Sodium hydrogen N-(1-oxododecyl)-L-glutamate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25, January 2021 to 24, February 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Quality Assesment is on page 4 of the attached report

Specific details on test material used for the study:

Batch no.: y191119 supplied by the sponsor
Aspect: white powder
Purity: 97.3%
Solubility: soluble in water
Storage condition of test material: room temperature (15 °C - 25 °C), stable for at least 20 months
Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium): stable for 12 months minimum
Solubility: soluble and stable in water
Reactivity of the test material with the incubation material used (e.g. plastic ware): not reactive
pH (sol. 5% 20 °C) = 5.10
More information can be found on the attached report

Analytical monitoring:

yes

Remarks:

Stability of test item was evaluated at the beginning and at the end of the test

Details on sampling:

Samples, properly diluted with deionised water with low TOC contents, have been analysed by means of an integrated sampling system directly from vials containing 40 ml volume. For each sample under test the corresponding blank is analysed. The result is expressed corrected from the control and the deionised used for dilutions at initial and final timing test.
CALCULATIONS
% stability: 100 x (TOC substance 72h - (TOC ctl 72h - TOC deionized water 72h))/
(TOC substance 0h - (TOC ctl 0h - TOC deionized water 0h))
Samples are considered stable if the % stability is between 80 and 120% of the value.
STABILITY
The samples were stable throughout the test period.
Results that are summarized in table 4 of the attached report confirm stability.
Treated (500.00 mg/l), at t0 TOC 267.31, at t72h 268.33, % stability 100
Treated (48.83 mg/l), at t0 27.20, at t72h 26.75, % stability 98
Treated (4.77 mg/l), at t0 3.12, at t72h 2.73, % stability 87

Vehicle:

yes

Details on test solutions:

Culture medium has been prepared by mixing 4 stocks solution in order to obtain the following concentation:
SOLUTION N.1
NH4CI 0,75 g, CaCl2 2H20 0,9 g, MgCl2 6H20 0,6 g, MgSO4 7 H20 0,75 g, KH2PO4 0,08 g (dissolved in 500 ml of deionized water)
SOLUTION N.2
FeCl3 6H20 0,032 g, Na2EDTA 2H20 0,05 g (dissolved in 500 ml of deionized water)
SOLUTION 3
H3BO3 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 2 H20 1 mL of the following solution: 25 mg in 500 mL of deionized water, then dilution 1 ml of it in 10 ml, Na2MoO4 2H2O 1 mL of the following solution: 35 mg in 10 mL of deionized water (dissolved in 500 ml of deionized water)
SOLUTION 4
NaHCO3 25 g (dissolved in 500 ml of deionized water).
Solutions 1,2,3 have been sterilised by autoclaving (120 °C, 15 min). Solution 4 has been sterilised via membrane filtration (0.22 micrometer). 10 ml of solution 1 and 1 ml of solutions 2,3,4 have been made up to 1000 ml with water.

Test organisms (species):

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

Details on test organisms:

Algal suspension kept in medium culture in sterile 250 mL Erlenmeyer
Temperature: 23 +/- 2°C
Lighting: > 4300 lux
Stirring: steady with electrical stirrer

Test type:

static

Water media type:

freshwater

Remarks:

Culture medium

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Observations were also done at 24 and 48 hours

Test temperature:

The results of temperature during the whole test are reported in Attachment N. 2 -Table N. 3 of the attached report and satisfy the requirements of the guideline (23 +/- 2°C)

pH:

The results of pH at the beginning and at the end ofthe test are reported in Attachment N. 2 - Table N. 2A and 2B of the attached report and satisfy the requirements of the regulation (pH in the control must not deviate by more than 1.5 units during the test).

Nominal and measured concentrations:

Range finding test: from 0.1 to 1000 mg/L test item (nominal concentration)
Definitive test: 500.00, 156.25, 48.83, 15.26, 4.77 mg/L test item (nominal concentration)

Details on test conditions:

