Introduction

The global production rate and usage of metal oxide nanoparticles (NPs) has been increased drastically over few decades. Among the engineered nanoparticles, cerium dioxide (CeO2) is used extensively for many commercial and industrial applications (Keller et al. 2010). Cerium nanoparticles are used in the production of fuel cells, glass, pigments, fuel additives and as a catalyst in the chemical process (Dahle and Arai 2015; The Project of Emerging Nanotechnologies, 2015 catalyst). It is certain that these nanoparticles can be inevitably released into the aquatic environment and cause serious health implications for living organisms and environment (Rogers et al. 2010). Nanoparticles can persist in the natural aquatic environment for a long time due to their aggregation, sedimentation, slow degradation rate, and this could increase their uptake, accumulation, and bio magnification in the food chain (García 2011).

The toxicity of CeO2 nanoparticles was studied over few decades on different test organisms. Cerium oxide particles are shown to cause membrane damage and ROS generation in bacterial and algal cells due to the cellular uptake/adsorption of particles. In crustaceans, the toxicity of CeO2 is due to NP ingestion, accumulation, and adhesion. The recent reports on ecotoxicity of CeO2 are mentioned in Table 1.

Table 1 Ecotoxicity of CeO2 on aquatic organisms
Full size table

Artemia salina is widely used as a food source in aquaculture industries due to its ease of availability, convenience, method of culturing, and lack of labor intensiveness. A. salina (pelagic organisms) has been considered as an important test organism to study the eco-toxicological implications of nanoparticles (Gambardella et al. 2014). The bioavailability of NPs and their accumulation in the food chain are important parameters to assess the risk of nanoparticles on aquatic organisms (Wang and Wang 2014). Despite of many reports on the toxicity of CeO2 on aquatic and terrestrial organisms, their mechanism of toxicity is still not clear. Besides, the previous report on changes in the enzymatic activity of CeO2 NPs by Gambardella et al. (2014) on A. salina, the current study is the first of its kind to quantify the uptake and accumulation of cerium oxide particles on A. salina.

The major objective of this study was to investigate the nano and bulk CeO2 toxicity on A. salina in natural seawater system. The dynamic behavior (sedimentation and stability) of CeO2 in seawater and its effect on the hatching rate of Artemia cyst were studied. The changes in the enzyme activities like CAT, GST, SOD, and AChE and the morphological characteristics of A. salina nauplii after exposure to nano and bulk CeO2 were investigated. Nevertheless, the current study would bring a better understanding of the eco toxicological implications (toxicity, accumulation, depuration, and changes in biochemical markers) of nano and bulk CeO2 particles on A. salina, a representative toxicity testing organism of the marine environment.

Materials and methods

Chemicals

Nano CeO2 particles (particle size < 50 nm, purity 99.8%) and bulk CeO2 (particle size > 5 μm, purity 99%) were procured from Sigma-Aldrich, India. All the chemicals used in the studies were of analytical grade.

Experimental medium

Natural seawater was obtained from Central Institute of Brackish water Aquaculture (CIBA, Chennai, Tamil Nadu, India). Natural seawater was utalized as an experimental medium for all the toxicity studies. Initially, seawater was filtered using Whatman no. 1 paper and further sterilized to remove the biological contaminants present. The physicochemical characterizations of seawater medium as reported in our previous study (Bhuvaneshwari et al. 2016) are as follows, pH (8.0), conductivity 14.65 mS/cm, TDS 12,000 mg/L, TOC 8.56 mg/L, and salinity 37 ppt. The presence of heavy metals was measured to be 0.020 (Ce), 0.140 ± 0.01 (Zn), 1.84 ± 0.02 (Fe), 0.145 ± 0.04 (Cu), 0.513 ± 0.03 (Al), 0.108 ± 0.02 (Ti), 0.189 (Mn), and 0.030 mg/L (Cr).

Primary characterization of nanoparticles

The stock of 100 mg/L of nano and bulk CeO2 was prepared in Milli-Q water and sonicated using the probe sonicator (Sonics, USA) at 130 W for 20 min. The hydrodynamic diameter of nano and bulk CeO2 (100 mg/L) in seawater medium was measured by a particle size analyzer (90 Plus Particle Size Analyzer, Brookhaven Instruments Corp., USA) using the dynamic light scattering method. The particle size measurement by Dynamic light scattering (DLS) method was based on the time-dependent variations on the scattering of laser light by the nanoparticles in Brownian motion. The scattered light is collected at 90° angle and directed to the multichannel correlator, which calculates the diffusion coefficient of the nanoparticles. The mean hydrodynamic size was calculated from the Stokes–Einstein equation, which correlates the diffusion coefficient calculated from the light scattering measurements with that of the particle size (Kaszuba et al. 2008).

The primary particle size and surface morphology of procured nano and bulk cerium oxide particles were imaged using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For TEM, the nano and bulk CeO2 particles were coated on the copper gird and subjected to imaging (TEM, Philips, CM12, Netherlands). For SEM imaging, the stock concentration was further diluted to 10 mg/L in Milli-Q water and coated on a glass side under dust-free environment. Further, this was subjected to gold sputtering and observed using a scanning electron microscope (EV018 Carl Zeiss, Germany). The average size of nano and bulk CeO2 particles were calculated by measuring 100 particles randomly from the TEM and SEM images with the help of an imaging software, Image J, Version 1.49. The crystallinity and purity of nano and bulk cerium oxide particles were analyzed using an X-Ray diffractometer (D8 Advanced X-ray Diffractometer, Bruker, Germany) with CuKα radiation of wavelength λ = 1.5406 in the 2θ scan range of 20–80°.

