Magnesium bis(dihydrogenorthophosphate)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category


1. Both substances are inorganic salts of a monovalent cation from Group 2 of the periodic table, calcium or magnesium, and phosphoric acid. Thus, they all share the Ca2+ or Mg2+ cation and the PO43- anion as common functional groups.
2. Both substances will ultimately dissociate into the common breakdown products of the Ca2+ or Mg2+ cations and the PO43- anion.
3. Calcium and magnesium orthophosphates have been shown to have a similar toxicological profile and physicochemical nature and therefore this data is considered to be adequate and reliable for use in read-across. Any further testing would not be scientifically justified as all substances will dissociate to their anionic and cationic forms in natural waters and these ions (Ca2+, Mg2+ and PO43-) are all ubiquitous and are not considered to pose a risk of ecotoxicity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and 100 mg/L. Samples were taken after 0 and 72 hours.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions were prepared by adding the test substance in nutrient medium.Test concentrations of 100, 45, 21 and 9 mg/L were prepared in 2 L beakers by adding the test substance of 100, 45, 21 and 9 mg in 1L of sterile nutrient medium, respectively.The test concentration of 4 mg/L was prepared by diluting the test solution of 9 mg/L.Total volume of 150 ml was used as a final test solution considering necessary amount for the analysis of the test substance in the test solution.The sterile nutrient medium not including test substance was used for a control.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Strain No. ATCC 22662; UTEX 1648
- Source (laboratory, culture collection): The test species was obtained from American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852) in March 3, 1994 and cultured at Korea Institute of Toxicology, KRICT.

ACCLIMATION
- Culturing media and conditions: An OECD culture medium was diluted to produce the biomass of 1×104 cells/mL and then sub-cultured continuously at intervals of 3 to 4 days. For pre-cultivation, the test algae were inoculated into the OECD culture medium at a biomass of 0.5-1.0×104 cells/mL 2 to 4 days prior to study initiation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.1 °C

pH:

test start: 7.50 - 8.34
test termination: 8.03 – 9.08

Nominal and measured concentrations:

Nominal test substance concentrations: control, 0.3, 1, 3.1, 9.8, 31.3, and 100 mg/L
Measured test concentrations at 0 h: 0.08-0.88 mg/L
Measured test concentrations at 72 h: 0.61-1.56 mg/L

Details on test conditions:

TEST SYSTEM
- Initial cells density: 1.0×10^4 cells/mL (2-4 days prior to test initiation)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1


GROWTH MEDIUM
- Standard medium used: yes


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


OTHER TEST CONDITIONS
- Light intensity and quality: 4,440-8,880 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Morphological changes of algal cells were observed including expansion, aggregation, atrophy, and decoloration at 72 hours after exposure. The cell densities were determined by direct counts using ×200 or ×400 microscope (Leica MPS 32)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: control, 0.1, 1, 10, and 100 mg/L.
- Results used to determine the conditions for the definitive study: tthe average specific growth rate was inhibited by 13.5% and by 51.4% for the area under growth curve method at the highest dose of 100 mg/L.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.56 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Algal cells were observed to maintain a similar shape to the control in all test solutions. In all the test concentrations, the growth rates of algal cells were higher than those of the control. Therefore, it was determined that the test substance would cause growth stimulation rather than growth inhibition.

Table 1. Growth rates

Nominal concentration (mg/L)	Growth rate	Relative growth rate (%)	Relative Inhibition (%)
Control	0.058	-	-
4	0.072	126	-26
9	0.063	110	-10
21	0.072	125	-25
45	0.069	120	-20
100	0.070	122	-22

Validity criteria fulfilled:

not specified

Conclusions:

In all the test concentrations, the growth rates of algal cells were higher than those of the control. Therefore, it was determined that the test substance would cause growth stimulation rather than growth inhibition.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, data originate in part from a secondary source

