Sodium chlorodifluoroacetate

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:

From 8-10-2022 to 14-10-2022

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

Details on sampling:

- Concentrations: 87.79, 131.69, 197.53, 296.30, 444.44, 666.67 and 1000.0 mg/L

Vehicle:

yes

Remarks:

OECD alga medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution was prepared by weighing test item in a beaker; OECD alga medium was added and stirredAND transferred to measuring cylinder, beaker was rinsed with OECD alga medium and transferred again to measuring cylinder. Rinsing process was repeated until the complete transfer of test contents.
The concentrations were prepared by adding stock solutions and made up to required volume using OECD alga medium. A homogeneous distributionwas achieved by stirring using magnetic stirrer shortly before preparation of the test vessels.
The test medium was prepared in the sterile OECD alga medium by adding exponentially growing culture of Raphidocelis subcapitata suspension.

- Controls: For the evaluation of the quality of the Raphidocelis subcapitata and the experimental conditions, 3, 5-Dichlorophenol 97% is tested at least twice a year to demonstrate satisfactory test conditions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection

ACCLIMATION
- Culturing media and conditions (same as test or not): The algal cell density of pre-cultures during dose range finding study and main study was 48750 and 49800 cells/mL at start.
- Any deformed or abnormal cells observed: Not reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None.

Test temperature:

22.3ºC-22.9ºC

pH:

8.10-8.33

Nominal and measured concentrations:

87.79, 131.69, 197.53, 296.30, 444.44, 666.67 and 1000.0 mg/L

Details on test conditions:

- Test vessel: erlenmeyer flask
- Material, size, headspace, fill volume: glass, 250 mL capacity, 100 mL fill volume.
- Aeration: Not specified.
- Initial cells density: The algal cell density of pre-cultures during dose range finding study and main study was 48750 and 49800 cells/mL at start
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable
GROWTH MEDIUM
- Standard medium used: yes (OECD alga medium)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the OECD medium was prepared as per the OECD guideline 201.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The temperature and light intensity was monitored once in a day during pre-culture and exposure. The pH of the test nominal loading rates was measured at the beginning (0 h) and at the end (72 h) of the exposure.
OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: 5218 to 5826 lux was maintained with a universal white type fluorescent lamp
EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: counting chamber (observation times: 24, 48 and 72 hours): Samples collected were loaded on a haemocytometer and cells were counted manually using microscope. Cell density was obtained by calculating the average cell count (by summating the number of cells in all the four large corner squares and divide the sum by 4) and multiplied with chamber
conversion factor (CCF) and dilution factor: Cell Density (cells/mL) = Average cell count × 10000(CCF) × Dilution factor.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor of 2.1
- Justification for using less concentrations than requested by guideline: Not applicable; 7 concentrations used for the main test.
- Range finding study
- Test concentrations: 0 (control), 0.00897, 0.0895, 0.897, 9.1, 44.9 and 89.7 mg/L.
- Results used to determine the conditions for the definitive study: At the tested concentrations of 0.00897, 0.0895, 0.897, 9.1, 44.9 and 89.7 mg/L, the inhibition growth rate was calculated to be 1.1, 2.8, 5.2, 35.2, 100 and 100% respectively.

Reference substance (positive control):

yes

Remarks:

3, 5-Dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

137.26 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

84.36 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

365.525 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

289.84 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

241.88 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

131.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

87.79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

381.179 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

289.84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

246.925 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

137.26 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

84.36 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

712.76 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

432.06 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

322.39 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

131.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

87.79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

664.554 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

432.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

336.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Not reported.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the 72 hours growth rate inhibition and percent inhibition in yield lie within the validity criteria acceptance range and establish the acceptability of test system response and test procedures followed.

Reported statistics and error estimates:

The 72 hours EC10, EC20 and EC50 values were calculated for growth rate and yield using the SigmaPlot, version 14 software. Regression curve was drawn for assessed parameters by plotting the percent inhibition values against the corresponding log concentration.

