[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate

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:

2017-06-12 to 2017-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP-study performed according to OECD 202 guideline. All validity criteria were successfull.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

2017-01-10

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

Duplicate samples for analysis were taken from the control and the test concentration of 100 mg.L-1 at the start and at the end of the test (t=48h).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The mixing vessel was a cylindrical glass bottle sealed with a screw cap. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and 1 L test water was added. Then 105.20 mg test item (gelled texture) was weighed on a glass slide that afterwards was placed under the surface of the test water contained in the mixing vessel through fishing wire. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 hours of gentle stirring in the dark and at approx. 40°C, the contents of the vessel was allowed to stand undisturbed for ca. 5 min at room temperature. Then the stock solution was directly added into test tubes (without headspace) that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless. The test was carried out without adjustment of the pH

- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

Total water hardness was approximately 250 mg/L (as CaCO3)

Test temperature:

between 20.8 and 20.9 °C throughout the test (average value: 20.9°C), and complied with the requirements (20°C ± 2°C, constant within 1°C).

pH:

7.62 - 8.61
pH requirements: 6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

6.33 - 8.03
All these dissolved-oxygen concentrations correspond to values > 70% of the air-saturation value.

Salinity:

None

Nominal and measured concentrations:

Nominal : 100 mg/L
Since the concentrations of the test item were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, the results were based on nominal values.

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: Static (stability of the test item was demonstrated in closed and static conditions during the range-finding test)
- No. of daphnids: 20 per treatment group (including controls), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Number of replicates: 4 replicates with daphnids per treatment group.
- Introduction of Daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
Test conditions were recorded with suitable instruments.
- Immobility: Eventual immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At the beginning and at the end of the test from all treatment groups.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Range finding study: Ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal concentrations of 1.0, 5.0,10.0, 20.0 and 74.0 mg test item.L-1 (stock solution directly added into test tubes) and to a control.
- Results used to determine the conditions for the definitive study: Since no effect was observed, a second range-finding test was performed using only one test concentration, at 100 mg.L-1 (nominal). No effect was also observed. After these range-finding studies, it has been decided to do a limit tests with at 100 mg test substance/L for the final test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the test concentration of 100 mg.L-1, confirming the observations of the range-finding test where results showed no immobilisation at any of the tested concentrations.
Based on these results, the 24 and 48-hour EC50 values were therefore > 100 mg test item.L-1.

Results with reference substance (positive control):

On April 4, 2017 (most recent test), the 24h-EC50 was 0.97 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

No statistical analysis was performed. Effective concentrations were determined directly from the raw data.

Analytical results

Duplicate samples taken fromthe control and the test concentration of 100 mg.L^-1 were analysed at the startand the end of the testin order to determine maintenance of actual concentrations of the test item.

The analytical results of this test showed that concentrations of test solutions prepared following the outlined procedures were reproducible. Indeed, analyses of samples taken fromthe test concentration of 100 mg/L at the start and the end of the test revealed that test item levels found were stable, with losses of test item < 20%. Moreover, since the concentrations of the test item were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, the results can therefore be based on nominal values.

Table 6.1.3/1: Concentrations of the test item (mg.L-1) in test water - Final test

Nominal concentration (mg test item.L^-1)	Start (t=0h)		End (t=48h)		Relative loss to initial value (t=0h - t=48h) (%)
		Mean		Mean
Control	Absence	Absence	Absence	Absence	N.A.
Control	Absence		Absence		N.A.
100	100.33	100.22	93.00	96.54	7
100	100.10		100.08		0

N.A.: not applicable

Absence: concentrations below the LOQ (1.04 mg.L^-1) and the LOD (0.31 mg.L^-1).

Table 6.1.3/2: Acute immobilisation of daphnids after 24 and 48 h in the final test

Nominal concentration(mg test item.L^-1)	Replicate	Number of daphnids exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
100	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0

Validity criteria of the study:

- Controls: In the control, no daphnids became immobilized nor trapped at the surface of the water or showed signs of stress.

- Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 60 % of the air-saturation value in controls and test vessels.

Thus the validity criteria have been fulfilled in the present study.

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal concentrations, the 48-hour EC50 was found to be higher than 100 mg test item/L and the highest nominal concentration without observed effects values was estimated to be at least 100 mg test item/L.

Executive summary:

A study was performed to assess the acute toxicity of test item QUESTICE L to Daphnia magna. The method followed was designed to be compliant with the OECD 202 Guideline and the study was conducted under GLP conditions.

 

A limit test was performed following the results of a range-finding test. Twenty daphnids (four replicates, five daphnids per replicate) were exposed tothe test item at a nominal concentration of 100 mg test item/L and to a control.The immobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours. Duplicatesamples for analysis were taken from the control and the test concentration of 100 mg/L at the start and at the end of the test (t=48h) in order to determine maintenance of actual concentrations.

The test item levels were found to be relatively stable throughout the test (within ± 20% of the initial and nominal concentration throughout the test). Thus, the evaluation of the effects on daphnids was based on the nominal concentrations. After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the test concentration of 100 mg/L, confirming the observations of the range-finding test where results showed no immobilisation at any of the tested concentrations.

Under the experimental conditions and based on nominal concentrations, the 48-hour EC50 was found to be higher than 100 mg test item/L and the highest nominal concentration without observed effects values was estimated to be at least 100 mg test item/L.

All validity criteria were fulfilled. This study is considered as valid for that endpoint.

Results Synopsis:

Test: Toxic effect of QUESTICE L to Daphnia magna according to OECD guidelines No. 202 (GLP).

48h-EC50: > 100 mg/L

Description of key information

Under the experimental conditions and based on nominal concentrations, the 48-hour EC50 was found to be higher than 100 mg test item/L and the highest nominal concentration without observed effects values was estimated to be at least 100 mg test item/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

An experimental study to assess the acute toxicity of test item QUESTICE L to Daphnia magna was available.The method followed was designed to be compliant with the OECD 202 Guideline and the study was conducted under GLP conditions.

 

A limit test was performed following the results of a range-finding test. The test item levels were found to be relatively stable throughout the test (within ± 20% of the initial and nominal concentration throughout the test). Thus, the evaluation of the effects on daphnids was based on the nominal concentrations.

After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the test concentration of 100 mg/L, confirming the observations of the range-finding test where results showed no immobilisation at any of the tested concentrations.

The study respected the requirements of the guideline. This study is considered acceptable for that endpoint.