N,N'-bis(2,3-dihydroxypropyl)-5-nitrobenzene-1,3 dicarboxamide

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:

27.1.2020 - 31.1.2020

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

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(2,3-dihydroxypropyl)-5-nitrobenzene-1,3 dicarboxamide

EC Number:

616-392-7

Cas Number:

76820-34-3

Molecular formula:

C14H19N3O8

IUPAC Name:

N,N'-bis(2,3-dihydroxypropyl)-5-nitrobenzene-1,3 dicarboxamide

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Not available

IUPAC Name:

Unknown impurities

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00601016
- Expiration date of the lot/batch: 03/2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.
- Stability under test conditions: Stable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The determination of the test item concentrations was carried out by HPLC method.
Preliminary test: The analytical determination of the test item concentrations was performed at the beginning of the test and at the end of the test. The samples for analysis were taken from the highest (100 mg·L-1) and the lowest test concentration (1.0 mg·L-1). The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples were analysed on the day of delivery. The samples for analysis at the end of the test (48 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analysed on the day of delivery.All samples were stored at laboratory temperature.
The analytical results showed, that the test item was stable in dilution water at the conditions

Test solutions

Details on test solutions:

Preparation of solutions of the test item
The stock solution of the test item was prepared in the dilution water. 50 mg of the test item was weighed into 500 mL of the dilution water for the preliminary test. The concentrations of solutions used in the preliminary test were obtained by dilution of the stock solution with dilution water.
Test solution
Stock solution of test item: 50.03 mg / 500 mL
Test concentrations: 100, 50, 10, 5, 1 mg·L-1
Conductivity of deionized water: 2.52 µS·cm-1
pH of dilution water: 8.0
Oxygen content of dilution water: 8.7 mg·L-1
Content of (Ca+Mg) in dilution water: 2.54 mmol·L-1

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna Straus were obtained by aperiodic parthenogenesis from own laboratory breeding. Daphnia were fed by a mixture of algae. The sensitivity of the test species is periodically monitored by testing with the reference item, potassium dichromate.

TEST ORGANISM
- Common name: Daphnia Magna
- Strain: Straus
- Source: own laboratory breeding
- Age at study initiation: young daphnia born in 24 hours
- Method of breeding: aperiodic parthenogenesis
- Feeding during test: no

ACCLIMATION
The grow-up gravid females are separated into dilution water. Young daphnia born in 24 hours are segregated to the handling container from which they entered the experiment.
- Type and amount of food: a mixture of algae
- Health during acclimation: no mortality observed

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

2.54 mmol·L-1

Test temperature:

202°C, with a variation of  1°C at each individual test

pH:

8

Dissolved oxygen:

8.7 mg·L-1

Conductivity:

2.52 µS·cm-1

Nominal and measured concentrations:

1, 5, 10, 50, 100 mg/L + C(control)
Determined concentration of test substance
0 hours: 0,99 ; 101,5 mg·L-1
48 hours: 0,96, 100,96 mg·L-1

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers 50ml
- Type (delete if not applicable): open
- Aeration: no
- Lighting: 16 hours light and 8 hours dark cycle
- Number of test animals: 20 for each concentration and control
- Number of replicates (per concentration): 4
- Number of test animals per replicate: 5
- Biomass loading rate: at least 2 ml per individual

The holding and culturing of the Daphnia is performed at conditions (light, temperature, medium) similar to those which are used in the test. For holding and culturing of the daphnia the M4 medium was used (according to Annex 2
of the EU method C.2 and according to Annex 3 of the OECD TG 202). For test was used dilution water (according to Annex 2 of the EU method C.2 and according to Annex 3 of the OECD TG 202). Dilution water was prepared just before the test by admixing 2.5 mL of stored solutions A, B, C and D into 1 000 mL of deionized water. Because culturing and test medium was different we included pre-test acclimation period: brood daphnids were maintained in dilution water for 48 hours prior to the start of the test
Dilution water
A. 117.6 g CaCl2·2H20 in 1L deionized water (Batch No.: PP/2018/07142, exp.: 04/2021, Lach-Ner, s. r.o.)
B. 49.3 g MgSO4·7H2O in 1L deionized water (Batch No.: PP/2018/08687, exp.: 04/2023, Lach-Ner, s. r.o.)
C. 25.9 g NaHCO3 in 1L deionized water (Batch No.: PP/2016/04662, exp.: 02/2020, Lach-Ner, s. r.o.)
D. 2.3 g KCl in 1L deionized water (Batch No.: PP/2019/10337, exp.: 02/2022, Lach-Ner, s. r.o.)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

0 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

0 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The sensitivity of the test species and correctness of test performance is periodically verified by testing with the reference item, potassium dichromate.
The results of the verification test with K2Cr2O7, carried out in period from 22.05. to 24.05.2019 are the following:
24 hour – EC50 = 1.34 mg·L-1 (95% confidence limit: 1.16 – 1.64 mg·L-1)
48 hour – EC50 = 0.78 mg·L-1 (95% confidence limit: 0.68 – 0.91 mg·L-1)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The experiment with the concentration of 100 mg·L-1 included in the preliminary test has been performed using 20 daphnia, therethrough the conditions of the limit test given in guidelines have been fulfilled. It was demonstrated that the EC50 is higher than this concentration.

Evaluation of results:
No immobilisation was observed in the test. Therefore exact values of EC50 could not be calculated and the values of EC are given in the form of a range. The EC0 and EC100 values are identified by direct observation. The nominal concentrations were used for all evaluations and results.
Test results:
24 hour – EC50 >100 mg·L-1 (nominal concentration)
48 hour – EC50 >100 mg·L-1 (nominal concentration)
24 hour – EC0 = 100 mg·L-1 (nominal concentration)
48 hour – EC0 = 100 mg·L-1 (nominal concentration)
24 hour – EC100 >100 mg·L-1 (nominal concentration)
48 hour – EC100 >100 mg·L-1 (nominal concentration)

Executive summary:

The test item, NBA-A, was tested in acute immobilisation test on Daphnia magna.

The test was performed according to method C.2 – Daphnia sp. Acute Immobilisation Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

The preliminary test was performed in a range of the test item nominal concentrations 1 – 100 mg·L-1. The test was performed as static.

Based on no toxicity of the test item found in the preliminary test, the study was terminated. The experiment with the concentration of 100 mg·L-1 included in the preliminary test has been performed using 20 daphnia, therethrough the conditions of the limit test given in guideline have been fulfilled. It was demonstrated that the EC50 is higher than this concentration.

There is stated in the guideline that, if evidence is available to demonstrate that the concentration of the test item has been satisfactorily maintained within ± 20 percent of the nominal or measured initial concentration throughout the test, the results can be based on nominal or measured initial values. In this study concentration of the test item has been satisfactorily maintained within ± 20 percent of the nominal concentration throughout the test. Further the nominal concentrations were used for all evaluation and results.

Test results:

24 hour – EC50 > 100 mg·L-1 (nominal concentration)

48 hour – EC50 > 100 mg·L-1 (nominal concentration)

24 hour – EC0 = 100 mg·L-1 (nominal concentration)

48 hour – EC0 = 100 mg·L-1 (nominal concentration)

24 hour – EC100 > 100 mg·L-1 (nominal concentration)

48 hour – EC100 > 100 mg·L-1 (nominal concentration)