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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March to April, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

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:

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, 18 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 (180 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, 40 mL were then introduced within 15 min into the four replicate test vessels for each treatment group. A control treatment was prepared by adding ISO water with Saponin only to the control vessels, while in the solvent control this was amended with 4
µl of acetone in each vessel.
As shown below, at the top test concentrations of 4 or 8 mg, the nominal concentrations clearly exceed measured concentrations and nominal concentrations are obviously above water saturation. No oily film was observed at the surface of the beakers which were used for medium preparations nor in the test vessels. Excess test substance most likely did coat the glass vessels as a non-soluble film.

The test vessels were glass 50 mL Erlenmeyer (conical). To minimize potential losses through volatility, the test vessels contained sufficient media (40 mL) to leave a low headspace. The vessels were capped with a glass stopper. This system constituted a ‘closed’ system.

Four test vessels were prepared for each test concentration as well as four control vessels. Each test and control vessel was amended with five neonates of Daphnia magna. After 24 h, the neonates were transferred to fresh solution of the test chemical (semi-static test).

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organism Daphnia magna, in the form of ephippia, was obtained from MicroBio Test Inc, Mariakerke (Gent), Belgium. Hatching was initiated 3 days prior to the start of the toxicity test. One vial of ephippia was poured into a microsieve and rinsed thoroughly with tap water to eliminate all traces of the storage medium.
The ephippia were transferred into the hatching petri dish in 15 mL Standard Freshwater pre- aerated by air bubbling. The petri dish was covered and incubated for 3 days, at 20-22°C, under continuous illumination of 6000 lux. With this incubation procedure (recommended by MicroBioTest), the daphnids are aged < 24 h at the start of the immobilization test.
A 2h pre-feeding with dry algae was then applied. One tube of Spirulina powder (MicroBio Test Inc.) was filled with Standard Freshwater and shaken thoroughly to homogenize the contents. The contents were transferred to the hatching petri dish 2 hours prior to collecting the neonates for the toxicity test, making sure to distribute the algal food evenly. After this feeding period the neonates were directly transferred to the toxicant solutions.
Daphnids sometimes tend to get trapped at the air-water interface. This is well described in the literature and occurs in our laboratory seasonally, and may be affected by highly pure water or other unknown factors. It can be prevented by low levels (0.5 mg/L) of natural saponins from Quillaja saponaria. Thus this low level of saponins was included in all treatments in this test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

between 21.2 and 21.8 °C

pH:

between 7.86 and 8.02

Dissolved oxygen:

between 8.64 and 8.91 mg/L

Nominal and measured concentrations:

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

Details on test conditions:

All flasks were incubated in the dark at room temperature. At the start of the test, the pH and the oxygen saturation of freshly prepared test water and the saturated solution were determined. The pH, temperature and the oxygen saturation in one replicate of each treatment were also determined at the start, the 24 h renewal time and at the end of the test. The laboratory temperature was set within the range 21 to 24°C and maintained within ± 2°C for the duration of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 25% immobility observed at the highest tested concentrations (clearly above water saturation limit)

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Mobility and other sub-lethal effects

Details on results:

The 48 hour NOEC is considered to be 0.56 mg/L and 0.60 mg/L, based on the mean exposure to parent and parent + delta-Damascone, respectively. After 48 hours 25 % immobility was recorded at the highest measured test concentration of 0.78 mg/L (4 mg/L, nominal) based on exposure to parent and 0.93 mg/L (4 mg/L nominal) based on the exposure to parent + delta-Damascone.
In terms of time-weighted mean measured concentrations, the 48-hour EC50 value was determined to be > 0.78 mg/L and > 0.93 mg/L, based on parent and parent + delta-Damascone, respectively and the LOEC to be 0.76 mg/L (parent) and 0.85 mg/L (parent + delta-Damascone). The 48 hour EC50 is, therefore, clearly greater than the aqueous saturation concentration of the test item.

The validity criteria for control immobility (≤10%) with no other observed physical or behavioural effects on the daphnids and dissolved oxygen (≥3 mg/L / > 50% saturation) were both satisfied. The test is, therefore, considered valid.

Results with reference substance (positive control):

Reference Test in presence of 0.5 mg/L Saponine

Immobilisation of Daphnia magna

Study Director: A. Natsch

Test Dates: 19-27 March 2018

Test Substance: Potassium dichromate, CAS 7778-50-9, Sigma-Aldrich P2588, Lot MKBF6527V

Test Medium: Reconstituted standard freshwater (ISO water) containing 0.5 mg/L Saponine from Quillaja saponaria. (CAS 8047-15-2; PC code: 1002545802; S4521; Saponin Quillaja sp. , Sapogenin content 20-35%)
Test Guideline: The study was designed in accordance with OECD Chemicals Testing Guideline No. 202. Daphnia sp., Acute immobilisation Test (revision adopted 13 April 2004).

Objective: The purpose of this study was to estimate the effects on the immobilization of Daphnia magna by exposing neonates to the reference substance during a 48-hour period.

