Reaction mass of N,N'-ethylenebis[N-methyl-2-[(1-oxo-9-octadecenyl)oxy]-N-[2-[(1-oxo-9-octadecenyl)oxy]propyl]propylammonium] dimethyl disulphate and [2-[bis(2-hydroxypropyl)amino]ethyl]bis(2-hydroxypropyl)(methyl)ammonium methyl sulphate, dioleate (ester)

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:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

Batch manufactured and supplied by : Stepan Company

Test item : AGENT 5088-127A

CAS No. : 65059-61-2 + 70773-61-4

Chemical name (IUPAC) : Reaction mass of [2-[bis(2-hydroxypropyl)amino]ethyl]bis(2-hydroxypropyl)(methyl)ammonium methyl sulphate,dioelate(ester) [&] N,N’-ethylenebis[N-methyl-2-[(1-oxo-9-octadecenyl)oxy]propyl]propylammonium]dimethyldisulphate

Physical appearance : Solid

Purity as per CoA : 100%

Lot No. : 5124-143-001

Manufactured date : 20 December 2017

Retest date : 20 December 2020

Recommended storage condition : Ambient (+15 to +25°C)

Note: a) Date of receipt of test item at test facility: 17 January 2018
b) Test Item code by test facility: S047-01

Analytical monitoring:

not specified

Details on test solutions:

The test medium was prepared by mixing 10 mL stock solution 1 and 1 mL each stock solutions 2, 3 and 4 with 500 mL of sterilized water and then making up the volume to 1 L using sterilized water. The pH of the medium was between 7.82 and 7.83 during the study. This medium was equilibrated with carbon dioxide by keeping it in laminar hood for two hours.

Details on test organisms:

Pseudokirchneriella subcapitata ATCC 22662

This algal species was used as the test system as it is a recommended algal species in the guideline.

Source
American Type Culture Collection
P.O.Box 1549
Manassas, VA 20108
USA

Culture Maintenance
Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between 21 and 25°C (±2°C) under a continuous illumination of 8000±1600 Lux.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

not specified

Test temperature:

ca 22 C

pH:

ca 7.8

Dissolved oxygen:

not specified

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

0.069 (G2), 0.207 (G3), 0.621 (G4), 1.863 (G5), 5.589 (G6), 16.767 (G7) and 50.301 (G8) mg/L

Details on test conditions:

Algal cells with test concentrations were incubated in the following conditions during range finding and the definitive test.

Parameters Range finding test Definitive test
Duration of the test 72 ± 1 hours 72 ± 1 hours
Test containers 250 mL conical flask 250 mL conical flask
pH of test concentration 7.80 to 7.83 7.80 to 7.83
Temperature of growth chamber (C) 22.1 to 22.5 22.1 to 22.5
Mean continuous light intensity (Lux) 7000 to 7458 7000 to 7458

Equipment
Equipment Model No. Manufacturer
Algal growth chamber PSM/AC-01 PSM Scientific, Bengaluru, India
Not applicable Leed Instrument, Bengaluru
Electronic Balance AG245, PB303 Mettler Toledo, Switzerland
Digital lux meter LX 1010B Mextech, India
pH meter Eutech pH/mv/ºC meter Eutech Instruments, Singapore
Microscope Eclipse 50i Nikon, Japan

Culturing apparatus was an incubator shaker fitted with white fluorescent tube lights for uniform continuous illumination which could give a light intensity of approximately 4000 to 10000 Lux. Conical flasks of 250 mL capacity was used as test containers.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.201 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.763 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.439 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

- Results with reference substance valid yes

Validity criteria fulfilled:

yes

Conclusions:

The EC values calculated for growth rate and yield on the basis of the nominal concentration of the test item are presented below:

At 72 hours EC values (mg/L)
ErC50 2.201
ErC20 0.7634
ErC10 0.439
EyC50 0.571
EyC20 0.2206
EyC10 0.1341

Note: ErC refers to growth rate and EyC refers to yield.

Executive summary:

The effect of AGENT 5088-127A was tested on the growth of freshwater single cell green algaPseudokirchneriella subcapitata.

 

Range finding test was carried out to select the test concentrations for the definitive test. In the range finding test, the tested concentration was 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L with single replicate. The test item exhibited1.95, 2.61, 13.68, 17.59, 53.09, 84.04, 92.51 and 100% reduction in cell biomass at the tested concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L at 72 h.

 

The alga was exposed to the test item at the nominal concentrations of 0.069, 0.207, 0.621, 1.863, 5.589, 16.767 and 50.301 mg/L (factor of about 3) along with a negative control and a reference substance (Potassium dichromate) at 0.9 mg/L. The cell growth was measured at 24, 48 and 72 hours after the initiation of the test.

 

The concentration/effect relationship was determined using two factors namely growth rate and yield at the end of the test (72 hours). The per cent inhibition of growth rate was 0.27, 4.92, 19.98, 42.69, 69.65 and 100% and the percent yield inhibition was 1.12, 19.25, 58.21, 85.05, 96.23 and 100% at the nominal concentrations of 0.069, 0.207, 0.621, 1.863, 5.589, 16.767 and 50.301 mg/L, respectively, when compared to the negative control. There was 21.83 % and 61.51 % inhibition of average growth rate and yield of alga, respectively, in the reference substance group at 0.9 mg/L. Observed cells were found morphologically normal.

 

The EC values calculated for growth rate and yield on the basis of the nominal concentration of the test item are presented below:

 

At 72 hours	EC values (mg/L)
ErC50	2.201
ErC20	0.7634
ErC10	0.439
EyC50	0.571
EyC20	0.2206
EyC10	0.1341

 

Note: E_rCrefers to growth rate and E_yC refers to yield. 

Description of key information

Based on the growth inhibition test on Pseudokirchneriella subcapitata showed an EC50 of 2.201 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.201 mg/L