Cerium dioxide

Toxicity to other aquatic organisms

Administrative data

Endpoint:

toxicity to other aquatic vertebrates

Remarks:

This data was reported here to be in the "aquatic toxicity" section, but it concerns an invertebrate organism

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

; however, it should be kept in mind that the chosen administration route of nano-CeO2 (i.e., forced ingestion) was not representative of environmental conditions.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

5-day exposure of Paracentrotus lividus to nanometric cerium dioxide (nano-CeO2) to assess the acute toxicity

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Cerium dioxide

EC Number:

215-150-4

EC Name:

Cerium dioxide

Cas Number:

1306-38-3

Molecular formula:

CeO2

IUPAC Name:

cerium dioxide

Test material form:

solid: nanoform

Details on test material:

- Name of test material: Nanometric cerium oxide (nano-CeO2)
- Supplier: None (in-house synthesis using wet chemistry method)
- Substance type: Monoconstituent substance
- Substance form: Nanoparticulate substance
- Primary particle size (SEM, XRD): Average diameter of 16-22 nm (data from De Marzi et al., 2013)
- Particle size distribution (SEM): Presence of ~40-nm clusters (data from De Marzi et al., 2013)
- Stability: Slight agglomeration/aggregation based on SEM observations (data from De Marzi et al., 2013)
- Specific surface area: No data available
- Surface charge (DLS): Zeta potential of -46.2 mV in water at pH 7.4 (data from De Marzi et al., 2013)
- Isoelectric point: No data available
- Shape (SEM): No data available
- Crystallinity (XRD): No data available
- Analytical purity / impurities: No data available
- Number density of nano-CeO2 in the suspension: No data available
- Cerium content in nano-CeO2 suspension: No data available
- Solubility: No data available
- Oxidation degree: No data available
- Surface properties (SEM, FTIR): Smoothed and non-porous surface; detection of Ce-O bond, O-H bond in water adsorbed on the sample surface and residual surfactant (CH2) (data from De Marzi et al., 2013)
- Lot/batch No.: No data available
- Expiration date of the lot/batch: No data available

Further explanations on the physico-chemical characterisation of CeO2 nanoparticles are presented below in "any other information on materials and methods incl. tables".

Specific details on test material used for the study:

- Name of test material: Nanometric cerium dioxide (nano-CeO2)
- Supplier: Nanostructured & Amorphous Materials, Inc (USA)
- Substance type: Monoconstituent substance
- Substance form: Nanoparticulate substance / nanomaterial in suspension
- Primary particle size: 50 to 105 nm calculated from surface specific area (SSA) value and transmission electron microscopy (TEM) observations
- Particle size distribution: No data available
- Stability: No data available
- Specific surface area: 8 to 15 m²/g (supplier's data)
- Surface charge: No data available
- Isoelectric point: No data available
- Shape: No data available
- Crystallinity: No data available
- Analytical purity / impurities: ≥ 98% purity
- Number density of nano-CeO2 in the suspension: No data available
- Cerium content in nano-CeO2 suspension: No data available
- Solubility: No data available
- Oxidation degree: No data available
- Surface properties: No data available
- Lot/batch No.: No data available
- Expiration date of the lot/batch: No data available

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The nano-CeO2 powders were diluted in sea water in the amount of 10E-2 and 10E-4 g/L, and mixed by a high speed vortex, up to complete dispersion.

Test organisms

Test organisms (species):

other: Paracentrotus lividus

Details on test organisms:

TEST ORGANISM
- Common name: Sea urchin
- Strain: No data available
- Source: Unpolluted Portofino Natural Protected Area (Italy)
- Age at study initiation: Adult specimens
- Weight at study initiation, method of breeding: No data available
- Length at study initiation: 50-60 nm
- Feeding during test: No data available

ACCLIMATION
- Acclimation period: One week
- Acclimation conditions: Sea urchins were maintained in tanks containing ultra-filtered and pasteurized sea water, in ration of 1 specimen per 10 L. The water was oxygenated and maintained at 16°C.
- Type and amount of food, feeding frequency, health during acclimation: No data available

QUARANTINE
No data available

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
No data available

Study design

Test type:

other: Not applicable

Water media type:

saltwater

Limit test:

no

Total exposure duration:

5 d

Post exposure observation period:

Not included

Test conditions

Hardness:

No data available

Test temperature:

No data available

pH:

No data available

Dissolved oxygen:

No data available

Salinity:

No data available

Nominal and measured concentrations:

10E-4 and 10E-2 g/L, corresponding to 0.1 and 10 mg/L (nominal nano-CeO2 conc.)

