Olivine, cobalt silicate blue

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:

supporting study

Study period:

2012-10-22 to 2012-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 2011-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on testing and assessment, Number 29. Guidance document on transformation/dissolution of metals and metal compounds in aqueous media. ENV/JM/MONO (2001)9, 23-July 2001.

Version / remarks:

According to OECD Series on Testing and Assessment # 29 (2001), “For hazard classification purposes the results of the dissolution/transformation protocol are compared with existing ecotoxicity data for metals and metal compounds. However, for purposes such as data validation, there might be cases where it may be appropriate to use the aqueous medium from a completed transformation test directly in an OECD 202 and 203 daphnia and fish ecotoxicity test. If the CaCl2.2H2O and MgSO4.7H2O concentrations of the transformation medium are reduced to one-fifth of the ISO 6341 medium, the completed transformation medium can also be used (upon the addition of micronutrients) in an OECD 201 algae ecotoxicity test.” Therefore, to assess the hazard potential of this pigment containing several different metals, an ecotoxicity validation test was performed applying the dissolved fraction collected after 7-d of transformation/dissolution of the pigment in the respective ecotoxicity medium.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2011-02-07

Specific details on test material used for the study:

not applicable

Analytical monitoring:

yes

Details on sampling:

- Sampling method/Sample storage conditions before analysis:
Dissolved cobalt concentrations were measured in all test solutions at the beginning of the test prior to the addition of the algae and in samples of the algae cultures taken from replicates pooled per treatment at the end of the test.
Duplicate samples of approximately 20 mL were taken. The samples were filtered (0.2 µm polyether sulphone membrane syringe filter) at room temperature (20-25°C), transferred into disposable polyethylene vials (Scintillation vials), acidified with HNO3 (final concentration 1 % HNO3), and stored at 4°C until chemical analysis. The concentrations of dissolved cobalt were determined by ICP-OES. The results were assured by recovery experiments and analysis of certified reference materials.

Results:
The limit of detection (LOQ) and the limit of quantification (LOQ) for olivine, cobalt silicate blue was 0.309 µg/Ldissolved Co were 0.257 μg/L and 0.770 μg/L, respectively.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
As the test item is a multi-component, sparingly soluble substance, its toxicity was examined using the dissolved fraction(s) after 7-d Transformation/dissolution (T/D fraction). The T/D fraction for the various test concentrations were prepared individually in accordance with the OECD guidance document on transformation/ dissolution of metals and metal compounds in aqueous media No. 29 (2001)*.

Amounts of 0.05, 0.5, 5.0, 50 and 500 mg test item were transferred into glass flasks and brought to the volume of 500 mL with growth medium to obtain T/D loadings of 0.1, 1.0, 10.0, 100 and 1000 mg/L, respectively. One flask was filled with 1000 mL of growth medium for the control. Flasks were shaken at 100 rpm for 7 days at room temperature (~20°C). For a preparation of test solutions the insoluble parts of each solution were separated from the aqueous phase by filtration through a 0.22 µm filter (Syringe Filters DIAFIL PS (Polyether sulfone, PES)) under sterile conditions. The filtrated T/D fractions were subsequently used for the algal growth test.

- Controls: growth medium only was used as control.

*Reference:
- OECD Series on testing and assessment, Number 29. Guidance Document on transformation/dissolution of metals and metal compounds in aqueous media. ENV/JM/MONO (2001)9, 23-July 2001

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Species: Pseudokirchneriella subcapitata, Chlorophycea, Chlorophyta
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Germany, Catalog No 61.81 SAG.

ACCLIMATION
- Culturing media and conditions: prior to testing, a pre-culture is established in the OECD 201 growth medium to obtain exponentially-growing algae for the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

no data

Test temperature:

22.0 to 22.5 °C

pH:

Controls: test start: 8.01; test end: 8.03 to 8.11
Test solutions: test start: 8.26 to 9.03; test end: 8.27 to 9.23

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: 0.1, 1.0, 10.0, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile 250 mL conical glass flasks covered with air-permeable silicone-sponge caps.
- Initial cells density: cell density of the inoculum culture was determined, and then an adequate aliquot of the inoculum culture (653.6 µL) was added to each replicate to obtain the required cell density of 10,000 cells/mL.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8
Test vessels were filled with 100 mL of the respective test solution.

GROWTH MEDIUM
The sterilised synthetic OECD medium according to OECD 201 (2011) was used as growth medium. The molar ratio of EDTA to iron slightly exceeds unity in this medium. This prevents iron precipitation and at the same time, chelation of heavy metal ions is minimised (molar ratio EDTA / Fe(III+) = 1.135). Considering the molar ratio and the stability constants of EDTA with Fe(III+) and Co(II+) ions, the chelation of cobalt remains negligible in comparison with nominal test concentrations. All stock solutions and the medium were prepared with purified water processed using a water purifier "Pure Lab Ultra". Please also refer to table 1 in the field "Any other information on materials and methods incl. tables" below.

OTHER TEST CONDITIONS
- Light intensity and quality: culture vessels were continuously illuminated with a light intensity (day light: OSRAM “cool white”) of 7089 to 7253 lux close to the surface of the liquid. Light measurements [lux] were made using a cosine (2 π) receptor (LI-189 with radiation sensor, LI-COR) each day.
- test culture temperature was measured daily in one test vessel per day.
- pH of the solutions was measured in one test vessel per test concentration, including the control, at the start of the test and in every test vessel at the end of the test.
- algae cultures were continuously resuspended by rotation on an orbital laboratory shaker (150 rpm) (INFORS, Switzerland).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter (CASY 1 Model TT, Roche Diagnostics GmbH, Germany)
The accuracy of the electronic counts was checked against manual cell counts under the microscope, as per internal standard operation procedures.
The cell concentrations were determined after 24, 48 and 72 hours in samples taken directly from the test vessels.

