Alkali metal tetrakis (perfluoro-alcoholato)metallate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13.4.2004

Deviations:

yes

Remarks:

switching from M4-medium (husbandry) to dilution water did not to cause any detrimental effects for test daphnia; therefore, no 48h acclimation period was performed and this deviation from OECD Guideline 202 can be considered uncritical.

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:

Sample preparation: 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 (100.6 % and 97.7 %) lay inside the demanded range of 100 ±15 % and were not taken into account. The QC recoveries were calculated using the following equation: QC recovery in % = measured concentration / nominal concentration *100 %

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna
Authority: STRAUS
Strain: Berlin
Sex: female
Age: between 0 and 24 hours
Origin: Umweltbundesamt Berlin
In-house breeding since 27. September 2007

Animal Husbandry: Daphnia magna is bred in the LAUS GmbH throughout the year. The animals are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical. The keeping is performed similar to the method described in the OECD guideline, following SOP 115 002 01 („Zucht und Hälterung von Daphnia magna STRAUS“), version 13 from 11. Nov. 2019.
Vessels: preserving glasses, nominal volume 2 L
Medium: M4-Medium (recipe of ELENDT)
Food: green algae (Desmodesmus subspicatus)
Medium renewal: twice a week
Photo period: 16/8 hours, using neon tubes
Temperature: 20 ±2 °C

Dilution water (Daphnia medium) with the following specification was used (stated in SOP 11800201), containing 293.80 mg/L CaCl2*2H2O, 123.30 mg/L MgSO4*7H2O, 64.80 mg/L NaHCO3 and 5.80 mg/L KCl. Deviations from the nominal weighted loads were less than 5 %. After preparation, the dilution water was aerated and the pH was measured. The pH was 7.6. As test vessels beakers, glass, nominal volume 50 mL were used.

Selection of Daphnia: 24 hours before the start of the test, the adult animals were separated from the young. 0.5 hours before test start, the adults were caught with the help of a glass tube, and the newborn daphnia (age < 24h) were sieved from the medium and immediately placed into a beaker containing dilution water. After the settling-in period, animals which showed no apparent damage were used for the test. Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to cause any detrimental effects for test daphnia and therefore, no acclimation period of 48 h was performed. This is a deviation from OECD Guideline No. 202 and EU-Method C.2, which can be considered uncritical.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 ±2 °C

pH:

7.6

Dissolved oxygen:

aerated

Nominal and measured concentrations:

0.1 / 0.22 / 0.46 / 1 / 2.2 mg/L (nominal concentration)

Details on test conditions:

Using a glass tube, the daphnia were caught and lifted from the beaker. They were put on a small sieve, and the medium surrounding the animals was sucked off using absorbent paper. Immediately after that, the animals were put into the respective test solution. The test vessels were left to stand for 48 hours. After 24 and 48 hours, the immobilised daphnia were counted. Daphnia are considered immobilised when they do not perform any movements or are only able to move their antennae when the beaker is gently agitated. Daphnia which are trapped at the surface of the test solution are also considered immobilised. The pH and the concentration of dissolved oxygen in the test vessels were measured at the beginning and at the end of the test. The content of Li in the test solutions was measured at the beginning and at the end of the test. The TOC content of the test item in the stock solution (c = 10 mg/L) was measured at the beginning and at the end of the test.
Experimental Conditions
Date of performance: 08. – 10. Jul. 2020
Treatments: 0.1 / 0.22 / 0.46 / 1 / 2.2 mg/L (nominal concentration), the concentrations to be tested were based on the result of a non-GLP pre-test.
Temperature: 20.4 – 21.0 °C
Photo period: 16/8 hours, using neon tubes
Duration: 48 hours
Observation times: 24 and 48 hours
Medium renewal: none
Test vessels: glass beakers, nominal volume 50 mL, tall shape
Replicates (Treatments): 4 vessels, each containing 20 ±5 mL test solution and 5 daphnia
Replicates (Blank control): 4 vessels, each containing 20 ±5 mL dilution water and 5 daphnia

Reference substance (positive control):

yes

Remarks:

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

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The study to determine acute toxicity toward invertebrates was performed using 5 concentrations ranging from 0.1 to 2.2 mg/L nominal concentration. For each test concentration and the blank control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.
Four concentrations showed toxicity between 55 and 100 % immobilisation. None of the animals was immobilised in the blank control.
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.
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 lay between 73 % and 100 % of the nominal concentrations at the beginning of the test and between 75 % and 100 % 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 202 (§ 23)).
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. A recalculation was not performed, because this additional analysis should only show that test item is present in the solutions.
No observations were made which might cause doubts concerning the validity of the study outcome.
All validity criteria were fulfilled:
- Immobilisation in the controls may not exceed 10 %. --> Immobilisation in the controls was 0 %.
- The concentration of dissolved oxygen at the end of the test must be at least 3 mg/L. --> The lowest concentration of dissolved oxygen at the end of the test was 8.6 mg/L.

Results with reference substance (positive control):

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study (202005R201). The 24h-EC50 value was determined as 2.01 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline.

Reported statistics and error estimates:

The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.3.0. The details of calculation are stated in an Annex to the study report. Statistical calculation using ToxRat® Professional 3.3.0.

Immobility: In the blank control and the lowest concentrated treatment, none of the daphnia was immobilised. 

The determined immobility is listed in the following table.