Table 1: Mean cell density after exposure during range finding study 

Group	Test Conc.  (mg/L)	Replicate	Mean Cell Density (10^4Cells/mL)
			Observation Periods
			24 hour	48 hour	72 hour
G1	0.0	1	4.50	33.75	103.40
		2	4.50	34.75	103.95
		3	4.50	35.00	102.58
		4	4.75	35.25	105.33
		5	5.00	32.50	108.90
		6	4.50	33.75	103.40
G2	0.01	1	4.25	34.50	106.70
		2	4.50	31.75	105.05
		3	4.50	33.00	103.13
G3	0.1	1	4.75	32.25	104.50
		2	4.50	32.75	108.63
		3	4.50	34.25	103.40
G4	1.0	1	4.50	32.25	108.08
		2	4.75	32.00	106.15
		3	4.50	34.75	108.08
G5	10.0	1	5.00	33.50	107.25
		2	4.50	34.00	103.13
		3	5.25	32.00	99.28
G6	50.0	1	5.00	34.75	103.68
		2	4.50	35.50	102.85
		3	5.25	32.00	102.85
G7	100.0	1	4.25	36.25	104.78
		2	4.25	34.25	98.73
		3	4.50	34.50	99.00

Table 2. Percent inhibition in cell density, growth rate and yield during range finding study 

Group	Test Conc. (mg/L)	% Inhibition in Yield	% Inhibition in Cell Density	% Inhibition in Growth Rate
G1	0.0	-	-	-
G2	0.01	-0.35	-0.35	-0.07
G3	0.1	-0.88	-0.88	-0.17
G4	1.0	-2.82	-2.80	-0.53
G5	10.0	1.32	1.31	0.26
G6	50.0	1.41	1.40	0.27
G7	100.0	3.61	3.59	0.71

Table 3. Mean cell density after exposure during main study 

Group	Test Conc.  (mg/L)	Replicate	Mean Cell Density (10^4Cells/mL)
			Observation Periods
			24 hours	48 hours	72 hours
G1	0.0	1	5.00	32.25	105.33
		2	4.75	33.00	103.95
		3	5.00	34.75	106.43
		4	5.00	32.75	105.33
		5	5.00	32.50	108.90
		6	4.75	33.25	103.40
G2	87.79	1	4.75	34.50	103.68
		2	4.75	31.75	101.75
		3	5.00	33.0	104.23
G3	131.69	1	4.50	32.25	97.90
		2	5.00	32.75	101.75
		3	5.00	34.25	102.03
G4	197.53	1	4.75	29.00	94.60
		2	5.00	30.50	92.13
		3	4.50	30.25	94.05
G5	296.30	1	4.00	33.50	78.65
		2	4.50	34.00	81.40
		3	4.50	32.00	82.23
G6	444.44	1	3.50	21.50	37.90
		2	3.25	24.25	32.18
		3	3.00	22.00	31.08
G7	666.67	1	2.25	9.00	7.70
		2	2.75	8.75	7.43
		3	2.75	9.25	7.98
G8	1000.0	1	1.25	1.50	1.65
		2	1.25	2.00	1.38
		3	1.50	1.75	1.93

