Alkali metal tetrakis (perfluoro-alcoholato)metallate

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:

July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28.7.2011

Deviations:

yes

Remarks:

The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: AM(pfa)4
Appearance: white powder
Homogeneity: homogeneous

Analytical monitoring:

yes

Details on sampling:

The TOC content of the test item in the stock solution (c = 10 mg/L) was measured in extra replicates without algae cells at the beginning and at the end of the test.
Sample preparation: At the start of the test 5 mL of each test item solution and the control were acidified with 50 µL HNO3 (65 % in water) and measured via ICP-OES. At the end of the test 10 mL of each test item solution and the control were acidified with 100 µL HNO3 (65 % in water) and measured via ICP-OES.
On each sampling day, validity of calibration was controlled by measuring QC samples (30 µg/L). The mean QC recoveries per sequence (119.6% and 117.0%) lay outside the demanded range of 100 ±15%. But this is only a minor deviation and no back calculation from alkali metal to the test item is possible. Therefore, this deviation can be considered as uncritical. The QC recoveries were calculated using the following equations: QC recovery in % = measured concentration / nominal concentration * 100%

Vehicle:

no

Details on test solutions:

Storage: The test item was stored in the test facility in a closed vessel at room temperature (17.3 – 23.0 °C), kept under inert gas.
A stock solution containing 10.2 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments (0.1 / 0.32 / 1 / 3.2 mg/L) were prepared by dilution of this stock solution with algal medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test System: Selection of the test system was made following the proposal of the guidelines with the following specification: Unicellular freshwater green alga, genus Desmodesmus, species subspicatus, SAG Strain Number 86.81, taxonomic position Chlorophyta – Chlorophyceae.
Origin and Culture: The culture of Desmodesmus subspicatus was obtained in May 2020 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature: 21.2 – 22.2 °C

pH:

7.2 - 8.2

Nominal and measured concentrations:

0.1 / 0.32 / 1 / 3.2 / 10 mg/L nominal concentration - The tested concentrations were based on non GLP pre-tests.

Details on test conditions:

Date: 06. – 09. Jul. 2020
Vessels: glass flasks total volume 65 mL
Lighting: within the specified range (4440 – 8880 Lux)
Replicates for treatments: 3 vessels, filled with 45 ±1 mL test solution and algae, 1 vessel, filled with 45 ±1 mL test solution without algae for analytical determination
Blank Control: 6 vessels, each filled with 45 ±1 mL algal medium and algae, 1 vessel filled with 45 ±1 mL algal medium without algae for analytical determination

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.96 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.61 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

The study was performed using 5 concentrations ranging from 0.1 to 10 mg/L (nominal). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.
Significant inhibition of algal growth was observed at the following concentrations: 0.32 / 1 / 3.2 / 10 mg/L nominal for growth rate and yield.
At the beginning and at the end of the test, the content of Alkali (A) as component of the test item in the test solutions was determined using ICP-OES-determination. Additional, at the start and at the end of the test, the content of the test item in the stock solution (c = 10 mg/L nominal) was estimated by determination of the total organic carbon (TOC) content in the test solutions using a carbon analyser.
Based on the molecular formula, a recalculation from the measured A concentrations to the whole test item was possible. The back calculated concentrations lay between 89 % and 136 % of the nominal concentrations at the beginning of the test and between 93 % and 144 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the mean of the measured concentrations (see OECD 201 (§ 39)).
The measured concentrations of the TOC analysis showed that the test item was present in the test solution of 10 mg/L (nominal) throughout the test.
The 72h-EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.
The pH of the blank control should not fluctuate by more than 1.5 units. There was no change of the pH in the blank control. All validity criteria were met. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Results with reference substance (positive control):

Positive Control: Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.
The results of the last reference study (202007R301) with the positive control K2Cr2O7 (performed under GLP conditions in May 2020) are presented as follows: 72h ErC50 was 1.00 mg/L (95% confidence interval 0.97 – 1.03 mg/L) and 72h EyC50 was 0.35 mg/L (95% confidence interval 0.32 – 0.39 mg/L).

Reported statistics and error estimates:

STATISTICS AND BIOLOGICAL RESULTS
Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software Tox-Rat® Professional, version 3.3.0. Statistical calculation was performed using ToxRat® Professional 3.3.0.

The cell numbers were determined with an electronic particle counter. The means and standard deviations of the cell numbers of the blank control and the treatments are presented in the following table:

 

Table 1: Cell Number/mL

Nominal conc. in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2620	11137	95980	576437
	SD	0	679	13975	24683
0.1	Mean	2620	9780	70907	621687
	SD	0	1693	16847	56361
0.32	Mean	2620	9340	81907	351633
	SD	0	1413	24625	41412
1	Mean	2620	7600	28293	132213
	SD	0	908	3668	10922
3.2	Mean	2620	7513	12380	23873
	SD	0	2162	1044	5300
10	Mean	2620	5300	4813	6693
	SD	0	710	613	656

SD = Standard Deviation

 

The light intensity is checked monthly and recorded in a control chart. The lighting corresponded to the specified range of 4440 - 8880 lux.

