4-bromo-3,3,4,4-tetrafluorobut-1-ene

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 01 2021 to June 21 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-bromo-3,3,4,4-tetrafluorobut-1-ene

EC Number:

242-440-8

EC Name:

4-bromo-3,3,4,4-tetrafluorobut-1-ene

Cas Number:

18599-22-9

Molecular formula:

C4H3BrF4

IUPAC Name:

4-bromo-3,3,4,4-tetrafluorobut-1-ene

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the test substance. Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to distribution into the test chambers. At exposure termination, samples were collected from the pooled remaining replicates from each treatment and control group. At each sampling interval, 10.0 mL samples of test solution were collected in VOA vials in duplicate with one set of samples processed immediately for analysis and the other set of samples were stored under refrigerated conditions for potential future analysis. In addition, 5.0 mL of test solution was sampled into 4 ounce glass vessels containing 95 mL of DMSO. The day 0 samples were collected in the incorrect vials and could not be analyzed as the size of the vial was incompatible with the instrument. Samples could not be transferred without loss of the volatilized test material within the vials. The additional samples collected in DMSO were used for verification of exposure concentration on day 0.

Test solutions

Vehicle:

no

Details on test solutions:

Test solutions were prepared at target nominal concentrations 5.3, 11, 21, 43, and 85 mg/L by direct addition of calculated weights of 4-Bromo-3,3,4,4-Tetrafluorobut-1-ene to freshwater AAP with additional sodium bicarbonate in 4 L glass aspirator bottles which were filled leaving no headspace. The required mass of test substance for each treatment group was weighed in tared 3 mL syringes fitted with an 18 gauge needle. The calculated amounts of test substance were not adjusted for test substance purity. The test solutions were stirred with a Teflon® lined stir bar on a magnetic stir plate for approximately 30 minutes. The test solutions appeared clear and colorless and were otherwise unremarkable at the time of preparation. The negative control solution contained freshwater AAP medium with additional sodium bicarbonate only

Test organisms

Test organisms (species):

other: Raphidocelis subcapitata

Details on test organisms:

The freshwater alga, Raphidocelis subcapitata, was selected as the test species for this study. The species is representative of an important group of freshwater algae and was selected for use in the test based upon a past history of use, and ease of culturing in the laboratory. Original algal cultures were obtained from the University of Texas at Austin and have been maintained in culture medium at Eurofins, Easton, Maryland since June 2017. Algal cells used in this test were obtained from Eurofins-Easton cultures that had been actively growing in culture medium under the same environmental conditions as used in this test for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh medium four days prior to test initiation.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 2°C

pH:

Refer to table 3 in field 'any other information on materials and methods in tables'

Nominal and measured concentrations:

Please refer to table 4 in field 'any other information on results, incl, tables'

Details on test conditions:

TEST SYSTEM
Test chambers were sterile, 300 mL glass bottles sealed with glass stoppers, and were completely filled, leaving minimal headspace. Each test chamber contained two glass marbles to promote mixing. Test chambers were labeled with the study number, test concentration and replicate, and were shaken continuously on mechanical shakers at approximately 100 rpm in an environmental chamber designed to maintain 24 ± 2°C throughout the exposure. Test chambers were indiscriminately positioned at test initiation, and indiscriminately re-positioned within the testing area after sampling each day during the 72-hour exposure. No Aeration.


- Initial cells density: 5,000 cells/mL
- Control end cells density: 558,549 cells/mL
- No. of vessels per concentration (replicates): 9
- No. of vessels per control (replicates): 18


GROWTH MEDIUM
- Standard medium used: yes, freshwater AAP medium.


TEST MEDIUM / WATER PARAMETERS
Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified well water (NANOpure® water). The test medium was then prepared by adding appropriate volumes of the stock nutrient solutions to NANOpure® water. Additional sodium bicarbonate was (35 mg/L, for a total concentration of 50 mg/L) added to the test medium to facilitate testing in a closed-system. The pH of the medium was adjusted to 7.5 ± 0.1 with 10% hydrochloric acid, and the medium was sterilized by filtration (0.22 µm) prior to use.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no (during actual test period)
- Photoperiod: continuous throughout test
- Light intensity and quality: 5559 to 6475 lux.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter, every 24 hours. See more details in field 'any other information on materials and methods, incl. tables'


TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately x2
- Range finding study: nominal concentrations of 0.85, 8.5, and 85 mg/L
- Test concentrations: Nominal 5.3, 11, 21, 43, and 85 mg/L and geometric mean measured 1.9, 4.3, 10, 21, and 71 mg/L


