Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and stearic acid

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-20 - 2021-02-26

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

sodium hydrogen octadecanoic acid (2R)-2-octadecanamidopentanedioate

EC Number:

939-201-1

Molecular formula:

unspecified

IUPAC Name:

sodium hydrogen octadecanoic acid (2R)-2-octadecanamidopentanedioate

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): Only trade name is given.
- Physical state: white solid
- Analytical purity: 97% AS
- Lot/batch No.: Ra-He8390-77

Specific details on test material used for the study:

Water solubility: 4.36 g/L (Final Report, Study code: 12-01886/1-4) *
Vapor pressure: ≤ 0.0057 Pa (Safety Data Sheet)
Storage conditions: Room temperature

* The results of this study do not match the observations made for the water solubility of the test item in the test media under testing conditions. Several pre-tests indicated a solubility of the test item in the test medium below 1mg/L. For the current study, all reasonable efforts were made to prepare a saturated fration of the test item in the test medium, which resulted in a markedly lower mean measured concentration than reported in study 12-01886/1-4. More details can be found in section 4.1 and in the final report for concentration control analysis in the Appendix.

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

TEST SUBSTANCE PREPARATIONS
The test solutions were prepared by directly adding the test substance to the test medium. Test substance was weighed in and made up with test medium to reach the nominal concentrations.
The test substance was weighed into a 2L volumetric flask, subsequently filled to the mark with test medium, and stirred on a magnetic stir plate for about 24 hours at room temperature.

Undissolved test substance was removed by filtration with a membrane filter (pore size 0.2 μm, regenerated cellulose). During filtration the first 50-100 mL of filtered solution were discarded (used to condition the filter).

Following, the aqueous fraction is inspected visually for the presence of any undissolved test substance by observing the scattering of a laser light through the test solution (Tyndall effect). Acceptable test solution pH is within the range ±0.5 of the control. If a test solution is outside of this range, it was adjusted to control media pH (±0.2) with HCl or NaOH prior to the start of exposure.

Fresh test solutions were prepared daily.
Following preparation all test solutions appeared colorless and clear, showing a negative Tydall effect. The pH of the stock test solutions was adjusted to 7.5 before use with 1 mol HCl. No pH adjustment was done during the exposure.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

Test species and strain: Lemna gibba Clone G3
Reason for selection of the test species:
Recommended Lemna species in the test guidelines
Age (on study day 0) An inoculum culture of Lemna gibba (8 days old at 24 °C ±2°) is used to initiate the test (study day 0).
Supplier: The stock culture was obtained from BASF SE, Agricultural Solutions – Ecology and Environmental Analytics, Speyerer Strasse 2, 67117 Limburgerhof, Germany.
Culture conditions: A culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared with sufficient colonies of Lemna transferred aseptically into fresh sterile 20X AAP
media. The inoculum culture is incubated under test conditions (see section 3.7) for 8 days prior to test initiation.
Reference substance testing: In order to verify that the Lemna cultures are responding normally to toxic stress, tests with a reference substance (3,5 Dichlorphenol) are conducted. Reference substance tests are
conducted according to OECD 221 guidelines and in accordance with GLP, but without a GLP status.
The results from the reference substance test are compared to 3,5 Dichlorphenol EC50 values published in OECD Guideline [Ref 1], which represent the typical response range for the Lemna species tested.The EC50(7d) of the reference substance
3,5 Dichlorphenol was 7.8 mg/L (experiment date: 21 Apr 2021, project number: 63E0308/02E014).
This result is within the range of 5.9 – 13.3 mg/L and indicates that the culture of Lemna gibba used in this study is responding normally to toxic stress.

Inoculum culture
Photoperiod: Continuous
Culture media: Aynthetic fresh water (20X AAP).
The test medium (20X AAP medium) was prepared as described in OECD Guideline for Testing of Chemicals, No. 221 Lemna sp. Growth inhibition Test, Mar 2006, Annex 4.
Temperature: 24 ±2°C

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test conditions

Test temperature:

23.6 – 24.2 °C

pH:

8.6-8.7

Nominal and measured concentrations:

Test groups: 0 (control), and 10 mg/L as nominal concentrations based on test substance mass without correction for purity or content.
0 (control), and 0.38 mg/L as mean measured concentrations (geomean), based on test substance mass without correction for purity or content.