Table 4. Average specific growth rate during main study 

Group	Test Conc. (mg/L)	Replicate	Growth Rate Observation intervals (Days)				Mean Growth Rate
G1	0.0	1	2.16	1.86	1.18	1.73	1.74
		2	2.10	1.94	1.15	1.73
		3	2.16	1.94	1.12	1.74
		4	2.16	1.88	1.17	1.73
		5	2.16	1.87	1.21	1.75
		6	2.10	1.95	1.13	1.73
		Mean	2.14	1.91	1.16	1.74
		±SD	0.03	0.04	0.03	0.01
		CV (%)	1.24	2.01	2.86	0.36
G2	87.79	1	2.10	1.98	1.10	1.73	1.72*
		2	2.10	1.90	1.16	1.72
		3	2.10	1.89	1.15	1.73
		Mean	2.12	1.92	1.14	1.73
		±SD	0.03	0.05	0.03	0.00
		CV (%)	1.40	2.70	2.96	0.24
G3	131.69	1	2.05	1.97	1.11	1.71	1.72*
		2	2.16	1.88	1.13	1.72
		3	2.16	1.92	1.09	1.72
		Mean	2.12	1.92	1.11	1.72
		±SD	0.06	0.04	0.02	0.01
		CV (%)	2.87	2.34	1.90	0.45
G4	197.53	1	2.10	1.81	1.18	1.70	1.70*
		2	2.16	1.81	1.11	1.69
		3	2.05	1.91	1.13	1.70
		Mean	2.10	1.84	1.14	1.70
		±SD	0.05	0.06	0.04	0.00
		CV (%)	2.50	0.03	3.41	0.27
G5	296.30	1	1.93	2.13	0.85	1.64	1.65*
		2	2.05	2.02	0.87	1.65
		3	2.05	1.96	0.94	1.65
		Mean	2.01	2.04	0.89	1.65
		±SD	0.07	0.08	0.05	0.01
		CV (%)	3.38	4.06	5.33	0.47
G6	444.44	1	1.80	1.82	0.39	1.34	1.33*
		2	1.72	2.01	0.28	1.34
		3	1.64	1.99	0.35	1.33
		Mean	1.72	1.94	0.34	1.33
		±SD	0.08	0.11	0.06	0.01
		CV (%)	4.48	5.55	16.44	0.45
G7	666.67	1	1.36	1.39	-0.16	0.86	0.86*
		2	1.56	1.16	-0.16	0.85
		3	1.56	1.21	-0.15	0.87
		Mean	1.49	1.25	-0.16	0.86
		±SD	0.12	0.12	0.01	0.01
		CV (%)	7.77	9.53	-5.09	1.38
G8	1000.0	1	0.77	0.18	0.10	0.35	0.35*
		2	0.77	0.47	-0.37	0.29
		3	0.95	0.15	0.10	0.40
		Mean	0.93	0.27	-0.06	0.35
		±SD	0.11	0.17	0.27	0.06
		CV (%)	12.69	65.01	-442.19	16.23

Table 6. Percent inhibition in cell densiy, growth rate and yield during main study 

Group	Test Conc. (mg/L)	% Inhibition in Growth Rate	% Inhibition in Yield	% Inhibition in Cell Density
G1	0.0	-	-	-
G2	87.79	0.43	2.23	2.21
G3	131.69	0.93	4.76	4.73
G4	197.53	2.31	11.40	11.33
G5	296.30	5.15	23.62	23.49
G6	444.444	23.10	70.34	69.95
G7	666.67	50.30	93.22	92.71
G8	1000.00	80.06	98.98	98.44

Tabl 7. Acceptance criteria for control during main study 

0 – 72 Hours Mean Coefficient of Variation of Section-By-Section Average Specific Growth Rate
Daily average growth rate	±SD	% CV out of the daily growth rates
1.74	0.53	30.08
1.74	0.56	32.01
1.73	0.57	32.89
1.74	0.56	32.39
1.75	0.50	28.27
1.74	0.53	30.08
Mean % of CV 0 – 72 hours		31.19
% Coefficient of Variation of Specific Growth Rate		0.42

 

BIOMASS INCREASE DURING THE WHOLE TEST PERIOD
Initial biomass (cells/mL)	Cell density on day 3 (cells/mL)	Biomass increase Factor
5657	1034000	182.79
5657	1039500	183.76
5657	1025750	181.33
5657	1053250	186.19
5657	1089000	192.51
5657	1034000	182.79
Mean	1045916.67	184.90

Table 8. Temperature and light intensity during pre-culture and exposure main study 