Details on test conditions:

TEST SYSTEM
- Test vessel: Tanks
- Type, material, size, headspace, fill volume, aeration, type of flow-through, renewal rate of test solution: No data available
- No. of organisms per vessel: 1 per vessel
- No. of vessels per experimental condition (replicates): See below

The introduction of the nano-CeO2 solutions was performed immediately after powder dispersion, to ensure homogeneous distribution of the powders into the sea urchin mouth by use of a syringe with a blunt needle. Groups of 5-10 animals were forced to ingest the nanoparticle dispersions in a volume of 1 mL and kept in separate tanks. Coelomocytes were harvested 5 days after treatment as an endpoint of acute exposure. Briefly, the coelomic fluid, containing about 107cells per mL, was drawn from each sea urchin by a syringe inserted in the peristomal membrane around the Aristotele’s lantern containing an equal volume of an anticoagulant buffer to prevent clotting.
At least 4 independent experiments were performed.

TEST MEDIUM / WATER PARAMETERS
No data available

OTHER TEST CONDITIONS
No data available

EFFECT PARAMETERS MEASURED:
- Uptake of nano-CeO2 in sea urchin coelomocytes: Cells were fixed with 2% paraformaldehyde and prepared for microscopic inspection or environmental scanning electron microscopy analyses coupled to dispersive X-ray spectroscopy (ESEM-EDS). Coelomocytes (50 mL) were smeared on acetate slides, permeabilized by methanol 100% and subsequently rehydrated. The nano-CeO2 present inside the coelomocytes was identified by ESEM, equipped with EDS. The electron microscope analyses were performed by catching Back Side Electrons (BSE), in order to obtain information on the chemical nature of the samples, rather than their morphology.

- Morphological changes of subcellular compartments: The smears of coelomocytes were stained for 3 min at room temperature in 4% propidium iodide in modified Tyrode (1912) physiological solution, in order to visualize cell nuclei. In parallel, cells were incubated for 30 min at room temperature in 0.1 mg/mL wheat germ agglutinin (WGA), conjugated with fluorescein isothiocyanate (FITC). As WGA binds terminal sialic or neuraminic acid of glycoconjugates, it is considered a good marker of the trans-Golgi, including lysosomes. Staining of the endoplasmic reticulum (ER) was performed for 3 min at room temperature, in the dark, by the use of 2.5 mg/mL 3,3’-Dihexyloxacarbocyanine iodide (DiOC6), a fluorescent dye which binds to the membranes via its hydrophilic groups. Images were acquired by a confocal laser scanning microscope.

- Alteration of cholinesterase activities: The collected cells (~10E7) were washed twice in ice-cold PBS, centrifuged at 10,000 rpm for 3 min at room temperature and the cell pellets were then homogenized, and centrifuged for 3 min at 10,000 rpm at room temperature. The supernatant was collected. AChE, BChE and PChE activities were measured on cells lysates corresponding to about 10 µg of proteins with a classical colorimetric assay in a final volume of 3 mL. The colorimetric reaction was recorded for 18 min by spectrophotometer readings at 412 nm. Negative control experiments included a 30 min pre-incubation with selective inhibitors. Each measurement was performed three times, and each time at least in triplicate.

