Cobalt Lithium Manganese Nickel Oxide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 January 2012 to 3 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OCSPP 850.4500: Algal Toxicity.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-390-0

EC Name:

-

Cas Number:

182442-95-1

Molecular formula:

LiCoNiMnO2 with the stoechiometry of Co+Ni+Mn equal to 1 and the ranges of the elements approximately as: Li: >0.90 - <1.20 Co: >0.0 - <0.50 Ni: >0.20 - <0.98 Mn: >0.0 - <0.50 O: 2

IUPAC Name:

Cobalt Lithium Manganese Nickel Oxide

Test material form:

other: solid (unspecified)

Details on test material:

- Name of test material (as cited in study report): Cobalt Lithium Manganese Nickel oxide
- Appearance: black solid
- Empirical formula: Li (Co 0.33 Mn 0.33 Ni 0.33) O₂
- Homogeneity: homogeneous
- Storage condition of test material: ambient; under nitrogen
- Storage stability: the stability of the test material under the storage conditions specified was guaranteed by the Sponsor for the duration of the test.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling was performed at the start of the exposure (0 hours) from the inoculated replicate 7 of each concentration and the control.
At the end of the exposure (96 hours) samples were taken from the combined inoculated replicates of the test concentrations and from the combined inoculated control group replicates. These samples were stored frozen until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Since the test material was considered to be poorly soluble in water, the test solutions were prepared following general guidance recommended by OECD 23 and US EPA OPPTS 850.1000 in order to achieve a saturated solution of the test material.
200.39 mg of test material was mixed with 2 L test medium. The mixture was stirred on a magnetic stir plate for about 7 days at room temperature. After this time, undissolved test material was removed by filtration with a membrane filter (Whatman OE66, pore width 0.2 µm). The filter was conditioned with several volumes of test solution before collecting the final solution. The control was treated in the same way. The exposure was started after separation of the undissolved test material by adding inoculum culture at a ratio of 1:100. The test solution after addition of algal inoculum was considered equivalent to the nominal concentration since no more than 1 % dilution occurred.
The water accommodated fraction (WAF) of the test solution appeared colourless and clear.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata KORSHIKOV (SAG 61.81)
- Age of inoculum (at test initiation): A stock algal culture was maintained continuously at the test facility. Before the exposure an inoculum culture was prepared from the stock culture and incubated for 4 days at 21 – 24 °C. After this time, the inoculum culture was in exponential growth phase and could be used to initiate the study.

CULTURE CONDITIONS
- Inoculum culture: An inoculum culture in exponential growth phase was prepared with an aliquot of the stock algal culture added to sterile test media to provide an initial cell density of 0.5 x 10⁴ cells/mL. The inoculum culture was incubated under test conditions for 4 days prior to test initiation. The increase in biomass was verified to ensure that growth was within the normal range and algal cells were examined microscopically to ensure normal morphology prior to use for test inoculation.
The inoculum culture cell density following 4 days of growth was 274 x 10⁴ cells/mL (548 fold increase). The inoculum culture morphology was normal and healthy.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Test temperature:

25.1 - 25.6 °C

pH:

7.6 (0 hours); 7.3 - 9.7 (96 hours)

Nominal and measured concentrations:

0 (test medium control) and 100 mg/L (nominal loading)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: closed; the flasks were plugged with gas permeable silicone sponge caps
- Fill volume: 100 mL
- Aeration: the vessels were continuously shaken at approximately 100 rpm
- Initial cell density: 0.5 x 10⁴ cells/mL
- No. of vessels per concentration (replicates): 6. An additional uninoculated (without algae) replicate per test group was included for background fluorescence correction.
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (AAP medium, as outlined in US EPA and ASTM guidelines)
- Detailed composition: 15.0 mg/L NaHCO3; 25.5 mg/L NaNO3; 12.16 mg/L MgCl2.6H2O; 4.41 mg/L CaCl2.2H2O; 14.6 mg/L MgSO4.7H2O; 1.044 mg/L K2HPO4; 0.160 mg/L FeCl3.6H2O; 0.300 mg/L Na2EDTA.2H2O; 0.186 mg/L H3BO3; 0.415 mg/L MnCl2.4H2O; 0.00327 mg/L ZnCl2; 0.00143 mg/L CoCl2.6H2O; 0.00726 mg/L Na2MoO4.2H2O; and 0.000012 mg/L CuCl2.2H2O.
The measured pH of the medium was 7.4. The medium was sterilised after preparation and prior to inoculation.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous illumination (artificial light, type universal white; OSRAM L 25). To minimise the potential effect of slight variations in illumination, the test vessels were rearranged daily.
- Light intensity and quality: Average 7081 lux (within ± 15 % variability) at a wavelength of 400 - 700 nm

EFFECT PARAMETERS MEASURED
The inoculum culture was observed microscopically at the start of the test to verify normal and healthy cells. At test termination, a pooled sample from each test concentration was examined and any abnormal appearance of the algae noted.
At the end of the exposure period cell density was determined by a direct microscopic count (two counts in a Neubauer haemocytometer).