Colloidal stability of nano and bulk CeO2 in seawater medium

The mean hydrodynamic diameter of different concentrations of nano (0.3, 3, 9, 18, 25, and 32 mg/L) and bulk (0.7, 7, 14, 28, 56, and 100 mg/L) cerium oxide particles at different time intervals of 0, 1, 2, 4, 8, 12, 24, and 48 h was measured by dynamic light scattering technique using a particle size analyzer. The stock of 200 mg/L of nano and bulk CeO2 was prepared in Milli-Q water and sonicated for 20 min using ultrasonics probe sonicator. For 0 h, the mean hydrodynamic diameter was measured immediately after the dilutions were made.

Sedimentation assay

The sedimentation of CeO2 particles was measured by the change in absorbance of the test medium at 345 nm using a UV–Visible spectrophotometer (U-2910, Hitachi, Japan). The nano and bulk cerium oxide particles of different concentrations were prepared in seawater medium and loaded into the new 50-mL falcon tubes (Gupta et al. 2016). One milliliter of samples were withdrawn from the top, bottom, and middle layer of the tubes at 0, 1, 2, 4, 8, 12, 24, and 48 h of time intervals using a pasture pipette. The change in absorbance of CeO2 particles was measured in the samples withdrawn from the different layers of the tube.

Dissolution of nano and bulk CeO2 in seawater medium

The dissolution of different concentrations of nano (0.3, 3, 9, 18, 25, and 32 mg/L) and bulk (0.7, 7, 14, 28, 56, and 100 mg/L) cerium oxide particles was measured after 48 h of interaction in seawater using ICP-OES (Inductively coupled plasma atomic emission spectroscopy, Perkin Elmer Optima 5300). After 48 h of interaction, the nano and bulk suspensions were centrifuged at 15,000 g for 30 min at a temperature of 4 °C (Bhuvaneshwari et al. 2017). The supernatant was filtered using a 0.1-μm filter, followed by a 5-kDa filtration (Ultra filtration disc, Millipore). The filtrate containing only the Ce ions was used to measure the dissolution of nano and bulk CeO2 in seawater.

Toxicity assessment

Hatching rate of brine shrimp

The effect of nano and bulk CeO2 particles on the hatching of brine shrimp cysts was measured according to OECD guidelines 236 (OECD 2013). Nano and bulk CeO2 concentrations of 10, 25, 50, 75, and 100 mg/L and control (devoid of CeO2 particles) were prepared in seawater. Ten brine shrimp cysts were added to different concentrations of CeO2 particle suspension taken in 12-well micro well plates. The rate of hatching at 48 h of incubation was noted. Hatching rate of brine shrimp cysts exposed to nano and bulk cerium particles was calculated according to the equation.

$$ H=N/C+{N}^{\ast }(100) $$

H corresponds to the hatching percentage, N is the number of hatched cysts, including the hatched organism in umbrella stage, and C is the decapsulated full cysts (Arulvasu et al. 2014).

Acute toxicity assessment

Acute toxicity tests were performed on both nano and bulk CeO2 particles according to the Organization for Economic Cooperation and Development (202) testing guidelines (OECD 2004). The brine shrimp cysts were allowed to hatch in the seawater with sufficient aeration at a temperature of 20 °C and 16:8-h light: dark condition. After 24 h, the first instar larvae (nauplii) were taken for toxicity studies. Nano and bulk CeO2 of different concentrations, 10, 20, 40, 80, 160, and 320 mg/L, were interacted with 24-h-old nauplii. The control sample containing only seawater was maintained. The number of live Artemia nauplii was counted after 48 h of interaction. The lethal concentrations (LC50) of nano and bulk CeO2 toxicity on A. salina nauplii were calculated.

The toxicity of dissolved Ce ions was measured according to Yang et al. (2017). After 48 h of interaction, the nano and bulk suspensions were centrifuged at 15,000g at 4 °C for 30 min twice using an ultracentrifuge. The obtained supernatant was filtered through a 0.1-μm filter, followed by a 5-kDa filtration, and the suspension containing Ce ions was exposed to the 24-h-old A. salina for 48 h. The number of live nauplii was counted after 48 h of interaction.

Biochemical changes

Depending on the median lethal (LC50) concentrations, the range of test concentrations of nano (0.3, 3, 9, 18, 25, and 32 mg/L) and bulk (0.7, 7, 14, 28, 56, and 100 mg/L) cerium oxide particles were chosen for further toxicity studies such as biochemical changes. The concentration of nano and bulk CeO2 mentioned above was interacted with the first instar A. salina nauplii for 48-h interaction. The interacted nauplii were harvested and washed twice with Milli-Q water. To the washed nauplii, 1 mL of 100 mM potassium phosphate buffer was added and sonicated for 3 min using a probe sonicator (Sonics, USA) in ice. After sonication, the homogenate was centrifuged at 15,000g for 15 min, and the supernatant was collected. The sample supernatant was further used for the following biochemical assays.

