[No public or meaningful name is available]

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VII, section 9.1.1, column 2 of REACH Regulation, long-term toxicity on invertebrate testing shall be proposed by the registrant when it is unlikely that short-term toxicity testing can provide a true measure of the intrinsic aquatic toxicity of the substance for instance for nanoforms with low dissolution rate.

The rate of dissolution gives information on how many ions/molecules are released from the particle over time. The ion(s)/ molecules released may also dictate the toxicity of the nanoforms, which will be an important aspect of the evaluation (ECHA Guidance, 2019).

The dissolution rate half-time (when half of the nanomaterial is left and half is dissolved) of the substance in phagolysosomal fluid at pH 4.5 ranges from 2.9 to 6 days. According to OECD No. 62 this dissolution rate is regarded as moderate to high.

The substance is composed of amorphous and crystalline silica and chalk (CaCO3). After dissolution calcium and carbonate ions are released, which are extremely common in all natural surface waters and are therefore ubiquitous in the environment. Even if the dissolution rate in environmental media at pH 7 would be lower the particles are expected to agglomerate in these media and no cellular uptake is expected. Furthermore, silica and chalk are natural constituents of different types of soil (e.g. sand) and can be found ubiquitous in nature as well.

Seawater contains approximately 400 ppm calcium and rivers generally contain 1-2 ppm calcium but in lime areas rivers may contain calcium concentrations as high as 100 ppm (Lenntech, 2010). One of the main reasons for the abundance of calcium in water is due to its natural occurrence in the Earth’s crust. These calcium-rich rocks undergo physical and/ or chemical weathering in the environment (http://csetprep.org/science/CycleCa.htm). Weathering of calcium carbonate is considered a congruent reaction in which the total mineral goes into solution. These calcium sediments are carried by water from the mountains to oceans or lakes as well as to the land portion of the Biosphere and thereby become part of the calcium and carbon cycles. This natural abundance of calcium and carbonate ions in the environment means that aquatic organisms including invertebrates are constantly exposed to calcium carbonate without suffering from any adverse or detrimental effects. Calcium carbonate is also directly applied to lakes to mitigate the effects of surface water acidification (Driscoll et al., 1987). Lake/ watershed systems that cannot completely neutralise strong acid inputs are characterised by low pH values and elevated concentrations of trace metals. Populations of fish and other aquatic biota are endangered by this phenomenon. However, the direct application of calcium carbonate can effectively mitigate the chemical effects of lake acidification and ensure the survival of aquatic species. Therefore, this use implies that calcium carbonate is not toxic to aquatic invertebrates following long-term exposure. Furthermore, both calcium and carbonate are essential constituents of living organisms, including invertebrates. Calcium is an essential element to crustaceans and other groups of animals with a calcified exoskeleton (Alstad et al., 1999). Soft waters are characterised by low calcium concentrations and the distribution and relative success of calcium demanding invertebrates can be limited by low calcium in extreme soft water localities. A further calcium depletion caused by reversed acidification could therefore seriously affect freshwater crustaceans. The use of calcium carbonate to mitigate the effects of surface water acidification and the fact that calcium is essential to species with a calcified exoskeleton indicates that calcium carbonate cannot be toxic to aquatic invertebrates following long-term exposure.

An acute toxicity study to Daphnia magna was performed according to OECD 202 with a saturated solution (100% v/v) of calcium carbonate (nano) (Priestly, 2010). No immobilisation or toxic effects were observed in any of the Daphnia magna exposed. As a result, calcium carbonate is considered not acutely toxic to aquatic invertebrates.

In conclusion, calcium carbonate nanoparticles show no toxicity to Daphnia magna. In general, agglomeration of particles is expected under environmental conditions which will hinder cellular uptake in vivo.

Based on this data an adverse effect on daphnia can be excluded for the substance and a long-term toxicity test is scientifically not justified.


References
Alstad NEW, Skardal L and Hessen DO (1999) Limnol. Oceanogr., 44(8): 2011-2017

Driscoll CT, Fordham GF, Ayling WA and Oliver LM (1987) The Chemical Response of Acidic Lakes to Calcium Carbonate Treatment, Lake and Reservoir Management: Volume III, Limnological Effects of Liming, Issue 1: 404-411

ECHA Guidance, 2019: Appendix R.6-1 for nanoforms applicable to the Guidance on QSARs and Grouping of Chemicals

Lenntech (2010) Calcium (Ca) and Water, www.lenntech.com/periodic/water/calcium/calcium-and-water.htm

OECD, “Series on the Safety of Manufactured Nanomaterials- No. 62 Considerations for Using Dissolution as a Function of Surface Chemistry to Evaluate Environmental Behaviour of Nanomaterials in Risk Assessments. ENV/JM/MONO(2015)44.,” 2015. [Online]. Available: http://www.oecd.org/env/ehs/nanosafety/publications-series-on safety-of-manufactured-nanomaterials.htm.