PHYSICAL PARAMETERS MEASURED (see Table 1 outlining the scheme of measurements)
pH measurements were taken at 0 and 96 hours. Temperature was continuously measured during the whole test period in the climate chamber in a separate deionised water flask. Fluorescence measurements were taken at 0, 24, 48, 72 and 96 hours using a Tecan Infinite 200Pro fluorometer in a 96-well flat bottom black plate.
After the end of the exposure the control replicates were mixed and serially diluted by a factor of 2. The fluorescence of aliquots from the undiluted mixture and the dilutions were measured. These data were compared with the cell density measurements to derive a linear correlation between fluorescence and cell density. Light homogeneity was also evaluated by measuring light intensity at 5 locations within the incubation area at the start and end of the test.

TEST CONCENTRATIONS
- Range finding study: In a preliminary range finding test (72 hours) a concentration of 100 mg/L (loading ratio), filtered to remove undissolved test material, caused no inhibition of algal growth relative to the control after 96 hours. Therefore a limit test was conducted in the main study using a nominal loading of 100 mg/L test material.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The test material had no inhibitory effect on the algal yield or growth rate after 96 hours of exposure.
All of the test solutions were visibly clear and colourless after preparation and remained so through the end of exposure. No undissolved test material was visible and there were no other remarkable observations.

VALIDITY CRITERIA
The test was compliant with the following criteria:
- The control criterion for cell multiplication factor in the untreated control is > 100 in 96 hours. the cell multiplication factor in the untreated control (all replicates mixed together) was 582-fold after 96 hours (microscopically counted cell density: 291 x 10⁴ cells/mL).
- The control criterion for the coefficient of variation for mean control yield is ≤ 15 %. The coefficient of variation for mean control yield was 7.44 %.
- The control criterion for the coefficient of variation for average specific growth rate is ≤ 15 %. The coefficient of variation for mean control yield was 1.91 %.

Results with reference substance (positive control):

- Results with reference substance valid? Yes (the EC50 value was within the range of 0.92 - 1.46 mg/L, as specified in ISO 8692).
- EC50 (72 hours) = 1.45 mg/L

Any other information on results incl. tables

Analytical Determinations

The analytically determined concentrations of Co and Ni in the test material solutions were:

Co content: 19.9 g/100 g

Ni content: 19.7 g/100 g

 

Table 2: Analytically measured concentrations

Nominal loading rate (mg/L)	0 hours			48 hours
	Co (mg/L)	Ni (mg/L)	Test material (mg/L)	Mn (mg/L)	Ni (mg/L)	Test material (mg/L)
0	< 0.03	< 0.03	< 0.2	< 0.03	< 0.03	< 0.2
100	< 0.03	< 0.03	< 0.2	< 0.03	< 0.03	< 0.2

 

Both cobalt and nickel concentrations were below the LOQ at the start and end of exposure. The estimated equivalent amount of test material in solution was < 0.2 mg/L. Nevertheless, the test solutions were considered saturated with all soluble components of the test material, in test media and under test conditions.

All reasonable efforts were used to generate a saturated solution of the test material in test media following guidance in OECD 23 and OECD 29. Since the test material is a mixed metal oxide, the test solution is considered a water accommodated fraction (WAF).

 

Table 3: Growth inhibition results

Test group (loading rate, in mg/L)	Mean cell density (cells/mL) x 10⁴ at 96 h	% Inhibition (as yield)*	% Inhibition (as growth rate)*
0	271	0	0
100	301	-11.1	-0.954

* Negative values indicate a stimulatory effect relative to the control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, a saturated solution of the test material prepared at a loading rate of 100 mg/L in test media had no observable phytotoxic effect on the algae. The 96-hour EL50 was therefore determined to be in excess of 100 mg/L for both yield and growth rate. The 96-hour NOELR was determined to be 100 mg/L for both yield and growth rate. The results of the study demonstrate that the test material is not toxic to algae up to the limit of solubility.

Executive summary:

The toxicity of the test material to freshwater algae was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 201, EU Method C.3 and OCSPP 850.4500.

During the study, cultures of Pseudokirchneriella subcapitata were exposed to the test material at nominal concentrations of 0 (control) and 100 mg/L, under static conditions, for 96 hours. At the end of the exposure period cell density was measured and any abnormal appearance in the algae was noted.

The content of cobalt and nickel in the test solutions was analysed during the study. Both cobalt and nickel concentrations were below the LOQ at the start and end of exposure. The estimated equivalent amount of test material in solution was < 0.2 mg/L. Nevertheless, the test solutions were considered saturated with all soluble components of the test material, in test media and under test conditions.

Since the algae were exposed to a saturated solution of test material at the given loading rate, the effect concentrations were based on the loading rate (as recommended in OECD 23).

Under the conditions of the study, a saturated solution of the test material prepared at a loading rate of 100 mg/L in test media had no observable phytotoxic effect on the algae. The 96-hour EL50 was therefore determined to be in excess of 100 mg/L for both yield and growth rate. The 96-hour NOELR was determined to be 100 mg/L for both yield and growth rate. The results of the study demonstrate that the test material is not toxic to algae up to the limit of solubility. The media pH, temperature and light intensity were all maintained within acceptable guideline specifications during the study.