Starting cellular density: 10exp4 cell/ml
N. of replications: 6 for control and 3 for all treated concentrations in range finding and definitive test
Concentrations: 5 for range finding test and 5 for definitive test
Culture medium was used as control
Lightning: Continuous, with fluorescent lamp (7000-8000 lux)
Stirring: Steady with electrical stirrer
Temperature: 23+/-2°C
Assay duration: 72+/-2 hours
The culture medium has been used as dilution water.
A solution at 1000 mg/L of the test item has been prepared in culture medium. To better solubilize the solution has been sonicated. Then 4 different concentrations have been prepared with ten-fold dilutions from 100 mg/L to 0.01 mg/L under agitation.
Tested concentrations have been 1000.00 mg/L, 100.00 mg/L, 10.00 mg/L, 1.00 mg/L and 0.1 mg/L.
The total volume of the culture medium and the concentration of the algae inoculum added were the same in all vessels.
After the range finding test the test item has been prepared at 1000 mg/l in culture medium. to better solubilize the solution has been sonicated. Then 5 different concentrations (see table 1 below in any other information). The total volume of the culture medium and the concentration of the algae inoculum were the same in all vessels.
Algal suspension from stock culture has 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.
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 at the beginning and every 24 hours.
At the end of the test the quality of algae appearance has been observed at microscopy.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

118.343 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

Cells/ml

Details on results:

Average growth rate:
The average growth rate for a specific period (nij) is calculated as the logarithmic increase in the number of cells from the following equation for each control and treated vessel.
Calculate the average specific growth rate over the entire period using the nominally inoculated number of cell/ml as the starting value (Xi)
nij = (ln Xj - ln Xi)/( tj - ti)
Where
nij = is the average specific growth rate from time i to time j
Xi= is the numberof cell /ml at time i
Xj = is the numberof cell/ml at time j
The percent inhibition of growth rate was calculated as follows:
%Ir = [(nc - nt): nc] x 100
Whereas:
%Ir= percent inhibition in average specific growth rate
nc = mean value for average specific growth rate in the control group
nt = mean value for average specific growth rate in the treated group
VALIDITY
The assay is valid if the following criteria are satisfied:
-the cell concentration in the control cultures should have increased by a factor of at least 16 within three days, that corresponds to a specific growth rate of 0.92 day -1 (day 0-1 = 1.73, day 1-2 = 2.24 and day 2-3 1.45, all > 0.92)
-the mean coefficient of variation for section by section (CVss) specific growth rates in the control cultures must not exceed 35% (day 0-3 22.46, < 35%)
- the coefficient of variation of average specific growth rates during the whole period (CVwp) in replicate control cultures must not exceed 7% (1.23%).
All data can be found in Attachment 2 of attached report (Validity Criteria of the test)

Reported statistics and error estimates:

EC50 value has been evaluated using XLSTAT 2021.1.1.1085 validated software.

Validity criteria fulfilled:

yes

Conclusions:

EC50 (72 h) of the nominal concentration of the test item “SODIUM HYDROGENN-(1-OXODODECYL)-L-GLUTAMATE”: 118.343 mg/L.

Executive summary:

On the test item “SODIUM HYDROGEN N-(1-OXODODECYL)-L-GLUTAMATE" the growth inhibition test on a culture of algae Pseudokirchneriella subcapitata has been performed on a culture in exponential growth phase.
This approach, according to OECD 201, allowed to calculate the inhibition concentration for 50% of theorganisms (lC50 or EC50).
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 an EC50 test has been performed. The algae have been exposed to 5 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 appearanceofthe 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.
Stability of the test item in the definitive test has been performed using TOC analyser. Organic carbon concentration has been measured at the beginning and at the end of the test in a pool derived from vessels at the higher, intermediate and lower tested concentration.
The obtained results, in compliance with test acceptability criteria, can be summarised as follow:
EC50 (72 h) of the nominal concentration of the test item “SODIUM HYDROGENN-(1-OXODODECYL)-L-GLUTAMATE”: 118.343 mg/L.

Description of key information

EC50 (72h) = 118.343 mg/L, (nominal) for Pseudokirchneriella subcapitata (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

118.343 mg/L

Additional information

On the test item “SODIUM HYDROGEN N-(1-OXODODECYL)-L-GLUTAMATE" the growth inhibition test on a culture of algae Pseudokirchneriella subcapitata has been performed on a culture in exponential growth phase.
This approach, according to OECD 201, allowed to calculate the inhibition concentration for 50% of theorganisms (lC50 or EC50).
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 an EC50 test has been performed. The algae have been exposed to 5 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 appearanceofthe 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.
Stability of the test item in the definitive test has been performed using TOC analyser. Organic carbon concentration has been measured at the beginning and at the end of the test in a pool derived from vessels at the higher, intermediate and lower tested concentration.
The obtained results, in compliance with test acceptability criteria, can be summarised as follow:
EC50 (72 h) of the nominal concentration of the test item “SODIUM HYDROGENN-(1-OXODODECYL)-L-GLUTAMATE”: 118.343 mg/L.