2,2,3,3,5,5,6,6-octafluoro-4-(trifluoromethyl)morpholine

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

Study period:

27 November 2012 - 30 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted under GLP.

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:

yes

Remarks:

Test substance maintained in test chambers well in excess of water solubility to ensure that the concentration remained at saturation during the entire 72-hour exposure period. Test substance is very volatile and very difficult to keep in water.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Perfluoromethylmorpholine (PMM)

IUPAC Name:

Perfluoromethylmorpholine (PMM)

Details on test material:

- Name of test material (as cited in study report): FC-3284
- Physical state: liquid
- Storage condition of test material: Stored at room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.1 mL
- Sampling method: The aliquot volume for all samples analysed was 0.1 mL, which was removed using a disposable 1.0 mL syringe inserted through the septum seal of the vial cap. The aliquot was then transferred to a capped septum sealed vial containing 10 mL of Milli Q water for analysis.
- Sample storage conditions before analysis: At test termination, all test chambers were collected without opening the vial and were shipped overnight for analysis. Samples were recieved December 1, 2012. Upon receipt of the samples, they were centrifuged at 1500 rpm for 90 minutes. After centrifugation, the samples were allowed to sit at ambient laboratory conditions for over 24 hours before analysis.

Test solutions

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions were prepared individually for each replicate in 40mL glass vials. Each vial had been filled with ~40 mL dilution medium. A 25-uL aliquot of the test substance was measured with a gas-tight glass microliter syringe and injected into the 40 mL dilution medium. The vials were then brought to volume with dilution medium until a positive meniscus was present and then sealed with bonded septa caps and secured with plastic stretchable tape. The vials were placed on a orbital shaker table (100 rev/min for 138 hours at 30 °C). After incubation, the sealed vials were centrifuged for 90 min at 1600 rpm to condense the unsolubilized test substance into a single droplet. Vials were inoculated by unsealing test vials, removing a 1.0 mL aliquot and adding a 1.0 mL of the appropriate inoculum, and resealing immediately to limit the loss of the test substance.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc): A clear ball of test substance was observed on the bottom of each replicate chamber.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Not Reported
- Source (laboratory, culture collection): University of Texas at Austin (UTEX)
- Age of inoculum (at test initiation): 4 days old
- Method of cultivation: The prepared cultures were maintained in a temperature-controlled environmental chamber under continuous light. Periodically, new cultures were cloned from an exisitng culture derived from the parent stock.

ACCLIMATION
-Acclimation period: None
-Culturing media and conditions (same as test or not): cultures maintained under the same conditions as those used for testing.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

n.a.

Test temperature:

22.3 - 24.7 °C

pH:

7.6 - 8.8

Nominal and measured concentrations:

Nominal limit concentration (loading rate): 25 µL/44 mL (971 mg/L)
Measured concentration: 0.583 mg/L

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: 40 mL VOA all-glass vial, closed with a bonded septa cap & secured with plastic stretchable tape
- Agitation: Yes, during incubation the algal cells were kept in suspension by continuous shaking on an orbital shaker at 100 rpm.
- Control end cells density: 62.5E+04 cells/mL
- No. of colonies per vessel: Initial cell density was 5.0E+03 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
1 replicate vessel containing just the test material with no algae and was subjected to test conditions
1 replicate vessel containing no algae or test material and was subjected to test conditions
3 replicate vessels containing just test material with no algae, was not subjected to test conditions but refrigerated
1 replicate vessel containing no algae, no test material and was not subjected to test conditions but refrigerated

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse-osmisis water is passed through a series of deionization tanks, a laboratory water purification system consisting of carbon, de-mineralization, and organic adsorption cartridges, and then through a 0.2 µm filter.

- Water quality parameters: all pesticides, PCBs and organophosphate concentrations were below method detection limit. Element and nitrate concentrations were either below method detection limit or within the laboratory historical range (1998 - 2012).

- Culture medium different from test medium: Yes. Test medium was supplemented with approximately 500 mg of NaHCO3/L to minimize pH shift during the exposure period.

