test vessels are mixed by shaking and two 0.2 mL samples were removed from the test vessels.

Sampling regime: Samples were taken and analyzed from two replicates at each concentration and at each time point.

Vehicle:

yes

Remarks:

Acetone, 100 microL/L

Details on test solutions:

At the start of the test, a solution of the test item at 80 mg/mL in acetone was prepared. Serial dilutions of this solution in acetone were made to final concentrations of 80, 40, 20, 10 and 5 mg/mL. From these solutions, 16 µL aliquots were introduced to individual 250 mL glass beakers. To achieve homogeneous distribution and to avoid that the chemical comes out of solution, the beakers were first filled with the growth media (160 mL per beaker), stirred at maximal speed on a magnetic stirrer and then the acetone solution was introduced into the vortex. With this procedure, nominal test concentrations of 0.5, 1, 2, 4 and 8.0 mg/L were prepared. From the beakers with the stock solution of each test item concentration, 50 mL were then introduced, within 15 min, into the three replicate test vessels for each treatment group. A control treatment was prepared by adding medium only to the control vessels, while in the solvent control this was amended with 5 µL of acetone in each vessel.

The test vessels were glass 50 mL Erlenmeyer (conical) flasks. To minimize potential losses through volatility, the test vessels contained sufficient media to leave as minimum a headspace as possible. The vessels were capped with a glass stopper. This system constituted a ‘closed’ system.
Three test vessels were prepared for each test concentration, four vessels for the control (algal nutrient medium only) and four vessels for the solvent control (algal nutrient medium and solvent only). Each test and control vessel was inoculated with sufficient Raphidocelis subcapitata cells to achieve a starting algae cell concentration of 1 x 10^4 cells/mL.
A media blank (OECD medium only) was prepared to establish background fluorescence on each sampling occasion. Background fluorescence was subtracted from the fluorescence results for each of the inoculated test vessels. The resulting fluorescence was then used to calculate the number of cells based on a calibration curve determined at time zero. The cell counts were then used to determine the yield and the corresponding specific growth rates. In addition, test vessels with the test compound but no algal cells were prepared and incubated both in the light and in the dark to determine degradation / losses of parent due to light and biological activity. These treatments were performed at a nominal concentration of 2 mg/L.

Test organisms (species):

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

Details on test organisms:

The test organism, Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata or Selenastrum capricornutum), was obtained from MicroBio Test Inc. (Algal beads: Selenastrum capricornutum.) This species is recommended for testing in accordance with the OECD guideline
201. Algae were grown in culturing medium and kept frozen at -80°C. One vial (1 mL) was thawed, suspended in growth medium, washed by centrifugation and then grown for 3 days under standard growth conditions.
Cells in this starter culture were harvested by centrifugation, resuspended in cell culture medium and counted in a cell counting chamber. The inoculum was diluted to a nominal concentration of 10^6 cells / mL (actual counted value 1.035 x 10^6), and this stock cell suspension was further diluted 100- fold in the test vessels (final inoculum of 10^4 cells / mL). All flasks for the test were inoculated from the same starter culture.
At the same time the 10^6 cells / mL stock solution was serially diluted and the fluorescence was determined in order to calibrate the fluorescence measurements vs. microscopically counted algal cell density.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

between 21.8 and 22.57°C

pH:

between 8.02 and 9.85

Although the pH in the Control (Blank) deviated by more than 1 .5 pH units, the deviation in the Solvent Control was less than 1.5 pH units.

Nominal and measured concentrations:

nominal test concentrations of 0.5, 1, 2, 4 and 8 mg/L

Details on test conditions:

Measured Light intensity (Lux): 9880

All flasks were incubated in a temperature-controlled shaker at 22.5°C with 150 rpm below an LED array emitting constant light. At the start of the test, the pH of freshly prepared test media was determined. The pH in one replicate of each test concentration was also determined at the start and the end of the test. The light intensity within the test area was monitored at the start and end of the test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.82 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: No effects observed on yield of algae up to and including the highest tested concentration (above saturation). EC50 > 0.82 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.82 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: No effects observed on yield of algae up to and including the highest tested concentration (above saturation). EC10 > 0.82 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.82 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: No effects observed on yield of algae up to and including the highest tested concentration (above saturation). NOEC > or = 0.82 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.82 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No effects observed on growth rate of algae up to and including the highest tested concentration (above saturation). EC10 > 0.82 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.82 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No effects observed on growth rate of algae up to and including the highest tested concentration (above saturation). NOEC > or = 0.82 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.82 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No effects observed on growth rate of algae up to and including the highest tested concentration (above saturation). EC50 > 0.82 mg/L

Details on results:

The test item demonstrated no significant effects on the inhibition of either yield or growth rate of the algae at up to and including the highest concentration tested, corresponding to or greater than the aqueous saturation concentration.
Based on time-weighted mean measured test concentrations for the parent and parent + d- Damascone, the 72-hour EyC50 and the 0-72 hour ErC50 values were calculated to be > 0.82 mg/L and > 1.04 mg/L, respectively.
The corresponding NOEC values for yield and specific growth rate, based on parent and parent + d- Damascone after 72 hours were, respectively, 0.82 mg/L and 1.04 mg/L.