- Expression of stress and inflammation biomarkers: Coelomocytes were collected, as a total cell population, from 80 sea urchins, 20 sea urchins treated with nano-CeO2 (10E4g/L) and 20 from controls. Specifically, specimens were obtained from four independent experiments: 5 specimens, for each experiment, and 5 for controls. About 10E7 collected cells from each sample were centrifuged at 10,000 rpm for 3 min. The pellets were homogenized in a lysis buffer. The protein contents of the extracts were quantified by the Bradford method (1976). Ten micrograms of protein per each cell extract were run on a 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with an antibovine brain HSC70 or a rabbit polyclonal antibody to GRP78 human synthetic peptide. Stress protein levels were normalized with actin levels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 100
- Justification for using less concentrations than requested by guideline, range finding study: No data available

Reference substance (positive control):

no

Results and discussion

Details on results:

Sea urchin adults were first monitored for their physiological conditions, in terms of motility, lack of spines and lethality. Falugi C et al. observed that none of the specimens treated with 10E-2 g/L CeO2 solution survived after 1-2 days (data not shown). On the contrary, sea urchins treated with 10-4 g/L CeO2 could be maintained for at least 5 days, with no major signs of a pathological state.
 
- NANO-CeO2 UPTAKE:
The target organs for observation of nano-CeO2 uptake and accumulation were: the digestive apparatus, the reproductive and immune systems. However, in their study, Falugi C et al. only described the results obtained at the level of the immune cells, namely coleomocytes. The authors detected cell groups which did not show any CeO2 uptake in control specimens. Nano-CeO2 was accumulated as agglomerates engulfed by coelomocytes. In the aqueous (saline) medium, particles of different dimensions were observed, including agglomerates ranging in size from 1-2 mm to about 10 mm (data not shown).
Considering these features, Falugi C et al. hypothesised that nanoparticles were internalized and highly accumulated into coelomocytes, via both phagocytosis and endocytosis. The EDS analysis confirmed the chemical composition of particles present inside the cells.
 
- MORPHOLOGICAL ALTERATIONS:
Control cells showed numerous strongly stained vesicles, rather uniformly distributed within the cytoplasm. Coelomocytes drawn from sea urchins fed with nano-CeO2 showed a severe reduction in the number of stained vesicles, the location of which seemed to be polarized at one side of the cell. The endoplasmic reticulum (ER), stained by DiOC6 showed a network of flat vesicles in controls, more dense around the nucleus. Cells from sea urchins fed with CeO2 presented strictly packed membranes, not resolved by the confocal imaging.

- IMPAIRMENT OF CHOLINESTERASE ACTIVITIES:
A statistically reduced enzymatic activity of the three different ChE isoforms (AChE, BChE and PChE) was found in all CeO2 exposed coelomocytes. The most significant reduction in the enzymatic activity was found for the PChE isoform.  
 
- EXPRESSION OF STRESS AND INFLAMMATION BIOMARKERS:
HSC70 was reduced in samples exposed to CeO2 nanoparticles (2.4-fold reduction compared to control levels). Similarly, the levels of GRP78 were reduced in samples exposed to nano-CeO2 (2.7-fold reduction with respect to controls). The mean values of HSC70 and GRP78 levels were always significantly different from controls, as determined by one-way ANOVA, followed by the Tukey multiple comparison test.

Reported statistics and error estimates:

Cholinesterase enzymatic activities were analysed for significance by One-way analysis of variance (ANOVA) followed by the Student T Test, and by the use of the nonparametric method of Mann and Whitney. The mean values of the immunoblotting bands intensities were compared using the ANOVA, followed by the multiple comparison Tukey test. The level of significance was set to P ≤ 0.05.

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

After forced ingestion, nano-CeO2 was found inside the immune cells (i.e., coelomocytes) of the sea urchin (Paracentrotus lividus). Nanoparticles internalization caused harmful modifications of the trans-Golgi and endosplasmic reticulum subcellular compartments. In parallel, the enzymatic activity of three ChE isoforms was inhibited and the expression levels of two stress proteins (HSP70 and GRP78) were reduced. The authors suggested that uptake into phagocytic coelomocytes might be a detoxification strategy used to cope with hazardous materials and to promote survival. Since biomarkers of innate immunity and endoplasmic reticulum stress were found to be down-regulated five days after exposure, it might be hypothesised that the suppression of immune signalling may be a useful feedback response for helping cells to survive tissue injury.

