(S)-2-(5-Oxo-3-propyl-1,2-dihydropyrrol-1-yl) butyramide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Feb 2019 - 21 Mar 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(S)-2-(5-Oxo-3-propyl-1,2-dihydropyrrol-1-yl) butyramide

Cas Number:

357338-13-7

Molecular formula:

C11H18N2O2

IUPAC Name:

(S)-2-(5-Oxo-3-propyl-1,2-dihydropyrrol-1-yl) butyramide

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off-white powder
- Storage condition of test material: In refrigerator protected from light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control
- Sampling method: 1.8 mL test solution were retrieved (by pipette) from the approximate centre of the test vessels at t=0 h, t=24 h and t=72 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤ -15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition of substance to test medium.
The test item was completey soluble in the test medium at all concentrations tested. Therefore, preparation of test solutions started with the highest concentration of 100 mg/L (corrected for purity) applying a 15 minute period of ultrasonic waves followed by one hour of magnetic stirring to accelerate the dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum at test initiation: 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Stock culture medium: M1 medium, according to NPR 6505
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium and test medium: M2 medium, according to OECD 201

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 23°C

pH:

At start (t=0 h): 7.9-8.2
At end (t=72 h): 8.3

Nominal and measured concentrations:

Nominal: control and 1.0, 3.2, 10, 32 and 100 mg/L (corrected for purity)
Measured: The measured concentrations were at the level of nominal (i.e. 94 – 103% relative to nominal concentrations) throughout the test. Effect parameters were therefore expressed in terms of analytically confirmed nominal concentrations.
See Table 1 in 'Any other information on results' for details on measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 10^4 cells/mL
- Control end cells density: 280 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Additional replicates: 1 extra replicate of each test group for sampling purposes after 24 hours of exposure; 1 or 2 replicates of each test concentration without algae

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using tap-water purified by reverse osmosis.
- Intervals of water quality measurement: pH: at the beginning and at the end of the test, for all test concentrations and the control. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod and light intensity: Continuously using TLD-lamps with a light intensity within the range of 83 - 88 μE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length =
10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test, microscopic observations were performed on the highest test concentration and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Combined Limit/Range finding study test concentrations: control and nominal concentrations of 0.1, 1.0, 10 and 100 mg/L (corrected for purity).
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Jan 2019)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: nominal concentration corrected for purity

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: nominal concentration corrected for purity

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: nominal concentration corrected for purity

Details on results:

- A well-defined EC50 for growth rate inhibition could not be determined because the observed effects were below 50%.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to nominally 100 mg/L (corrected for purity) when compared to the control.
- No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
- Analytical results: The measured concentrations were at the level of nominal (i.e. 94 – 103% relative to nominal) throughout the test. The concentrations measured in the samples without algae were comparable to the concentrations in the samples incubated with algae. It should be noted that a low amount of test item was detected in the control at 24 hours of exposure. Considering the stability of the test item in the other test concentrations and that no test item was detected in the control at the start and at the end of the test, it is likely that the sample had been contaminated during sampling.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The EC50 for growth rate inhibition (72h-ErC50) was 1.30 mg/L with a 95% confidence interval ranging from 1.29 - 1.32 mg/L.
The 72h-ErC50 was within the expected range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012, and the historical range of 0.86 and 2.3 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.

Reported statistics and error estimates:

- Statistical significance: Dunnett`s Multiple t-test Procedure, α=0.05, one-sided, smaller.
- ECx calculation: ECx-values could not be determined because the observed effects were below 10%.
- ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Any other information on results incl. tables

Table 1: Final Test: Test Samples

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	n.d.	n.a.
	1.0	0.989	99
	3.2	3.19	100
	10	9.77	98
	10 [1]	9.94	99
	32	31.4	98
	100	96.7	97
24	0	0.034 [2]	n.a.	n.a.
	1.0	0.996	100	101
	3.2	3.19	100	100
	10	9.74	97	100
	10 [1]	9.74	97	98
	32	30.7	96	98
	100	99.1	99	102
72	0	n.d.	n.a.	n.a.
	1.0	1.01	101	102
	3.2	3.26	102	102
	10	9.93	99	102
	10 [1]	9.37	94	94
	32	32.1	100	102
	100	103	103	106

[1]    Without algae.

[2]    Estimated value, calculated by extrapolation of the calibration curve.Ecotoxicological samples might have been contaminated during sampling. The maximum contribution to the lowest sampleat this date of sampling was 3.9% based on peak area.

n.d. Not detected.

n.a.  Not applicable

Table 2: Growth Rate and Percentage Inhibition for the Total Test Period

Test item Nominal conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.877	0.0267	6
1.0	1.871	0.0180	3	0.32
3.2	1.884	0.0168	3	-0.38
10	1.894	0.0101	3	-0.89
32	1.895	0.0183	3	-0.94
100	1.877	0.0096	3	0.0010

Table 3: Growth Rate and Percentage Inhibition at Different Time Intervals

Test item Nominal conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.584		1.585		1.462
1.0	3	2.564	0.78	1.594	-0.53	1.456	0.44
3.2	3	2.545	1.5	1.673	-5.5	1.435	1.9
10	3	2.593	-0.35	1.623	-2.4	1.465	-0.21
32	3	2.596	-0.45	1.642	-3.6	1.446	1.1
100	3	2.565	0.73	1.643	-3.6	1.424	2.6

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria

Conclusions:

The 72h-ErC50 and 72h-ErC10 of the substance for growth rate inhibition of Pseudokirchneriella subcapitata were beyond the range of concentrations tested,
i.e. exceeded an analytically confirmed nominal exposure concentration of 100 mg/L (corrected for purity).
The 72h-NOEC was set at 100 mg/L (corrected for purity) based on statistical significance and biological relevance.