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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition 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
- 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:

Dilution water
The culture medium has been used as dilution water.
The culture medium has been prepared by mixing the 4 stock solutions in order to obtain final concentrations specified in the following table
SOLUTION N NUTRIENT QUANTITY TO BE DISSOLVED IN 500 ML OF DEIONIZED WATER

1 NH4CI 0,75 g
CaCl2 2H20 0,9 g
MgC12 6H20 0,6 g
MgSO4 7 H2O 0,75 g
KH2PO4 0,08 g

2 FeCl3 6H20 0,032 g
Na2EDTA 2H20 0,05 g


3 H3B03 0,0925 g
MnCl2 4H20 0,207 g
ZnCl2 1 mL of the following solution: 150 mg in 100 mL of deionized water
CoCl2 6H20 1 mL of the following solution: 75 mg in 100 mL of deionized water
CuCl2 2H20 1 mL of the following solution: 25 mg in 500 mL of deionized water, then diluting 1 mL of it in 10 mL
Na2MoO4 2H20 1 mL of the following solution: 35 mg in 10 mL of deionized water

4 NaHCO3 25 g

Solutions 1, 2, 3 have been sterilised by autoclaving (120°C, 15 min). Solution 4 has been sterilised via membrane filtration (0.22 um). 10 mL of solution 1 and 1 mL of solutions 2, 3, 4 have been made up to 1000 mL with deionised water.
For higher volumes the medium has been prepared according to the same proportion.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata SAG 61.81
Test organism: Green alga
Genus: Pseudokirchneriella
Specie: subcapitata
Family: Chlorophyceae
Order: Chlorococcalles

The algal suspension in breeding in Eurofins Biolab has been kept in culture medium in sterile 250 mL
Erlenmeyer flasks in the following conditions until the beginning of the test:

Temperature: 23°C ± 2°C
Lighting: lighting greater than 4300 lux
Stirring: Steady with electrical stirrer

Test type:

not specified

Water media type:

other: cell culture medium

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23 °C ± 2°C

pH:

7.8- 8

Details on test conditions:

Definitive test
After the range finding test a solution of the active ingredient of the test item at 1000 mg/L has been prepared in culture medium (corresponding to 2044 mg/L of the test item). 5 dilutions have been performed and tested (see Table below).
The total volume of the culture medium and the concentration of the algae inoculum added were the same in all vessels.
Group Number Cellular density
(cells/ml) Administered solution
Nominal concentration of
the active ingredient Cell observation times
(hours)
Treated 3 104 1000.00 mg/L 0-24-48-72
Treated 3 104 500.00 mg/L 0-24-48-72
Treated 3 104 250.00 mg/L 0-24-48-72
Treated 3 104 125.00 mg/L 0-24-48-72
Treated 3 104 62.50 mg/L 0-24-48-72
Control 6 104 Culture Medium 0-24-48-72

TREATMENT
Algal suspension from stock culture, have been inoculated in the receivers containing the defined
concentration of the test sample.
Erlenmeyer flasks containing treated organisms and Erlenmeyer flasks containing control organisms,
have been kept in suspension by steady stirring at 23°C + 2°C under continuous light for 72 + 2 hours.
OBSERVATIONS
The test and control samples were incubated for a period of 72 ± 2 h during which the cell density in
each test and control solution was measured every 24 h by Burker chamber.
pH has been measured at the beginning and at the end of the assay.
Temperature of the ambient has been recorded every 24 hours.
At the end of the test the quality of algae appearance has been observed at microscope.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 62.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

430.011 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

62.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Cellular concentration
Algae densities for treated and control during the assay are reported here below:

ALGAE DENSITY (cells/mL) IN TREATED GROUPS
Tempo (Time) (days) 1 2 3
Gruppo TRATTATO (TREATED Group) 1000.00 mg/L
0 1.0E+04 1.0E+04 1.0E+04
1 1.5E+04 1.3E+04 1.3E+04
2 2.8E+04 2.8E+04 3.5E+04
3 7.3E+04 8.0E+04 8.0E+04
Gruppo TRATTATO (TREATED Group) 500.00 mg/L
0 1.0E+04 1.0E+04 1.0E+04
1 2.3E+04 2.2E+04 2.3E+04
2 5.2E+04 5.3E+04 5.7E+04
3 1.3E+05 1.5E+05 1.4E+05
Gruppo TRATTATO (TREATED Group) 250.00 mg/L
0 1.0E+04 1.0E+04 1.0E+04
1 3.5E+04 3.7E+04 3.5E+04
2 1.0E+05 9.3E+04 1.1E+05
3 2.2E+05 2.1E+05 2.3E+05
Gruppo TRATTATO (TREATED Group) 125.00 mg/L
0 1.0E+04 1.0E+04 1.0E+04
1 4.5E+04 5.3E+04 4.3E+04
2 2.0E+05 2.1E+05 2.3E+05
3 4.1E+05 4.7E+05 4.4E+05
Gruppo TRATTATO (TREATED Group) 62.50 mg/L
0 1.0E+04 1.0E+04 1.0E+04
1 6.0E+04 5.5E+04 5.7E+04
2 3.3E+05 3.4E+05 3.2E+05
3 7.0E+05 7.3E+05 8.0E+05