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: based on the results of the range-finder test (concentrations tested: 1, 10, and 100 mg/L), the loadings of the test item were selected.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at specified loading after 7 days (T/D fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95%-CL: 14 - 25

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 10 mg/L after 7 days (T/D fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at specified loading after 7 days (T/D fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95%-CL: 81 - 97

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 100 mg/L after 7 days (T/D fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at specified loading after 7 days (T/D fraction)

Basis for effect:

other: yield

Remarks on result:

other: 95%-CL: 6 - 17

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at loading of 10 mg/L after 7 days (T/D fraction)

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at specified loading after 7 days (T/D fraction)

Basis for effect:

other: yield

Remarks on result:

other: 95%-CL: 18 - 53

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 100 mg/L after 7 days (T/D fraction)

Basis for effect:

other: yield

Details on results:

- a loading- dependent inhibiting effect on the cell number increase of P. subcapitata was observed at the highest loadings of 100 and 1000 mg/L. Corresponding microscopy revealed normal appearances of the algae cells.
- growth rate: a loading- dependent inhibition was observed after exposure to the test item for 72 hours at the two highest treatments. The inhibition of growth rate was 54.4 and 95.3 % at the nominal loading of 100 mg/L and 1000 mg/L, respectively.
- NOEL of 10 mg/L was estimated for growth rate inhibition. The EL50 was determined to be 89 mg/L.
- inhibition of yield at the two highest loadings of 100 and 1000 mg/L was 95.9 and 99.9 % after 72 hours of exposure, respectively.
- NOEL of 10 mg/L was estimated for yield. The EL50 was determined to be 28 mg/L.
Please also refer to "Any other information on results incl. tables" and "Attached background material" below.

Results with reference substance (positive control):

The nominal ErC50 value of 2.42 mg/L (95 % confidence limits 1.95 – 3.03 mg/L) is in good agreement with the results of an international ring test with ErC50 of 3.38 ± 1.30 mg/L (International Organisation for Standardisation, 1993)*.

*Reference:
- International Organisation for Standardisation (1993). ISO 8692 Water quality - Algal growth inhibition test.

Reported statistics and error estimates:

- The evaluation of the concentration-effect relationship and calculations of effect concentrations were based on loadings of the test item.
- The mean value of the cell counts for each treatment was used for plotting growth curves.
- Calculation of the percent inhibition of growth rate [r] and yield [y] was performed according to the guideline OECD 201 (2011) and was plotted as a function of the loading.
- Data showing inhibition around 50 % was statistically analysed to determine EL50, EL20 and EL10 values together with 95 % confidence intervals using Probit-analysis (OECD, 2000)* assuming log-normal distribution of the data. Individual replicate responses were used for the regression analysis.
- The NOEL and LOEL values for growth rate and yield were determined using the Williams t-test (Williams, 1972)*.
- The computer program ToxRat Professional was used for all statistical evaluations (ToxRat® Professional 2.10.)*.

*References:
- OECD (2000). OECD guidance document on aquatic toxicity testing of difficult substances and mixtures. Series on testing and assessment, number 23. Organisation for Economic Co-operation and Development, Paris. ENV/JM/MONO (2000).
- Williams, D.A. (1972): The comparison of several dose levels with a zero dose control. Biometrics 28, 519-531.
- ToxRat® Professional 2.10. ToxRat® Solutions GmbH, Naheweg 15, 52477 Alsdorf (http://www.toxrat-solutions.de).

Table: Percent inhibition of growth rate and yield at different loadings compared to controls after 72 h.

loading [mg/L]	% Inhibition of growth rate	% Inhibition of yield
Control	0.0	0.0
0.1	-0.8	-5.6
1.0	1.2	6.0
10	0.7	4.0
100	54.5	95.9
1000	95.3	99.9

Validity criteria fulfilled:

yes

Conclusions:

A loading-dependent inhibition of the growth rate of P. subcapitata was observed in an algae growth inhibition test according to OECD 201. A NOEC of 10 mg/L was estimated for the growth rate inhibition whereas the respective EC10 and EC50 amount to 20 and 89 mg/ (all: dissolved fraction at specified loading after 7 days (T/D fraction)).

Description of key information

Results of a supporting study with Reaction mass of cobalt olivine and crystalline silicon dioxide (Olivine, cobalt silicate blue pigment) indicate a low potential for bioavailability and acute toxicity to fish. The 72-h EC10 and EC50 for the effect of Reaction mass of cobalt olivine and crystalline silicon dioxide on growth rate of freshwater alga (Pseudokirchneriella subcapitata) derived in a GLP-conform OECD 201 test amount to 20 and 89 mg/L based on testing the dissolved fractions of individual loadings after 7 days of dissolution, respectively (Wenzel, 2013). Nevertheless, read-across to the assessment entity soluble cobalt substances is applied since cobalt cations determine fate and toxicity in the environment.

Key value for chemical safety assessment

EC50 for freshwater algae:

89 mg/L

EC10 or NOEC for freshwater algae:

20 mg/L