Reaction mass of (S)-2-[(R)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide and (S)-2-[(S)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 February 2008 - 18 March 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

21 September 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10706:2000, “Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea)”, 2000-03-30.

Version / remarks:

These study procedures were in conformity with the OECD guideline for testing of chemicals No. 211.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 16 May 2007

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-(2-oxo-4R-propylpyrrolidin-1-yl)butanamide

Cas Number:

357336-20-0

IUPAC Name:

(2S)-2-(2-oxo-4R-propylpyrrolidin-1-yl)butanamide

Test material form:

solid: particulate/powder

Details on test material:

- Description: White to off-white powder
- Storage conditions: At room temperature protected from light

Specific details on test material used for the study:

Highly soluble in water

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and the controls.
- Sampling method: At the beginning and at the end of two intervals of 48 hours (nominal days 6 and 8, 13 and 15) and one interval of 72 hours (nominal days 0 and 3) samples of 1.5 mL were taken. At the end of the refreshment period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: In a freezer.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: The test substance was completely soluble in test medium at the test concentrations. Preparation of test solutions started with a loading rate of 100 mg/L applying vigorous shaking to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the loading rate in test medium.
- Controls: Test medium without test substance or other additives.
- Evidence of undissolved material: No, the final test solutions were all clear and colourless.
- Other relevant information: The test substance was kept from direct light as much as possible during weighing and preparation of test solutions.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, 1820
- Age at study initiation: <24 hours
- Stage and instar at study initiation: at least third generation
- Method of breeding: acyclical parthenogenesis under specified breeding conditions (see below for details).
- Source: in-house laboratory culture with a known history
- Age of parental stock: >2 weeks old
- Feeding during test : yes, daily: 0.25 mL of Chlorella pyrenoidosa suspension containing 1.6 x10^8 cells, corresponding to 0.2 mg C/Daphnia/day.

ACCLIMATION : no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Each batch was started with newborn daphnids, i.e. less than 3 days old, by placing them individually in 50 mL M7-medium (18-22°C). The maximum age of the cultures was 4 weeks. Three times a week the young were counted and the parental daphnids were transferred to new media. Daphnids were fed daily with a suspension of fresh water algae.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

143 - 179 mg CaCO3/L throughout the test

Test temperature:

18.8 - 19.7°C throughout the test

pH:

7.6 - 8.2 throughout the test

Dissolved oxygen:

7.7 - 9.5 mg O2/L throughout the test

Nominal and measured concentrations:

- Nominal concentrations: 4.6, 10, 22, 46 and 100 mg/L
- Measured concentrations: see table 1 in 'any other information on materials and methods'. The measured concentrations were in agreement with nominal concentrations (94-114%) and stable during the two or three-day refreshment periods (98-108% of initial).
- Effect parameters were thus based on nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL (6 x Ø 3.5 cm), containing a minimum of 50 ml test solution; vessels are all-glass covered with a Perspex plate.
- Renewal rate of test solution: three times a week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20, containing only test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by Reverse Osmosis
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature, oxygen and pH were measured at the start of the test and after each renewal

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h, daily
- Light intensity: intensity at the start: 638-690 lux (10-11 μE.m^-2.s^-1); intensity at the end: 642-717 lux (10 μE.m^-2.s^-1)

EFFECT PARAMETERS MEASURED:
- Parental daphnids: number of living, immobile and dead parental daphnids (every workday and upon renewal on non-workdays); presence of eggs in the brood pouch (every workday and upon renewal on non-workdays); body length (at the end of the test).
- F1 generation: appearance of first brood (when observed); number of newborn daphnids and their condition (every workday and upon renewal on non-workdays); presence of unhatched eggs (when observed); incidence of immobility (when observed).
- Test medium: temperature, oxygen and pH (at the start of the test and just before and after each renewal); hardness (once a week in fresh and old media); light (at the start of the test).