- Intervals of water quality measurement: Interval not provided. Most recent report dated February 2012

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous light with a cool-white fluorescent bulb that had a light intensity ranging from 8,318 - 8,283 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Other: Replicates were sampled for cell density determination using a gas-tight microliter syringe and piercing the septa cap. Minimal volume of ~10 µL was removed at each sampling event (24, 48, & 72 hours, ± 1 hour). Cell densities were determined using a hemacytometer and an optical microscope. In addition, microscopic examination was conducted to evaluate for any morphological or physical effects on algal cells.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.583 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 971 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.583 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

971 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Exponential growth in the control: Yes (Figure 1)
- Growth in controls (validity criteria):
- cell density increase through 72-hours: factor of >16
- % coefficient of variance in section-specific growth rates to hour 72: 4% (Table 1)
- % coefficient of variance in average specific growth rate at hour 72: 0% (Table 3)

Any visual signs of phytotoxicity (abnormalities): Observation was done at 24, 48, and 72 hours (± 1 hour) and no abnormalities were noted in the report.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test substance is very volatile and very difficult to keep in water. Test substance maintained in test chambers well in excess of water solubility. This was done to ensure that the concentration remained at saturation during the entire 72-hour exposure period.
- Effect concentrations exceeding solubility of substance in test medium: yes

Reported statistics and error estimates:

All statistical analyses were performed with SAS software. The NOEC values, based on growth rate from time zero and yield, were estimated using a oneway analysis of variance (ANOVA) procedure and a one-tailed Dunnett’s test (p = 0.05) where the alternate hypothesis was the mean for the growth parameter was reduced in comparison to the control. Prior to the Dunnett’s test, a Shapiro-Wilk’s test and a Levene’s test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. If the results from the Shapiro-Wilk’s and Levene’s tests indicated normality and insignificant heterogeneity (i.e., p > 0.01), the analysis was performed on the non-transformed raw data. In instances of non-normality or heterogeneity (i.e., p < 0.01), a square root transformation was performed. If both the non-transformed raw data and the transformed data exhibited non-normality or inequality of variance, a non-parametric analysis of variance was performed on the ranks of the raw data values. A parametric analysis was performed on all specific growth rate from time zero data and all yield data.

The specific growth rate in each treatment was calculated for each period, i.e., 0 to 24, 0 to 48, and 0 to 72 hours. Yield in each treatment was calculated for each period, i.e., 24, 48, and 72 hours.

The ErC50 and EyC50 values were estimated to be greater than the FC-3284 treatment level based on the lack of statistically significant reduction in growth rate from time zero or yield compared to the control treatment.

Any other information on results incl. tables

Table 1: Cell Density and Growth Rate Reductions
Measured concentration	Replicate	Individual Cell Densities, (cells/mL x 10^4)			Growth Rate (µ)	Growth Rate Reduction (%)
		24∙h	48∙h	72∙h	0 – 72∙h	0 – 72∙h
Control	A	2.33	12.8	63.0	0.0672	---
	B	2.44	11.5	61.3	0.0668
	C	2.67	12.3	61.8	0.0669
	D	2.44	11.5	63.0	0.0672
	E	2.33	11.8	62.8	0.0671
	F	2.44	11.8	63.0	0.0672
	Mean				0.0671
					0 %CV
FC-3284 0.583 mg/L	A	2.44	11.8	64.3	0.0675	-1
	B	2.44	12.3	60.8	0.0667
	C	2.56	12.0	64.0	0.0674
	D	2.33	13.0	72.5	0.0691
	E	2.22	12.5	65.8	0.0678
	F	2.44	12.3	66.5	0.0679
	Mean				0.0677
					1 %CV

Table 2: Cell Yields and % inhibition
Measured concentration	Replicate	Daily Cell Yields, (cells/mL x 10^4)			% CV	Inhibition (%)
		24∙h	48∙h	72∙h	72∙h	0 – 72∙h
Control	A	1.83	12.3	62.5	1	---
	B	1.94	11.0	60.8
	C	2.17	11.8	61.3
	D	1.94	11.0	62.5
	E	1.83	11.3	62.3
	F	1.94	11.3	62.5
	Mean	1.94	11.5	62.0
FC-3284 0.583 mg/L	A	1.94	11.3	63.8	6	-5
	B	1.94	11.8	60.3
	C	2.06	11.5	63.5
	D	1.83	12.5	72.0
	E	1.72	12.0	65.3
	F	1.94	11.8	66.0
	Mean	1.91	11.8	65.2