The control cell density in the solvent control cultures increased with an average growth rate of 1.83 (i.e. 246- fold growth of cells over 72 h). Therefore the validity criterion of achieving an increase in control cell density by at least a factor of 16 (average growth rate > 0.92) within the 72-hour test period was achieved. The environmental conditions of the test were considered acceptable. The mean coefficient of variation for section-by-section specific growth rates (0-24, 24-48 and 48-72 hours) in the solvent control culture was 14.3 % and therefore did not exceed 35 %. The coefficient of variation for average specific growth rate during the whole test period in replicate solvent control cultures was 1.5 % which did not exceed 7 %. Therefore, the validity criteria outlined in the OECD guideline were all met.
The average 0-72 h growth rate in the control without solvent was at 1.84 while it was at 1.83 with solvent, the difference is not statistically different (p> 0.05), indicating there is no effect of the solvent on the growth of the test organism.
The growth rate in the control tests showed that the use of the modified test system to reduce losses of test substance through volatility (filled and sealed test vessels) has no significant effect on growth rate, as vigorous growth and amplification of cell numbers by a factor of 245.9 fold, well above the requirements of the guideline, were observed.

Results with reference substance (positive control):

Reference Test Summary Report

Inhibition of Growth to the Alga Raphidocelis subcapitata

Study Director: A. Natsch

Test Substance: 3,5-dichloro-phenol (CAS 591-35-5; Aldrich D70600; LOT MKBH4843V)

Test Medium: EC media amended with 0.5 g/L of NaHCO3. This additional NaHCO3 was added to provide a source of carbon dioxide for algal growth under conditions without headspace.
Test Guideline: The study was designed in accordance with OECD Testing Guideline, No. 201 : Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006, Annex 5 corrected 28 July 2011), closed Vessel Test Design

Objective: The purpose of this study was to estimate the effects of the reference substance against algal growth by exposing the green alga, Pseudokirchneriella subcapitata, to the reference substance during a 72-hour period.

Test Species: Pseudokirchneriella subcapitata
Treatment Doses: Control, 1.5, 3, 6, and 12 mg/L
Study Duration: 72 hours
Test System: Starting cell concentration of 1 × 104 cells/mL
Replication: 3 replicates per exposure concentration with 4 replicate controls

Control Validity Criteria:
Cell density increase: 80.8-fold (passed, required > 16-fold)
Mean coefficient of variation for section-by-section specific growth rates: 30.7% (passed, required  35%)
Coefficient of variation for average specific growth rate: 1% (passed, required  7%)

The ErC50 and EyC50 values were determined to be 4.15 and 3.49 mg/L, respectively.
The corresponding NOEC values were 1.5 mg/L.

The result of the internal ring trial cited in ISO norm 8692 for ErC50 is 3.38 ±1.3 mg/L. Thus, these results were within the expected range.


.

Reported statistics and error estimates:

Statistical analysis of the inhibition curves was performed using the Graph pad prism program v
6.09. Difference between control treatment and chemical treatments for specific growth rate (µ) and final yield over specified time intervals were analysed using Student’s T test to determine the no observed effect concentration (NOEC).
Four parameter curve fit analysis was performed by plotting the logarithmic concentration levels vs. the % inhibition in GraphPad using the normalized response and variable slope algorithm in order to estimate EC10, EC20 and EC50 values and their 95% confidence limits. (The EC10, EC20 and EC50 are defined as the concentrations that result in a 10%, 20% and 50% mean reduction, respectively, relative to the control of a given parameter). To distinguish between ECx values, estimated using final yield and growth rates, the symbols EyCx and ErCx were used, respectively, whereby x reflects the inhibition threshold in %. All other calculations were performed in Microsoft Excel.

The data were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.

Validity criteria fulfilled:

yes

Conclusions:

The effects of GR-87-0307 on the growth of the unicellular green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition toxicity test conducted in accordance with OECD Chemicals Testing Guideline No. 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011). All validity criteria were met, therefore the test was considered valid.

At all tested concentrations, no effect on algal growth was observed. The maximal test concentrations were clearly above the limit of aqueous solubility. However, as this is a labile molecule releasing a volatile molecule, absolute solubility is not easily measurable, at least not with the slow stirring or percolation method, as the molecule is constantly breaking down. Nevertheless, the results indicate that even when dosed at nominal concentrations clearly above the solubility limit, no effect on algal growth is observed, even if, as expected there is a continuous release of the fragrant molecule and a slow decay of the initially dissolved concentration.

