3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane

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:

06 April 2018 - 16 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

In a range finding test, Pseudokirchneriella subcapitata cells were exposed to nominal concentrations of 0.1, 1.0, 10 and 100 mg/L. Based on the results of the range finder, the following concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Reseacrh Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10(+4) to 10 (+5) cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not stated

Test temperature:

24 ±1 °C

pH:

Start of test: 7.5 to 7.9
End of test: 8.7 to 9.2

Dissolved oxygen:

Not measured

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

Samples were taken from the control and each test group at 0 hours and 72 hours and analysed using the GC-MS conditions described above.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Closed vessel: Plugged with polyurethane foam bungs and incubated
- Initial cells density: measured using a Coulter Multisizer Particle Counter. After 72 hours, the cell density of the flask was measured again.
- Control end cells density: measured at beginning and end of test
- No. of vessels per concentration (replicates): two
- No. of vessels per control (replicates): two
- No. of vessels per vehicle control (replicates): Not applicable

CULTURE MEDIUM
- Standard medium used: yes

TEST SOLUTIONS/CONCENTRATIONS: A nomimal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (5.3 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.

The control group was maintained under identical conditions but not exposed to the test item.

RANGE FINDER TEST:
In the range finder test, a sample of each test and control culture was removed and the cell density determined. The flasks were then plugged with polyurethane foam bungs and incubated at 24 ±1 °C under continuous illumination provided by warm white lighting (380 to 730 nm) and constantly shaken for 72 hours at 150 rpm. After 72 hours, the cell density was determined again.

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours.

DEFINITIVE TEST:
200 mg of test item was dissolved in culture medium and the volume adjusted to 2 liters to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 56, 32, 18 and 10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (2.6 mL) to give the required test concentrations of 10, 18, 32, 56 and 100 mg/L. 100mL of the test preparation was then added to a conical flask, plugged with polyurethane foam bungs and incubated at 24 ±1 °C under continuous illumination (380 to 730 nm) and constant shaking at 150 rpm for 72 hours.

Samples were taken at 30, 49 and 72 hours and the cell densities determined. Three determinations were made for each sample. The shape and size fo the algal cells were also inspected microscopically and any abnormalities recorded.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 80 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

80 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- The growth rate and yield of Pseudokirchneriella subcapitata were not affected by the presence of the test item over the 72 hour exposure period.
- There were no abnormalities detected in any of the control or test cultures at 72 hours.
- The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 9.2 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units and therefore within the limits given in the test guideline.
-At the start of the test, all control and test cultures were observed to be clear colourless solutions. After 72 hours, all control and test cultures were observed to be green dispersions.
- Analysis of the test preparations at 0 hours showed measured test concentrations to be near nominal with the exception of the 56 mg/L test preparation where a measured concentration of 76% of nominal was obtained. A significant decline in the measured test concentration was observed in the test preparations at 72 hours in the range of 17% to 27% of nominal.

Analysis of additional samples incubated along side the test which contained no algal cells showed measured concentrations at 72 hours to range from 71% to 84% of the nominal indicating that the losses observed were primarily attributed to adsorption of the test item to the algal cells present. As a result, geometric means were used to determine the result of the test. The geometric mean measured test concentrations were determined to be 8, 15, 24, 42 and 80 mg/L.

Table 1       Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentrations (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 hours	72 hours	Growth Rate	Yield
Control	R1	7.57E+03	1.14E+06	-	-
	R2	7.98E+03	1.15E+06
	Mean	7.77E+03	1.15E+06
0.10	R1	6.69E+03	9.34E+05	[1]	9
	R2	7.27E+03	1.16E+06
	Mean	6.98E+03	1.05E+06
1.0	R1	6.57E+03	1.01E+06	[3]	[2]
	R2	7.25E+03	1.32E+06
	Mean	6.91E+03	1.17E+06
10	R1	6.07E+03	8.53E+05	[3]	5
	R2	6.75E+03	1.33E+06
	Mean	6.41E+03	1.09E+06
100	R1	8.36E+03	9.16E+05	4	24
	R2	6.98E+03	8.36E+05
	Mean	7.67E+03	8.76E+05