Table 3: Section-by-section Growth Rates
	Replicate	Sec-by-Sec specific growth rate (cells/mL/hour)			% Coefficient of Variance
		0-24 h	24-48 h	48-72 h	Replicate	Overall
Control	A	0.0641	0.0710	0.0664	5	4
	B	0.0660	0.0646	0.0697	4
	C	0.0698	0.0636	0.0673	5
	D	0.0660	0.0646	0.0709	5
	E	0.0641	0.0676	0.0697	4
	F	0.0660	0.0657	0.0698	3

Table 4, Measured Concentrations of FC-3284 in Test Samples
Samples	Underwent test conditions (Y/N)	Sample replicate	Sampling volume (mL)	Measured Concentration (ng/mL)
FC-3284 971 mg/L (Loading rate)	Y	A	0.1	591
	Y	A, duplicate	0.1	602
	Y	A, LMS	0.1	106% Recovery
			Average	596
			%RPD	2.0
	Y	B	0.1	793
	Y	C	0.1	511
	Y	D	0.1	471
	Y	E	0.1	485
	Y	F	0.1	641
			Average Total (A-F)	583
			%RSD	21
Control – FC-3284, no algae	Y	G	0.1	552
	Y	G, duplicate	0.1	452
	Y	G, LMS	0.1	82.4 % recovery
			Average	502
			%RPD	20
	N	H	0.1	330
	N	I	0.1	556
	N	J	0.1	257
			Average (B-E)	381
			%RSD	41
Negative Control – Algae, no FC-3284	Y	A	0.1	<LOQ
	Y	B	0.1	<LOQ
	Y	C	0.1	<LOQ
	Y	D	0.1	<LOQ
	Y	E	0.1	<LOQ
	Y	F	0.1	<LOQ
			Average (A-F)	<LOQ
			%RSD	NA
Negative Control – no FC-3284 or algae	N	G	0.1	<LOQ
	Y	H	0.1	<LOQ
Laboratory Control Spikes	---	Low¹ 1	0.1	101% recovery
	---	Low¹ 2	0.1	114% recovery
	---	High² 1	0.1	121% recovery
	---	High² 2	0.1	117% recovery
			Average	113
			%RSD	7.8
LOQ: Limit of Quantitation = 13.5 ng/mL 1) 6.74 ng FC-3284 spike 2) 64 ng FC-3284 spike

Table 5. Effective concentrations and nominal concentrations of FC-3284
Inhibition Parameter	Nominal loading rate (mg/L)	Measured Concentrations (mg/L)
NOEC (growth rate)	971	0.583
72h EC50 (growth rate)		> 0.583
NOEC (yield)		0.583
72h EC50 (yield)		> 0.583

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Cell density increased within 72 hours by average factor of >16. Control cultures mean coefficient of variation section-by-section specific growth rates < 35%. Control cultures coefficient of variation of specific growth rates during whole test < 7%.

Conclusions:

The 72-hour EC50 of FC-3284 to Pseudokirchneriella subcapitata is greater than 0.583 mg/L. The 72-hour NOEC of FC-3284 to Pseudokirchneriella subcapitata is 0.583 mg/L.

Executive summary:

Using a loading rate of 971 mg/L for FC-3284, no statistically significant reduction in yield were observed after 72 hours of exposure. The test substance was maintained in closed test chambers well in excess of water solubility. This was done to ensure that the concentration remained at saturation during the entire 72-hour exposure period. This test substance is very volatile and very difficult to keep in water. The 72-hour EC50 of FC-3284 to Pseudokirchneriella subcapitata was greater than 0.583 mg/L based on the effect parameter of growth rate inhibition.

The study was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice (GLP) standards. This study is reliable without restrictions and the results are suitable for purposes of Risk Assessment, Classification & Labeling, and PBT Analysis.

Results Synopsis

Test Type: Static

72-hr EC50 > 0.583 mg/L

72-hr NOEC = 0.583 mg/L

The EC50 and NOEC result corresponds to a loading rate of 971 mg/L of FC-3284.