N,N'-bis[2-hydroxy-3-(2,2,3,3-tetrafluoropropoxy)propyl]-N,N,N',N'-tetramethylethane-1,2-diaminium dichloride

Toxicity to aquatic algae and cyanobacteria

Administrative data

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: GLP study carried out to internationally recognised standard OECD Guideline 201

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis[2-hydroxy-3-(2,2,3,3- tetrafluoropropoxy)propyl]-N,N,N',N'-tetramethylethane-1,2- diaminium dichloride

IUPAC Name:

N,N'-bis[2-hydroxy-3-(2,2,3,3- tetrafluoropropoxy)propyl]-N,N,N',N'-tetramethylethane-1,2- diaminium dichloride

Constituent 2

Reference substance name:

1,1,2,2,17,17,18,18-octafluoro-6,13-dihydroxy-8,8,11,11-tetramethyl-4,15-dioxa-8,11-diazaoctadecane-8,11-diium dichloride

EC Number:

700-737-4

Cas Number:

1220100-43-5

Molecular formula:

C18H34F8N2O4.2Cl

IUPAC Name:

1,1,2,2,17,17,18,18-octafluoro-6,13-dihydroxy-8,8,11,11-tetramethyl-4,15-dioxa-8,11-diazaoctadecane-8,11-diium dichloride

Details on test material:

[14C] Radiolabelled test substance was received and stored frozen in the original container.
Non Radiolabelled test substance was received and stored at room temperature in the original container in a dessicator.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Exposure Solution Preparation

A 10 mg/L primary stock solution was prepared prior to test initiation by placing 0.02006 g of the non-radiolabeled test substance (0.02000 g as active ingredient) in a 2-L volumetric flask and adding 0.50 mL (0.570 mg) of the 1.14 mg/mL primary radiolabled stock solution. The solution was then brought to a final volume of 2.0 L with AAP medium. The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance following mixing for approximately 5 minutes using a Teflon®- coated stir plate and magnetic stir bar. The exposure solutions were prepared from dilutions of the 10 mg/L primary stock solution as follows:

Primary Stock Volume Stock Diluted to (mL) Nominal
Concentration Used (mL) with AAP Conc (mg/L)
(mg/L)
10 300 NA 10
10 400 1000 4.0
10 160 1000 1.6
10 64 1000 0.64
10 26 1000 0.26
10 10 1000 0.10
NA = Not Applicable

All exposure solutions were observed to be clear and colorless with no visible undissolved test substance after mixing by shaking and inversions.

Additional untreated AAP medium was used for the control. Six flasks for the control, as required by the OECD guideline, were maintained under the same conditions as the treatment levels and contained no test substance.

In order to estimate the impact that the presence of algal biomass had on the test substance concentration, an additional replicate flask (D) of the 0.64 mg/L nominal treatment level was prepared. This flask, which was not inoculated with algae, was analyzed after 72 hours of exposure for test substance concentration. The result of this analysis was compared with the 72 hour result for the 0.64 mg/L solution containing algae.

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

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

Details on test organisms:

The alga used in this toxicity test was the freshwater green alga Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), strain 1648, Class Chlorophyceae. The alga was obtained from University of Texas, Austin, Texas and was maintained in stock culture at Smithers Viscient culture facility.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23 deg C

pH:

pH 6.8 to 7.0 at test initiation
pH 7.1 to 7.6 at 72 hrs exposure

Nominal and measured concentrations:

Nominal test concentrations: 0.10, 0.26, 0.64, 1.6, 4.0 and 10 mg/L
Mean measured test concentrations: 0.10, 0.24, 0.59, 1.5, 3.8 and 10 mg/L

Details on test conditions:

The test was conducted in an environmental chamber designed to maintain the test conditions specified in the protocol: a temperature of 23 ± 2 ºC, continuous light intensity of 4400 to 5900 lux (420 to 550 footcandles) and photosynthetically-active radiation (PAR) range of 60 to 120 µE/m2/s. An orbital shaker table provided a shaking rate of 100 ± 10 rpm.