CULTURING APPARATUS
Test chambers were sterile, 300 mL glass bottles sealed with glass stoppers, and were completely filled, leaving minimal headspace. Each test chamber contained two glass marbles to promote mixing. Test chambers were labeled with the study number, test concentration and replicate, and were shaken continuously on mechanical shakers at approximately 100 rpm in an environmental chamber designed to maintain 24 ± 2°C throughout the exposure. Test chambers were indiscriminately positioned at test initiation, and indiscriminately re-positioned within the testing area after sampling each day during the 72-hour exposure.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% CI - 7.2 to 12 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CI - 7.2 to 12 mg/L

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% CI - 9.7 to 15 mg/L

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CI - 9.7 to 15 mg/L

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI - 10 to 48 mg/L

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% CI - 17 to 22 mg/L

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CI - 16 to 22 mg/L

Effect concentrations 11

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Cell density, Yield and Growth rate

Details on results:

For details on cell density, growth rate and yield, refer to tables 5 and 6 in field 'any other information on results incl. tables'

Reported statistics and error estimates:

NOEC values were based on statistical comparisons (Dunnett’s test; p ≤ 0.05) between treatment and negative control data.
The 72 hour yield and growth rate data were evaluated for normality and homogeneity of variance (α = 0.01) using Shapiro-Wilk’s and Levene’s tests, respectively. All data met assumptions of normality and homogeneity of variance.
The calculation of cell densities, yield, growth rates and percent inhibition values, as well as all statistical analyses, were conducted using “The SAS System for Windows, Version 9.4” .

Refer to document "Growth and Conc-Effect curves" in field 'any other information on results incl. tables" for growth curves and concentration-effect relationship.

Any other information on results incl. tables

Table 4

Measured Concentrations of 4-bromo-3,3,4,4-tetrafluorobut-1-ene in Test Solution Samples

Nominal Test Concentration (mg/L)		Sample Number (783P-106-)	Sampling Time (Day)	Measured Concentration (mg/L)^1,2	Percent of Nominal^2	Mean Measured Concentration (mg/L)^2	Mean Measured Percent of Nominal^2
Negative		1C	0	< LOQ	--	< LOQ	--
Control		7	3	< LOQ	--
5.3		2C	0	2.30	43.4	1.9	36
		8C	3	1.53	28.9
11		3C	0	4.96	45.1	4.3	39
		9C	3	3.65	33.2
21		4C	0	13.2	63.1	10	48
		10C	3	8.32	39.6
43		5C	0	29.2	67.8	21	49
		11C	3	15.5	36.0
85		6C	0	87.2	103	71	84
		12C	3	58.0	68.2
	^1   The limit of quantitation (LOQ) of 4-bromo-3,3,4,4-tetrafluorobut-1-ene (BTFB) in freshwater AAP with NaHCO3 is set at 0.0100 mg/L, defined as the lowest nominal concentration of matrix fortification samples with a mean recovery within 70-110% ^2   Results were generated using Excel 2010 in full precision mode.  Manual calculations may differ slightly.

 

Table 5

Cell Density and Yield by Replicate Over the 72-Hour Exposure Period

Geometric Mean, Measured Test Concentration (mg /L)		Replicate		Cell Density ^1 (cells/mL)						Yield (cells/mL)^2
				24 Hours		48 Hours		72 Hours		72 Hours
Negative Control	A		39,618		163,231		607,937		602,937
(0.0)	B		36,890		156,713		589,186		584,186
	C		39,352		136,114		470,823		465,823
	D		42,115		160,769		484,308		479,308
	E		41,796		124,820		570,152		565,152
	F		60,479		145,838		628,885		623,885
	Mean		43,375		147,914		558,549		553,549
	S.D.		8,590		15,193		65,829		65,829
1.9	A		26,617		134,697		638,284		633,284
	B		29,286		161,567		520,133		515,133
	C		29,129		144,162		468,273		463,273
	Mean		28,344		146,809		542,230		537,230
	S.D.		1,498		13,629		87,133		87,133
4.3	A		29,191		147,745		564,673		559,673
	B		31,358		138,576		563,374		558,374
	C		32,988		148,625		517,961		512,961
	Mean		31,179		144,982		548,669		543,669
	S.D.		1,905		5,565		26,602		26,602
10	A		25,867		127,996		396,598		391,598
	B		25,218		106,476		364,551		359,551
	C		25,628		110,196		432,589		427,589
	Mean		25,571		114,889		397,913		392,913
	S.D.		328		11,502		34,038		34,038
21	A		23,860		82,081		238,844		233,844
	B		24,008		95,578		265,931		260,931
	C		25,020		98,045		282,108		277,108
	Mean		24,296		91,901		262,294		257,294
	S.D.		631		8,594		21,860		21,860
71	A		8,675		5,843		0		0
	B		8,256		7,006		0		0
	C		7,760		5,359		1,574		0
	Mean		8,230		6,069		525		0
	S.D.		458		847		909		0
^1The initial cell density of the stock culture was determined and an inoculum volume was administered to each test chamber to yield a cell density of approximately 10,000 cells/mL at test initiation (0 hours). ^2   Yield was calculated as the final biomass (cell density) in the exposure period minus the initial biomass (cell density).

