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REACH

Calcium Manganese Titanium oxide

EC number: - | CAS number: -

Life Cycle description
Uses advised against

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

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:

20 - 23 July 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study conducted under GLP meeting guideline validity criteria. Substance was not detectable after 72 hours due to low solubility of the test item and degradation/transformation/flocculation in the test system. This was deemed acceptable and unavoidable. The presence of EDTA may have also impacted chemical availability to the organism.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Annex 5 corrected: 28 July 2011

Deviations:

Qualifier:

according to guideline

Guideline:

other: OECD Guideline No. 23 "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

September 2000

Deviations:

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark.
- Stability under test conditions: See analytical results section.
- Solubility and stability of the test substance in the solvent/vehicle: Solubility of the test item in the test media was determined to be 0.960 mg/L. See analytical results section for stability information.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Seperate water accomodated fractions ( WAF's) prepared for each dose rate (nominal dose rate: 3.1, 6.3, 13, 25, 50 and 100 mg/L). The test sample and medium were mixed with a magnetic stirrer for 48 h. The suspension was then filtered with a membrane filter (GV, 0.22 µm, Merck Millipore).
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: See above.
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Seperate water accomodated fractions ( WAF's) prepared for each dose rate.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A

OTHER SPECIFICS: N/A

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Individual WAF's prepared at each nominal dose rate; 3.1, 6.3, 13, 25, 50 and 100 mg/L

- Sampling method:
The test item is composed of CaO, MnO2 and TiO2. Each metal ion was individually quantified, and the concentration of metals deriving the test item were calculated to their oxidation number. The sum of these concentrations was defined as the test item concentration.

In the 100 mg/L dose rate test group, the concentration of the test item was measured at the start of exposure, 24, 48 and 72 h after the start of exposure. Other dose groups were measured at the start and end of the exposure period (i.e. 0 and 72 h).

Subsamples were taken (100 mL) were taken from all test solutions at 0 h. 100 mL was taken from sacrificial tests flasks at 24 and 48 h timepoints in the 100 mg/L test group. 90 mL was taken from each test flask and pooled for each respective test group at 72 h.

- Sample storage conditions before analysis:
Not reported

Vehicle:

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Individual WAF solutions prepared for each of the nominal loading rate. Weighed amounts of test item were added to test medium to prepare nominal loading rates of 3.1, 6.3, 13, 25, 50 and 100 mg/L. Solutions were stirred using a magnetic stirrer for 48 h. The suspensions were then filtered through a membrane filter (GV, 0.22 µm pore size, Merck Millipore) by suction. The filrate was used as the representative test solution and was divided into separate test replicates.
- Eluate: Algae media was prepared in purified water in accordance with OECD 201.
- Controls: Algae media only, treated in the same manner as the test solutions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, at the start of the experiment all solutions appeared clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata / green algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): Laboratory culture
- Age of inoculum (at test initiation): 72 h - pre-culture incubated under test conditions for 3 days prior to test initiation.
- Method of cultivation: Passage cultured under sterile conditions in the laboratory. The pre-culture was incubated under the same conditions as the test for 3 days prior to test initiation.

ACCLIMATION
- Acclimation period: The pre-culture was incubated under the same conditions as the test for 3 days prior to test initiation.
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

Total exposure duration:

72 h

Hardness:

Test temperature:

22.2-22.5 ºC

pH:

7.8-7.9

Dissolved oxygen:

Salinity:

Conductivity:

Nominal and measured concentrations:

Nominal: 3.1, 6.3, 13, 25, 50 and 100 mg/L
Measured: Test item was only detected in the 100 mg/L nominal test solution through the course of the study. Based on CaO, MnO2 and TiO2 concentrations the geometric mean concentration for the 100 mg/L nominal test group was 0.200 mg/L. Using the geometric spacing factor between concentrations the 3.1, 6.3, 13, 25 and 50 mg/L (nominal) test concentrations were estimated to be 0.00625, 0.0125, 0.0250, 0.0500 and 0.100 mg/L, respectively, based on the geometric mean measured concentrationof the top concentration group i.e. 0.200 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Not reported
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: Filled with 100 mL test solution. Size and type of vessel not reported.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 0.75E4
- Control end cells density:
- No. of vessels per concentration (replicates): 3 (4 for 100 mg/L as additional sacrificial vessel was prepared for analysis at 24 and 48 h)
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): No

GROWTH MEDIUM
- Standard medium used: Yes
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory prepared with purified water in accordance with OECD 201.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH = test start and end. Temperature and light intensity = test start, 24, 48 h and test end.

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH:
- Photoperiod: Continuous
- Light intensity and quality: 90 µmol/m2/sec in the wavelength 400-700 nm (actual: 86-89 µmol/m2/sec)
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell growth measured every 24 h
- Determination of cell concentrations: Electonic particle counter (Coulter Z2, Beckman Coulter Inc., Instrument No. CC-006)
- Chlorophyll measurement: Not measured
- Other: No

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: 0.1, 0.57, 3.2, 18 and 100 mg/L nominal
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is routinely tested. The results from the previous positive control test with potassium dichromate were within the normal ranges for this reference item.