Temperature was measured continuously with a VWR minimum/maximum thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shaker were recorded daily. Light intensity was measured at four locations around the perimeter of the test area with a VWR Traceable light meter at 0-hour and at each 24 hour interval during the exposure period. Light intensity was measured in footcandles and converted to lux based on 1 footcandle = 10.76 lux. Photosynthetically-active radiation (PAR) of the test area was measured at test initiation using a Licor Model LI-189 photometer and sensor model LI-190SA. Test flasks were randomly placed on the shaking table at test initiation based on computer-generated random numbers. Following each observation interval, the test flasks were assigned new random positions based on computer-generated random numbers.

Water quality parameters (pH and conductivity) were measured at test initiation, and at the termination of the 72-hour exposure period. Measurements at test initiation were conducted on the test solution remaining after the individual test flasks had been filled. At test termination, after cell counts were completed, replicate solutions for each treatment and control were respectively composited for pH and conductivity measurements. Test solution pH was measured with a Yellow Springs Instrument (YSI) Model pH100 pH meter, and conductivity was measured with a YSI Model 3100-115V salinity-conductivity-temperature meter.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.083 to 0.12 mg/L at 95% CL

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.11 to 0.15 mg/L at 95% CL

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.17 to 0.23 mg/L at 95% CL

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.11 to 0.18 mg/L at 95% CL

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.15 to 0.27 mg/L at 95% CL

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.40 to 1.3 mg/L at 95% CL

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72-Hour acute toxicity test with freshwater green alga, Pseudokirchneriella subcapitata, following OECD Guideline 201 has provided the following data on the test substance.

72-Hour Yield
EC10 (95% Confidence Intervals) = 0.12 mg/L (0.083-0.12)
EC20 (95% Confidence Intervals) = 0.14 mg/L (0.11-0.15)
EC50 (95% Confidence Intervals) = 0.19 mg/L (0.17-0.23)
NOEC = 0.10 mg/L

Growth Rate
EC10 (95% Confidence Intervals) = 0.15 mg/L (0.11-0.18)
EC20 (95% Confidence Intervals) = 0.19 mg/L (0.15-0.27)
EC50 (95% Confidence Intervals) = 0.58 mg/L (0.40-1.3)
NOEC = 0.10 mg/L

A recovery phase was conducted to determine the algistatic or algicidal properties of the test substance.
The results of this recovery phase indicated that the test substance has an algistatic, rather than algicidal effect on the growth of Pseudokirchneriella subcapitata at a nominal concentration of 10 mg/L.

Executive summary:

The objective of this study was to determine the effect of the test substance on the growth of the freshwater green alga Pseudokirchneriella subcapitata, formerlySelenastrum capricornutum. The results are based on mean measured concentrations of test substance and are reported as the 72‑hour EC10, EC20 and EC50 values for biomass expressed as yield and average growth rate data calculated from the 0‑hour to 72‑hour cell density counts, denoted as E_yC10, E_yC20 and E_yC50 and E_rC10, E_rC20 and E_rC50, respectively, and the No-Observed-Effect Concentration (NOEC) values for total yield and average growth rate.

Biological Parameter	Based on Mean Measured Concentrations (mg/L)
	EC10 (95% Confidence Intervals)	EC20 (95% Confidence Intervals)	EC50 (95% Confidence Intervals)	NOEC
72-Hour Yield	0.12 (0.083 – 0.12)	0.14 (0.11 – 0.15)	0.19 (0.17 – 0.23)	0.10
72-Hour Average Growth Rate	0.15 (0.11 – 0.18)	0.19 (0.15 – 0.27)	0.58 (0.40 – 1.3)	0.10

A recovery phase was conducted to determine the algistatic or algicidal properties of the test substance. The results of this recovery phase indicated that the test substance has an algistatic, rather than algicidal effect on the growth of Pseudokirchneriella subcapitataat a nominal concentration of 10 mg/L.