2,4-Imidazolidinedione, 5-(2-methylpropylidene)-, (5Z)-

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:

2020-11-24 to 2020-11-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(5Z)-5-(2-methylpropylidene)imidazolidine-2,4-dione

EC Number:

700-726-4

Cas Number:

1369499-44-4

Molecular formula:

C7H10N2O2

IUPAC Name:

(5Z)-5-(2-methylpropylidene)imidazolidine-2,4-dione

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling schedule: All concentration levels and the control were analytically verified via LC-MS at the start (0 hours) of the exposure (72 hours) with algae.
- Sampling and pre-treatment: At the start, samples were taken from one additional replicate of each test item concentration and the control and at the end samples were taken from pooled replicates.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the stock solutions
A stock solution with a nominal concentration of 250 mg test item/Lwas freshly prepared with dilution water and treated with ultrasound for 20 minutes at room temperature. The Tyndall effect was negative.

Preparation of the test item concentrations
Out of the stock solution five concentrations were tested in a geometrical series with a dilution factor of 2: 15.6 - 31.3 - 62.5 -125 - 250 mg/L

- Controls: Six replicates (without test item) were tested under the same test conditions as the test item replicates.
- Test container: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
- Test volume : 100 mL

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 hours per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

37 mg CaCO3/L]

Test temperature:

22 - 23,5 °C

pH:

7.54 to 9.22

Conductivity:

142 μS/cm

Nominal and measured concentrations:

Nominal 250 – 125 – 62.5 – 31.3 – 15.6 mg/L

The measured concentrations of the test item (5Z)-5-(2-methylpropylidene)imidazolidine-2,4-dione (Z-IBH) in the fresh media were in the range of 103 to 113% of the nominal values at the start of the exposure (0 hours). The measured concentrations of the test item in the old media were in the range of 112 to 117% of the nominal values.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL
- Type (delete if not applicable): closed, sealed with cotton wool plugs
- Material, size, headspace, fill volume: Sterile glass Erlenmeyer flasks with 250 mL volume and 100 mL fill volume
- Initial cells density: Nominal:approximately 5 x E+03 - E+04 cells/mL; Actual: 5375 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:
TEST MEDIUM / WATER PARAMETERS
Concentration [mg/L]
NH4Cl 15
MgClg x 6 H2O 12
CaCl2 x 2H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA X 2 H2O 0.1
H3BO3 0.185
MnCl x4 H2O 0.415
ZnCl2 3E-03
Na2MoO4 x 2 H2O 7E-03
COCl2 x 6 H2O 1.5E-03
CuCl2 x 2 H2O 1E-05
NaHCO3 50
pH 8.1 ± 0.2
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 24h day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self -fluorescence was found in the preliminary range finding test at the saturated solution
- Other: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations Range finding study: 1.0, 10.0, and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

107 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (92.3 - 120)

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 250 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 15.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

125 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: (109 - 149)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Adherence to test vessels: not reported
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: growth: 0.903 (0.858 – 0.946); valid range: 0.775 ± 0.558
- EC50: yield: 0.441 (0.421 – 0.462); valid range: 0.421 ± 0.339

Reported statistics and error estimates:

The NOEC / LOEC was determined by calculation of statistical significant differences of growth rate and yield using Dunnett’s Method. A Shapiro-Wilk’s Normality test and a Brown-Forsythe’s Equal Variance test were done first. P-values for both Normality
and Equal Variance tests are 0.05. The α -value (acceptable probability of incorrectly concluding that there is a difference) is α = 0.01.
The ECx-value of yield inhibition after 72 hours was calculated by sigmoidal dose-response regression.

Any other information on results incl. tables

MeasuredConcentration of the test item in the Definitive Test with Algae

Sampling date

Fresh media, 0 hours

Old media, 72 hours

(5Z)-5-(2-methylpropylidene)imidazolidine2,4-dione (Z-IBH)
Nominal Test item concentration [mg/L]]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]
250	284	113	283	113
125	134	107	143	114
62.5	69.8	112	70.1	112
31.3	34.5	110	36.8	117
15.6	16.1	103	17.5	112

Control

<LOQ

<LOQ

Meas. conc.= measured concentration of the test item, dilution taken into account

%                  = percentage of the initial measured concentration of the test item

LOQ             = limit of quantification (1 mg/L of the test item)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, (5Z)-5-(2-methylpropylidene)imidazolidine-2,4-dione (Z-IBH) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 250 mg/L and 125 (109 -149) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 31.3 mg/L.