Estimation of total protein content

The difference in the total protein content of control and nanoparticle-treated samples was measured using a Bradford assay (Bradford 1976). About 10 μL of the sample was added to the 96-well plates, to which 200 μL of Bradford reagent was added under dark condition. The samples were incubated at room temperature for 5 min, and the absorbance was measured at 595 nm using the UV–vis spectrophotometer (Hitachi U2910).

Reactive oxygen species generation (ROS)

The reactive oxygen species (ROS) generation was determined using a non-fluorescent dye 2′,7′-dichloro dihydrofluorescein diacetate (DCFH-DA) according to Ulm et al. (2015). A. salina nauplii interacted with nano and bulk CeO2 particles were washed with Milli-Q water. A total of 10 washed nauplii were transferred to 24-well plates containing 10 μM DCFH-DA dye and incubated for 30 min at 37 °C. After incubation, the nauplii were washed twice using PBS of pH 7.4 and homogenized using a probe sonicator. The fluorescence intensity (DCF) was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a fluorescence spectrophotometer (SL174, ELICO, India). The ROS generation was further confirmed by visualizing the control and treated nauplii incubated with DCFH-DA dye using fluorescence microscopy (DM2500, Leica Microsystems, Germany).

Catalase assay (CAT)

The decomposition of hydrogen peroxide (H2O2) to H2O was measured at 240 nm according to the protocol adopted by Jemec et al. (2008). Fifty microliters of protein supernatant was added to 950 μL of hydrogen peroxide solution (10.8 mM) prepared in 50 mM of potassium phosphate buffer (pH 7.0). The absorbance was measured for 3-min interval at 240 nm using the UV- Visible spectrophotometer.

Superoxide dismutase assay (SOD)

Superoxide dismutase activity was measured according to method reported by Kono et al. (1979). The reaction mixture contained 1.3 mL sodium carbonate buffer, 500 μL (4-Nitro Blue tetrazolium chloride) NBT, and 100 μL Triton X-100, to which 100 μL hydroxylamine hydrochloride was added to initiate the reaction. After 2 min, 70 μL of the sample extract was added. The decrease in absorbance at 540 nm is a measure of NBT reduction by the SOD enzyme.

Acetylcholinesterase assay (AChE)

Acetylcholinesterase (AChE) is an enzyme involved in the hydrolysis of the neurotransmitter acetylcholine (ACh) to acetate and choline (Ellman et al. 1961). Ellman’s reagent was prepared without substrate (100 mM phosphate buffer, 500 μM 5, 5′-Dithiobis-(2-nitrobenzoic acid)). 10 μL of sample supernatant was taken in a 96-well plate to which 180 μL of Ellman’s reagent was added and incubated for 30 min. After incubation, 10 μL of the substrate (acetylthiocholine iodide 20 mM) was dispensed to the above cocktail, and the absorbance difference was measured for 3 min at 405 nm using the UV–vis spectrophotometer.

Glutathione S-transferase (GST)

Glutathione S-transferase activity was measured according to the protocol developed by Mannervik and Nise (1969). One milliliter of enzyme cocktail was prepared using 980 μL of PBS, 10 μL of 100 mM (1-chloro-2,4-dinitrobenzene) CDNB, and 10 μL of 100 mM glutathione. Nine hundred microliters of above enzyme cocktail and 100 μL of sample supernatant was taken in 1 ml cuvette and the absorbance was measured at 340 nm using UV–vis spectroscopy.

Uptake and accumulation of nano and bulk CeO2

Twenty four-h-old nauplii were exposed to different concentrations of nano and bulk CeO2 particles for 48 h (Ates et al. 2013). After 48 h of interaction, 10 nauplii were washed thoroughly with Milli-Q water and transferred to pre-weighted tubes. The washed nauplii were dried at 80 °C in a hot-air oven, after which, the dry mass of the nauplii was measured. To determine the uptake of nano and bulk CeO2, the dried nauplii were acid digested with an excess of concentrated HNO3 acid. The samples were analyzed for Ce content using ICP-OES (Perkin Elmer Optima 5300), and the sensitivity of this instrument is about 0.004 mg/L. The ingested particles were allowed to depurate for 48 h in fresh medium. The nauplii were then separated and dried in the pre-weighted tubes. Further, it was acid digested using nitric acid, and the samples were subjected for measurement of the accumulated Ce concentration present after depuration.

Statistical analysis

All experiments were carried out in the triplicates and expressed as the mean with the corresponding standard deviation (SD). Significance in the difference between the treatment groups of nanoparticles was calculated using two-way ANOVA (Bonferroni post-test) by Graph Pad Prism (Version 5) software. The lethal concentration (LC50) values were calculated using a nonlinear regression model. The statistical significance between the control and test group was accepted at a level of p < 0.05.

Results

Characterization of nanoparticles

The primary size and morphology of nano and bulk CeO2 particles were determined using TEM and SEM. The primary particle size measured using TEM was found to be 15 ± 3.5 and 582 ± 50 nm for nano and bulk CeO2, respectively (Fig. 1a, b). SEM micrograph showed the average size of nano and bulk CeO2 as 130 ± 20 nm and 530 ± 60 nm, respectively (Fig. S1 A and B). The mean hydrodynamic diameter of nano and bulk CeO2 particles at 100 mg/L in seawater medium was measured to be 510 ± 80 and 1092 ± 14 nm at 0 h, respectively. The purity and crystalline structure of procured CeO2 particles were analyzed from the X-ray diffraction pattern (Fig. S2). The progressive scan range from 20 to 80° showed the polycrystalline nature of particles with intense peaks. The sharp high peak at 28.53° corresponding to the 111 crystalline plane confirmed the presence of CeO2 in the sample. The other sharp and intense peak at 33.09°, 47.5°, and 56.26° corresponded to 200, 220, and 311 crystalline planes, respectively. The crystalline size was measured to be 49 and 119 nm for nano and bulk CeO2, respectively. The obtained crystalline planes were in a match with JCPDS (Joint Committee on Powder Diffraction Standards) No: 34-0394 for cerium oxide particles. All the crystalline planes correspond to the pure form and cubic fluorite structure of cerium oxide particles.