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category


1. Both substances are inorganic salts of a monovalent cation from Group 2 of the periodic table, calcium or magnesium, and phosphoric acid. Thus, they all share the Ca2+ or Mg2+ cation and the PO43- anion as common functional groups.
2. Both substances will ultimately dissociate into the common breakdown products of the Ca2+ or Mg2+ cations and the PO43- anion.
3. Calcium and magnesium orthophosphates have been shown to have a similar toxicological profile and physicochemical nature and therefore this data is considered to be adequate and reliable for use in read-across. Any further testing would not be scientifically justified as all substances will dissociate to their anionic and cationic forms in natural waters and these ions (Ca2+, Mg2+ and PO43-) are all ubiquitous and are not considered to pose a risk of ecotoxicity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and 100 mg/L. Samples were taken after 0 and 72 hours.
The concentration oftest substance in the test solution was analyzed at the beginning (0 hr) and at the end (72 hrs) ofthe study. About 15 mL was taken from triplicate flask of each test concentration and pooled. 30 mL sample (10 mL x 3 replicate) was taken from pooled solution and put into 20 mL volume vial. 0.3 mL of nitric acid solution (61%) was added into the samples then the samples were sent to the chemical analysis laboratory at Korea Research Institute of Chemical Technology for analysis of calcium. Samples at 0 hour were not filtered additionally because the undissolved test substance in the test solutions were filtered using 0.45 um membrane filter (advantec, Inc., USA) when the test solutions were prepared. Samples at 72 hours were centrifuged slightly (5000 rpm, 10 min) to remove grown algae and preparation process was same as above.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: After 100 mg of the test substance (purity: 99.9%) was added in 1,000 mL beaker and dilution water (OECD nutrient medium) was added up to the mark of the beaker to produce the nominal concentration of 100 mg/L. For the control group, only the dilution water was used.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Strain No. ATCC 22662; UTEX 1648
- Source: The culture collection of algae, The University of Texas at Austin, USA (UTEX)- Strain No.: UTEX1648

ACCLIMATION
- Culturing media and conditions: An OECD culture medium was diluted to produce the biomass of 1×104 cells/mL and then sub-cultured continuously at intervals of 3 to 4 days. For pre-cultivation, the test algae were inoculated into the OECD culture medium at a biomass of 0.5-1.0×104 cells/mL 2 to 4 days prior to study initiation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.7 ± 0.5 ℃

pH:

Control: 9.06 (0 hour) - 8.36 (72 hours)
0.3 mg/L: 8.83 (0 hours) - 8.39 (72 hours)
1.0 mg/L: 8.84 (0 hours) - 8.37 (72 hours)
3.1 mg/L: 8.87 (0 hours) - 8.35 (72 hours)
9.8 mg/L: 8.89 (0 hours) - 8.30 (72 hours)
31.3 mg/L: 8.79 (0 hours) - 8.32 (72 hours)
100.0 mg/L: 8.57 (0 hours) - 8.44 (72 hours)

Nominal and measured concentrations:

Nominal test substance concentrations: control, 0.3, 1, 3.1, 9.8, 31.3, and 100 mg/L
Measured test concentrations at 0 h: 0.3, 0.4, 0.3, 0.8, 1.7, and 4.4 mg/L
Measured test concentrations at 72 h: n.d., n.d., n.d., 0.3, 1.6, and 4.7 mg/L.

Details on test conditions:

TEST SYSTEM
- Initial cells density: 0.5-1.0×104 cells/mL (2-4 days prior to test initiation)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1


GROWTH MEDIUM
- Standard medium used: yes


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


OTHER TEST CONDITIONS
- Light intensity and quality: 4,440-8,880 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Morphological changes of algal cells were observed including expansion, aggregation, atrophy, and decoloration at 72 hours after exposure. Cell densities were determined by direct counts using a BECKMAN COULTER Multisizer 3 particles analyzer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: control, 0.1, 1, 10, and 100 mg/L.
- Results used to determine the conditions for the definitive study: tthe average specific growth rate was inhibited by 13.5% and by 51.4% for the area under growth curve method at the highest dose of 100 mg/L.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 4.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Algal cells were observed to maintain a similar shape to the control in all test solutions.