Day	Temperature of the surrogate (ºC)	Light intensity (Lux)				Revolutions per minute (rpm)
		1	2	3	4
During Pre-culture & Exposure
0	22.3	5994	5825	5831	5925	100
1	22.4	5846	5726	5923	5845	100
2	22.4	5968	5993	5910	5901	100
3	22.7	5697	5939	5967	5994	100
4	22.9	5831	5866	5921	5871	100
5	22.4	5995	5814	5726	5859	100
6	22.7	5999	5962	5896	5971	100
Mean	22.5	5904	5875	5882	5909	100
±SD	0.2	116.2	94.9	79.9	56.9	0.0
Min	22.3	5697				-
Max	22.9	5999				-

 

Table 9. pH of the test medium during main study 

pH
		0 hours						72 hours
Group	Test Conc. (mg/L)	R1	R2	R3	R4	R5	R6	R1	R2	R3	R4	R5	R6
G1	0.00	8.18	8.10	8.11	8.18	8.16	8.14	8.21	8.13	8.14	8.21	8.24	8.19
G2	87.79	8.10	8.21	8.26	-	-	-	8.11	8.23	8.23	-	-	-
G3	131.69	8.22	8.25	8.23	-	-	-	8.31	8.36	8.31	-	-	-
G4	197.53	8.20	8.23	8.21	-	-	-	8.30	8.33	8.38	-	-	-
G5	296.30	8.10	8.26	8.29	-	-	-	8.34	8.31	8.34	-	-	-
G6	444.44	8.24	8.22	8.27	-	-	-	8.30	8.31	8.34	-	-	-
G7	666.67	8.23	8.26	8.29	-	-	-	8.33	8.35	8.31	-	-	-
G8	1000.00	8.33	8.30	8.32	-	-	-	8.39	8.38	8.31	-	-	-
	Minimum	8.10						8.11
	Maximum	8.33						8.41

Validity criteria fulfilled:

yes

Remarks:

(increase of biomass in control during 72h>16 fold, coefficient of variation of the mean specific growth rate among replicates in control (t0-t72)<7%; the mean of the replicate coefficients of variation in section-by-section growth rate in control < 35%)

Conclusions:

The toxicity of test item was tested on alga Raphidocelis subcapitata, the growth rate inhibition effect concentrations were determined to be 72 h-EC50=664.55 mg/L, 72 h-NOEC= 87.79 mg/L and 72 h-LOEC= 131.59 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Raphidocelis subcapitata on test item over a period of 72 h in static conditions according to OECD Guideline 201, following GLP.In a preliminary range finding study the average growth inhibition rates were 1.1, 2.8, 5.2, 35.2, 100 and 100% at the tested rates of0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, respectively. Based on these results, the main test was conducted at rates of 87.79, 131.69, 197.53, 296.30, 444.44, 666.67 and 1000.0 mg/L along with control group. The algal growth (cell
density) was assessed at 24, 48 and 72 hours post-exposure. Exponentially growing alga cells (5792 cells/mL) were exposed to the selected nominal loading rates in 3 replicates for the treatment groups and 6 replicates for the control group. Positive control consisted of test medium alone and a solution of 3, 5-Dichlorophenol was used as reference substance. A growth rate inhibition of -0.07, -0.17, -0.53, 0.26, 0.27, 0.71 % was observed at loading rates 0.01, 0.1, 1.0, 10.0. 50.0 and 100.0 mg/L respectively. Similarly, inhibition in yield of -0.35, -0.88, -2.82, 1.32, 1.41 and 3.61% was observed at the loading rates0.01, 0.1, 1.0, 10.0. 50.0 and 100.0 mg/L respectively respectively. All validity criteria were fulfilled. Based on these results, the growth rate inhibition effect concentrations were determined to be72 h-EC50=664.55 mg/L, 72 h-NOEC= 87.79 mg/L and 72 h-LOEC= 131.59 mg/L respectively based on growth rate.

Description of key information

Key study. Test method according to OECD 201, GLP study. The growth rate inhibition effect concentrations in Raphidocelis subcapitata were determined to be 72 h-EC50=664.55 mg/L, 72 h-NOEC= 87.79 mg/L and 72 h-LOEC= 131.59 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

664.55 mg/L

EC10 or NOEC for freshwater algae:

336.64 mg/L