Fig. 1
figure 1

Transmission electron micrographs of nano CeO2 (a) and bulk CeO2 particles (b)

Full size image

Dissolution of ions from the nano and bulk CeO2 in seawater medium was measured using ICP OES, and the results are represented in Fig. 2a, b. The dissolution of CeO2 particles was found to be dependent on the exposure concentrations (p < 0.05). The dissolution of Ce ions from the lower exposure concentrations of 0.3 mg/L (nano CeO2) and 0.7 mg/L (bulk CeO2) were found below the detection limit (BDL). The maximum concentration of dissolved Ce was found to be 2.3 ± 0.2 and 4.6 ± 0.5 mg/L from 32 and 100 mg/L of nano and bulk CeO2, respectively. Further, the toxicity of dissolved Ce ions from nano and bulk CeO2 on A. salina was studied, and the results are represented in Fig. 2c, d.

Fig. 2
figure 2

Dissolution of nano and bulk CeO2 particles in seawater medium (a and b). Toxicity of dissolved Ce ions on Artemia salina (c and d). The symbols “δ” (p < 0.001) represents the level of significance between different time intervals

Full size image

Stability of nano and bulk CeO2 in seawater

The colloidal stability of nano and bulk cerium oxide particles in seawater medium was measured at different concentrations and time intervals (0, 1, 2, 4, 8, 12, 24, and 48 h) using the dynamic light scattering method (Fig. 3). The concentration-dependent increase in the mean hydrodynamic diameter of nano CeO2 in seawater medium was observed for all kinetic intervals of exposure (p < 0.001). At 0 h, the mean hydrodynamic diameter of 0.3 mg/L nano CeO2 was found to be 244 ± 3.1 nm, and it increased to 281 ± 11, 381 ± 10, 318 ± 15, 420 ± 35, 450 ± 9, and 460 ± 10, and 480 ± 21 nm at 1, 2, 4, 8, 12, 24, and 48 h of interaction, respectively. At higher concentration (32 mg/L), the mean hydrodynamic diameter increased from initial size of 492 ± 5.0 to 759 ± 5.2, and 998 ± 20 nm at 0, 4, and 48 h of interaction, respectively.

Fig. 3
figure 3

Hydrodynamic diameter of different concentrations of nano (a) and bulk (b) CeO2 particles in seawater medium at different time intervals (0, 1, 2, 4, 8, 12, 24, and 48 h). The data are presented as mean ± SD, n-3. The symbols “δ” (p < 0.001) represents the level of significance between different time intervals

Full size image

In contrast to nano CeO2, the bulk particles showed a significant increase in the mean hydrodynamic diameter for all the concentrations at all kinetic exposure intervals (p < 0.001). The mean hydrodynamic diameter of bulk CeO2 (0.7 mg/L) was found to be 689 ± 38, 767 ± 20, 967 ± 22, 901 ± 28, 980 ± 31, 1065 ± 50, 1186.4 ± 49, and 1303.1 ± 80 nm at 0, 1, 2, 4, 8, 12, 24, and 48 h of interaction, respectively. The mean hydrodynamic diameter of particles at 100 mg/L bulk CeO2 was found to be increasing from 1053 ± 30 (0 h) to 3055 ± 87 and 4413.1 ± 200 nm at 12 and 48 h of interaction, respectively. As the interaction time increases, the mean hydrodynamic diameter increased significantly (p < 0.001) for all concentrations. At 0 h of exposure, bulk CeO2 (28 mg/L) particles reached micron size in seawater medium. Nano CeO2 was observed to be in submicron (< 1 μm) size till 48 h of interaction at all the exposure concentrations, except 32 mg/L.

Sedimentation of nano and bulk CeO2 in seawater

The sedimentation profile of nano and bulk CeO2 in seawater medium is represented in Fig. S3 A and B. The absorbance of nano and bulk CeO2 particles at three different layers of the tube (upper, middle, and lower) was measured at different kinetic intervals (0, 1, 2, 4, 8, 12, 24, and 48 h). A concentration-dependent increase in the absorbance (p < 0.001) was observed for nano CeO2 till 8 h of interaction, with no significant difference between all three layers (p > 0.05). At 12, 24, and 48 h of interaction, no change in the absorbance was noticed for all three layers of 0.3, 3, 9, and 18 mg/L concentrations. However, the absorbance of upper and middle layers decreased for 25 and 32 mg/L concentrations. Thus, the sedimentation of bulk CeO2 was faster in seawater as compared to the nano CeO2.

