Potassium hexafluorophosphate

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:

2016-05-31 to 2016-06-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council Regulation (EC) No. 761/2009/

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Potassium hexafluorophosphate

EC Number:

241-143-0

EC Name:

Potassium hexafluorophosphate

Cas Number:

17084-13-8

Molecular formula:

F6P.K

IUPAC Name:

potassium hexafluoro-λ⁵-phosphanuide

Test material form:

solid: crystalline

Details on test material:

- State of aggregation: solid
- Other: crystalline powder,
Colour: white
hygroscopic

Specific details on test material used for the study:

Appearance
White crystalline powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
nominal 100mg/l

- Sampling method:
The limit test concentration was diluted factor 10 with HPLC water prior to analysis. Before dilution the samples were centrifuged at 3000 upm for 5 minutes at 20°C. The control was used as blank solution.

- Sample storage conditions before analysis:
All original samples were stored at 6 ± 2 °C. Prepared samples were stored at room temperature until analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

stock solution of 100 mg/L was freshly prepared with dilution water.
Media preparation: direct addition of the test item and shaking until the test item was completely dissolved.
one limit concentration of 100 nng/L was tested. The limit concentration is based on the results of a preliminary range finding test

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name:
Pseudokirchneriella subcapitata HINDÁK,
- Strain:
SAG 61.81

- Source (laboratory, culture collection):
Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen

- Age of inoculum (at test initiation):
A four day old preculture, prepared in dilution water, was used as inoculum.

- Method of cultivation:
Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE •m-2 •s-1 for 24 hours per day.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
Culture medium: Nutrient medium Z according to LÜTTGE etal. (1994).
Test medium: OECD medium

- Any deformed or abnormal cells observed:
not reported

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

no

Test conditions

Hardness:

0.24 mmol Ca+Mg/L (OECD medium)

Test temperature:

22-23 °C, mean 22.5 °C

pH:

100mg/L test item conc.
0h: 8.13; 72h: 9.68

Control: 0h: 8.22; 72h: 9.44

Dissolved oxygen:

not reported

Salinity:

freshwater was used

Conductivity:

not reported

Nominal and measured concentrations:

nominal: 100mg/L
measured: 0h: 82.8mg/L, 72h: 105mg/L (mean of 3 replicates each)

Details on test conditions:

TEST SYSTEM
- Test vessel:
sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs

- Type (delete if not applicable): open

- Material, size, headspace, fill volume:
glas, 100mgl test volume

- Aeration:
no
- Type of flow-through (e.g. peristaltic or proportional diluter):
static

- Renewal rate of test solution (frequency/flow rate):
static

- Initial cells density:
6305/ml (mean)

- Control end cells density:
start: 6305/ml (mean) end 2323516

- No. of organisms per vessel:
-

- No. of vessels per concentration (replicates):
6

- No. of vessels per control (replicates):
6

- No. of vessels per vehicle control (replicates):
no vehicle

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
OECD medium

- Culture medium different from test medium:
Culture medium: Nutrient medium Z according to LÜTTGE etal. (1994).

- Intervals of water quality measurement:
at least start and end of test

OTHER TEST CONDITIONS

- Adjustment of pH:
no (with OECD medium

- Photoperiod:
24h/day light

- Light intensity and quality:
Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1
fluorescent tubes, OSRAM L 36 W/865, cool daylight


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter

- Chlorophyll measurement:
cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm.
Dilution water was used as background signal. No self fluorescence was found in the range finding test at the concentration of 100 mg/L.

- Other:
Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
limit test 100mg/L

- Justification for using less concentrations than requested by guideline:
limit test due to effect <50% inhibition at range finding test

- Range finding study
yes

- Test concentrations:
1, 10, 100 mg/L

- Results used to determine the conditions for the definitive study:
nominal conc. 1 / 10 / 100 mg/L
growth rate inhibition: -2 / 1 / 9 %

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: nominal concentration analytically confirmed

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

the effect of 3% inhibition or growth rate is statistically significant but is considered to be not biologcically significant

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

nominal concentration analytically confirmed

Details on results:

- Exponential growth in the control (for algal test): yes
increase of cell growth: 369 fold (specific growth rate 1.97 day-1

- Observation of abnormalities (for algal test):
no (microscopic observation at start and end)

- Unusual cell shape:
no morphological abnormalities.