Table 1. Growth inhibition rates based on mean value during the test period

(1) Average growth rate

Nominal concentration (mg/L)	Mean measured concentration (mg/L)	Average growth rate	Relative inhibition (%)	Mean relative inhibition (%)
Control	ND	1.7038	-	-
0.3	0.3	1.7084	-0.3	0.0
		1.6861	1.0
		1.7180	-0.8
1	0.4	1.6385	3.8	4.2
		1.598	6.2
		1.6619	2.5
3.1	0.3	1.7464	-2.5	-3.4
		1.8030	-5.8
		1.7356	-1.9
9.8	0.8	1.8378	-7.9	-3.4
		1.7358	-1.9
		1.7090	-0.3
31.3	1.7	1.7674	-3.7	-2.2
		1.7312	-1.6
		1.7263	-1.3
100.0	4.4	1.7609	-3.4	-3.3
		1.7747	-4.2
		1.7453	-2.4

Description of key information

One key study to assess the toxicity of magnesium bis(dihydrogenorthophosphate) to aquatic algae exists, this study is conducted on an analogous substance (see justification below). On this basis sodium, potassium, calcium and magnesium phosphates are not considered to be toxic to algae. 
In addition, sodium, potassium, calcium, magnesium and phosphates are essential nutrients for both terrestrial and aquatic plants and are an integral component of the growth media for the algal inhibition test (OECD 201, sodium as NaHCO3, potassium and phosphate as KH2PO4, magnesium as MgCl2.6H20 and MgSO4.7H2O, calcium as CaCl2.2H2O). Phosphates are known to increase the growth of aquatic plants and aquatic toxicity is unlikely to occur therefore testing for this endpoint is not justified.
The additional supporting data provided were not considered to be adequate or reliable to fulfil neither the guideline nor the regulatory requirements. In addition, there is no evidence within this data to suggest that essential validity criteria are met.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Data on the acute toxicity of magnesium bis(dihydrogenorthophosphate) to aquatic algae are not available. The assessment was therefore based on studies conducted with the analogue substances tricaclcium bis(orthophosphate) (CAS 7758-87-4) and calcium hydrogen orthophosphate (CAS 7757-93-9) as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5.

Tricalcium bis(orthophosphate):

The acute toxicity of tricalcium bis(orthophosphate) to aquatic algae was investigated in a study following OECD guideline 201 (Kim et al. 2013). Pseudokirchnerella subcapitata was exposed to nominal test substance concentrations of 0.3, 1, 3.1, 9.8, 31.3, and100 mg/L. The mean measured test concentrations at test start were 0.3, 0.4, 0.3, 0.8, 1.7, and 4.4 mg/L. Based on the algal growth rate an EC50(72 h) >4.4 mg/L (measured) was determined (nominal: EC50(72 h) >100 mg/L).

Calcium hydrogenorthophosphate:

The acute toxicity of calcium hydrogen orthophosphate to aquatic algae was investigated in a study following OECD guideline 201 (Kim et al. 2013). Pseudokirchnerella subcapitata was exposed to nominal test substance concentrations of 0.3, 1, 3.1, 9.8, 31.3, and100 mg/L. The mean measured test concentrations at test start were 0.3, 0.4, 0.3, 0.8, 1.7, and 4.4 mg/L. Based on the algal growth rate an EC50(72 h) >4.4 mg/L (measured) was determined (nominal: EC50(72 h) >100 mg/L).

Based on the study results for the read across substance calcium hydrogen orthophosphate the assessed substance magnesium hydrogen orthophosphate is considered not acutely toxic to aquatic algae.