Details on test conditions:

The test medium (20X AAP medium) was prepared as described in OECD Guideline for Testing of Chemicals, No. 221 Lemna sp. Growth inhibition Test, Mar 2006, Annex 4. Growth medium intended for testing was prepared at least 2 days before use to allow the pH to stabilize. The pH of growth medium was checked prior to use and readjusted if necessary, by the addition of 1 M HCl. The test media was sterilized after preparation and prior to inoculation.

Test replicates: 6 replicates for the Control.
6 replicates for the limit test substance concentration
1 additional replicate per test group for initial concentration control analysis only.
1 additional uninoculated (without Lemna) replicate per test group to determine the influence of the test design on test substance stability.

Reason for selection of test concentrations:
The test concentrations were selected on the basis of a range finding test (experimental conduct in accordance with GLP but without a GLP Status). The results of the 7 days range finding test were (as nominal concentrations):
ErC50: >10 mg/L
According to the OECD-guideline, the highest suggested test concentration is 100 mg/L for a limit test. This test will be carried out at 10 mg/L since preliminary tests revealed a limited solubility of the test substance in the test media under testing
conditions (< 1 mg/L).
The raw data of the range finding test is archived together with the raw data of this study.

TEST SUBSTANCE PREPARATIONS
The test solutions were prepared by directly adding the test substance to the test medium. Test substance was weighed in and made up with test medium to reach the nominal concentrations according to the table below. The test substance was weighed into a 2L volumetric flask, subsequently filled to the mark with test medium, and stirred on a magnetic stir plate for about 24 hours at room temperature. Undissolved test substance was removed by filtration with a membrane filter (pore size 0.2 μm,
regenerated cellulose). During filtration the first 50-100 mL of filtered solution were discarded (used to condition the filter). Following, the aqueous fraction is inspected visually for the presence of any undissolved test substance by observing the scattering of a laser light through the test solution (Tyndall effect).
Acceptable test solution pH is within the range ±0.5 of the control. If a test solution is outside of this range, it will be adjusted to control media pH (±0.2) with HCl or NaOH prior to the start of exposure. Fresh test solutions were prepared daily.
Following preparation all test solutions appeared colorless and clear, showing a negative Tydall effect. No pH adjustment was done during the exposure.

Stability and homogeneity
The stability of the test substance as a solution in test media and under testing conditions was determined by concentration control analysis.

Concentration control analysis
Sampling was done for three representative renewal intervals, according to the sampling scheme below. Samples for concentration control analysis were taken from each freshly prepared solution (0h) without Lemna and at the end of each exposure interval (approx.24 hours) from all test vessels (pooled replicates), as well as from the uninoculated (without Lemna) test vessel of each concentration group.
For each concentration, two retained samples were taken at each time point. Retain samples were stored at the Laboratory ECO in a freezer (at -20 °C). Analysis of these samples was performed in case of equivocal analytical results with the original (primary) samples or after loss of/damage to original samples after agreement by the Study Director. Analysis is documented and justified in both, the raw data and report.


Test duration: 7 days
Test vessels: Glass petri dishes covered with glass plates (nominal volume 300 mL) (colorless glass with a minimum water depth of 20 mm)
Test media: 20X AAP media
Test volume: 160 mL
Initial colonies: 10 fronds/ test vessel at the start of exposure
Test chamber: Infors HT Multitron Pro controlled climate cabinet.
Test temperature: 23.6 – 24.2 °C
In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels.
Illumination: Artificial light, type universal white (OSRAM L 25), permanent illumination. To minimize the potential effect of slight variations in illumination.
Light intensity: 6921 - 7580 lux (mean: 7119 lux, coefficient of variation: 3.57%) at a wavelength of 400 - 700 nm.

Route of exposure: Semi-static exposure via the test medium.
Reason: In order to ensure constant exposure conditions this study was conducted as a static-renewal exposure. The renewal of the test solutions was done daily
Randomization: The test vessels were placed in the test chamber according to a randomization plan prepared by using a program of the laboratory data evaluation group of the testing facility. The test vessels were rearranged daily.
Test endpoint parameter: Growth as frond number and weight
Test parameter: The frond number in the test vessels were counted at the start of exposure, after day 2, day 4 and at test termination (day 7).
Dry weight was determined at the start of exposure in a comparable colony and at the end of exposure in all replicates.

Test initiation, maintainance and TOC measurement
Colonies consisting of 2 to 4 visible fronds were transferred from the inoculum culture and randomly assigned to the test vessels under aseptic conditions. Each test vessel should contain a total of 9 to 12 fronds. The number of fronds and colonies were the same in each test vessel. The test vessels were impartially distributed in an incubator and continuous illumination. Vessel positions were altered after each assessment. The temperature was monitored within the incubator and in a separate vessel filled with deionized water. At the end of the test, the pH was measured in all replicates of the control and treatment groups; the pH at test initiation was measured in the respective bulk solutions.
The TOC values of all the test solutions were measured at the start and end of one renewal interval as an additional water quality parameter.