 

In the following table, the pH values measured at the start and the end of the test are stated.

 

Table 2: pH values

Nominal Concentration in mg/L	0 h	72 h
Blank control	7.7	7.7
0.1	7.9	8.2
0.32	7.9	7.6
1	7.3	7.5
3.2	7.3	7.4
1	7.2	7.4

 

Microscopical Observations: In the following table, the appearance of the algae at the end of the test is stated.

Table 3: Microscopical Observations

Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
0.1	Yes
0.32	Yes
1	Yes
3.2	No cells visible
10	No cells visible

 

Analytical Determination: At the beginning and at the end of the test, the content of Alkali (A) as component of the test item in the test solutions was determined using ICP-OES-determination. Additional, at the start and at the end of the test, the content of the test item in the stock solution (c = 10 mg/L nominal) was estimated by determination of the total organic carbon (TOC) content in the test solution using a carbon analyser.

Based on the molecular formula, a recalculation from the measured A concentrations to the whole test item was possible. The back calculated concentrations were between 89 % and 136 % of the nominal concentrations at the beginning of the test and between 93 % and 144 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the mean of the measured concentrations (see OECD 201 (§ 39)) and details are given in the following tables:

 

Table 4: Measured Concentrations of A at 0 h

Nominal conc.	Intensity	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration
mg/L	--	µg/L	%	--	µg/L
QC sample (30 µg/L)	413242.0	35.92	119.7	--	--
Blank control	6446.7	-0.03	119.6	1.01	< LOQ
0.1	17499.3	1.15	119.6	1.01	0.97
0.32	41324.3	3.63	119.6	1.01	3.07
1	113618.0	10.77	119.6	1.01	9.09
3.2	416320.0	36.15	119.6	1.01	30.54
10	1024140.0	75.39	119.6	1.01	63.68
QC sample (30 µg/L)	411977.0	35.82	119.4	--	--

LOQ = 1 µg/L

 

Table 5: Measured Concentrations of A at 72 h

Nominal conc.	Intensity	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration
mg/L	--	µg/L	%	--	µg/L
QC sample (30 µg/L)	400896.0	34.99	116.6	--	--
Blank control	6208.6	-0.05	117.0	1.01	< LOQ
0.1	17869.0	1.19	117.0	1.01	1.03
0.32	40744.0	3.57	117.0	1.01	3.08
1	125174.0	11.86	117.0	1.01	10.24
3.2	421333.0	36.53	117.0	1.01	31.54
10	1048690.0	76.77	117.0	1.01	66.29
QC sample (30 µg/L)	403645.0	35.19	117.3	--	--

LOQ = 1 µg/L

 

Table 6: Content of Chemical Elements in the Test Item

Based on the known molecular formula of the test item, the individual amounts of element weight, relative to total weight could be calculated and used for analysis, as outlined in a table in the original study report.

 

Table 7: Calculated test item concentrations

Nominal conc. mg/L	Calculated Concentration in mg/L		% of Nominal Concentration		Geometric Mean
	0 h	72 h	0 h	72 h	mg/L
Blank control	--	--	--	--	--
0.1	0.14	0.14	136	144	0.14
0.32	0.43	0.43	134	135	0.43
1	1.28	1.44	128	144	1.35
3.2	4.29	4.43	134	138	4.35
10	8.94	9.30	89	93	9.12

 

The measured concentrations of the TOC analysis show that the test item was present in the test solution of 10 mg/L (nominal) throughout the test. A recalculation was not performed, because this additional analysis should only show that test item is present in the solutions.

 

Table 8: Measured Concentrations IC and TC

Nominal conc. Test Item	Measured TC t = 0 h	Measured TC t = 72 h	Measured IC t = 0 h	Measured IC t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	8.11	8.02	7.37	7.45
10	9.86	9.62	7.12	7.16

LOQ (Limit of quantification) TC = 254.7 µg/L

LOQ (Limit of quantification) IC = 99.94 µg/L

 

Table 9: Calculated Concentrations TOC

Nominal conc. Test Item mg/L	Calculated TOC (TC-IC) t = 0 h	Calculated TOC (TC-IC)t = 72 h
	mg/L	mg/L
Blank control	0.75	0.57
10	2.74	2.46

 

Growth Rate, Yield

From the cell numbers, the Growth Rate µ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table.

 

Table 10: Growth Rate µ, Yield

Nominal conc. in mg/L	Parameter	Growth Rate (0-72 h) [day-1]	Yield (0-72 h) [Cell Concentration/mL]
Blank control	Mean	1.80	573817
	SD	0.01	24683
0.1	Mean	1.82	619067
	SD	0.03	56361
0.32	Mean	1.63	349013
	SD	0.04	41412
1	Mean	1.31	129593
	SD	0.03	10922
3.2	Mean	0.73	21253
	SD	0.07	5300
10	Mean	0.31	4073
	SD	0.03	656

SD = Standard Deviation

 

Inhibition: The following mean inhibition values were calculated for the Growth Rate µ and the Yield.