Toxicity assessment

Effect of nano and bulk CeO2 on hatching of brine shrimp cysts

The toxic effect of nano and bulk cerium oxide particles on hatching of brine shrimp cysts in seawater was studied (Fig. 4a). The control group showed (devoid of nanoparticles) 80–90% hatching of cyst. As the concentration increased, the hatching of brine shrimp cysts decreased significantly (p < 0.001) at all exposure concentrations. The complete hatching inhibition was observed at 100 mg/L of both nano and bulk CeO2 particles. There was no significant difference observed between nano and bulk cerium particles on the hatching of brine shrimp Artemia cysts (p > 0.05).

Fig. 4
figure 4

a Hatching rate of Artemia salina brine shrimp cysts exposed to nano and bulk CeO2 particles in seawater medium. The level of significance between control and CeO2 particle-treated group are represented as “α”, corresponding to p < 0.001***. Symbol “ns” corresponds to p > 0.05 between nano and bulk CeO2 treatment. The data are presented as mean ± SD, n = 3. b Acute toxicity (LC50) of nano and bulk CeO2 particles on Artemia salina in seawater medium. The concentrations of CeO2 are expressed in log concentration. The data are presented as mean ± SD, n = 3

Full size image

Acute toxicity of nano and bulk CeO2 A. salina nauplii

The mortality of A. salina nauplii interacted with nano and bulk cerium oxide particles are shown in Fig. 4b. No sign of mortality was observed in the control (only seawater) group. Both the nano and bulk cerium oxide showed a concentration-dependent increase in the toxicity for the 24-h-old nauplii. Nano CeO2 showed significantly higher toxicity as compared to that of bulk particles (p < 0.01). The mortality of 24-h-old nauplii was observed to be 35 ± 5, 45 ± 15, 45 ± 5, 50 ± 2, 65 ± 5, and 100 ± 2% upon interaction with various concentrations (10, 20, 40, 80, 160 and 320 mg/L) of nano CeO2. Similarly, the mortality was found to be 10 ± 2, 20 ± 4, 30 ± 10, 40 ± 10, 50 ± 5, and 98 ± 2% for the bulk CeO2 concentrations of 10, 20, 40, 80, 160, and 320 mg/L, respectively. All the treated concentrations of nano and bulk cerium oxide particles were lethal to nauplii. The LC50 value of nano and bulk CeO2 was found to be 38.0 mg/L (with 95% CI of 27.32 to 52.85 mg/L) and 92.26 mg/L (with 95% CI of 73.82 to 115.3 mg/L), respectively. The median lethal concentration (LC50) of nano CeO2 on 24-h-old nauplii was observed to be significantly lower as compared to the bulk particles (p < 0.001). Dissolved ions from nano (32 mg/L) and bulk (100 mg/L) CeO2 caused only 10% mortality on A. salina (Fig. 2b, c).

Biochemical changes

Assessment of reactive oxygen species generation

ROS generation in nauplii exposed to nano and bulk CeO2 are represented in Fig. 5a, b. The fluorescence intensity was found to be significanlty increased from 9 mg/L for nano CeO2 with respect to the control (p < 0.001). Bulk CeO2 particles of 0.7, 7, and 14 mg/L did not show any significant increase in the fluorescence intensity with respect to the control (p > 0.05). On other hand, nano CeO2 showed significantly higher ROS generation as compared to the bulk particles, though the concentration of exposed bulk CeO2 was higher. Further, the ROS generation was confirmed by staining the nauplii with DCFH-DA (Fig. S4), which shows the in vivo reactive oxygen species generation. The nano CeO2 (25 and 32 mg/L) and bulk CeO2 treatment (28 and 100 mg/L) showed green fluorescence due to the production of intracellular reactive oxygen species.

Fig. 5
figure 5

ROS generation in Artemia salina treated with nano (a) and bulk (b) CeO2. SOD activity of Artemia salina treated with nano and bulk CeO2 (c and d). The level of significance between control and CeO2 particle-treated group are symbolized by p < 0.05*, p < 0.01**, and p < 0.001***. The data are presented as mean ± SD, n = 3

Full size image

Superoxide dismutase activity

As the nano and bulk CeO2 concentration increases, the antioxidant enzyme activity (SOD) was found to be decreased significantly, as compared to control. However, there was no significant difference (p > 0.05) seen between nano and bulk CeO2 (Fig. 5c, d). Higher exposure concentrations of nano (32 mg/L) and bulk CeO2 (100 mg/L) showed similar SOD activity at 48 h of interaction, despite the differences in the concentration of bulk CeO2 as compared to nano CeO2.

Acetylcholinesterase activity

The acetylcholinesterase activity in A. salina nauplii exposed to nano and bulk cerium oxide particles are represented in Fig. 6a, b. The concentration-dependent increase in acetylcholinesterase activity was observed upon exposure to nano and bulk CeO2 particles (p < 0.01) as compared to the control group. A significant difference (p < 0.05) in the activity of AChE in nauplii treated with nano and bulk cerium particles was observed. The nano CeO2 treated samples showed significantly higher acetylcholinesterase activity as compared to the bulk particle treatment (p < 0.05). Even though the bulk CeO2 exposure concentrations were higher than nano CeO2 particles, AChE activity was found to be higher in nauplii treated with nano CeO2.