Frond number
The frond number in the test vessels were counted at the start of the test, after day 2, day 4 and after end of exposure (day 7) in each replicate. Every frond visibly projecting beyond the edge of the parent frond was counted. Observations on the appearance of the fronds included necrosis, chlorosis, changes in plant size or shape and root growth were documented. The TOC values of all the test solutions were measured at the start and end of one renewal interval as an additional water quality parameter.

Dry weight
The biomass based on dry weight was determined at the start of exposure from a sample of the inoculum culture representative of what was used to begin the test, and at the end of the test with the plant material from each treatment and control vessel. All colonies were collected from each of the test vessels and rinsed with deionized water. They were blotted to remove excess water and then dried at 60°C for 3 days to a constant weight. Any root fragments were included.

VALIDITY CRITERIA
This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.
• The doubling time of frond number in the control was 1.52 days (<2.5 days).

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: based on dry weight

Effect concentrations 2

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

frond number

Remarks on result:

other: based on dry weight

Effect concentrations 3

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: based on dry weight

Effect concentrations 4

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Remarks on result:

other: based on dry weight

Details on results:

Since the measured concentrations deviated markedly from the nominal concentrations, the effect concentration based on the mean measured concentrations should be preferably used for the evaluation of the test substance.
No toxic effects were observed up to the solubility limit of the test substance in the test medium under testing conditions.

The duckweed population in the control vessels showed exponential growth, increasing from 10 fronds per vessel to an average of 214.8 fronds per vessel in the control after 7 days (corresponding to an average specific growth rate of 0.438 d-1). The dry weight increased from 1.3 mg (sample of the inoculum culture at start of exposure) to an average of 34.5 mg per vessel in the control at test termination.

Results with reference substance (positive control):

Reference substance testing:
In order to verify that the Lemna cultures are responding normally to toxic stress, tests with a reference substance (3,5 Dichlorphenol) are conducted. Reference substance tests are conducted according to OECD 221 guidelines and in accordance with GLP, but without a GLP status.
The results from the reference substance test are compared to 3,5 Dichlorphenol EC50 values published in OECD Guideline [Ref 1], which represent the typical response range for the Lemna species tested.
The EC50(7d) of the reference substance 3,5 Dichlorphenol was 7.8 mg/L (experiment date: 21 Apr 2021, project number: 63E0308/02E014).
This result is within the range of 5.9 – 13.3 mg/L and indicates that the culture of Lemna gibba used in this study is responding normally to toxic stress.

Any other information on results incl. tables

Mean specific growth rate inhibition of Lemna gibbain each test group relative to the control

Nominal concentration [mg/L]	Average specific growth rate [d-1] inhibition over 7-days
	Frond number, mean (SD)	% Inhibition	Dry weight, mean (SD)	% Inhibition
0 (control)	0.438 (0.005)	0%	0.468 (0.005)	0%
100	0.444 (0.006)	0%	0.472 (0.008)	0%

Inhibition greater than 100% or lower than 0% were replaced by 100% and 0%, respectively.

Effect concentrationsbased on nominal concentrations(mg/L) after 7 days for the response variable average specific growth rate:

Effect concentration [mg/L]	Average specific growth rate based on frond number [mg/L]	Average specific growth rate based on dry weight [mg/L]
ErC10 (95% CL)	>10 (–)	>10 (–)
ErC20 (95% CL)	>10 (–)	>10 (–)
ErCL50 (95% CL)	>10 (–)	>10 (–)
NOErC	≥10	≥10
LOErC	>10	>10

–insufficient data for calculation

Effect concentrationsbased on nominal concentrations(mg/L) after 7 days for the response variable average specific growth rate:

Effect concentration [mg/L]	Average specific growth rate based on frond number [mg/L]	Average specific growth rate based on dry weight [mg/L]
ErC10 (95% CL)	>0.38 (–)	>0.38 (–)
ErC20 (95% CL)	>0.38 (–)	>0.38 (–)
ErCL50 (95% CL)	>0.38 (–)	>0.38 (–)
NOErC	≥ 0.38	≥ 0.38
LOErC	>0.38	>0.38

–insufficient data for calculation

Mean yield inhibition ofLemna gibbain each test group relative to the Control

Nominal concentration [mg/L]	Average specific growth rate [d-1] inhibition over 7-days
	Frond number, mean (SD)	% Inhibition	Dry weight, mean (SD)	% Inhibition
0 (control)	204.8 (8.0)	0%	33.2 (1.3)	0%
100	213.7 (9.5)	0%	34.2 (1.9)	0%

Inhibition greater than 100% or lower than 0% were replaced by 100% and 0%, respectively.