Executive summary:

Falugi C et al. (2012) used the sea urchin (Paracentrotus lividus) as an in vivo model to assess the toxicological effects of nanometric cerium dioxide (nano-CeO2) at the cellular and molecular levels. There was no mention regarding the application of a particular guideline or GLP.

 

Adult urchins (50 to 60 mm) were collected in unpolluted areas, and maintained for one week before testing in laboratory tanks containing ultra-filtered and pasteurized seawater at 16 °C. Nano-CeO2 suspensions at 0.1 and 10 mg/L were prepared in seawater by mixing with a high speed vortex. Groups of 5 to 10 urchins were forced to ingest the suspensions using a syringe with a blunt needle that was introduced into the urchin mouth. The organisms were kept individually in 1 L tanks. The same number of controls was kept in separate tanks under identical conditions but without nanoparticles. Coelomocytes were harvested five days after treatment, and the following parameters were evaluated:

-     Uptake and accumulation into coelomocytes: the nanoparticles present inside the coelomocytes were identified by ESEM-EDS analyses.Electron microscope analyses were performed to obtain information on the chemical nature of the samples.

-     Impact on coelomocytes subcellular compartments: trans-Golgi apparatus and endoplasmic reticulum were examined after staining with WGA-FITC (wheat germ agglutinin conjugated with fluorescein isothiocyanate) and DiOC6 (dihexyloxacarbocyanine iodide), respectively.Images were acquired by a confocal laser scanning microscope.

-     Cholinesterase (AChE, BChE, PrChE) enzymatic activity: these molecules being modulated under inflammation and stress, they are considered as a good biomarker of environmental stress.AChE, BChE and PrChE were measured using a colorimetric method.

-     Stress proteins (HSC70 and GRP78) levels: HSC70, heat shock protein 70, is one member of the stress protein family.It acts as a generic stress response element but also serve as signalling molecule of the innate immunity. The glucose-regulated protein (GRP78) is another member of the HSP70 family which plays a central role in the endoplasmic reticulum (ER) homeostasis, due to its involvement in protein folding, ER calcium binding and control of ER stress via the activation of trans-membrane sensors. Both proteins were quantified by the Bradford method.

 

Using this experimental protocol, the following results were obtained:

-     Uptake and accumulation into coelomocytes: none of the urchins treated with 10 mg/L survived after 1 to 2 days. As a result, the following observations were conducted on individuals exposed to 0.1 mg/L. Nano-CeO2 agglomerates engulfed by coelomocytes were observed.In the aqueous medium, particles of different dimensions were observed, including agglomerates from 1 to 10 µm.

-     Impact on coelomocytes subcellular compartments: the trans-Golgi apparatus of controls coelomocytes showed numerous strongly stained vesicles, rather uniformly distributed within the cytoplasm. In urchins treated with nano-CeO2, a severe reduction in the number of stained vesicles was observed, their location seeming to be polarized at one side of the cell.The endoplasmic reticulum of controls showed a network of flat vesicles, more dense around the nucleus.Cells from urchins treated with nano-CeO2 presented strictly packed membranes, not resolved by the confocal imaging.

-     Cholinesterase (AChE, BChE, PrChE) enzymatic activity: a significant reduction of the enzymatic activity of the three ChE isoforms (AChE, BChE, PrChE) was reported.

-     Stress proteins (HSC70 and GRP78) levels: in groups exposed to nano-CeO2, HSC70 and GRP78 were reduced 6.4- and 2.7-fold compared to controls, respectively.

 

The authors suggested that uptake into phagocytic coelomocytes might be a detoxification strategy used to cope with hazardous materials and to promote survival. Moreover, since biomarkers of innate immunity and endoplasmic reticulum stress were found to be down-regulated five days after exposure, it might be hypothesized that the suppression of immune signalling may be a useful feedback response for helping cells to survive tissue injury.