2-Propenamide, N-[4-[[(4,6-dimethyl-2-pyrimidinyl)amino]sulfonyl]phenyl]-2-methyl-

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=72 h
Volume 3.0 mL
Storage 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.
Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the limit concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test samples were stored in the freezer (≤ -15°C). Storage stability of samples under these conditions was demonstrated in project 516986.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The batch of V123109 tested was a white crystalline powder with a purity of >95% and completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a 3-minutes treatment with ultrasonic waves followed by a 3-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was filtered through a 0.45 μm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS 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 104 cells/mL.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 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 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Method of cultivation:
Stock culture: 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.
Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap- water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

pH:

7.4-8.1
The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit).

Nominal and measured concentrations:

1.0, 10 and 100% of the SS prepared at loading rate of 100 mg/L.
Analyses of the samples taken from the undiluted SS showed an actual concentration of 77 mg/L at the start of the test. This concentration remained stable throughout the test duration, i.e. was 102% of initially measured throughout the test duration. Based on these results, effect parameters were based on the measured concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 10^4 cells/ml
- Control end cells density: 330 * 10^4 cells/ml
- No. of organisms per vessel: 10^4 cells/ml
- No. of vessels per concentration (replicates): 3 (6 for highest concentration)
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 1 or 2

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24h
- Light intensity and quality: TLD-lamps with a light intensity within the range of 82 to 85 μE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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 (pathlength =20 mm).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 77 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 77 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

77 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

based on biological relevance

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.99 mg/L for growth inhibition

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) was beyond the range tested, i.e. exceeded a measured concentration of 77 mg/L being considered the highest soluble in test medium.
The 72h-NOEC for growth rate inhibition based on statistical significance could not be determined, while based on biological relevance was set at 77 mg/L.

Executive summary:

The objectiveofthe study was to evaluate V123109 for its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀and EC₅₀for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of V123109 tested was awhite crystalline powderwith a purity of>95%and not completely soluble in test medium at the loading rate initially prepared.

A combined limit/range-finding test was performed. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the limit concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

 

Six exponentially growing algal cultures per group, were exposed to an untreated control and the limit concentration, i.e. undiluted saturated solution. In addition, three replicates per group were exposed to solutions containing 1.0 and 10% of the saturated solution in a combined range-finding test. The initial algal cell density was 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Analyses of the samples taken from the undiluted SS showed an actual concentration of 77 mg/L at the start of the test. This concentration remained stable throughout the test duration, i.e. was 102% of initially measured throughout the test duration. Based on these results, effect parameters were based on the measured concentration.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)	NOEC	EC10	EC20	EC50
Growth rate	<77*, 77^#	>77	>77	>77
Yield	<77	<77	<77	>77

* - based on statistical significance,^#- based on biological relevance

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, the EC₅₀for growth rate inhibition (72h-E_RC₅₀) and the EC₅₀for yield inhibition (72h-E_YC₅₀) was beyond the range tested, i.e. exceeded a measured concentration of 77 mg/L being considered the highest soluble in test medium.

The 72h-NOEC for growth rate inhibition based on statistical significance could not be determined, while based on biological relevance was set at 77 mg/L.

The 72h-NOEC for yield inhibition could not be determined in this study.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

77 mg/L