 

Table 11: Inhibition Values

Nominal conc. in mg/L	% Inhibition Growth Rate (0-72 h)	% Inhibition Yield (0-72 h)
Blank control	0	0
0.1	-1.4	-7.9
0.32	9.2	39.2
1	27.3	77.4
3.2	59.3	96.3
10	82.7	99.3

Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

 

STATISTICS AND BIOLOGICAL RESULTS

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software Tox-Rat® Professional, version 3.3.0. Statistical calculation was performed using ToxRat® Professional 3.3.0.

 

The following values were determined.

Table 12: Biological Results of the Test Item

Parameter	Value	95 % confidence interval
NOEC (Growth Rate) 72 h	0.14 mg/L	--
NOEC (Yield) 72 h	0.14 mg/L	--
LOEC (Growth Rate) 72 h	0.43 mg/L	--
LOEC (Yield) 72 h	0.43 mg/L	--
72h ErC10	0.55 mg/L	0.49 mg/L – 0.61 mg/L
72h EyC10	0.17 mg/L	0.11 mg/L – 0.26 mg/L
72h ErC50	2.96 mg/L	2.82 mg/L – 3.11 mg/L
72h EyC50	0.61 mg/L	0.49 mg/L – 0.75 mg/L

 

 

VALIDITY

Increase Factor: The cell concentration in the blank control should increase by a factor of at least 16 within 72h.

Daily Growth Rates: Mean coefficient of variation of daily growth rates in the blank controls should be 35 % at the most. Coefficient of variation of average growth rate during the whole test period should be 7 % at the most.

Values and assessment

The daily growth rates of the blank controls were calculated. Means, standard deviations and coefficients of variation were determined. Values and assessment can be found in the following tables.

 

Table 13: Daily Growth Rates of the blank controls

Growth rates	days 0 – 1	days 1 – 2	days 2 – 3	CV of sectional daily growth rates	days 0 – 3
Replicate 1	1.438	2.087	1.833	18 %	1.786
Replicate 2	1.384	2.083	1.940	20 %	1.803
Replicate 3	1.444	2.033	1.959	18 %	1.812
Replicate 4	1.377	2.198	1.867	23 %	1.814
Replicate 5	1.503	2.185	1.693	20 %	1.794
Replicate 6	1.527	2.297	1.509	25 %	1.778
Mean	1.446	2.147	1.800	21 %	1.798
SD	0.061	0.097	0.171	--	0.014
CV	4 %	5 %	10 %	--	1 %

CV = Coefficient of Variation

 

Table 14: Assessment

Parameter	Validity criteria	Observed value	Assessment
Increase factor biomass	factor 16 in 72 h	220	Valid
Mean coefficient of variation of daily growth rates	max. 35 %	21 %	Valid
Coefficient of variation of aver-age growth rate during the whole test period	max. 7 %	1 %	Valid

 

The growth curve shows that during the test exponential growth in the blank controls was given.

Validity criteria fulfilled:

yes

Conclusions:

Growth inhibition to Desmodesmus subspicatus through the test item was observed and the NOEC for growth rate was found 0.14 mg/L (72 h), whereas the ErC50 was determined as 2.96 mg/L. The LOEC for growth rate was 0.43 mg/L.

Executive summary:

The study was performed using 5 concentrations ranging from 0.1 to 10 mg/L (nominal). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following concentrations: 0.32 / 1 / 3.2 / 10 mg/L nominal for growth rate and yield.

At the beginning and at the end of the test, the content of Alkali (A) as component of the test item in the test solutions was determined using ICP-OES-determination. Additional, at the start and at the end of the test, the content of the test item in the stock solution (c = 10 mg/L nominal) was estimated by determination of the total organic carbon (TOC) content in the test solution using a carbon analyser.

Considering the molecular formula, a recalculation from the measured Alkali concentrations to the whole test item was possible. The back calculated concentrations lay between 89 % and 136 % of the nominal concentrations at the beginning of the test and between 93 % and 144 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the mean of the measured concentrations (see OECD 201 (§ 39)).

The measured concentrations of the TOC analysis also showed that the test item was present in the test solution of 10 mg/L (nominal) throughout the test. A recalculation was not performed, because this additional analysis should only show that test item is present in the solutions.

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test and the values were found within the range of the laboratory (growth rate 0.65 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

 

The following results for the test item AM(pfa)4 were determined:

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	0.14 mg/L	0.43 mg/L	0.55 mg/L	2.96 mg/L
Yield	0.14 mg/L	0.43 mg/L	0.17 mg/L	0.61 mg/L

Description of key information

Growth inhibition to Desmodesmus subspicatus through the test item was observed and the NOEC for growth rate was found 0.14 mg/L (72 h), whereas the ErC50 was determined as 2.96 mg/L. The LOEC for growth rate was 0.43 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.96 mg/L

EC10 or NOEC for freshwater algae:

0.14 mg/L