Toxicity of TiO₂, SiO₂, ZnO, CuO, Au and Ag engineered nanoparticles on hatching and early nauplii of Artemia sp.

Preparation of nanoparticles and their characterization

All the nanoparticles used in the study were obtained from the consortium of the NANOVALID (Nordmiljö AB, Sundsvall, Sweden) project. SiO₂ nanoparticles (powder form) were supplied by Nanologica AB (Södertälje, Sweden), silver nanoparticles were obtained as a suspension of 4% from Colorobbia, Firenze, Italy; gold nanoparticles as a suspension of 0.006% from INMETRO (Rio de Janeiro, Brazil); copper oxide nanoparticles (powder form) from Intrinsiq Materials (Hampshire, UK); zinc oxide nanoparticles (powder form) from Nanogate (Quierschied-Göttelborn, Germany) and TiO₂ particles were synthesized in house using the existing protocol (Reddy et al., 2004). The nanoparticles obtained in powder form were made into a suspension. A suspension of NPs was made by suspending SiO₂, TiO₂, CuO and ZnO NPs at a concentration of two mg/mL. The suspension was sonicated at ∼30 W with pulse of 50% for 10 min. Silver nanoparticles were obtained as suspension at a concentration of 4% with polyvinylpyrrolidone (PVP) as a stabilizing agent, gold nanoparticles in water at a concentration of 0.006%. Working dilutions of the nanoparticles were prepared in salt water (SW) (Oceanfish marine salt; Prodac International, Cittadella PD, Italy) for the experiments.

The nanoparticles used in this study were characterized in the NANOVALID consortium in round robin manner. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used in this study for basic characterization of nanoparticles before every experiment. In TEM studies the nanoparticle suspension of 50 μg/mL was applied to formvar coated copper grids and air dried. The grids were examined in JEOL 2010 TEM at 100 kV using 20 μ aperture, about 10 images were taken using GATAN camera and later analyzed using GATAN software. The images of about 100 particles were used for analysis. The nanoparticle suspensions at concentration of 25 μg/mL in milliQ water (mQW) and SW were measured in DLS system (Horiba Nanopartica SZ100, Kyoto, Japan). Each suspension was analyzed thrice up to 48 h and the data was represented as mean ± SEM. Solvents used to prepare the nanoparticles were used as controls for size determination.

Hatching of Artemia

For every one L culture, one g of Artemia cysts were used (San Francisco Bay Strain, UT, USA). These cysts were hydrated in distilled water under aeration for 45 min and then bleached to decapsulate the cysts wall using 20% sodium hypochlorite solution for 15 min. The bleached Artemia eggs were then washed for six times with distilled water and allowed to hatch in 2.5% SW (pH 8.0) with continuous light and aeration for 24 h (Sorgeloos, 1973). The temperature was maintained at 28 °C, the optimum temperature for hatching of Artemia cysts. Nanoparticles were added to the culture in concentrations of 100 and 10 mg/L. pH of the SW containing nanoparticles was always determined using pH meter after setting the pH meters with two reference standards at pH 7 and 9.

Toxicity studies using hatching rate as end point

Six types of nanoparticles (TiO₂, SiO₂, ZnO, CuO, Au and Ag) are tested for their toxicity on brine shrimp hatching. The Artemia cysts were allowed to hatch under standard culture conditions and in presence of two different concentrations of each nanomaterial (100 and 10 mg/L). The hatching rate, mortality and viability were checked after 24 h for each Artemia cultures exposed to nanoparticle. Three aliquots of 100 μL was taken from each flask after thorough mixing and counted under the stereo-microscope. Both, hatched Artemia (immotile vs. motile) (h), and unhatched (u) cysts were counted. Hatching rate was expressed as percent of total number of fully hatched Artemia in comparison to total Artemia (H) in the aliquot. The experiment was repeated six times. The average and SEM was calculated for each sample.

Standard Artemia toxicity test

In a second set of experiments, following the protocol of Solis et al. (1993), Artemia hatched under normal conditions in SW were exposed to various nanoparticles. A total of 10 nauplii were transferred to six-well plates and the nanoparticles at 100, 10, 1, 0.1 and 0.01 mg/L concentrations were added. The number of live larvae was counted at intervals of 6, 12, 18, 24, 36 and 48 h. Mortality of the larvae was used as end point. Mortality was recorded as cessation of swimming or any movement by nauplii. LC₅₀ was calculated for each particles using Graphpad (www.graphpad.com/quickcalcs/).

Oxidative stress test

Stresses due to reactive oxidation species can be observed by treating the nauplii exposed to different concentrations with 10 μM dichloroflourescien diacetate (DCFDA). DCFDA or with 20 μM dihydroethidium (DHE), both of which are chemiluminiscent probes and react with ROS generated due to oxidative stress. The nanoparticle treated nauplii exposed to DCFDA or DHE are imaged in Zeiss Axiovert imager using appropriate filters.

Validity of results

The hatching studies have been conducted six times and the validity criterion used is, low mortality in the control. The data was analyzed for statistic validation using Graphpad (www.graphpad.com/quickcalcs/). In the Artemia toxicity tests the validity criterion was mortality below 10% in 24 h period. The correlation test was performed for both sets of data using on line software for correlation coefficient and comparing data using Microsoft excel.

