N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 21 July 2020 to 24 September 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the freshly prepared control and test group from the bulk test preparation at 0 and 24 hours and from the old test media (pooled replicates) at 24 and 48 hours for quantitative analysis. The 0-Hour fresh sample was sent for analysis on the day of sampling and the remaining samples were stored frozen prior to being sent for analysis.

Duplicate samples were taken and stored frozen for further analysis if necessary.

All sample bottles were fortified by the addition of 10 mL of acetonitrile in order to stabilise the test item.

Vehicle:

no

Remarks:

Saturated solution used

Details on test solutions:

Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019) and a preliminary media preparation trial was conducted as follows:

A nominal amount of test item (550 mg) was dispersed, in duplicate, in 11 litres of Elendt M4 media with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:

• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Gelman Acrocap filter (approximately 100 mL used to pre-condition the filter was discarded)
• Filtration through a 0.2 μm Gelman Acrocap filter (approximately 500 mL used to pre-condition the filter was discarded)


Based on the information obtained as part of the preliminary media preparation trial for the main study the test item was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL discarded) to give a nominal test concentration of approximately 0.11 mg/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: in house cultures
- Feeding during test : None during exposure to test substance.
- Food type: Each culture was fed with a mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension.
- Frequency: Daily

ACCLIMATION
- Acclimation period: none


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:

Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 medium in a temperature-controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16-hours light and 8 hours darkness cycle with 20-minute dawn and dusk transition periods. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

2.5 mmol/L (250 mg/L as CaCO3)

Test temperature:

19 °C to 21 °C

pH:

pH range: 7.7 to 8.2 over test period

Dissolved oxygen:

Oxygen level range: 8.9 to 9.1 mg O2/L over test period

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 ml glass vessels
- Type (delete if not applicable): open (but covered to prevent evaporation)
- Material, size, headspace, fill volume: Each vessel contained 100 mL Elendt M4 medium
- Aeration: No
- No. of organisms per vessel: Five
- No. of vessels per concentration (replicates): Four
- No. of vessels per control (replicates): Four

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium in reconstituted water

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Light intemsity: 200 to 1200 lux
- Temperature: temperature-controlled room maintaining the water temperature at 18 to 22 °C

VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: Yes

TEST ITEM CONCENTRATION
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No sub-lethal effects of exposure were observed throughout the test.

Results with reference substance (positive control):

The positive control was run as a separate study used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. The positive control was conducted between 23 January 2020 and 07 February 2020.

The results from the positive control with potassium dichromate were within the normal range for the reference item.

Verification of Test Concentrations

Chemical analysis of the fresh test preparations showed a measured test concentration of 0.091 mg/L at 0 hour and 0.13 mg/L at 24 hours. Analysis of the old test preparations showed a measured test concentration of 0.12 mg/L at 24 hours and 0.14 mg/L at 48 hours. It was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.12 mg/L.

 

Immobilisation Data

There was no immobilisation in 20 daphnids exposed to a test concentration of 0.12 mg/L for a period of 48 hours. The immobilisation data is given in the following table:

Time (hours)	EC50 (mg/l)
24	>0.12
48	>0.12

  The NOEC after 24 and 48 hours exposure was 0.12 mg/L.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colourless solutions.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item gave 48-hour EC50 values based on the geometric mean measured test concentration of greater than 0.12 mg/L. The No Observed Effect Concentration (NOEC) was 0.12 mg/L.

The study showed that there were no toxic effects at saturation.

Executive summary:

Introduction

The study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the following Guideline:

• OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test"
• Method C.2 of Commission Regulation (EC) No. 440/2008 (Acute Toxicity for Daphnia)

 

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g., ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.11 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a nominal concentration of 100% v/v saturated solution for 48 hours at a temperature of 19°C to 21°C under semi-static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 µm to give a 100% v/v saturated solution of the test item. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

 Chemical analysis of the fresh test preparations showed a measured test concentration of 0.091 mg/L at 0 hour and 0.13 mg/L at 24 hours. Analysis of the old test preparations showed a measured test concentration of 0.12 mg/L at 24 hours and 0.14 mg/L at 48 hours. It was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.12 mg/L.

There was no immobilisation in 20 daphnids exposed to a test concentration of 0.12 mg/L for a period of 48 hours. The immobilisation data is given in the following table:

Time (hours)	EC50 (mg/l)
24	>0.12
48	>0.12

  The NOEC after 24 and 48 hours exposure was 0.12 mg/L.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colourless solutions.

Conclusion

Exposure of Daphnia magna to the test item gave 48 -hour EC50 values based on the geometric mean measured test concentration of greater than 0.12 mg/L. The No Observed Effect Concentration (NOEC) was 0.12 mg/L.

The study showed that there were no toxic effects at saturation.

Description of key information

Acute toxicity to aquatic invertebrates data summary.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 0.12 mg/L

Additional information

Exposure of Daphnia magna to the test item gave 48-hour EC50 values based on the geometric mean measured test concentration of greater than 0.12 mg/L. The No Observed Effect Concentration (NOEC) was 0.12 mg/L.

The study showed that there were no toxic effects at saturation.