ALGAE DENSITY (cells/mL) IN CONTROL GROUPS
Tempo (Time) (days) 1 2 3 4 5 6
Gruppo CONTROLLO (CONTROL Group (cells/ml)
0 1.0E+04 1.0E+04 1.0E+04 1.0E+04 1.0E+04 1.0E+04
1 7.3E+04 8.7E+04 7.5E+04 7.7E+04 8.0E+04 6.8E+04
2 7.8E+05 7.8E+05 8.5E+05 7.5E+05 7.5E+05 7.8E+05
3 2.3E+06 2.4E+06 2.3E+06 2.5E+06 2.5E+06 2.4E+06

% INHIBITION OF GROWTH RATE
Parametri Trattato (Treated)
1000.00 mglL 500.00 mglL 250.00 mglL 125.00 mglL 62.50 mglL
%IR 62.53 52.09 43.38 30.87 21.26

pH
The results of pH at the beginning and at the end of the test are reported in the following table and satisfy the requirements of the regulation.

Temperature
The results of temperature during the whole test are reported in Attachment N. 2 -Table N. 3 and satisfy
the requirements of the guideline (23 ± 2°C).
Recovery of the test item
In the table below nominal concentration vs measured concentrations detected by chemical analysis
have been reported.
Nominal conc. (mg/L) T=0 Measured conc (mglL) T=0 % OF RECOVERY
1000.00 954.84 95.48
500.00 486.19 97.24
250.00 244.59 97.84
125.00 121.37 97.10
62.50 57.85 92.56

Stability of the test item
The sample is considered stable because the measured concentration of the test item at the end of the
test has been maintained within ± 20 % to the concentration at the measured concentrations at the
beginning of the test.
The percentages of stability are reported in the table below.
The test item resulted stable during the test, therefore the statistical analysis (EC50) has been performed
using the nominal concentrations at T=0.

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=72 h % Stability on measured concentration Stability
1000.00 mg/L 954.84 928.87 97.28 Stable
500.00 mg/L 486.19 462.31 95.09 Stable
250.00 mg/L 244.59 224.91 91.95 Stable
125.00 mg/L 121.37 117.52 96.83 Stable
62.50 mg/L 57.85 56.62 101.33 Stable

Validity criteria fulfilled:

yes

Conclusions:

Algae EC50 of the the test item "Trisodium (2s)-2,6-bis(3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt" after 72 hours is equal to 430.011 mg/L. LOEC is 62.50 mg/L and NOEC is < 62.50 mg/L.

Executive summary:

A growth inhibition test on a culture of algae Pseudokirchneriella subcapitata was performed

on the test item "Trisodium (2s)-2,6-bis(3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt " according to OECD guideline 201.

During the range finding test the algae have been exposed to 5 different dilutions of the test item for 72 hours. After the results of the range finding test a definitive test has been performed. The algae have been exposed to 5 different dilutions of the test item for 72 hours. Algae have been measured using a Burker chamber.

At the end of the test, microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae.

At the beginning and at the end of the test (72 hours) pH has been measured in all the vessels. Temperature of the ambient has been recorded every day.

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 follows:

EC₅₀of the active ingredient of the test item at T= 72 h (measured concentration) = 430.011 mg/L

LOEC of the active ingredient of the test item = 62.50 mg/L

NOEC of the active ingredient of the test item < 62.50 mg/L

Description of key information

Algae EC50 of the the test item "Trisodium (2s)-2,6-bis(3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt" after 72 hours is equal to 430.011 mg/L. LOEC is 62.50 mg/L and NOEC is < 62.50 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

430.11 mg/L

EC10 or NOEC for freshwater algae:

62.5 mg/L