1,1'-(benzene-1,3-diyldimethanediyl)bis(1H-pyrrole-2,5-dione)

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-18 to 2018-02-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-({3-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]phenyl}methyl)-2,5-dihydro-1H-pyrrole-2,5-dione

EC Number:

695-930-2

Cas Number:

13676-53-4

Molecular formula:

C16H12N2O4

IUPAC Name:

1-({3-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]phenyl}methyl)-2,5-dihydro-1H-pyrrole-2,5-dione

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC-DAD

Details on sampling:

- Concentrations: 100, 50.0, 25.0, 12.5 and 6.25 % of a saturated solution were analytically verified
- Sampling method: The samples and the control were diluted with acetonitrile containing 0.2% H3PO4 directly after sampling (dilution factor 1.43). At the start of the exposure interval the lower concentrated samples (6.25% to 25.0% of the saturated solution) and the control were analyzed without further dilution. The other concentrations (50.0% and 100% of the saturated) were diluted with dilution medium. Additionally, all samples from the end of the exposure interval were analyzed without dilution.

Test solutions

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution with a nominal loading of 100 mgIL of the test item was prepared one day prior to the start of the exposure and the renewal of the test solutions (at -24 and 0 hours). An appropriate amount of the test item (100 mg/L was weighed out) was transferred with an appropriate amount of the dilution water into a glass flask. This dispersion was treated with ultrasound for 5 minutes at room temperature and mixed by constant stirring with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). 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 10 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 as the highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water. During filtration, the filter was always be kept covered. The undiluted saturated solution and four dilution levels prepared out of the saturated solution in a geometric series with a separation factor of 2 by dilution of the saturated solution with dilution water, were tested as follows: 6.25 - 12.5 - 25 - 50.0 - 100% of the saturated solution. The test concentrations were selected based on the results of a non-GLP preliminary range finding test.

- Controls: Negative Control: Dilution water without test item incubated under the same conditions as the test groups. Positive control: A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in ISO test water (acc. to OECD 202, Annex 3) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The reference substance is potassium dichromate.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna (Clone 5)
The daphnids were not fed during the study.

ACCLIMATION
- Acclimation period: Acclimatization was not necessary, because the dilution water was equivalent to the culture medium.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Medium modiefied to achieve total hardness of 160 -180 mg CaCO3/L

Test temperature:

18 - 22 °C, constant within ± 1 °C

Nominal and measured concentrations:

nominal: 0, 6.25, 12.5, 25.0, 50.0, 100% of saturated solution
geometric mean: 0.221, 0.322, 0.446, 1.19, 3.60 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Material, size, headspace, fill volume: Glass beakers (4 (lD) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
- Renewal rate of test solution (frequency/flow rate): The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Same as culture medium
- Total organic carbon: 160-180 mg CaCO3/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Prior to the start of the exposure (0 hours) and at renewal (24 hours), the water quality parameters (i.e. pH-value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured.

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilization was determined in all groups after 24 and 48 hours. An animal was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other adverse effects did not appear.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: saturated solution at a loading rate of 100 mg/Land two further dilution levels prepared by diluting the saturated solution by factor 10 and 100 with dilution water
- Results used to determine the conditions for the definitive study: At a loading of 100 mg/L all daphnids of the preliminary study were dead within 24 h, thus, the saturated solution was diluted with a factor 2.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.415 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.415 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

1.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

1.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: yes, EC50 = 2.10 mg/L CI (1.92-2.43 mg/L)
Ranges of validity
EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5),
EC50 (24 hours): 0.6 - 2.1 mgIL, according to OECD 202 (clone A)

Reported statistics and error estimates:

The EC10- and the EC50-values (after 24 and 48 hours of exposure) were calculated by sigmoidal dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism. The concentration-effect relationships are shown graphically. The EC50- value for the reference item with 95% confidence limits was calculated accordingly.

Any other information on results incl. tables

Table 1: Immobilization Rates after 24 and 48 hours of Exposure during the Definitive Test

Geometric mean measured test item concentration [mg/L]	Immobilization [%]
	24 h					48 h
	Replicates					Replicates
	1	2	3	4	mean	1	2	3	4	mean
3.60	100	100	100	100	100	100% Immobilization (mortality) after 24h
1.19	100	100	100	100	100
0.446	80	60	60	60	65	80	60	60	60	65
0.322	0	0	40	0	10	0	0	40	0	10
0.221	0	0	0	0	0	0	0	0	0	0
control	0	0	0	0	0	0	0	0	0	0

Table 2: Absolute Numbers of immobile Daphnids after 24 and 48 h of Exposure in the Definitive Test

Geometric mean measured test item concentration [mg/L]	NUMBER OF IMMOBILE DAPHNIDS / TOTAL NUMBER OF DAPHNIDS
	24 H					48 H
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
3.60	5/5	5/5	5/5	5/5	20/20	5/5	5/5	5/5	5/5	20/20
1.19	5/5	5/5	5/5	5/5	20/20	5/5	5/5	5/5	5/5	20/20
0.446	4/5	3/5	3/5	3/5	13/20	4/5	3/5	3/5	3/5	13/20
0.322	0/5	0/5	2/5	0/5	2/20	0/5	0/5	2/5	0/5	2/20
0.221	0/5	0/5	0/5	0/5	0/20	0/5	0/5	0/5	0/5	0/20
control	0/5	0/5	0/5	0/5	0/20	0/5	0/5	0/5	0/5	0/20

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item (m-Xylylenebismaleimide), the 48 hours-EC50 for Daphnia magna was 0.415 mg/L (95% confidence limits: 0.388 - 0.436 mg/L).