Table 6 

Growth rate by replicate over the 72-hr exposure period

Geometric Mean, Measured Test Concentration (mg/L)	Replicate	Growth Rate (per hour)
		0 - 24 Hours	0 – 48 Hours	0 – 72 Hours
Negative Control	A	0.0862	0.0726	0.0667
(0.0)	B	0.0833	0.0718	0.0662
	C	0.0860	0.0688	0.0631
	D	0.0888	0.0723	0.0635
	E	0.0885	0.0670	0.0658
	F	0.1039	0.0703	0.0671
	Mean	0.0894	0.0705	0.0654
	S.D.	0.0073	0.0022	0.0017
1.9	A	0.0697	0.0686	0.0674
	B	0.0737	0.0724	0.0645
	C	0.0734	0.0700	0.0631
	Mean	0.0723	0.0704	0.0650
	S.D.	0.0022	0.0019	0.0022
4.3	A	0.0735	0.0705	0.0657
	B	0.0765	0.0692	0.0656
	C	0.0786	0.0707	0.0645
	Mean	0.0762	0.0701	0.0652
	S.D.	0.0026	0.0008	0.0007
10	A	0.0685	0.0676	0.0607
	B	0.0674	0.0637	0.0596
	C	0.0681	0.0644	0.0619
	Mean	0.0680	0.0652	0.0608
	S.D.	0.0005	0.0020	0.0012
21	A	0.0651	0.0583	0.0537
	B	0.0654	0.0615	0.0552
	C	0.0671	0.0620	0.0560
	Mean	0.0659	0.0606	0.0550
	S.D.	0.0011	0.0020	0.0012
71	A	0.0230	0.0032	0.0000
	B	0.0209	0.0070	0.0000
	C	0.0183	0.0014	0.0000
	Mean	0.0207	0.0039	0.0000
	S.D.	0.0023	0.0028	0.0000

 

Table 7

Section-by-Section Growth Rate for Negative Control

Control Replicate	0-24 Hour Growth Rate	24-48 Hour Growth Rate	48-72 Hour  Growth Rate	% Coefficient of Variation^1
A	0.0862	0.0590	0.0548	25.6
B	0.0833	0.0603	0.0552	22.6
C	0.0860	0.0517	0.0517	31.3
D	0.0888	0.0558	0.0459	35.3
E	0.0885	0.0456	0.0633	32.8
F	0.1039	0.0367	0.0609	50.7
			Mean:	33.1
^1Coefficient of Variation  = S.D./mean *100 Results were generated using Excel 2010 in the full precision mode.  Manual calculations may differ slightly.

 

Table 8

Average Specific Growth Rate for the Negative Control

Control Replicate	0-72 Hour Growth Rate
A	0.0667
B	0.0662
C	0.0631
D	0.0635
E	0.0658
F	0.0671
Mean= Standard Deviation= % COV=	0.0654 0.0017 2.6

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Mean cell density in the negative control flasks increased by >16x. CoV of average specific growth rate was <7%. Mean %CoV for section-by-section specific growth rate was <35%. Refer to tables 5, 7 and 8 in 'any other information on results incl. tables'

Conclusions:

The freshwater alga, Raphidocelis subcapitata, was exposed to a geometric series of five treatment levels of 4-bromo-3,3,4,4-tetrafluorobut-1-ene ranging from 5.3 to 85 mg/L, based on nominal (1.9 to 71 mg/L, based on geometric mean), concentrations of 4-bromo-3,3,4,4-tetrafluorobut-1-ene. All control validity criteria were achieved. The measured concentrations of 4-bromo-3,3,4,4-tetrafluorobut-1-ene on Day 0 in the 5.3, 11, 21, 43, 85 mg/L treatment groups were 43.4, 45.1, 63.1, 67.8, and 103% of nominal, respectively. The measured concentrations of 4-bromo-3,3,4,4-tetrafluorobut-1-ene on Day 3 in the 5.3, 11, 21, 43, 85 mg/L treatment groups were 28.9, 33.2, 39.6, 36.0, and 68.2% of nominal, respectively. Toxicity of 4-bromo-3,3,4,4-tetrafluorobut-1-ene to R. subcapitata was assessed based on effects on cell density, growth rate, and yield relative to the negative control group. Because the test substance is volatile, rigorous precautions were taken to minimize losses through evaporation during the tests and collection and analysis of samples to avoid losses through volatilization. Despite the special handling and precautions, the measured concentrations resulted in concentrations well below the nominal concentrations, therefore nominal concentrations are more representative of exposure to the test system. The 72-hour measured EC50 values for cell density, growth rate, and yield were determined to be 19, 25, and 19 mg/L, respectively. The 72-hour nominal EC50 and EC10 value for growth rate were 53 and 18 mg/L, respectively. The 72-hour nominal NOEC was determined to be 11 mg/L.