Effect concentrationsbased on nominal concentrations(mg/L) after 7 days for the response variable average specific yield rate

Effect concentration [mg/L]	Average specific growth rate based on frond number [mg/L]	Average specific growth rate based on dry weight [mg/L]
ErC10 (95% CL)	>10 (–)	>10 (–)
ErC20 (95% CL)	>10 (–)	>10 (–)
ErCL50 (95% CL)	>10 (–)	>10 (–)
NOErC	≥10	≥10
LOErC	>10	>10

–insufficient data for calculation

Effect concentrationsbased on nominal concentrations(mg/L) after 7 days for the response variable average specific yield rate

Effect concentration [mg/L]	Average specific growth rate based on frond number [mg/L]	Average specific growth rate based on dry weight [mg/L]
ErC10 (95% CL)	>0.38 (–)	>0.38 (–)
ErC20 (95% CL)	>0.38 (–)	>0.38 (–)
ErCL50 (95% CL)	>0.38 (–)	>0.38 (–)
NOErC	≥ 0.38	≥ 0.38
LOErC	>0.38	>0.38

–insufficient data for calculation

The OECD 221 notes that toxicity values calculated by using the response variables average specific growth rate and yield are not comparable. The effect values based on average specific

growth rate (ErCx)will be generally higher than those based on yield (EyCx). However, the differences are due to the mathematical basis of the calculations and should not be interpreted

as a difference in sensitivity between the two variables. The results based on yield are influenced by several factors including the Lemna species and clone, control growth rate as

well as the slope of the concentration response curve. Results calculated based on average specific growth rate are independent of these variables and are the scientifically preferred basis

to estimate toxicity. The preferred endpoint for this test is the lowest average specific growth rate ErC(L)50 value.

No toxic effects were observed up to the solubility limit of the test substance in the test medium under testing conditions. No additional adverse effects or abnormal behavior were observed in

any of the test groups. The reported effect concentrations are based on expert judgement.

Since the measured concentrations deviated markedly from the nominal concentrations, the effect concentration, which is based on the mean measured concentrations should be preferably used for the evaluation of the test substance.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a 7-day growth inhibition study, Lemna gibba were exposed to Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-l-glutamate and stearic acid at nominal concentrations of 0 (control), and 10 mg/L, wherereas mean measured concentrations (geomean) accounted for 0 and 0.38 mg/L. The test organism was exposed to the test item under daily-renewal conditions in accordance with the OECD 221 guideline. The water pH and temperature were within acceptable guideline specifications. Plant growth (as frond number) and phytotoxic effects were observed on day 2, 4 and test termination (day 7). Percent growth inhibition relative to the control was calculated for each test concentration based upon growth rates and final yield for the parameters frond number and plant dry weight.
The 7-day ErC50 values determined based on nominal concentrations / mean measured (geomean) concentrations in this toxicity study were:
ErC50 growth rate:> 10 mg/L nominal and > 0.38 mg/L mean measured
ErC50 yield :> 10 mg/L nominal and > 0.38 mg/L mean measured

Executive summary:

In a 7-day growth inhibition study, Lemna gibba were exposed to Reaction mass of sodium  hydrogen N-(1-oxooctadecyl)-l-glutamate and stearic acid at nominal concentrations of 0 (control), and 10 mg/L, wherereas mean measured concentrations (geomean) accounted for 0 and 0.38 mg/L. The test organism was exposed to the test item under daily-renewal conditions in accordance with the OECD 221 guideline. The water pH and temperature were within acceptable guideline specifications. Plant growth (as frond number) and phytotoxic effects were observed on day 2, 4 and test termination (day 7). Percent growth inhibition relative to the control was calculated for each test concentration based upon growth rates and final yield for the parameters frond number and plant dry weight.

The 7-day ErC50 values determined based on nominal concentrations / mean measured (geomean) concentrations in this toxicity study were:

ErC50 growth rate:> 10 mg/L nominal and  > 0.38 mg/L mean measured

ErC50 yield :> 10 mg/L nominal  and > 0.38 mg/L mean measured