2,4-Imidazolidinedione, 5-(phenylmethylene)-, (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 2020-11-30, with the definitive exposure phase from 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-(phenylmethylidene)imidazolidine-2,4-dione

EC Number:

700-728-5

Cas Number:

74805-60-0

Molecular formula:

C10H8N2O2

IUPAC Name:

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

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.686 – 2.19 – 6.66 – 20.7 – 65.7 mg/L (geometric mean measured test item concentrations)
- Sampling method: All loading rates and the control were analytically verified via LCMS at the start (0 hours) of the exposure (72 hours) with algae.
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 AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Preparation of the stock solutions
A saturated solution with a nominal loading rate of 100 mg test item/L was freshly prepared and treated with ultrasound for 5 minutes at room temperature. Thereafter the solution was stirred for 4 hours (room temperature, 1100 rpm) with a magnetic stir rer. Undissolved particles were removed by membrane filtration The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered. The Tyndall effect was positive.

Preparation of the test item concentrations
Out of the saturated solution five dilution levels were tested in a geometrical series with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100% of the saturated solution, corresponding to the geometric mean measured test item concentrations of: 0.686 –
2.19 – 6.66 – 20.7 – 65.7 mg test item/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).

- Controls: Six replicates (without test item) were tested under the same test conditions as the test item replicates.

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 2°C

pH:

7.59 to 9.02

Conductivity:

142 μS/cm

Nominal and measured concentrations:

Saturated Solution of the test item [% GL] 100 – 31.6 – 10 – 3.16 – 1 mg/L
Geometric mean measured: 65.7- 20.7 - 6.66 - 2.19 - 0.686 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- 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:Out of the saturated solution five dilution levels were tested in a geometrical series with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100% of the saturated solution
- Range finding study
- Test concentrations: 0, 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.:

2.19 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.:

5.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (4.11 – 7.58)

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 24.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (22.5 – 27.0)

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.19 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.:

< 0.686 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.:

6.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: (3.61 – 14.0)

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.

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-benzylideneimidazolidine-2,4-dione (Z-BH)
Saturated Solution of the test item [% GL]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	[%]	Geometric mean Test item concentration [mg/L]
100	63.0	68.5	109	65.7
31.6	19.3	22.1	114	20.7
10.0	6.10	7.27	119	6.66
3.16	2.00	2.40	120	2.19
1.00	0.627	0.751	120	0.686

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 (0.500 mg/L of the test item)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, the effect values to the test item on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h were determined as follows (based on geometric mean measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 h were 24.4 mg/L and 6.68 mg/L, respectively. The NOEC for both growth rate and yield was 2.19 mg/L, respectively.