Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

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:

From November 12, 2019 to December 11, 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)

Deviations:

yes

Remarks:

The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

The content of the test item in the test solutions was estimated by calculation based on the organic carbon content of the test item and TOC measurement. TOC (Total organic carbon) was determined as TC (total carbon) minus IC (inorganic carbon).

Details on sampling:

The content of TOC in the test vessels was measured at the start and at the end of the
test. Samples for the analytical determination were taken from the replicates without algae.

Vehicle:

no

Details on test solutions:

Concentrations tested: 0, 0.01, 0.032, 0.1, 0.32, 1, 3.2 and 10 mg/L nominal concentration.
The concentrations to be tested were based on non-GLP pre-tests

Vessels: glass flasks total volume 65 mL

Replicates
Treatments: 3 vessels, filled with 45 ±1 mL test solution and algae
1 vessel, filled with 45 ±1 mL test solution without algae for analytical determination
Blank Control: 6 vessels, each completely filled with algal medium and algae
1 vessel completely filled with algal medium without algae for analytical
determination
Duration: 72 hours
Lighting: within the specified range (4440 – 8880 Lux)
Temperature: 21.4 – 23.6 °C

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Specification
Unicellular freshwater green alga.
Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae

Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge
GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

21.4 – 23.6°C

pH:

6.9-7.6

Nominal and measured concentrations:

0.01, 0.032, 0.1, 0.32, 1, 3.2, 10 mg/L

Details on test conditions:

CHEMICALS
All solutions were sterilized before use.
Composition of the Solutions:

STOCK SOLUTION I
NH4Cl: 1500 mg
MgCl2*6H2O: 1200 mg
CaCl2*2H2O: 1800 mg
MgSO4*7H2O: 1500 mg
KH2PO4: 160 mg
H2O deionised ad: 1000 mL

STOCK SOLUTION II
FeCl3*6H2O: 64 mg
Na2EDTA*2H2O: 100 mg
H2O deionised ad: 1000 mL

STOCK SOLUTION III
H3BO3: 185 mg
MnCl2*4H2O: 415 mg
ZnCl2: 3 mg
CoCl2*6H2O: 1.5 mg
CuCl2*2H2O: 0.01 mg
Na2MoO4*2H2O: 7 mg
H2O deionised ad: 1000 mL

STOCK SOLUTION IV
NaHCO3: 50 g
H2O deionised ad: 1000 mL

ALGAL MEDIUM
Stock solution I 10 mL
Stock solution II 1 mL
Stock solution III 1 mL
Stock solution IV 1 mL
H2O deionised ad 1000 mL

Stock solutions I and III were sterilized using an autoclave. The deionised water was sterilized
using sterilfiltration. Stock solutions II and IV were sterilized using filtration. The algal
medium corresponds to the OECD TG 201 medium.
Deviations from the nominal weighted loads were less than 5 %.

PERFORMANCE OF THE STUDY
For each treatment, 200 mL of the respective test item solution was mixed with the necessary
amount of algal pre-culture (0.341 mL) to achieve a cell concentration of 2.3 *103
cells/mL. For the blank control, 350 mL nutrient medium was used instead of test item solution
and mixed with the necessary amount of algal pre-culture (0.597 mL). In this mixture,
the pH-value was measured.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated
open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an
orbital shaker to keep the algae in suspension. Before the start of incubation and every 24
hours, the cell number was determined with an electronic particle counter. After the test
the pH value in treatments and blank control was measured again.
At the end of the test, the treatments were examined microscopically in order to assess the
appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test
item).
The content of TOC in the test vessels was measured at the start and at the end of the
test. Samples for the analytical determination were taken from the replicates without algae.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7, CAS No. 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.08 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.09 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.09 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the EyC50 and ErC50 (0-72 h) were 0.19 mg/L and 0.43 mg/L, respectively. The ErC10, EyC10, values were determined at 0.08mg/L and 0.13 mg/L, respectively.

Executive summary:

A study was conducted to determine the toxicity of the test substance to aquatic algae, according to OECD Guideline 201 resp. EU C.3, in compliance with GLP. The freshwater algae Desmodesmus subspicatus was exposed for 72 h to nominal concentrations of 0.01, 0.032, 0.1, 0.32, 1, 3.2, 10 mg/L of the test substance.Three replicates were tested for each of the test substance concentrations and six replicates for the control. Analytical dose verification was performed.The cell concentration of each replicate was determinded by measuring the cell numbers every 24 hours. From the cell number, growth rate and yield were determined. Significant inhibition of algal growth was observed at the following nominal concentrations: 0.32 – 10 mg/L (growth rate and yield). With the exception of the stock solution (100 mg/L), and the two highest tested concentrations (3.2 and 10 mg/L), the measured TOC values were the same low range as the blank value. Therefore, the measured initial concentration of the 100 mg/L stock solution and the respective dilution factor were used to calculate the results. This is permitted as it is a soluble test item and the test solutions were prepared by dilution of a stock solution. The EC10 for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 0.08 and 0.13 mg/L, respectively. The EC50 for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 0.43 and 0.19 mg/L, respectively. Under the study conditions, the NOEC specific for growth rate and yield after 72 h were 0.09 mg/L for both the growth rate and yield (Walter, 2019).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.43 mg/L

EC10 or NOEC for freshwater algae:

0.08 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance to aquatic algae, according to OECD Guideline 201 resp. EU C.3, in compliance with GLP. The freshwater algae Desmodesmus subspicatus was exposed for 72 h to nominal concentrations of 0.01, 0.032, 0.1, 0.32, 1, 3.2, 10 mg/L of the test substance.Three replicates were tested for each of the test substance concentrations and six replicates for the control. Analytical dose verification was performed.The cell concentration of each replicate was determinded by measuring the cell numbers every 24 hours. From the cell number, growth rate and yield were determined. Significant inhibition of algal growth was observed at the following nominal concentrations: 0.32 – 10 mg/L (growth rate and yield). With the exception of the stock solution (100 mg/L), and the two highest tested concentrations (3.2 and 10 mg/L), the measured TOC values were the same low range as the blank value. Therefore, the measured initial concentration of the 100 mg/L stock solution and the respective dilution factor were used to calculate the results. This is permitted as it is a soluble test item and the test solutions were prepared by dilution of a stock solution. The EC10 for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 0.08 and 0.13 mg/L, respectively. The EC50 for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 0.43 and 0.19 mg/L, respectively. Under the study conditions, the NOEC specific for growth rate and yield after 72 h were 0.09 mg/L for both the growth rate and yield (Walter, 2019).