Olivine, cobalt silicate blue

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-12-10 to 2012-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 2004-04-13

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

adopted 2008

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:

The concentrations of dissolved cobalt (cobalt content of the test item is 41.4%) were measured in samples of all bulk test solutions at the beginning of the test and in pooled solution samples from each treatment level after 48 hours of exposure. Duplicate samples of approximately 20 mL were taken. Solutions were examined for soapy bubbles or particles floating on the surface. The samples were filtered (0.22 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 approximately 4°C until further analysis.
The concentration of dissolved cobalt was determined by ICP-OES. The results were assured by recovery experiments and analysis of certified reference materials.

Vehicle:

no

Details on test solutions:

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

Amounts of 1000, 320, 100, 32, and 10 mg test item were transferred into glass flasks and brought to the volume of 1 L with copper-reduced dilution water to obtain the nominal loadings. The contents of the bottles were agitated at 100 rpm for 7 days at 21.5°C in an incubation shaker. After this time the insoluble parts of the solutions 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 as test media in the acute toxicity test.

- Controls: control test media consisting of copper-free dilution water was taken directly from the tap immediately before use.

*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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Species: Daphnia magna
- Source: German Federal Environment Agency, Institute for Water, Soil, and Air Hygiene (Berlin)
- Age: 4-24 hours old (juvenile)
- Method of holding: batches of 30 to 50 daphnids were held at room temperature in approximately 1.8 L of dilution water for one week. For use in the test, newborn Daphnia were removed with a wide-bore pipette (to avoid damage) and isolated in fresh dilution water for at least 1 hour prior to being added to the test vessels.
- Food type/Amount: daphnids were fed with an algal suspension (Desmodesmus subspicatus containing Artemio® Fluid JBL). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a minimal volume. Thirty millilitresof this algal suspension were added to 1 L of medium. The medium was changed once per week.

The daphnids were not fed during the test.
- Frequency: daily

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

total hardness (holding and dilution water): 1.1 mmol/L

Test temperature:

19.8°C at test start and 20.4°C at test end

pH:

test start: 8.47 – 8.52
test end: 8.51 – 8.58

Dissolved oxygen:

Oxygen saturation:
test start: 5.3 – 7.7 mg O2/L
test end: 8.2 – 8.5 mg O2/L

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: 10, 32, 100, 320, and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL glass beakers
Approximately 50 mL of test medium was added to each replicate test vessel followed by five D. magna less than 24 hours old.
The test vessels were not aerated during the test.
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated local tap water was used in accordance with the OECD guideline 202 (2004). The tap water was sourced from the Schmallenberg district water production plants, mostly fed by small springs and percolation. The purification process on site at the laboratory included filtration with activated charcoal, passage through a lime-stone column, and aeration to the point of oxygen saturation. To avoid copper contamination, plastic water pipes were used in the test facilities.

Chemical properties of the dilution and holding water used during the test:
- Conductivity: 212.5 µS/cm
- pH: 8.9
- dissolved oxygen: 9.1 mg/L
- nitrate: 1.1 mg/L
- nitrite: <0.005 mg/L
- ammonium: <0.010 mg/L
- phosphate: 1.57 mg/L
- total hardness: 1.1 mmol/L
- alkalinity: 2 mmol/L
- calcium: 0.8 mmol/L
- magnesium: 0.3 mmol/L
- chromium: <0.091 µg/L
- cadmium: < 0.112 µg/L
- copper: <2.02 µg/L
- iron: < 2.20 µg/L
- manganese: <2.99 µg/L
- nickel: < 1.92 µg/L
- lead: < 1.11 µg/L
- zinc: <0.47 µg/L
- total residual chlorine: < 0.02 mg/L
- free chlorine: <0.02 mg/L
- bound chlorine: <0.02 mg/L
- DOC: 0.537 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 light / 8 dark
- Light intensity: 495 lux at test start and 486 lux at test end
The test was conducted in a temperature- and light-controlled incubator.