- Colour differences:
no

- Flocculation:
no

- Adherence to test vessels:
no

- Aggregation of algal cells:
no
- Other:
agglutination of algae cells: no

- Any stimulation of growth found in any treatment:
no

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
no

- Effect concentrations exceeding solubility of substance in test medium:
no

Results with reference substance (positive control):

- Results with reference substance valid?
yes
(potassium dichromate)
- EC50:
72h-ErC50: 0.460 mg/L (valid range (average ± 3 x SD): 0.780 ± 0.473 mg/L
- Other:

Reported statistics and error estimates:

Statistical Significance (NOEC/LOEC) - Growth Rate

t-test

Normality Test (Shapiro-Wilk) Passed (P = 0.319)
Equal Variance Test: Passed (P = 0.513)

Group Name N Missing Mean Std Dev SEM
Control 6 0 1.967 0.0507 0.0207
100 mg/L 6 0 1.900 0.0396 0.0161

Difference 0.0663

t = 2.527 with 10 degrees of freedom. (P = 0.030)
95 percent confidence interval for difference of means: 0.00784 to 0.125
The difference in the mean values of the two groups is greater than would be expected by chance; there is a statistically significant difference between the input groups (P = 0.030).
Power of performed test with alpha = 0.050: 0.551
The program states a significant difference for the nominal concentration of 100 mg/L compared to the control. A statistically significant difference was found but an inhibition of 3 % is considered to be not biologically significant.

Any other information on results incl. tables

Cell Densities

 

Nominal test item concentration [mg/L	Replicate No.	0 hours	Cell density 24 hours	[cells/mL] 48 hours	72 hours
	1	6305	28107	298680	1919233
	2	6305	36443	365773	1870916
	3	6305	44034	385111	2085723
100	4	6305	31529	253881	1637841
	5	6305	41948	374433	1666143
	6	6305	46758	377579	2206546
	Mean	6305	38137	342576	1897734
	1	6305	55874	491241	2612369
	2	6305	38983	358973	1796494
	3	6305	52596	443915	2254863
Control	4	6305	45926	453241	2091249
	5	6305	50087	500329	2615639
	6	6305	49847	509384	2570479
	Mean	6305	48886	459514	2323516

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to

control values are marked(+)‚ not significant differences are marked(^-).

Nominal test item concentration [mg/L]	Replicate No.	Growth rate [d^-1]	Growth rate inhibition [Vo]	Yield [cells/mL]	Inhibition of yield [Vo]
	1	1.91	3	1912928	17
	2	1.90	4	1864611	20
	3	1.93	2	2079418	10
100	4	1.85	6	1631536	30
	5	1.86	5	1659838	28
	6	1.95	1	2200241	5
	Mean	(+)^1   1.90	3	(+) 1891429	18
	1	2.01		2606064
	2	1.88		1790189
	3	1.96		2248558
Control	4	1.94		2084944
	5	2.01		2609334
	6	2.00		2564174
	Mean	1.97		2317211

 

1 = A statistically significant difference was found but an inhibition of 3 % is considered to be not biologically significant

Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours)

 

	Replicate No.	Specific growth rate [del] section-by-section				Mean (0 - 72 hours)		SD +		CV Fol	Mean CV Fol
		0 - 24 hours	24 - 48 hours		48 - 72 hours
Control	1 2 3 4 5 6	2.18 1.82 2.12 1.99 2.07 2.07	2.17 2.22 2.13 2.29 2.30 2.32		1.67 1.61 1.63 1.53 1.65 1.62	2.01 1.88 1.96 1.94 2.01 2.00		0.293 0.310 0.290 0.383 0.328 0.357		14.6 16.4 14.8 19.8 16.3 17.8	16.6
				Mean			1.97
				SD ±			0.05
				CV [%]			2.58

SD = Standard deviation     CV = Coefficient of variation

Measured Concentrations and Percent of Nominal Concentration

Sampling date	2016-05-31 Start of the exposure, 0 hours		2016-06-03 End of the exposure, 72 hours
Start of analysis	2016-05-31		2016-06-03
Nominal concentration of the test item [mg/L]	PU-2015-627
	Meas. conc. [mg/L]		Meas. conc. % [mg/L]	%
100	82.8*	83	105	105
Control	< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account

%                       = percent of the nominal concentration of the test item

LOG                  = limit of quantification of theanalytical method(5.00 mg/L of the test item)

*                = reanalysed on 2016-06-06 (mean value of 3 replicates

Validity Criteria

Validity Criterium	Required	This study
Increase of the cell growth in the control cultures	Exponentially > 16-fold corresponding to a specific growth rate of 0.92 day-1	369 fold (specific growth rate 1.97 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days  0-1, 1-2 and 2-3) in the control cultures	<35 %	16.6 %
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	<7 %	2.58 %

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

for details see end of "any other information on results incl. tables"

Conclusions:

nominal 72h-ErC50 > 100 mg/L
nominal 72h-NOEC = 100 mg/L

Executive summary:

Potassium hexafluorophosphate was tested according to OECD201, static with Pseudokirchneriella subcapitata in a limit test with analytically confirmed nominal concentration.

A slight but statistically significant effect difference to control was found but an inhibition of 3 % of groth rate is considered to be not biologically significant.

nominal 72h-ErC50 > 100 mg/L

nominal 72h-NOEC = 100 mg/L