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R = Replicate

- = Not applicable

[ ] = increase in growth compared to control

Table 2     Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 hours	0 hours	49 hours	72 hours	72 Hours
Control	R1	7.9	4.30E+04	2.16E+05	1.02E+06	9.2
	R2		4.56E+04	2.08E+05	1.07E+06
	R3		5.21E+04	2.48E+05	1.44E+06
	R4		4.94E+04	2.46E+05	1.31E+06
	R5		4.25E+04	2.14E+05	1.12E+06
	R6		4.29E+04	2.10E+05	1.28E+06
	Mean		4.59E+04	2.24E+05	1.21E+06
8.0	R1	7.8	5.24E+04	2.59E+05	1.36E+06	9.0
	R2		5.59E+04	2.86E+05	1.28E+06
	R3		5.83E+04	2.49E+05	1.25E+06
	Mean		5.55E+04	2.65E+05	1.29E+06
15	R1	7.7	5.78E+04	2.70E+05	1.48E+06	8.9
	R2		5.07E+04	2.23E+05	1.19E+06
	R3		5.35E+04	2.34E+05	1.38E+06
	Mean		5.40E+04	2.42E+05	1.35E+06
24	R1	7.6	5.05E+04	2.44E+05	1.45E+06	8.7
	R2		4.73E+04	2.51E+05	1.65E+06
	R3		4.99E+04	2.34E+05	1.27E+06
	Mean		4.93E+04	2.43E+05	1.46E+06
42	R1	7.6	4.19E+04	1.95E+05	9.70E+05	8.7
	R2		4.94E+04	2.25E+05	1.36E+06
	R3		5.02E+04	2.15E+05	1.33E+06
	Mean		4.72E+04	2.12E+05	1.22E+06
80	R1	7.5	4.43E+04	2.14E+05	1.32E+06	8.9
	R2		4.43E+04	2.16E+05	1.04E+06
	R3		4.81E+04	2.12E+05	1.17E+06
	Mean		4.56E+04	2.14E+05	1.18E+06

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R = Replicate

Table 3     Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 hours	% Inhibition	0 to 72 hour	% Inhibition*
Control	R1	0.074	-	1.01E+06	-
	R2	0.075		1.07E+06
	R3	0.079		1.43E+06
	R4	0.077		1.31E+06
	R5	0.075		1.11E+06
	R6	0.077		1.28E+06
	Mean	0.076		1.20E+06
	SD	0.002		1.64E+05
8.0	R1	0.078	[3]	1.35E+06
	R2	0.077	[1]	1.27E+06
	R3	0.077	[1]	1.24E+06
	Mean	0.077	[2]	1.29E+06	[7]
	SD	0.001		5.72E+06
15	R1	0.079	[4]	1.48E+06
	R2	0.076	0	1.18E+06
	R3	0.078	[3]	1.38E+06
	Mean	0.078	[2]	1.35E+05	[12]
	SD	0.002		1.52E+05
24	R1	0.079	[4]	1.44E+06
	R2	0.081	[7]	1.65E+06
	R3	0.077	[1]	1.26E+06
	Mean	0.079	[4]	1.45E+06	[21]
	SD	0.002		1.91 E+06
42	R1	0.073	4	9.65E+05
	R2	0.078	[3]	1.36E+06
	R3	0.078	[3]	1.33E+06
	Mean	0.076	[1]	1.22E+06	[1]
	SD	0.003		2.18E+05
80	R1	0.077	[1]	1.32E+06
	R2	0.074	3	1.03E+06
	R3	0.076	0	1.16E+06
	Mean	0.076	1	1.17E+06	3
	SD	0.002		1.41E+05

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated. 

R = Replicate

SD = Standard Deviation

- = Not applicable

[ ] = increase in growth compared to control

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and based on the geometric mean measured test concentrations gave EC50 values of greater than 80 mg/L. The No Effect Concentration was 80 mg/L.

Executive summary:

A study was conducted to assess the effect of the test item on the growth of the green algae, Pseudokirchneriella subcapitata in line with OECD 201.

Following a preliminary range finder, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L (three replicate flasks per concentration) for 72 hours under constant illumination and shaking at a temperature of 24 ±1 °C.

Analysis of the test preparations at 0 hours showed measured test concentrations to be near nominal with the exception of the 56 mg/L test preparation where a measured concentration of 76% of nominal was obtained. A significant decline in the measured test concentration was observed in the test preparations at 72 hours in the range of 17% to 27% of nominal.

Analysis of additional samples incubated along side the test which contained no algal cells showed measured concentrations at 72 hours to range from 71% to 84% of the nominal indicating that the losses observed were primarily attributed to adsorption of the test item to the algal cells present. As a result, geometric means were used to determine the result of the test.

In conclsuion, the effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and based on the geometric mean measured test concentrations gave EC50 values of greater than 80 mg/L.  The No Effect Concentration was 80 mg/L.

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and based on the geometric mean measured test concentrations gave EC50 values of greater than 80 mg/L.  The No Effect Concentration was 80 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

80 mg/L