Analysis of nanoparticles

The size of nanoparticles both in distilled water and SW was measured. The nanoparticles showed reduced size in SW by TEM. The TEM studies showed no apparent change in aggregation or dispersion of particles (Fig. 1). Measurement of particle size by DLS which allows examination of particles sizes as well as aggregates when in suspension revealed that nanoparticles exhibited different aggregation profiles in mQW and SW. TiO₂ (7,082 nm in mQW: 8,009 nm in SW), CuO (329 nm in mQW: 468 nm in SW) and ZnO (117 nm in mQW: 185 nm in SW) nanoparticles showed higher values for aggregate sizes in SW as shown in Fig. 2. Silver (169 nm in mQW: 101 nm in SW), gold (5,542 nm in DW: 3,712 nm in SW) and SiO₂ (7,704 nm in DW: 6,096 nm in SW) had smaller aggregates in SW as compared to particles in mQW (Fig. 2). Visible flocculation was observed in case of copper and zinc oxide nanoparticles. DLS measurements for these nanoparticles gave very high values corroborating the flocculence seen (Fig. 2). Though size changes in the mean size of particles in mQW and SW was noticed both by TEM and DLS, it was not found to be significant in statistical analysis (Table 1).

The nanoparticles suspended both in mQW and 2.5% NaCl solution (SW) were measured up to 48 h to observe change, if any in their dispersion pattern (Fig. S1). Silver NPs, TiO₂ and SiO₂ did not show much difference in dispersion in mQW over 48 h but the particles showed higher aggregate sizes in SW in 48 h. In CuO and ZnO NPs visible flocculation and sedimentation was seen in case of both MilliQ and salt solution by 48 h.

Hatching studies

Brine shrimp Artemia were allowed to hatch in filtered sterile SW kept at continuous aeration that also keeps the particles in suspension throughout the experiment. Brine shrimp showed a hatching rate of 74% ± 5.5 in mQW and the viability of these hatched nauplii was about 97%. The hatching rate of Artemia altered drastically in presence of nanoparticles (Table 2; Fig. 3). The hatching rate dropped with increase in concentration of nanoparticles in SW. The highest effect was seen in presence of silver and copper oxide nanoparticles. The hatching rate dropped to about 29% in presence of these two nanoparticles at a concentration of 100 mg/L; of these only 50% were motile and viable at a concentration of 100 mg/L (Table 2). The hatching rate remained lower at 10 mg/L of silver nanoparticles but the viability of hatched nauplii was good at 85%. With copper oxide also hatching rate was low (43.23%) with better viability around 64%. Similarly, with silver nitrate or in presence of capping agent PVP the hatching slightly improved (33% and 41%, respectively) with good viability of 70% and 84%, and about 60% in case of copper sulphate with viability of 33%.

Hatching rate in presence of other nanoparticles (gold, SiO₂, TiO₂, copper oxide and zinc oxide) was low at about 50–60% as compared to control (Table 2; Fig. 3) and posthatching mortality was not very high. In presence of zinc oxide nanoparticles, 60% of the nauplii hatched but the cysts had a tendency to clump together. In TiO₂ at 100 mg/L, hatching rate was 41.50% with 67.8% viability; the hatching rate was at 54% with ∼90% viability in 10 mg/L TiO₂ dioxide NPs. Gold and SiO₂ nanoparticles displayed very good viability after hatching (Fig. 3). SiO₂ nanoparticles at a concentration of 10 mg/L displayed hatching rate and viability equivalent to the controls.

The pH of SW did not change upon addition of nanoparticles and remained around 8.3–8.5 up to 48 h. Live hatched nauplii appeared normal and swimming vigorously. There were no visible changes in morphology of nauplii hatched in presence of nanoparticles. Some of the nauplii had emerged but were immotile/dead; some were trapped in the membrane and still-dead (Figs. S2.1–S2.6). Silver and copper oxide nanoparticles which had maximum toxic effect on hatching rate also did not show any gross morphological abnormalities. Staining of hatched Artemia with DCFDA revealed oxidative stress in a few still trapped nauplii and the gut of dead nauplii (Fig. S3).

Larval toxicity test

Following the ARC (Artemia Reference Center) test protocol, the larvae hatched in SW were exposed to nanoparticles for up to 48 h and the viability recorded every 6 h for first 24 h and subsequently every 12 h. Artemia were not fed during the test period. The nauplii showed variability in mortality to various nanoparticles. The most toxic was the silver nanoparticles and silver in ionic form (Table 3). Silver in ionic form was highly toxic as 80% of the nauplii were dead in 24 h in silver nitrate solution (Fig. 4). The LC₅₀ of the silver nanoparticles was around 10.12 mg/L at 24 h. Other nanoparticles used in the study were nontoxic initially but after 30 h (Table 3), Many Artemia died in concentration-dependent manner (Fig. 4). LC₅₀ of gold was 7.54 mg/L at 48 h. LC₅₀ of SiO₂, TiO₂ and CuO was 23.59; 18.94 and 20.92 mg/L at 48 h. In presence of zinc oxide nanoparticles, around 36% nauplii perished in 48 h in the highest concentration used with the LC₅₀ above 100 mg/L at around 259.34 mg/L. Ionic forms of silver, copper and zinc were found to be more toxic than their corresponding nanoforms having LC₅₀ Ag⁺ (8.19 mg/L), Cu⁺⁺ (13.5 mg/L) and Zn⁺⁺ (0.769 mg/L).

In the ARC test; we do see sedimentation and flocculation of nanoparticles by 48 h. The nauplii maintained in SW containing nanoparticles showed accumulation of NPs in the gut of Artemia by end of 24 h (Fig. 5). In case of silver nanoparticles, the nauplii that were dead within 24 h, the gut does not show accumulation of material (Fig. 5), even the nauplii appeared stunted. Staining of the nauplii exposed to nanoparticles showed signs of stress when stained with DCFDA for ROS and DHE for Superoxide (Fig. 5). Nauplii exposed to silver nanoparticles showed highest staining, followed by TiO₂ and SiO₂. Nauplii exposed to other nanoparticles do not show any signs of stress did show accumulation of nanoparticles in the gut (Fig. 5).