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

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:

2018-2019

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)

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: 21/11/2023

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: 5 years
- Solubility and stability of the test substance in the solvent/vehicle: soulble in water

Analytical monitoring:

yes

Remarks:

HPLC

Vehicle:

no

Details on test solutions:

Medium preparation
As dilution water, reconstituted water has been used; it has been prepared dissolving the following salts in
1 litre of deionised water:
N. SOLUTION REAGENTS FORMULA QUANTITY (g/L)
A Bihydrated calcium chloride CaCl2 2H20 11.76
B Eptahydrated magnesium sulphate MgSO4 7H20 4.93
C Acid sodium bicarbonate NaHCO3 2.59
D Potassium chloride KCI 0.23

25 mL of the solutions A, B, C, D have been taken to 1000 mL volume with deionised water. Medium has been prepared according to the same proportion and reported on internal logbook.

Assay sample preparation
Range finding test
The test item has been prepared at 1000 mg/L of the active ingredient (corresponding to about 2044 mg/L of the test item) in culture medium. 5 different concentrations have been prepared and tested with ten-fold dilutions from 1000 mg/L to 0.1 mg/L.


Definitive test- Limit test
After the range finding test a solution of the active ingredient of the test item at 100 mg/L has been prepared in culture medium (corresponding to 204 mg/L of the test item). The test has been performed on this nominal solution

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna straus, Crustacean Cladocero
- Age at study initiation (mean and range, SD): 6-24 hours
- Source: Breeder in Eurofins Biolab Test facility
- Feeding during test: None during the course of the test.

ACCLIMATION
- Acclimation conditions (same as test or not): 20°C ± 2°C, Photoperiod:16h of light, 8 hours of darkness
- Type and amount of food: Green alga Pseudokirchneriella subcapitata and Saccharomices cerevisiae

Test type:

not specified

Water media type:

other: reconstituted dilution water

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

at 24 and 48 h

Hardness:

between 140 and 250 mg CaCO3/l

Test temperature:

20°C ± 2°C

Dissolved oxygen:

> 3 mg/I

Nominal and measured concentrations:

After the range finding test a solution of the active ingredient of the test item at 100 mg/L has been prepared in culture medium (corresponding to about 2044 mg/L of the test item)

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

TABLE N. 1: Number of immobilised Daphnia at 24 and 48 hours in treated (test item) and control groups in the definitive test
Control 24 hours 48 hours
CONTROL replication N.1 0/5 0/5
CONTROL replication N.2 0/5 0/5
CONTROL replication N.3 0/5 0/5
CONTROL replication N.4 0/5 0/5
Concentration: 100 mg/I
24 hours 48 hours
TREATED replication N.1 0/5 0/5
TREATED replication N.2 0/5 0/5
TREATED replication N.3 0/5 0/5
TREATED replication N.4 0/5 0/5

TABLE N. 2: Concentration of dissolved oxygen (mglL) in treated and in control at the beginning and at the end of the test (48 hours) in the definitive test
Control Beginning of test End of test
CONTROL N.1 6.56 5.93 _
CONTROL N.2 6.79 5.77
CONTROL N.3 6.34 5.94
CONTROL N.4 6.57 5.88
Concentration: 100 m /L
Treated Beginning of test End of test
TREATED replication N.1 6.50 5.96
TREATED replication N.2 6.44 5.90
TREATED replication N.3 6.31 5.85
TREATED replication N.4 6.37 5.69

TABLE N. 3: Determination of pH at the beginning and at the end of the test (48 hours) in treated and in control in the definitive test
Control Beginning of test End of test
CONTROL N.1 7.82 7.76
CONTROL N.2 7.84 7.78
CONTROL N.3 7.82 7.76
CONTROL N.4 7.82 7.76
Concentration: 100 m /L
Treated Beginning of test End of test
TREATED replication N.1 7.86 7.83
TREATED replication N.2 7.85 7.84
TREATED replication N.3 7.87 7.87
TREATED replication N.4 7.86 7.85
TABLE N. 4: Determination of temperature (°C) in the environment where the assay is performed at the beginning and at the end of the test (48 hours) in the definitive test
Time TEMPERATURE
Beginning of test 20.0
End of test 20.0
TABLE N. 5: Concentration of the test item at the beginning and at the end of the test (48 hours) in treated sample in the definitive test
Nominal conc. Measured conc (mg/L) T=0 Measured conc. (mg/L) T=48 h °A Stability on
measured concentration
Stability
100.00 mg/L 101.47 89.22 87.93 Stable

Validity criteria fulfilled:

yes

Conclusions:

The obtained results, in compliance with assay validity criteria, showed that Daphnia magna EC50, after 48 hours, of the active ingredient of the test item "Trisodium (2s)-2,6-bis(3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt" is >100 mg/L.

Executive summary:

The toxicity of the test item, on the cladocero Daphnia magna has been evaluated according to OECD guideline N. 202.

After the results of the range finding test a limit test has been performed. The Daphnia have been exposed to 100 mg/L of the active ingredient present in the test item for 48 hours.

Daphnia have been kept under observations for a period of 48 hours and the number of immobilised organisms and/or possible abnormal behaviours both in control and in treatment group vessel have been observed.

At the beginning and at the end of the test (48 hours) dissolved oxygen and pH have been measured in the highest concentration and in control group. Temperature of the assay environment have been recorded at the beginning and at the end of the test.

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, of the active ingredient of the test item can be summarised as follow:

EC₅₀of the active ingredient in the test item at 48 h > 100 mg/L

Description of key information

EC₅₀of the substance "Trisodium (2s)-2,6-bis(3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt" in the test item at 48 h > 100 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L