Fig. 6
figure 6

AChE activity of Artemia salina treated with nano and bulk CeO2 (a and b), GST activity of Artemia salina treated with nano and bulk CeO2 (c and d), CAT activity of Artemia salina treated with nano and bulk CeO2 (e and f). The level of significance between control and CeO2 particle-treated group is symbolized by p < 0.05*, p < 0.01**, and p < 0.001***. The data are presented as mean ± SD, n = 3

Full size image

Glutathione S-transferase activity

GST is an antioxidant enzyme involved in metabolic inactivation of toxic substances that enters the cell. The concentration-dependent inhibition of GST activity was observed after exposure to both nano and bulk CeO2 particles (Fig. 6c, d). As compared to the control group, a significant decrease in GST activity was observed in all the treated concentrations of nano and bulk particle (p < 0.001), except for 0.3 mg/L (P > 0.05). There were no significant difference in GST activity observed between nano and bulk CeO2 particle treatment (P > 0.05).

Catalase activity

As compared to the control group, a significant decrease in CAT activity (p < 0.001) was observed for both nano and bulk cerium oxide-treated naupliis (Fig. 6e, f). However, lower concentrations of nano (0.3 mg/L) and bulk (0.7 and 7 mg/L) CeO2 particle-treated nauplii showed an insignificant decrease in the CAT activity when compared to the control. Nano CeO2-treated samples showed significantly lesser CAT activity as compared to the bulk particle treatment (p < 0.05). No significant difference was observed between higher exposure concentrations of nano 32 mg/L and bulk 100 mg/L (P > 0.05).

Uptake and accumulation of nano and bulk CeO2

The uptake and accumulation of different concentrations of nano and bulk CeO2 exposed to A. salina nauplii are represented in Fig. 7a, b. The uptake and accumulation of nano and bulk CeO2 were quantified based on the dry weight of A. salina nauplii after 48 h of interaction. Uptake of both nano and bulk CeO2 into A. salina was increased significantly with respect to the concentrations (p < 0.001). A. salina nauplii treated with nano CeO2 showed significantly higher uptake as compared to bulk particles (p < 0.05).

Fig. 7
figure 7

Uptake and accumulation of nano CeO2 exposed to Artemia salina (a), uptake and accumulation of bulk CeO2 exposed to Artemia salina (b). The symbol “δ” (p < 0.001) represents the level of significance between uptake and accumulation. The data are presented as mean ± SD, n = 3

Full size image

The accumulated nano and bulk CeO2 was quantified after 48 h of depuration as illustrated in Fig. 7a, b. Concentration-dependent increase in accumulation was observed for nano CeO2. On other hand, lower exposure concentrations of bulk CeO2 did not show any significant increase in accumualtion (7, 14, and 28 mg/L), except at 52 and 100 mg/L (p < 0.01). Despite the higher concentration of bulk CeO2, the uptake and accumulation was found to be higher in nauplii treated with nano CeO2.

Discussion

Primary size of nano and bulk CeO2

The TEM images revealed that the particle size of nano CeO2 was significantly lesser than its bulk particles. Smaller sized nano CeO2 particles exhibit higher reactivity as compared to its bulk counterpart due to their higher surface area to volume ratio. The hydrodynamic diameter of nano (510 ± 80 nm) and bulk (1053 ± 14 nm) CeO2 at 100 mg/L showed significant size differences in seawater medium.

Stability and sedimentation of nano and bulk CeO2 in seawater

Nanoparticles tend to aggregate once it is exposed to the natural system (Adam et al. 2015) and their colloidal stability play a significant role in the toxicity (Pauluhn 2009). The stability and aggregation of nano and bulk CeO2 was influenced by the physicochemical characteristics, such as surface charge, shape, particle size, and chemical reactivity in seawater medium. In this study, it was seen that as the interaction time increased, the hydrodynamic diameter showed a significant increase (p < 0.001) at all exposure concentrations for both nano and bulk CeO2 particles. The particle size of nano CeO2 was observed to be in the range of 460–750 nm till 4 h and reached the submicron size range of 800–900 nm at 24 h for all exposure concentrations. After 48 h, nano CeO2 reached a micron size of 998 nm on exposure to 32 mg/L, whereas lower exposure concentrations remained in the submicron size range of 800–900 nm. In contrast to the nano CeO2, the bulk CeO2 was in the submicron size (700–850 nm) at 0 h for the concentrations of 0.7 to 52 mg/L and micron size (1053 nm) for 100 mg/L. As the interaction time increased, the size of bulk particles showed a significant increase to >1 μm at 1 h, 2 μm at 8 h, and reached 4 μm at 48 h, respectively.

In our study, both nano and bulk particles formed distinctive aggregates and sizes in seawater medium. The results also revealed faster aggregation of bulk particles in seawater medium as compared to nano cerium. Surface-area-to-unit-volume ratio decreases due to aggregation; hence, larger size particles are formed. Aggregation could be due to the direct adsorption of NOM (natural organic matters) and humic acid present in the natural water (Chinnapongse et al. 2011). Wang et al. (2009a) reported the aggregation of Ag and TiO2 NPs particles due to higher salinity of seawater medium, which reduces the electrostatic repulsion between the particles. Higher electrolyte concentrations of monovalent Na+ and K+ and divalent ions Ca2+ and Mg2+ in the seawater facilitate the aggregation of metal oxide nanoparticles (Badawy et al. 2010; Stebounova et al. 2011). These electrolytes induce the aggregation of nanoparticles by reducing the electrostatic force of the electric double layer present between the nanoparticles (Zhang et al. 2008).