Test Species: Daphnia magna

Treatment Doses: Control, 0.5, 1, 2, and 4 mg/L

Study Duration: 48 hours

Test System: Closed Vessel Test Design

Replication: 4 replicates per exposure concentration and 4 replicate controls, 5 neonates per replicate

Control Validity Criteria:
Less than 10% immobilization in control incubations (5% immobilization observed in control, passed)
with no other observed physical or behavioural effects on the daphnids

The EC50 was determined to be 0.92 mg/L after 24 h and at 0.66 mg/L after 48 h. The result of the internal ring trial cited in OECD TG 202 for EC50 is 0.6 mg/L – 2.1 mg/L after 24 h. Thus, these results were within the expected range. Also, this result in presence of 0.5 mg/L Saponin does not deviate from historical data.

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour NOEC is considered to be 0.56 mg/L and 0.60 mg/L, based on the mean exposure to parent and parent + delta-Damascone, respectively. After 48 hours 25 % immobility was recorded at the highest measured test concentration of 0.78 mg/L (4 mg/L, nominal) based on exposure to parent and 0.93 mg/L (4 mg/L nominal) based on the exposure to parent + delta-Damascone.
In terms of time-weighted mean measured concentrations, the 48-hour EC50 value was determined to be > 0.78 mg/L and > 0.93 mg/L, based on parent and parent + delta-Damascone, respectively and the LOEC to be 0.76 mg/L (parent) and 0.85 mg/L (parent + delta-Damascone). The 48 hour EC50 is, therefore, clearly greater than the aqueous saturation concentration of the test item.

Executive summary:

The objective of the study was to determine the effects of GR-87-0307 on the immobilization of Daphnia magna, during a 48-hour semi-static test. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 202. Daphnia sp., Acute Immobilization Test (revision adopted 13 April 2004).

 

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 (0.49, 0.60, 0.85, 0.93 and 0.92 mg/L based on time-weighted mean measured concentrations for parent). A control group and a solvent control group were also included.

 

Four test vessels per treatment (50 mL conical flasks) were prepared which were sealed and which contained sufficient media (40 mL) to leave a low headspace to minimize any potential losses due to volatility. Each test vessel was amended with 5 freshly hatched Daphnia and incubated at 21 - 24°C for 48 hours. After 24 h Daphnia were transferred to fresh media and immobilization was recorded both at 24 and 48 h. The Daphnia magna were not fed during the test.

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, and 48 hours, and analysis was made both for parent molecule and the anticipated released fragrance molecule. Analysis of the test media samples was conducted at 0 h, 24 h before medium exchange, at 24 h after medium exchange and at 48 h. Analysis of the test samples at the start of the exposure intervals (0 and 24 hours) showed measured concentrations to range from 0.49 – 1.26 mg/L (sum of parent +d-Damascone) and at the end of the exposure intervals (24 and 48 hours) to range from 0.32 – 0.77 mg/L (sum of parent +d- Damascone). Increasing concentrations of the liberated fragrant d-damascone were observed during the incubation intervals.

 

Due to the loss over time of the parent and accumulation of the liberated d-damascone, results have been based on time-weighted mean (TWM) measured concentrations based on either parent or the sum of parent and liberated d-damascone.  

 

The EC50 values and the corresponding NOEC and LOEC values are presented based on time- weighted mean (TWM) measured concentrations based on either parent or the sum of parent and liberated d-damascone in the following Table.

 

	Effect concentrations expressed as time-weighted mean measured concentration (mg/L)
	Based on parent	Based on parent +d-Damascone
	24hours            48hours	24 hours	48 hours
EC50	>0.85                  >0.78	> 0.97	> 0.93
LOEC	>0.85                   0.76	> 0.97	0.85
NOEC	0.85                    0.56	0.97	0.60

 

The 48 hour, NOEC is considered to be 0.56 mg/L and 0.60 mg/L, based on the mean exposure to parent and parent +d-Damascone, respectively. After 48 hours, 25 % immobility was recorded at the highest measured test concentration of 0.78 mg/L (4 mg/L, nominal) based on exposure to parent and 0.93 mg/L (4 mg/L nominal) based on the exposure to parent +d-Damascone. In terms of time-weighted mean measured concentrations, the 48-hour EC50value was determined to be > 0.78 mg/L and > 0.93 mg/L, based on parent and parent +d-Damascone, respectively and the LOEC to be 0.76 mg/L (parent) and 0.85 mg/L (parent +d-Damascone). The 48 hour EC50 is, therefore, clearly greater than the aqueous saturation concentration of the test item.

 

The validity criteria for control immobility (≤10%), with no observed signs of disease or other effects on behavior, and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

Description of key information

The objective of the study was to determine the effects of GR-87-0307 on the immobilization of Daphnia magna, during a 48-hour semi-static test. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 202. Daphnia sp., Acute Immobilization Test (revision adopted 13 April 2004).

The 48 hour, NOEC is considered to be 0.56 mg/L and 0.60 mg/L, based on the mean exposure to parent and parent + d-Damascone, respectively. After 48 hours, 25 % immobility was recorded at the highest measured test concentration of 0.78 mg/L (4 mg/L, nominal) based on exposure to parent and 0.93 mg/L (4 mg/L nominal) based on the exposure to parent + d-Damascone.

In terms of time-weighted mean measured concentrations, the 48-hour EC50 value was determined to be > 0.78 mg/L and > 0.93 mg/L, based on parent and parent + d-Damascone, respectively and the LOEC to be 0.76 mg/L (parent) and 0.85 mg/L (parent + d-Damascone). The 48 hour EC50 is, therefore, clearly greater than the aqueous saturation concentration of the test item.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.78 mg/L

Additional information

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).