The test item demonstrated no significant effects on the inhibition of either yield or growth rate of the algae at up to and including the highest concentration tested, corresponding to or greater than the aqueous saturation concentration.
Based on time-weighted mean measured test concentrations for the parent and parent + d- Damascone, the 72-hour EyC50 and the 0-72 hour ErC50 values were calculated to be > 0.82 mg/L and > 1.04 mg/L, respectively.
The corresponding NOEC values for yield and specific growth rate, based on parent and parent + d- Damascone after 72 hours were, respectively, 0.82 mg/L and 1.04 mg/L.

Executive summary:

The objective of the study was to determine the effects of GR-87-0307 on the growth of the green alga,Raphidocelis subcapitata, during a 72-hour growth inhibition test. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).

 

The test substance was known to have limited solubility and limited stability in the test medium. It was therefore considered that the most appropriate method of dissolution of the test substance was to prepare dilution series in acetone and to directly dose finite amounts of the substance with a solvent spike experiment (final level of acetone 100 µL / L).

 

The test was conducted at nominal test concentrations of 0.5, 1, 2, 4 and 8 mg/L (0.22, 0.29, 0.49, 0.82 and 0.48 mg/L based on time-weighted mean measured concentrations for parent). A control group and a solvent control group were also included.

Test vessels (50 mL glass Erlenmeyer flasks) were prepared which were sealed and contained sufficient media (50 mL) to leave as minimum a headspace as possible in order to minimize any potential losses through volatility. Each test vessel was inoculated with 1 x 104 algae cells/mL and incubated at 22.5°C for 72 hours with biomass deter mination and analytical determination of substance concentration at 24-hour intervals.

 

The test chemical is a labile precursor designed to liberate a fragrant molecule (d-damascone). Analysis of the test media samples was conducted at 0, 24, 48 and 72 hours, and analysis was made both for the parent molecule and released fragrance molecule. Analysis of the test samples at 0 hours showed measured concentrations of parent molecule to range from 0.49 – 1.03 mg/L. At 72 hours, analysis of the test samples showed measured concentrations 0.15 – 0.38 mg/L. Increasing concentrations of the liberated fragrantd-damascone were measured.

 

Due to the loss over time of the parent and accumulation of the liberatedd-damascone, results have been based on time-weighted mean (TWM) measured concentrations based on the parent and also based on the sum of parent and liberatedd-damascone. The 72-hour yield (EyCx) and growth rate (ErCx) toxicity values, with corresponding NOEC values are presented. NOEC is reported as the concentration at which there is no statistically significant effect on the parameter when compared to the controls.

 

The test item demonstrated no significant effects on the inhibition of either yield or growth rate of the algae at up to and including the highest concentration tested, corresponding to or greater than the aqueous saturation concentration.

Based on time-weighted mean measured test concentrations for the parent and parent + d- Damascone, the 72-hour EyC50 and the 0-72 hour ErC50 values were calculated to be > 0.82 mg/L and > 1.04 mg/L, respectively.

The corresponding NOEC values for yield and specific growth rate, based on parent and parent + d- Damascone after 72 hours were, respectively, 0.82 mg/L and 1.04 mg/L.

All validity criteria were met, therefore the test was considered valid.

Description of key information

The objective of the study was to determine the effects of GR-87-0307 on the growth of the green alga,Raphidocelis subcapitata, during a 72-hour growth inhibition test. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).

At all tested concentrations, no effect on algal growth was observed. The maximal test concentrations were clearly above the limit of aqueous solubility. However, as this is a labile molecule releasing a volatile molecule, absolute solubility is not easily measurable, at least not with the slow stirring or percolation method, as the molecule is constantly breaking down. Nevertheless, the results indicate that even when dosed at nominal concentrations clearly above the solubility limit, no effect on algal growth is observed, even if, as expected there is a continuous release of the fragrant molecule and a slow decay of the initially dissolved concentration.

The test item demonstrated no significant effects on the inhibition of either yield or growth rate of the algae at up to and including the highest concentration tested, corresponding to or greater than the aqueous saturation concentration.

Based on time-weighted mean measured test concentrations for the parent and parent + d- Damascone, the 72-hour EyC50 and the 0-72 hour ErC50 values were calculated to be > 0.82 mg/L and > 1.04 mg/L, respectively.

The corresponding NOEC values for yield and specific growth rate, based on parent and parent + d- Damascone after 72 hours were, respectively, 0.82 mg/L and 1.04 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.82 mg/L

EC10 or NOEC for freshwater algae:

0.82 mg/L

Additional information

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test).

According to the EU CLP regulation (No 1272/2008 and its adaption 286/2011), Scentaurus Berry doesn't need to be classified as Hazardous to the Aquatic Environment Acute 1 classification (Acute results greater than the aqueous saturation concentration of the test item).