Aggregation can enhance the gravitational settling of particles in the experimental medium. Most of the pelagic and benthic organisms are highly exposed to sediment particles; hence, it is necessary to study the sedimentation profile of CeO2. Sedimentation of both nano and bulk particles increased with the exposure concentration. Bulk CeO2 was found to be sedimented within 2 h of interaction in the experimental medium. The rate of sedimentation increased with respect to time. Keller et al. (2010) reported the higher rate of sedimentation of CeO2 nanoparticles due to lower TOC (Total Organic Carbon) and higher ionic strength in seawater. It is important to consider the aggregation of particles in test medium because the direct uptake/ingestion and accmulation of particles have significant impact on the survival of the organism.

Uptake and accumulation of CeO2 into A. salina

Ingestion is considered as the primary route of uptake of nanoparticles and the leading cause of toxicity in most of the aquatic organisms like fishes, micro and macro-crustaceans, and zooplanktons (Zhu et al. 2010, Ward and Kach 2009). Being the filter-feeding pelagic organism, A. salina can ingest particles in the size range of 7 to 50 μm (Hund-Rinke and Simon 2006). Stability results show the submicron (< 1 μm) size of nano cerium till 48 h in seawater, whereas the initial size (0 h) of bulk particles was in the micron size range. Despite the higher exposure concentrations of bulk CeO2, the uptake was found to be more for nano form at all the exposure concentrations. Due to smaller particle size, the ingested nano CeO2 can be localized in the midgut epithelial cells, gastrointestinal tract, and exoskeleton of A. salina. Arnold et al. (2013) reported that the ingested CeO2 particles could translocate to the epithelial cells and cross the intestinal barriers. The ingested polystyrene nanoparticles (Rosenkranz et al. 2009) and QD (Feswick et al. 2013) was reported to be translocated into the lipid-storage droplets of Daphnia magna, suggesting their penetration into the gut epithelial cells depending on the particle size. Also, the smaller particles are reported to have longer gut retention time than the larger particle aggregates, indicating their transportation to the digestive gland (Ward and Kach 2009). These factors could facilitate higher uptake and accumulation of nano CeO2 into A. salina. In case of bulk particles, the subcellular localization may not be possible due to larger aggregates. Apart from the size of the particle aggregates, there are other exogenous factors like concentration, physicochemical properties, fate and behavior of particles, (Moiseenko and Kudryavtseva 2001) that can influence the uptake of CeO2 particles (Garaud et al. 2015). The accumulation of ingested particles after depuration was found to correlate with the exposure concentration. The uptake of both nano and bulk CeO2 particles was higher than the accumulation of particles, which signifies the depuration of ingested particles by A. salina. The uptake of nano and bulk CeO2 was found to be 3.5 and 4.0 fold higher than particle accumulation and were concurrent with the acute toxicity results of both the particles. Further, the ingestion of aggregated CeO2 particles into the gut of A. salina was confirmed using microscopic studies (Fig. S5).

Acute toxicity of nano and bulk CeO2 on nauplii and cyst

Acute toxicity of nano and bulk CeO2 particles on Artemia salina nauplii was evaluated. The observed LC50 for nano CeO2 (38.0 mg/L) was much lower than that of the bulk CeO2 (92.26 mg/L). Bulk CeO2 showed 50% mortality on A. salina only when its concentration increased to 3 fold as compared to nano CeO2. The toxicity was found to be dependent on the particle size and exposure concentration. Acute toxicity results suggested that the interaction of nano sized CeO2 particles occurs predominantly with A. salina than bulk particles. Similar to our study, the toxicity of nano TiO2 on nematode Caenorhabditis elegans (Wang et al. 2009a) and to D. magna (Zhu et al. 2009) was reported to be significantly higher than the toxicity of bulk TiO2. Further clarity in the mechanism of toxicity was sought by studying the effect of dissolved ions on the toxicity to A. salina (Fig. 2b, c). The dissolution percentage was in the range of 0.8–4% for nano and bulk particles in the concentration range of 32–100 mg/L. The obtained results indicated the poor dissolution properties of CeO2 particles in the experimental system. Similarly, few studies reported that the dissolved Ce (III) ions do not exert any toxic effect on the algae, P. subcapitata (Pulido-Reyes et al. 2015; Rogers et al. 2010). Röhder et al. (2014) suggested that the low intrinsic toxicity of Ce ions does not account for the observed toxicity of nano CeO2 on Chlamydomonas reinhardtii. Another report showed the CeO2 dissolution into ions has not been shown to play a significant role in its toxicity on earthworm Eisenia fetida (Lahive et al. 2014).

The effect of nano and bulk CeO2 on the hatching of brine shrimp cyst was studied. Both nano and bulk particles showed similar trend and a concentration-dependent inhibition in the hatching of cysts Nanoparticles can inhibit the hatching of cyst by direct adsorption onto the outer chorion layer of the cysts and interfere in the oxygen intake and development of embryos (Bai et al. 2010; Sano et al. 2008; Arulvasu et al. 2014). Casals et al. (2010) stated that the nanoparticles could cause inactivation of proteins like metalloproteinase resulting in the inhibition of the hatching process. Therefore, from the study findings, it can be assumed that the higher toxicity of nano CeO2 as compared to its bulk counterpart was due to particle size rather than the dissolved ions.

