Tricobalt bis(orthophosphate)

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:

March-July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Subsamples were collected from the control, the lowest and the highest test dilutions prior to test commencement and at test termination for chemical analysis. All samples were stored at 4°C in the dark prior to being transported to The Shepherd Color Company for analysis.
Subsamples of the test solutions were sent to The Shepherd Color Company for chemical analysis of the definitive test solutions for confirmation of exposure
concentrations.

Vehicle:

no

Details on test solutions:

The test sample was considered poorly water soluble. Accordingly, preparation of the test solutions followed OECD's Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD, 2000). A nominal concentration of 100 mg/L was selected as the maximum concentration of the test item to be tested for all range-finding and definitive tests (OECD, 2006). Furthermore, all nominal test solutions were prepared based on the whole product, without correction for purity.

Range Finding Test Solution Preparation:
To prepare the test solution a 1.5L solution of the highest concentration (i.e., 100 mg/l) was prepared by adding 150mg of test item into 1.5L of freshwater algal growth media in a glass beaker and stirred at 700 rpm for 1 hour at room temperature. This solution was allowed to settle for approximately 72 hours.
Preparation of the remaining test solutions was completed at this time. A total of 1,400 ml of the 100 mg/l test solution was decanted for testing leaving the settled solids behind. All lower concentrations of the test item (10, 1, 0.1, 0.01) were prepared by serial dilution.

Definitive Test Solution Preparation:
To prepare the test solution a 1.5L solution of the highest concentration (i.e., 100 mg/l) was prepared by adding 150mg of Violet 92 into 1.5L of freshwater algal growth media in a glass beaker and stirred at 700 rpm for 1 hour at room temperature. This solution was allowed to settle for approximately 72 hours.
Preparation of the remaining test solutions was completed at this time. A total of 1,400 ml of the 100 mg/l test solution was decanted for testing leaving the settled solids behind. All lower concentrations of the test item (20, 10, 5, 2.5, 1.25) were prepared by serial dilution.

The dilution water was freshwater algal growth medium with the following composition:
25.5 mg/L Sodium nitrate
12.16 mg/L Magnesium chloride (hexahydrate)
4.41 mg/L Calcium chloride (dihydrate)
14.7 mg/L Magnesium sulfate (heptahydrate)
1.044 mg/L Potassium chloride
15 mg/L Sodium bicarbonate
0.3 mg/L EDTA
0.186 mg/L Boric acid
0.415 mg/L Manganese chloride (tetrahydrate)
0.16 mg/L Ferric chloride (hexahydrate)
3.27 µg/L Zinc chloride (anhydrous)
1.43 µg/L Cobalt chloride (hexahydrate)
7.26 µg/L Sodium molybdate (dihydrate)
12 ng/L Cupric chloride (dihydrate)

Test organisms (species):

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

Details on test organisms:

The unicellular green algae P. subcapitata is cultured at 23 to 25°C under 24:0 photoperiod at Hydrosphere Research lab. Lighting cool-white fluorescent at 360 to 440 foot-candles. Cultures are reset weekly. The strain is unknown.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.7 - 23.6°C

pH:

7.1 - 7.9

Nominal and measured concentrations:

nominal: 0, 1.25, 2.5, 5, 10, 20 mg/L of test substance

Details on test conditions:

- Test type: Static algal flask test
- Test duration: 72-hour
- Agitation during testing: Hand swirled twice daily
- Incubation chamber: Temperature and light controlled water bath
- Temperature: 23 ± 1 °C, as recorded daily with a maximum/minimum thermometer
- Light quality: Cool-white fluorescent
- Light intensity: 360 to 440 fc
- Photoperiod: Continuous (24 hours)
- Test vessel size: Clear glass 250-ml Erlenmeyer flasks covered with non- toxic foam plugs
- Test replicates: Four per dilution
- Testing medium: Freshwater algal growth medium

Reference substance (positive control):

yes

Remarks:

Potassium chloride

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

The bioassay tests were acceptable tests based on controls and test conditions specified in the methods. The test met all the test validity criteria. The coefficient of variance in the control at 72 hours was 18.6%, below the 20% required by the method. The results indicate that the 72-hour Effect Load at the 50% level (EL50 based on cell yield) for P. subcapitata was 1.60 mg/L.

Results with reference substance (positive control):

The last toxic response obtained on the reference item Potassium chloride (July 5, 2022) gives an IC25-96h of 40 mg/L, indicating that the algae sensitivity is correct.

Reported statistics and error estimates:

For all concentrations and the controls, cell yield (72-hour) and average specific growth rate (0 to 3 days) were calculated for each replicate and then averaged. Percent inhibition and % C.V. were also calculated for cell yield and average specific growth rate. For the control flasks, the section-by-section specific growth rates (e.g., 0-1, 1-2, and 2-3 days) and mean % C.V. were determined. Concentration-response curves were plotted for cell yield and average specific growth rate. Due to the low water solubility of the test item, test results are reported in terms of the Effective Loading Rate (EL50), referring to the loading rate that causes an adverse effect (in this case, immobility) in 50% of the exposed population) within a specified period (i.e., 24, 48, 72 hours).

P.subcapitata Toxicity Test Results

	Cells/mL (average count x 10e4)
Concentration (mg/L)	24 hours	48 hours	72 hours
0	4.31	13.38	63.25
1.25	2.94	11.63	42.94
2.5	1.63	6.06	8.00
5	1.44	1.50	2.08
10	1.38	1.31	1.06
20	1.25	0.94	0.81
EL50	1.95 mg/L	2.34 mg/L	1.60 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The results indicate that the 72-hour EL50 based on cell yield for P. subcapitata was 1.60 mg/L expressed in nominal test item concentration..

Description of key information

Under the experimental conditions of a test conducted according to OECD guideline 201, the results obtained for the test item indicate a 72-hour EL50 based on cell yield for P. subcapitata of 1.60 mg/L expressed in nominal test item concentration.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.6 mg/L