EFFECT PARAMETERS MEASURED:
- number of immobile daphnids in each treatment was visually determined daily and immobile daphnids were removed.
- any abnormalities in appearance and behaviour
- daphnids were classed as immobile when they did not swim 15 seconds after perturbation.

TEST CONCENTRATIONS
- Range finding study:
Two non-GLP range-finding tests were performed with dissolved fractions (T(D fractions) of loadings of 1 mg/L, 10 mg/L and 100 mg/L, and a control. Four replicates per treatment with five daphnids per replicate were exposed to the test item for a period of 48 hours. A chemical analysis was not performed. An immobilisation of 0, 100, and 0% was observed after 48 hours at the T/D loadings of 100, 10, and 1 mg/L, respectively, in the first pre-test. To adress the apparent inconsistency in the dose-related response, a 2nd pre-test was conducted. In the second pre-test, daphnids remained unaffected, i.e. mobile, in the control as well as at 10 mg/L and 1 mg/L (T/D loadings). An immobilisation of 15% was observed at the highest loading of 100 mg/L after 48 hours.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 1000 mg/L after 7 days (T/D fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 1000 mg/L after 7 days (T/D fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

dissolved fraction at a loading of 1000 mg/L after 7 days (T/D fraction)

Basis for effect:

mortality

Details on results:

Behavioural abnormalities/ other biological observations:
- mortality/immobilisation of Daphnia magna in control treatments was not observed after 24 and 48 hours, and it was also not observed after 24 hours at any of the treatments.
- after 48 hours, an average immobilisation of 5, 10 and 10% compared to the control group was measured at loadings of 100, 300 and 1000 mg/L, respectively (not statistically significant).
- no signs of disease or stress were observed.
- all surviving specimens appeared to be in a healthy condition.

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

The endpoint of the test was immobilisation, evaluated quantitatively by ELx, NOEL, and LOEL determination. The NOEL and LOEL were calculated by ANOVA followed by parametric pairwise comparisons of the treatments to the control using a Fishers Exact test with Bonferroni correction [OECD, 2006]*. Probit analysis was performed for the estimate of the EL50 and dose-response curve, assuming log-normal distribution of the values, when 50% immobilisation for treatments compared to the control was observed. The size of the effect detectable is reported. The computer software ToxRat® Professional was used for statistical evaluations [ToxRat® Professional 2.10]*.

*References:
- OECD (2006). OECD current approaches in the statistical analysis of ecotoxicity data: a guidance to application. Organisation for Economic Co-operation and Development, Paris.
- ToxRat® Professional 2.10. ToxRat® Solutions GmbH. Dr. Monika M. Ratte, Naheweg 15, 52477 Alsdorf, Germany.

Cumulative mortality after 24 and 48 h exposure to Co₂SiO₄.

Nominal conc. [mg/L]	0-24 h  % immobilisation	0-48 h % immobilisation
Control	0	0
10.0	0	0
32.0	0	0
100	0	5
320	0	10
1000	0	10

^*estimated based on combined cobalt measurements. 

Validity criteria fulfilled:

yes

Conclusions:

Based on results of this OECD 202 test, the 48-h EC50 for immobilisation of Daphnia magna by olivine, cobalt silicate blue is >1000 mg/L, and the respective NOEC is ≥ 1000 mg/L (all: dissolved fractions at a loading of 1000 mg/L 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 invertebrates. The respective 48-h EC50 for the effect of Reaction mass of cobalt olivine and crystalline silicon dioxide on mobility of freshwater invertebrates (Daphnia magna) derived in a GLP-conform OECD 202 test is estimated with > 1000 mg/L (dissolved fraction at a loading of 1000 mg/L after 7 days of dissolution) (Schlechtriem, 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

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

dissolved fraction at a loading of 1000 mg/L after 7 days of dissolution

Effect concentration:

> 1 000 mg/L