ROS generation and antioxidant enzymes

Ingested (up taken and accumulated) particles could bring out deleterious changes in the physiological process of A. salina. One of the major mechanisms proposed for metal oxide nanoparticle toxicity was the ion-mediated toxicity due to the ROS generation in the cells. From the ionic toxicity results (Fig. 2b, c), it is evident that Ce ions are not likely to be responsible for the toxicity in A. salina. Park et al. (2008) reported that the CeO2 particles can cause oxidative damage to cells or organisms. Similarly, our results showed a significant increase in the ROS generation with respect to exposure concentration and particle size of nano and bulk CeO2. Potential redox cycling of CeO2 particles with peroxide are reported to generate the damaging oxygen radicals (Heckert et al. 2008) in cells. The reduction of Ce (IV) to Ce (III) attains oxygen vacancy on CeO2 particle surface, which is the most possible active site for ROS generation (Lin et al. 2006). The higher ROS generation by nano CeO2 was responsible for the higher toxicity than that of its bulk particles.

The primary antioxidant enzymes like SOD and CAT act as defensive barriers to protect the cells against oxidative stress. Higher levels of ROS radicals (.O2 and H2O2) can inhibit the human extracellular SOD activities (Gottfredsen et al. 2013). In this study, SOD activity decreased with increasing CeO2 concentration. Hao et al. (2009) also reported the inhibition of SOD activity in the gills, liver, and brain of carp exposed to 100 and 200 mg/L of nTiO2 as an indication of oxidative stress. The minimum concentration of bulk CeO2 (100 mg/L) required to cause a significant decrease in the SOD and CAT activities was 3 folds higher than the concentration of nano CeO2 (32 mg/L) tested in this study. On other hand, the inhibition of CAT and SOD activities by a variety of toxicant and consequent oxidative stress have been reported in few studies (Bagnyukova et al. 2005, Vander et al. 2003). The exposure of Ni (nickel) NPs to fish (Oreochromis mossambicus) showed a concentration-dependent decrease in SOD activity, which contributes to the oxidative stress condition (Jayaseelan et al. 2014). Afifi et al. (2016) and Saddick et al. (2015) also suggested the inhibition of CAT and SOD activities in fish cells (Oreochromis niloticus and Tilapia zillii) exposed to Zn and Ag NPs. Such inhibition of SOD and CAT activities upon exposure to CeO2 would enhance the generation of ROS (Fig. 5a, b) and produce serious oxidative damage to tissues. Further, the decrease in GST activity was found to be dependent on the concentration of nano and bulk CeO2. Our results can be corroborated with the study by Ali and Ali (2015), wherein they have reported the inhibition of GST activity in a freshwater snail, Lymnea luteola, upon exposure to CuO NPs as a result of the oxidative stress in the cells. Thus, the exposure of CeO2 particles induces significant changes in the antioxidant defense system of A. salina in response to the uptake and accumulation of these particles in body.

AChE activity

AChE activity is used as a biomarker of nanoparticle toxicity (Wang et al. 2009b). The ingestion and accumulation of CeO2 particles could have caused internal damage to tissues, and as a result, this might have direct influence on the biochemical enzymes. Our results showed the concentration- and particle size-dependent increase in AChE activity. An apparent increase in the AChE activity was detected in D. magna (Ulm et al. 2015) and Rainbow trout (Johari et al. 2013), following exposure to Ag NPs. They reported that the elevated AChE activity upon NP exposure could deplete the neurotransmitter level, which interacts with the acetylcholine receptor. Similarly, scallops (Chlamys farreri) exposed to TiO2 NPs had elevated AChE activity in the gills and digestive gland (Xia et al. 2017). Gamberdella et al. 2013 reported a dose-dependent increase in the AChE activity in larvae of sea urchin. The increase in AChE activity led to tissue damage upon exposure to CeO2 particles. Impairment of AChE activity can cause both behavioral and physiological consequences on A. salina. Behavioral response is the ability of organisms to manage with its surrounding environment to reproduce and survive (Engenheiro et al. 2005). Increase in AChE activity causes alteration in swimming behavior of A. salina (Gamberdella et al. 2014). Xuereb et al. (2009) reported that the impairment of feeding rate and locomotion bevaiour of Gammarus fossarum directly correlates with the AChE activity. Changes in feeding behavior as a result of AChE activity was observed by Baird et al. 2007. These effects can have direct consequences on the organism. Based on our results, it can be concluded that nano and bulk CeO2 exerted size- and dose-based toxicity response in A. salina. Further confirmation at the molecular level would shed light on the detailed mechanism of toxicity. These findings provide a fundamental understanding of the behavior and toxicity of CeO2 particles in the marine environment.

Conclusion

This study aims to assess the toxic effect of nano and bulk cerium oxide particles towards A. salina. The primary particle size (TEM and SEM) and stability studies clearly revealed that the size of nano CeO2 was lesser than the bulk particles. Higher uptake and accumulation of nano CeO2 induced deleterious changes to the physiological process of A. salina. ROS generation was observed as the major toxicity mechanism exhibited by both nano and bulk CeO2 particles. Higher cholinesterase expression in Artemia tissues exposed to cerium particles was seen. As a result, higher toxicity of nano CeO2 was observed on A. salina as compared to the bulk form of particles. This study adds knowledge on the differential effect of nano and bulk CeO2 on A. salina based on stability and exposure concentration. The observed results indicated that the toxicity was found to be dependent on the concentration of CeO2 particles and its stability in seawater. Future studies will focus on the effect of cerium oxide particles on different stages of A. salina and trophic transfer potential in the ecosystem.