trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate

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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 23ADB/50
- Expiration date of the lot/batch: 5 years

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water

Analytical monitoring:

yes

Remarks:

HPLC

Vehicle:

no

Details on test solutions:

Sample preparation
Range finding test
The test sample has been prepared at 1000 mg/L of the active ingredient in water (corresponding to about 2044 mg/L of the test item). 5 dif erent concentrations, have been tested with ten-fold dilutions from 100 mg/L to 0.01 mg/L of active ingredient.
The test has been performed on the diluted solutions.
N° of groups: 5 treated + 1 control
N° of animals: 3/tank

Definitive test (Limit test)
After the range finding test a solution of the active ingredient of the test item at 1000 mg/L has been prepared in water. Then the solution has been diluted to of 100 mg/L of active ingredient corresponding to about 204 mg/L of the test item. The test has been performed on this solution in the below conditions:
N° of groups: 1 treated + 1 control
N° of animals: 7/tank
14 fish have been used for the definitive test subdivided as follows:
GROUP Animals (N°) Administered solution Nominal concentration of the active ingredient Check point chemical- physical parameters (hours)
Control 7 Water 0-24-48-72-96
Treated 7 100 mg/L 0-24-48-72-96

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Brachydanio rerio
- Source: Supplier: Aquarium Center
- Length at study initiation (length definition, mean, range and SD): 2 cm ± 1 cm
- Biomass-density: < 1 g/L

Maintenance
During quarantine period fish have been kept in glass tanks with a capacity of 60 litres.
The water used for the assay has been allowed to stabilize for 48 hours to greatly reduce chlorine
presence, in the event it was present if used for purification.
Hardness: 10-250 mg CaCO3/L
Temperature: 23°C ± 2°C
Aeration: continuous with porous stones
Tanks have been lighted with white light lamps with 12 h photoperiod daily.
Feeding
Type: Feedstuff in flakes
Frequency: 3 times a week with suspension 24 hours before the beginning of the test.


QUARANTINE
Before the beginning of the study, fish have been kept in quarantine for 12 days. During this period, fish
have been daily observed. No mortality or symptoms have been recorded during quarantine period.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

10-250 mg CaCO3/L

Test temperature:

23 °C ± 2 °C

pH:

6.0-8.5

Dissolved oxygen:

>60 %

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

TABLE N. 1: Number of dead fish in control and treated groups during the definitive test
TIME
GROUP 3 hours 6 hours 24hours 48 hours 72 hours 96 hours
CONTROL 0/7 0/7 0/7 0/7 0/7 0/7
TREATED 0/7 0/7 0/7 0/7 0/7 0/7

TABLE N. 2: Symptomatology of fish in control and treated groups during the definitive test
TIME
GROUP 3 hours 6 hours 24hours 48 hours 72 hours 96 hours
CONTROL 0 0 0 0 0 0
TREATED 0 0 0 0 0 0

LEGENDA
0 Nothing abnormal R Changes to respiration
E Equilibrium loss P Changes to pigmentation
N Changes to swimming A Other
N. A. Not applicable, fish dead

TABLE N. 6: Stability of the test item in treated groups in the definitive test
Nominal conc. Measured conc (mg/L) T=0 Measured conc. (mg/L) T=96 h % Stability on measured concentration Stability
100.00 mg/L 96.24 108.29 112.52 Stable

Validity criteria fulfilled:

yes

Conclusions:

The obtained results, in compliance with assay validity criteria, interpreted in accordance to OECD 203, showed that the Brachydanio rerio LC50 of the active ingredient of the test item "Trisodium (25)-2,6-bis(3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt” after 96 hours is >100 mg/L.

Executive summary:

The toxicity of the test item, "Trisodium (2s)-2,6-bis(3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt", on the fish,Brachydanio rerio,has been evaluated according to OECD guideline N. 203

In particular, the assay of acute toxicity with fish Brachydanio rerio has been performed by means of a static assay in which the solution has not been changed for the whole test period.

After a range finding test, a limit test has been performed using 14 fish have been used, 7 of them treated with the test item at 100 mg/L of the active ingredient and 7 fish used as control, kept in the same assay conditions without adding the test item.

Fish have been observed for a period of 96 hours and mortality and/or possible anomalous behaviours have been observed both in control and in treated.

Temperature, pH and percentage of saturation of oxygen in the water of each tank have been measured at the beginning of the treatment and with intervals of 24 hours.

Results are referred to the active ingredient present in the test item (48.9% active ingredient).

Stability of the active ingredient of the test item in the definitive test has been performed using HPLC technique equipped with UVs detector

The active ingredient present in the test item has been measured at the beginning and at the end of the test in the control and in the treated sample.

The obtained results, in compliance with test acceptability criteria, for the active ingredient of the test item "Trisodium (2s)-2,6-bis(3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt" can be summarised as follow:

LC₅₀of the active ingredient in the test item at 96 h > 100 mg/L

Description of key information

The obtained results, in compliance with assay validity criteria, interpreted in accordance to OECD 203, showed that the Brachydanio rerio LC50 of the active ingredient of the test item "Trisodium (25)-2,6-bis(3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt” after 96 hours is >100 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L