PRELIMINARY STUDY: as no data were available from an acute toxicity test, a seven day preliminary test was performed with test conditions similar to the final test.
- Test concentrations: 0, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, one parental daphnid died after three days, at the highest test concentration (100 mg/L) while no daphnids died at any of the lower concentrations. The percentage of daphnids carrying eggs at the tested concentrations was comparable to the control. Although the mortality rate and the observations did not give reason to suspect significant effects on reproduction in a full test, a range of concentrations was used for the 21-day reproduction test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: parental mortality

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Parental daphnids:
- Mortality: 20% in the control, 0% in the highest tested concentration (100 mg/L) and 10-20% in the lower test concentrations. Based on these observations, it was concluded that mortality of the parental daohnids was not treatment related.
- Presence of eggs: In the control, the presence of eggs in the brood pouch was recorded for the first time on day 6 and the first brood appeared on day 8. The first recording of the presence of eggs in the brood pouch was similar to the control in treated solutions up to and including 100 mg/L. Hence, no delay in appearance of the first brood was observed in the treatments when compared to the control.
- Reproduction: There were no recordings of immobile young or appearance of unhatched (aborted) eggs at any of the test concentrations throughout the 21-day test period. No significant differences were recorded between the values for total reproduction at any of the test concentrations when compared to the values of the control.
- Body length: No significant reduction of average body length compared to the controls was observed (< 3% at all tested concentrations).
- Mortality of control: yes, but not exceeding 20%.

Experimental conditions: measurements of temperature, pH, dissolved oxygen, hardness and dissolved organic carbon content were all within the prescribed limits as laid down in the protocol.

For a summary of effect parameters, see table 4 in 'Any other information on results'.

Reported statistics and error estimates:

Total reproduction on day 21 was tested for normality and for homogeneity of variance and statistically tested using an ANOVA test followed by a mean comparison test (Bonferroni t Test). The software was TOXSTAT Release 3.5.

Any other information on results incl. tables

Table 2 Cumulative mortality of parental daphnids during the exposure period

Concentration (mg/L)	Number exposed	Cumulative number of dead parental daphnids on day:						Mortality %
		1	3	13	15	20	21
control	20	0	0	1	2	3	4	20
4.6	10	0	0	0	0	1	1	10
10	10	0	0	0	0	1	1	10
22	10	0	0	0	0	1	2	20
46	10	0	0	0	0	1	1	10
100	10	0	0	0	0	0	0	0

Table 3 Average body lengths of parental daphnids

Concentration (mg/L)	Average length (mm)	SD	% Reduction
control	4.35	0.09
4.6	4.31	0.15	1
10	4.36	0.10	0
22	4.24	0.25	3
46	4.36	0.07	0
100	4.26	0.13	2

Table 4 Overview of effect parameters

Parameter	Concentration (mg/L)
21-day LC50 (parental mortality)	> 100
NOEC for parental mortality	100
21-day EC50 for reproduction	> 100
21-day LOEC for reproduction	> 100
21-day NOEC for reproduction	100
21-day LOEC for parental body length	> 100
21-day NOEC for parental body length	100
Overall LOEC	> 100
Overall NOEC	100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1). The mortality of the parent animals (female Daphnia) did not exceed 20% at the end of the test (20%). 2). The average cumulative number of young per female in the controls after 21 days was ≥60 (117 ± 13).

Conclusions:

The results of a Daphnia reproduction study, performed according to OECD 211 and GLP, show that the substance did not affect reproduction of Daphnia magna up to and including the highest test concentration of 100 mg/L after 21 days of exposure (overall-NOEC).

Executive summary:

In a 21 -day semi-static reproduction study, the toxicity of the test item to aquatic invertebrate Daphnia magna was assessed according to OECD guideline no. 211 and GLP principles. Nominal test concentrations were prepared at a loading rate of 100 mg/L with further dilutions to achieve the following test concentrations: 4.6, 10, 22, 46 and 100 mg/L. Per test concentration 10 vessels were used, whereas for the control 20 vessels were used. In each vessel, one daphnid (<24 hours of age) was placed. Every three days test solutions were renewed. The test concentrations were analytically verified and the results showed that the test concentrations were in agreement with nominal concentrations (94-114%) and stable during the two or three-day refreshment periods (98-108% of initial). There was no dose dependent effect of the test substance on mortality, reproduction or body length of the parental daphnids. Therefore it was concluded that the test item was not toxic for the reproduction of Daphnia magna (NOEC 100 mg/L). All criteria for acceptability of the test were met according to OECD guideline 211 and the study is considered to be reliable without restrictions.