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.025 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): Cells slightly enlarged/ bloated at 72 h in the 50 and 100 mg/L (nominal) test groups.
- Unusual cell shape: As above.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: EL50 and NOELR both exceed the solubility of the test item in the test solution. Solubility of the test item was established as up to 0.960 mg/L in the preliminary study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 1.3 mg/L
- Other: No

Reported statistics and error estimates:

EL50 / EC50 (growth rate): These were estimated as greater than the highest tested concentrations since 50 % inhibition was not observed at the highest tested concentration.
NOELR / NOEC (growth rate): Bartletts test was done to determine the homogeneirty of variance for the data. Then one-way analysis of variance and Dunnett's multiple comparison test was used to determine the significant difference between the control and the exposure levels. The statistical analysis was conducted using Microsoft Excel.

Table 1 Test solution pH values

Nominal loading concentration (mg/L)	pH
Nominal loading concentration (mg/L)	Test start	Test end
Control	7.9	7.8
3.1	7.9	7.8
6.3	7.9	7.8
13	7.9	7.8
25	7.9	7.8
50	7.9	7.8
100	7.9	7.8

Table 2 Cell Number at each timepoint

Nominal loading concentration (mg/L)	No.	Cell concentration (x10⁴cells/mL)
Nominal loading concentration (mg/L)	No.	0 h	24 h	48 h	72 h
Control	A	0.75	3.7	18	73
	B	0.75	4.1	18	66
	C	0.75	3.7	18	67
	D	0.75	3.9	18	74
	E	0.75	4.0	18	61
	F	0.75	3.6	18	60
	Mean	0.75	3.8	18	67
	SD	0	0.20	0.24	5.9
3.1	A	0.75	4.0	17	58
	B	0.75	4.0	18	51
	C	0.75	3.5	17	49
	Mean	0.75	3.8	17	52
	SD	0	0.33	0.79	4.6
6.3	A	0.75	3.9	17	45
	B	0.75	4.1	16	36
	C	0.75	4.1	17	43
	Mean	0.75	4.0	17	41
	SD	0	0.14	0.24	4.9
13	A	0.75	3.9	17	54
	B	0.75	3.8	19	70
	C	0.75	3.8	18	62
	Mean	0.75	3.9	18	62
	SD	0	0.085	0.94	8.2
25	A	0.75	4.1	15	29
	B	0.75	4.0	13	25
	C	0.75	4.4	15	34
	Mean	0.75	4.2	14	29
	SD	0	0.21	0.95	4.2
50	A	0.75	3.9	8.3	17
	B	0.75	3.9	8.8	17
	C	0.75	3.9	9.2	19
	Mean	0.75	3.9	8.8	18
	SD	0	0.014	0.44	1.1
100	A	0.75	4.0	10	22
	B	0.75	3.8	9.7	19
	C	0.75	3.5	9.3	18
	Mean	0.75	3.8	9.8	20
	SD	0	0.26	0.49	2.1

Table 3 Growth rate and growth rate inhibition

Nominal loading concentration (mg/L)	No.	Growth rate (0-3 d)	Growth rate inhibition (%)
Control	A	1.53	-
	B	1.49	-
	C	1.50	-
	D	1.53	-
	E	1.47	-
	F	1.46	-
	Mean	1.50	-
	SD	0.0294	-
3.1	A	1.45	3.3
	B	1.41	5.9
	C	1.39	7.1
	Mean	1.42	5.5*
	SD	0.0289	1.9
6.3	A	1.37	8.7
	B	1.29	14
	C	1.35	9.8
	Mean	1.34	11**
	SD	0.0407	2.7
13	A	1.43	4.7
	B	1.51	-1.2
	C	1.47	1.6
	Mean	1.47	1.7
	SD	0.0442	3.0
25	A	1.21	19
	B	1.18	21
	C	1.27	15
	Mean	1.22	19**
	SD	0.0473	3.2
50	A	1.04	31
	B	1.05	30
	C	1.08	28
	Mean	1.06	29**
	SD	0.0198	1.3
100	A	1.13	25
	B	1.07	28
	C	1.07	29
	Mean	1.09	27**
	SD	0.0339	2.3

* significant difference vs control (p<0.05)

** significant difference vs control (p<0.01)

Table 4 Measured concentrations of calcium in test item solutions

Nominal loading concentration (mg/L)	Measured concentration (mg/L)
Nominal loading concentration (mg/L)	At the start	24 hours	48 hours	At the end
Control a	4.75	4.91	4.89	4.61
Control b	4.69	4.90	4.81	4.67
3.1	4.68	-	-	4.61
6.3	4.73	-	-	4.52
13	4.67	-	-	3.97
25	4.72	-	-	3.97
50	4.81	-	-	4.21
100	4.90	5.11	4.98	4.26

n.d.: 1.26 mg/L