Priestly SL (2010) Calcium carbonate (nano): Acute Toxicity to Daphnia magna, Harlan Laboratories Ltd, Report No. 2974/0013

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1 February 2010 - 26 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Uncoated nano CaCO3

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- The test item did not form a solution using conventional methods such as ultrasonication and high shear mixing. Therefore, a saturated solution of the test item was prepared by stirring an excess (50 mg/L) of test item with reconstituted water for 48 hours at approximately 21 °C, then removing the undissolved test material by filtration through a pre-conditioned filter (0.2 µm) to give a saturated solution (100% v/v).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: Laboratory culture
- Age at study initiation (mean and range, SD): Less than 24 hours old
- Method of breeding: Culture conditions ensured that reproduction was by parthenogenesis.
- Feeding during test: No
- Food type: Suspension of algae (Chlorella sp.)
- Frequency: Fed daily during culturing

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test temperature:

Control: 19-21 °C
Test vessels: 20-21 °C

pH:

Control: 7.8-7.9
Test vessels: 7.9-8.4

Dissolved oxygen:

Control: 96-100% ASV (air saturation value)
Test vessels: 95-100% ASV

Control: 8.7-9.3 mg O2/L
Test vessels: 8.6-9.1 mg O2/L

Nominal and measured concentrations:

Nominal concentration: 100% v/v saturated solution of test material

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 250 mL glass jars
- Aeration: Not during the test. During preparation, the reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
- No. of organisms per vessel: 5 daphnids/ vessel
- No. of vessels per concentration (replicates): 4 replicates/concentration
- No. of vessels per control (replicates): 4 replicates


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water
- Conductivity: <5 µS cm-1
- Culture medium different from test medium: No
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: Only if necessary during preparation of the reconstituted water, with NaOH or HCl (to pH 7.8 ± 0.2)
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: No data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Based on the results of the range-finding test a limit test was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no immobilisation or adverse reactions to exposure were observed.
- Range finding study
- Test concentrations: 1.0, 10 and 100% v/v saturated solution of test material
- Results used to determine the conditions for the definitive study: No immobilisation was observed at the test concentrations of 1.0 and 10% v/v saturated solution. A single immobilised daphnid was observed at the 100% v/v saturated solution test concentration after 48 h exposure. This was considered to be due to natural causes rather than a toxic effect and was considered not to be significant given that only 10% immobilisation was observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

other: saturated solution of test material

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

other: saturated solution of test material

Basis for effect:

other: zero immobilisation

Details on results:

There was no immobilisation in 20 daphnids exposed to a test concentration of 100% v/v saturated solution for a period of 48 hours.

The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

The study showed that there were no toxic effects at saturation.

Results with reference substance (positive control):

24 h EC50: 0.84 mg/L (95% confidence limits 0.72-0.97 mg/L)
48 h EC50: 0.65 mg/L (95% confidence limits 0.58-0.72 mg/L)
48 h NOEC: 0.32 mg/L

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were satisfied.

Conclusions:

The acute toxicity of calcium carbonate (nano) to Daphnia magna has been investigated and gave a 48 h EC50 of >100% v/v saturated solution. The NOEC was 100% v/v saturated solution.
The study showed that there were no toxic effects at saturation.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

water solubility

Remarks:

dissolution rate

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

other: ISO 19057:2017 „Continous Flow System“ (CFS)

GLP compliance:

no

Type of method:

other: Continous Flow System (CFS)

Key result

Remarks on result:

other: The dissolution rate halftime ranges from 2.9 to 6 days.

Details on results:

The halftime of residual mass T1/2 is directly related to the dissolution rate k by k=ln2/(BET*T1/2). Here we compare the CSH nanoforms on their halftimes, because we anticipate less systematic errors by this descriptor. The halftimes were determined for the dissolution of Si and Ca as the main inorganic elements of CSH. However, only for Si the biological background is sufficiently low to detect the CSH dissolution, whereas the Ca background in the phagolysosomal simulant fluid at pH4.5 is nominally 8 mg/L,(Stefaniak, Guilmette et al. 2005) and the Ca dissolution from CSH nanoforms is not detectable on top of this background.

Conclusions:

Compared to the physiologically relevant timescales, which span a logarithmic range of hours to years, represented by benchmark materials of quick dissolution (e.g. ZnO NM110) to very slow dissolution (e.g. TiO2 NM105), the dissolution halftime is similar amongst all CSH nanoforms. The span ranges from 2.9 to 6 days.