3-((C12-18)-acylamino)-N-(2-((2-hydroxyethyl)amino)-2-oxoethyl)-N,N-dimethyl-1-propanaminium chloride

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Directive 92/69/CEE, Méthode C3

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: none

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

Species and scientific name:
freshwater unicellular green algae : Chlorella vulgaris CCAP 211/11b
Justification for the choice of species:
species easily bread and tested under laboratory conditions, rapidly growing (test performed in several generations), and required by the international Standards.
Supplier:
Aquarium de Trouville (17 rue de Paris - F - 14360 TROUVILLE).
Culture age:
approximately 72-hour old algal sub-culture.

Algal stock cultures: subcultures made at regular intervals.
Containers used for the pre-culture and test:
Erlenmeyer flasks of 250 ml capacity, provided with a cotton-wool plug.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24.2-26°C

pH:

6.9-7.2

Nominal and measured concentrations:

Preliminary test!
0/ 1 / 5 / 10 / 50 / 100 mg/L

Main test:
0 / 0.1 / 0.2 / 0.5 / 1.0 / 2.2 mg/L

Details on test conditions:

Triplicates cultures were used for control and treated group during the preliminary and the main test

Experimental observations and measurements:
cell concentration
24, 48 and 72 hours after the start of the test, the cell concentration was measured for each control and test flask by direct counting of cells with microscope.
pH values in test media
pH measurements were performed in total volumes of control and test media related to each concentration at the start of the test, and then approximately 72 hours later.
Observation of the test media
Observation of the test media was performed during the test ; test medium abnormalities, if any, were recorded.
Test substance concentration level sampling
Each medium was once sampled (at the beginning and at the end of the test).
The frozen samples were transmitted to the analytical testing facility at the end of the test, in order to have test substance concentration levels determined : although it was tried to decrease the limit of quantification, it was not possible to quantify the test item in aqueous solutions since an interferent peak occurs at 220 nm. The results are therefore based on nominal concentrations.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.14 mg/L

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.52 mg/L

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.1 mg/L

Basis for effect:

biomass

Results with reference substance (positive control):

Periodicity of validation: Every 6 months or for each new strain
ECA 50-72 h of the strain used: 1.1 mg/L
Conformity: yes, the batch was accepted.

Validity criteria fulfilled:

yes

Conclusions:

Inhibition of the growth of the unicellular green algae (IA with areas under the growth curves in 72 hours of exposure):
ECA (I) 50 - 72 hours, for freshwater unicellular algae, Chlorella vulgaris:
0.14 mg/L

Inhibition of the growth rate of the unicellular green algae (Iu with growth rates at 72 hours of exposure):
ECu (I) 50 - 72 hours, for freshwater unicellular algae, Chlorella vulgaris:
0.52 mg/L

No Observed Effect Concentration (N.O.E.C.):
Growth: <0.1 mg/L
Growth rate : 0.1 mg/L

Executive summary:

ALGAL INHIBITION TEST (72 HOURS) - STATIC SYSTEM - Chlorella vulgaris

Compliance with OECD Guideline No. 201 (1984) and EEC Directive 92/69 Method C.3 (1992).

 

METHOD

The algal cultures (Chlorella vulgaris) were exposed to a test substance for 72 hours.

A preliminary test (phase I) was performed using the nominal concentrations of 1, 5, 10, 50 and 100 mg of test substance per litre of test medium (3 cultures per concentration). The control group (3 cultures not exposed to any substance) was employed under the same experimental conditions.

A main test (phase II) was then performed using nominal concentrations of 0.1, 0.2, 0.5, 1.0 and 2.2 mg of test substance per litre of test medium (3 cultures per concentration). A control group (3 cultures flot exposed to any substance) was also employed under the same experimental conditions.

Test media were continuously mechanically stirred, and randomly rotated each test day.

Algal cell concentrations were measured approximately 24, 48 and 72 hours after the start of the test. Inhibition percentages at the end of the test were calculated with the methods using respectively the inhibition of the growth (IA by comparison of the areas under the growth curves during 72 hours of exposure), and the inhibition of the growth rate by comparison of the growth rates (i.e. by comparison of the areas under the growth at 72 hours of exposure).

Measurements of pH were performed for each test and control medium, at the start and at the end of the test.

Each medium was once sampled (at the beginning and at the end of the test). The frozen samples were transmitted to the analytical testing facility at the end of the test, in order to have test substance concentration levels determined: although it was tried to decrease the limit of quantification, it was not possible to quantify notified chemical in aqueous solutions since an interferent peak occurs at 220 nm. The results are therefore based on nominal concentrations.

The quality criterion for the test as notified in the OECD and EEC protocols relative to the growth in the control group was respected.

 

RESULTS

Within 72 hours of exposure (nominal concentrations):

1)-Inhibition of the growth of the unicellular green algae (IA with areas under the growth curves in 72 hours of exposure):

- EC42 -72 hours (tested concentration): 0.1 mg/L

- EC61 -72 hours (tested concentration): 0.2 mg/L

- EC72 - 72 hours (tested concentration): 0.5 mg/L

- EC93 - 72 hours (tested concentration): 1.0 mg/L

- EC95 - 72 hours (tested concentration): 2.2 mg/L

 

- Lowest observed concentration inducing complete inhibition: > 2.2 mg/L

- Highest observed concentration inducing no significant inhibition: <0.1 mg/L

- Confidence limits for the ECA (I) 50- 72 hours value (95%): 0.09 - 0.20 mg/L

 

ECA50 - 72 hours, for freshwater unicellular algae, Chlorella vulgaris:

0.14 mg/L

 

2)-Inhibition of the growth rate of the unicellular green algae (i.e. with growth rates at 72 hours of exposure):

- EC14 - 72 hours (tested concentration) : 0.1 mg/L

- EC29 -72 hours (tested concentration) : 0.2 mg/L

- EC49 - 72 hours (tested concentration) : 0.5 mg/L

- EC100 - 72 hours (tested concentration) : 1.0 mg/L

- EC122 - 72 hours (tested concentration) : 2.2 mg/L 

- Lowest observed concentration inducing complete inhibition: 1.0 mg/L

- Highest observed concentration inducing no significant inhibition: 0.1 mg/L

-Confidence limits for the EC50- 72 hours value (95%): 0.33 - 0.81 mg/L

 

EC50 - 72 hours, for freshwater unicellular algae, Chlorella vulgaris: 0.52 mg/L

 

3)-No Observed Effect Concentration (NOEC):

Growth: <0.1 mg/L

Growth rate: 0.1 mg/L

Description of key information

Inhibition of the growth of the unicellular green algae (IA with areas under the growth curves in 72 hours of exposure):
ECA (I) 50 - 72 hours, for freshwater unicellular algae, Chlorella vulgaris: 0.14 mg/L

Inhibition of the growth rate of the unicellular green algae (Iu with growth rates at 72 hours of exposure):
ECu (I) 50 - 72 hours, for freshwater unicellular algae, Chlorella vulgaris: 0.52 mg/L

No Observed Effect Concentration (N.O.E.C.):
Growth: <0.1 mg/L
Growth rate : 0.1 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

0.52 mg/L

EC10 or NOEC for freshwater algae:

0.1 mg/L