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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 21 July 2021 to 16 October 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

Two minor deviations from the study plan occurred 1) type of tank used to house the fish and 2) the specification of the carbon filter used in the dechlorination unit. Niether was considered to have affected the purpose or of validity of the study.

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

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 0.083 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.

In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA. Using this approach, the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. As both tests showed no adverse effects of exposure to a 100% v/v saturated solution of the test item, the test was conducted at a single concentration of 100% v/v saturated solution.


PREPARATION OF TEST SUBSTANCE SOLUTION
A nominal amount of test item (1100 mg) was dispersed in 22 L of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 L used to pre-condition the filter was discarded) to give a 100% v/v saturated solution.

The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0, 24, 48 and 72 hours and of the old media at 24, 48, 72 and 96 hours.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Source: Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK.
- Age at study initiation (mean and range, SD): Not stated
- Length at the end of the definitive test: mean standard length of 5 cm (standard deviation = 0.13)
- Weight at the end of the definitive test: mean body weight of 0.95 g (standard deviation = 0.08)

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets which was discontinued approximately 26 hours prior to the start of the definitive test.
- Health during acclimation (any mortality observed): No mortaliy in the 7 days prior to the start of the test

FEEDING DURING TEST
- Food type: no food was given during the test

PHOTOPERIOD
16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hour

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Approximately 140 mg/L as CaCO3.

Test temperature:

13 to 14 °C

pH:

7.7 to 8.0

Dissolved oxygen:

9.8 to 10.5 mgO2/L

Conductivity:

513 μS/cm at 20 °C (typical value for site water supply)

Nominal and measured concentrations:

Analysis of the fresh test preparations at 0, 48 and 72 hours showed measured test concentrations to range from 0.086 to 0.099 mg/L. The measured test concentration at 24 hours was excluded as it was considered to be anomalous (the measured value of 0.056 mg/L was not consistent with the other data and was lower than the measured value of the old sample taken at 48 hours). Analysis of the old or expired test solutions at 24, 48, 72 and 96 hours showed measured test concentrations to range from 0.088 to 0.10 mg/L. Therefore the results are based on 0-Hour measured test concentrations only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 25L glass vessels containing 20L of test media
- Type: vessels covered to reduce evaporation
- Aeration: Yes, via narrow bore glass tubes
- Renewal rate of test solution: daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the definitive test was the same as that used to maintain the stock fish. Laboratory tap water was dechlorinated by passage through an activated carbon filter (Fleck 2750 Duplex Dechlorination unit) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours dark with 20 minute dawn to dusk transition period

EFFECT PARAMETERS MEASURED: Any mortalities and sub-lethal effects of exposure were recorded at 1, 3, 6, 24, 29, 48, 53, 72, 78 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.


TEST CONCENTRATIONS
The test was conducted at a single concentration of 100% v/v saturated solution.

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.099 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.099 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Reported statistics and error estimates:

No statistical analysis was conducted. An estimate of the LC50 values was given by inspection of the mortality data.

Sublethal observations / clinical signs:

There were no sub-lethal effects of exposure observed in the test.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave 96-hour LC50 values based on the 0-Hour measured test concentration of greater than 0.099 mg/L. The No Observed Effect Concentration (NOEC) was 0.099 mg/L.

Executive summary:

Introduction

The study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with thefollowign guideline:

• OECD Guidelines for Testing of Chemicals (2019) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

 

 Method

 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 0.083 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.

 

 In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA. Using this approach, the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. As both tests showed no adverse effects of exposure to a 100% v/v saturated solution of the test item, the test was conducted at a single concentration of 100% v/v saturated solution.

 

Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100% v/v saturated solution for a period of 96 hours at a temperature of approximately 13 ºC to 14 °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 (0.2 µm Sartorius Sartopore filter, first approximate 1 L used to pre-condition the filter was discarded) to produce a saturated solution of the test item. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3, 6, 24, 29, 48, 53, 72, 78 and 96 hours after the start of exposure.

 

Results and Conclusion

 

Analysis of the fresh test preparations at 0, 24, 48 and 72 hours showed measured test concentrations to range from 0.086 to 0.099 mg/L. Analysis of the old or expired test solutions at 24, 48, 72 and 96 hours showed measured test concentrations to range from 0.088 to 0.10 mg/L. Therefore, the results are based on 0-Hour measured test concentrations only.

 

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave 96-hour LC50 values based on the 0-Hour measured test concentration of greater than 0.099 mg/L. The No Observed Effect Concentration (NOEC) was 0.099 mg/L.

Description of key information

Acute toxicity to fish data summary.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 0.099 mg/L

Additional information

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave 96-hour LC50 values based on the 0-Hour measured test concentration of greater than 0.099 mg/L. The No Observed Effect Concentration (NOEC) was 0.099 mg/L.