Magnesium fluoride

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:

7 February 2017- 10 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

resp. EU C.3

Deviations:

yes

Remarks:

The temperature in the experiment was in a range of 20.9 – 22.6 °C and partialy outside the range stated in the guideline. Because normal growth was observed and the deviation was very slightly, this can be stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Materion / lot 662599-1
- Expiration date of the lot/batch: 31-Jul 2017
- Purity test date: 18- Jan-2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: RT, away from moisture
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: low solubility
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not expected

Analytical monitoring:

yes

Details on sampling:

Samples were WAFs. This was done by weighing the nominal load 100 mg/L (real load 100.1 mg/L), adding tap water and stirring for 24 hours. The resulting solution was filtrated through 0.45 µm Nylon filters. Sampling intervals were 0, 24H 48h and 72h. After filtration the required amounts of minerals were added

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

For each treatment, 200 mL of the respective test item solution was mixed with the neces-sary amount of algal pre-culture (0.520 mL) to achieve a cell concentration of 4.5 *103 cells/mL. In this mixture, the pH-value was measured. Samples for the analytical determi-nation were taken.
For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal pre-culture (0.911 mL). In this mixture, the pH-value was measured.
The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
The test vessels were filled with 45 mL of the respective test solution and incubated open (covered with perforated plastic foil) for 72 hours, shaken on an orbital shaker. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and blank control was meas-ured again.
At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The concentration of Magnesium in the test vessels was measured at the start and at the end of the test

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater green alga, Desmodesmus subspicatus
- SAG Strain Number: 86.81
- Taxonomic position: Chlorophyta - Chlorophyceae
- Source (laboratory, culture collection): The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
- Origen and Culture: 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 perma-nent culture, the pre-culture was prepared.


ACCLIMATION
Four days before the start of each test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20.9 – 22.6 °C

pH:

7.6-8.7

Nominal and measured concentrations:

Nominal concentrations of 4.6, 10, 22, 46 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks total volume 65 mL
- Type: closed
- Aeration: No
- Initial cells density: 4.5x10*3 cells/mL
- No. of vessels per concentration (replicates): 3 replicates for each treatment
- No. of vessels per control (replicates): 6 replicates for the blank control

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: A stock solution containing 100.4 mg/L in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments were prepared by dilution of this stock solution with nutrient medium.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No needed. It was measured with a pH-meter 340i wtw.
- Photoperiod: The algae were held under continuous lighting throughout the test.
- Light intensity and quality: The target light intensity was 4500 Lux (Luxmeter LT lutron LX-101). Light intensity was measured at test solution level at four occasions (0h, 24h, 48h and 72h).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell numbers were determined with an electronic particle (Cell Counter Beckman Z2)

TEST CONCENTRATIONS
- Nominal concentrations used in the range-finding test: 4.6, 10, 22, 46 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

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

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): Yes, changes in mean cell density in the negative control replicates over the 72-hour exposure period indicated that exponential growth of cells occurred in those replicates.
- Observation of abnormalities (for algal test): No changes in cell morphology was observed when compared to the control replicates during the microscoping examinations of the cells.
- Findings: The cell numbers were determined with an electronic particle. The means and standard deviations of the cell numbers of the blank control and the treatments are presented in the table 4. The appearance of the algae at the end of the test is stated in the table 5. 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 table 6. The mean inhibition values were calculated for the Growth Rate µ and the Yield, see details in the table 7.

Results with reference substance (positive control):

The results of the last study with the positive control K2Cr2O7 are presented in the section "Any other information on results incl. tables" (table 8). The study was performed under GLP conditions in October 2016 and 72h EC50 (growth rate) was 0.80 mg/L, 95% confidence interval, 0.78 - 0.83 mg/L.

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software ((Microsoft Excel®). As inhibition in all concentrations was lower than 0, indicating a stimulation of algal growth compared to the blank control, the NOEC was stated as being equal or higher than the highest tested concentration of 100 mg/L nominal concentration. Accordingly the EC values and the LOEC were stated as being higher than the highest tested concentration of 100 mg/L nominal concentration

Table 4. Cell Number/mL
Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control*	Mean	4560	11192	64148	251884
Blank control*	SD	0	1382	6116	41088
4.6	Mean	4560	16467	71220	281760
	SD	0	6366	3229	42488
10	Mean	4560	11207	79633	327447
	SD	0	733	11665	72967
22	Mean	4560	11053	65740	306520
	SD	0	820	11811	38134
46	Mean	4560	11600	81600	336507
	SD	0	1463	6834	40072
100	Mean	4560	11593	66833	283807
	SD	0	497	7977	63917

SD = Standard Deviation* Due to extremely low algal growth in the 1^streplicate of the blank control, this replicate was excluded from further calculations

Table 5. Microscopical Observations
Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
4.6	Yes
10	Yes
22	Yes
46	Yes
100	Yes

Table 6. Growth Rate µ, Yield
Nominal Concentration in mg/L	Parameter	Growth Rate	Yield (0-72h)
		(0-72h) [day-1]	[Cell Concentration/mL]
Blank control*	Mean	1.33	247324
	SD	0.06	41088
4.6	Mean	1.37	277200
	SD	0.05	42488
10	Mean	1.42	322887
	SD	0.08	72967
22	Mean	1.4	301960
	SD	0.04	38134
46	Mean	1.43	331947
	SD	0.04	40072
100	Mean	1.37	279247
	SD	0.08	63917
SD = Standard Deviation
* Due to extremely low algal growth in the 1st replicate of the blank control, this replicate was excluded from further calculations.

Table 7. Inhibition Values
Nominal Concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control*	0	0
4.6	-2.89	-12.08
10	-6.38	-30.55
22	-5.05	-22.09
46	-7.4	-34.22
100	-2.8	-12.91
Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour toxicity of alga (Desmodesmus subspicatus) to the registered substance was determined according to OECD 201 guideline in compliance with GLP. The study was conducted based on nominal concentrations. Nominal concentrations of 4.6 mg/l, 10 mg/l, 22 mg/l, 46 mg/l and 100 mg/L were used. As a result, EC10, EC50, NOEC, LOEC values were determined.

Executive summary:

The 72-hour toxicity of alga (Desmodesmus subspicatus) to the registered substance was determined according to OECD 201 guideline in compliance with GLP.

The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L nominal concentration. Incubation time 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 not observed in any concentration. At the start and a the end of the test, the content of Magnesium in the test solution was determined using ICP.OES.

The 72h-EC50s of potassium dichromate (K₂Cr₂O₇, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

As a result, EC10, EC50, NOEC, LOEC values were determined.

Description of key information

The 72-hour toxicity of algae (Desmodesmus subspicatus) to the registered substance was determined according to OECD 201 guideline in compliance with GLP. The study was conducted based on nominal concentrations. Nominal concentrations of 4.6 mg/l, 10 mg/l, 22 mg/l, 46 mg/l and 100 mg/L were used. As a result, EC10, EC50, NOEC, LOEC values were determined. No toxicity was observed at the concentrations tested. No toxicity was observed at the concentrations tested. Therefore, based on the available data, the lowest value (EC50) was selected as a key value.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC50 for marine water algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

EC10 or NOEC for marine water algae:

100 mg/L

Additional information

The short-term toxicity to aquatic invertebrates results obtained from the supporting read-across substance (sodium fluoride) has been provided.