 

Table 1: Immobility

Nominal Conc. in mg/L	Immobility 24 hours					Immobility 48 hours
	absolute				in %	absolute				in %
Blank control	0	0	0	0	0	0	0	0	0	0
0.1	0	0	0	0	0	0	0	0	0	0
0.22	3	0	5	0	40	5	1	5	0	55
0.46	4	3	4	4	75	5	5	5	5	100
1	2	5	4	5	80	5	5	5	5	100
2.2	4	4	4	4	80	5	5	5	5	100

 

pH and O2

The pH values and the concentration of dissolved oxygen in the treatments and the blank control are given in the following table.

 

Table 2: pH and O2-values

Nominal Conc. in mg/L	pH		O2-Concentration in mg/L
	0 h	48 h	0 h	48 h
Blank control	7.6	7.5	8.8	8.6
0.1	7.6	7.6	8.9	8.6
0.22	7.6	7.6	8.8	8.6
0.46	7.6	7.6	8.8	8.7
1	7.6	7.6	8.8	8.7
2.2	7.6	7.6	8.8	8.7

 

Analytical Determinations

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 lay between 73 % and 100 % of the nominal concentrations at the beginning of the test and between 75 % and 100 % 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 202 (§ 23)).

 

Table 3: Measured Concentrations of A at 0 h

Nominal Concentration	Intensity	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration
mg/L	--	µg/L	%	--	µg/L
QC sample (30 µg/L)	412598.0	30.28	100.9	--	--
Blank control	6339.4	< LOQ	--	1.01	< LOQ
0.1	16170.8	0.51	--	1.01	0.52
0.22	27222.0	1.34	--	1.01	1.35
0.46	49803.1	3.04	--	1.01	3.07
1	103073.0	7.04	--	1.01	7.11
2.2	208780.0	14.97	--	1.01	15.12
QC sample (30 µg/L)	409839.0	30.07	100.2	--	--

LOQ = 1 µg/L

 

Table 4: Measured Concentrations of A at 48 h

Nominal Concentration	Intensity	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration
mg/L	--	µg/L	%	--	µg/L
QC sample (30 µg/L)	398965.0	29.25	97.5	--	--
Blank control	6297.4	< LOQ	--	1.01	< LOQ
0.1	16357.0	0.53	--	1.01	0.53
0.22	27422.9	1.36	--	1.01	1.37
0.46	50769.8	3.11	--	1.01	3.14
1	103553.0	7.07	--	1.01	7.14
2.2	210343.0	15.09	--	1.01	15.24
QC sample (30 µg/L)	400544.0	29.37	97.9	--	--

LOQ = 1 µg/L

 

Table 5: Content of Chemical Elements in the Test Item

Element	Amount in the Test Item	Molecular Mass (g/mol) per Element	Total Molecular Mass (g/mol) per Element in the Test Item	% per Element in the Test Item
C	16	12.01	192.17	19.73
F	36	19.00	683.94	70.22
Li	1	6.94	6.94	0.71
Al	1	26.98	26.98	2.77
O	4	16.00	64.00	6.57
Molecular Mass of the whole Test Item			974.03

 

Table 6: Calculated test item concentrations

Nominal Concentration	Calculated Concentration in mg/L		% of Nominal Concentration		Geometric Mean
mg/L	0 h	48 h	0 h	48 h	mg/L
Blank control	--	--	--	--	--
0.1	0.07	0.07	73	75	0.07
0.22	0.19	0.19	86	87	0.19
0.46	0.43	0.44	94	96	0.44
1	1.00	1.00	100	100	1.00
2.2	2.12	2.14	96	97	2.13

 

The measured concentrations of the TOC analysis show, that the test item was pre-sent 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 7: Measured Concentrations IC and TC

Nominal Test Item Concentration	Measured TC t = 0 h	Measured TC t = 48 h	Measured IC t = 0 h	MeasuredIC t = 48 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	11.23	11.11	10.12	10.35
10	11.87	12.80	9.80	9.97

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

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

 

Table 8: Calculated Concentrations TOC

Nominal Concentration Test Item	Calculated TOC (TC-IC) t = 0 h	Calculated TOC (TC-IC) t = 48 h
mg/L	mg/L	mg/L
Blank control	1.11	0.76
10	2.06	2.83

 

Validity criteria fulfilled:

yes

Conclusions:

The test item AM(pfa)4 was tested for acute toxicity to Daphnia magna for 48 hours and the following results were derived: 48h-NOEC = 0.07 mg/L, 48h-LOEC = 0.19 mg/L, 24h-EC50 = 0.35 mg/L, 48h-EC50 = 0.19 mg/L

Executive summary:

The study to determine acute toxicity toward invertebrates was performed using 5 concentrations ranging from 0.1 to 2.2 mg/L nominal concentration. For each test concentration and the blank control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.

Four concentrations showed toxicity between 55 and 100% immobilisation. None of the animals was immobilised in the blank control.

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.

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.

Because of the given molecular formula by the sponsor, a recalculation from the measured Alkali concentrations to the whole test item was possible. The back calculated concentrations lay between 73% and 100% of the nominal concentrations at the beginning of the test and between 75% and 100% 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 202 (§ 23)).

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 following results were determined for the test item AM(pfa)4 (species: Daphnia magna).

48h-NOEC = 0.07 mg/L

48h-LOEC = 0.19 mg/L

24h-EC50 = 0.35 mg/L

48h-EC50 = 0.19 mg/L

Description of key information

The test item AM(pfa)4 was tested for acute toxicity to Daphnia magna for 48 hours and the following results were derived: 48h-NOEC = 0.07 mg/L, 48h-LOEC = 0.19 mg/L, 24h-EC50 = 0.35 mg/L, 48h-EC50 = 0.19 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.19 mg/L