Introduction

Globally, ZnO nanoparticle is the third highest annually produced nanoparticles (550 tons per year) after silica dioxide and titanium dioxide, respectively (Piccinno et al. 2012). ZnO nanoparticles possess exotic piezoelectric and pyroelectric properties, a wide bandgap, high exciton binding energy, and wurtzite structure lacking the center of symmetry; these properties make them unique and suitable for versatile usage (Wang 2004). A comprehensive range of ZnO nanoparticle applications covers almost every mainstream sector of mankind needs like the following: as nano-fertilizers and as other nano-agrochemicals in agriculture (Raliya et al. 2017); as UV filter/absorber in textiles and cosmetics (Lu et al. 2015); as nano-photocatalyst for wastewater treatment and in electronics (Sharma et al. 2017); as nano-composite film in the food industry for packaging purposes (Ejaz et al. 2018); and as an antimicrobial agent (Alswat et al. 2016) and biosensors (Lupan et al. 2016) in pharmaceuticals for targeted drug delivery (Rasmussen et al. 2010; Ghaffari et al. 2017) and even for other biomedical purposes (Mishra et al. 2017). From these nano-based products, ZnO nanoparticles will eventually get released into the environment and ultimately sink into our aquatic bodies (Wang et al. 2018). Even models predicting the environmental fate of nanoparticles concluded the necessity to evaluate the toxicity of nanoparticles including the aquatic flora and fauna (Sun et al. 2016). For that reason, it is very imperative to assess the impact of ZnO nanoparticles on aquatic ecosystems. As alga is the primary source of food in aquatic ecosystem, any sort of damage to algal flora could lead to the disturbance in the complete food chain and ultimately to the aquatic ecosystem (Nowack and Bucheli 2007). Alga is the integral component of the aquatic ecosystem and being the primary producer, its impact would affect the whole food chain of the aquatic ecosystem; therefore, algae are the ideal model organisms to examine the effect of nanoparticles (Cattaneo 2018; Espinasse et al. 2018).

Earlier studies reported that a ZnO nanoparticle concentration between 0.06 and 100 mg L−1 imparts toxicity to most of the algae (Franklin et al. 2007; Miao et al. 2010; Chen et al. 2012; Li et al. 2017; Bhuvaneshwari et al. 2018). The reported toxicity is found different regarding the different species, particle nature, and test methods (Merdzan et al. 2014). ZnO nanoparticles are kinetically very active and undergo numerous transformations rapidly that prominently influence itsThe two prime factors imparting toxicity on the basis of earlier toxicological impact. The two prime factors imparting toxicity on the basis of earlier studies were dissolution and aggregation (Chen et al. 2012; Ma et al. 2013). Reactive oxygen generation production is also thought to be an important factor in mediating toxic responses (Klaine et al. 2008; Fu et al. 2014). Reduction in photosynthetic pigment (Hazeem et al. 2016), algal growth inhibition (Manzo et al. 2013; Li et al. 2017), and lipid peroxidation (Ji et al. 2011; Suman et al. 2015) as responses to ZnO nanoparticles have also been reported earlier. But the straightforward relationships between these factors, responses, and exposed organism have not yet been established. This avenue is further being explored to understand the overall impact of ZnO nanoparticles to the algal physiology. The purpose of the study is to monitor the ever-lasting impact of ZnO nanoparticles on Coelastrella terrestris which could help us predict the role of algae in aquatic nano-ecotoxicology. In the present study, we comparatively assessed the effect of ZnO nanoparticles and bulk form on Coelastrella terrestris on growth kinetics. We have monitored the dynamics of toxicity over a period of 25 days; therefore, this is relatively a long-term study exploring the impact of ZnO exposure on the life cycle of Coelastrella terrestris. We have tried to establish a straightforward relationship between the dose concentration and the number of exposure days by estimating IC50. Overall changes bought by the inclusion of ZnO in nano- and bulk form during the growth of the algae are monitored and the metabolic, physiological, and morphological changes under the treatment during the growth phases are assessed.

Material and methods

Algal cultures

Axenic culture of Coelastrella terrestris (NCBI accession number: MK294227.1) has been established in lab-made BG-11 media (pH 7.4; Rippka et al. 1979) in 250-mL Erlenmeyer flasks and cultures were kept at 25 ± 1 °C temperature and 14.5 W m−2 light intensity from the samples collected from Fateh Sagar, a freshwater lake in Udaipur, Rajasthan. Cultures were maintained on a regular basis through frequent media change under the same conditions and were subjected to harvest during their exponential phase after measuring the protein value. All the experiments were carried out under the same experimental conditions in triplicates.

Chemicals, particle dispersions, and nanoparticle characterization

ZnO nanoparticles and the bulk counterpart were purchased from Sigma-Aldrich (CAS: 1314-13-2-030-013-007) and Central Drug House Private Limited (CAS: 1314-13-2), respectively. The size of the ZnO nanoparticles was confirmed further through TEM (Tecnai, G-20 (FEI), USA) and FTIR analysis (Bruker Alpha Model, laser class-1). Stock solutions for both ZnO nano- and bulk forms were prepared with 10 mg in 100 mL deionized water. In the case of ZnO nanoparticles, stock suspension was sonicated for 30 min at 40 Hz by using a Probe sonicator (Q-500, Qsonica, USA.). Further, dilutions were made in BG-11 medium for toxicological assessment between 0.1- and 1-mg L−1 concentrations. Test suspensions were vortexed mildly before use. One milligram per liter of ZnO nanoparticle concentration was used further to determine the zeta potential of suspended nanoparticles and hydrodynamic diameters through dynamic light scattering using Zetasizer Nano (ZS90, Malvern).

Algal growth kinetics

Protein values from homogenously growing axenic algal cultures in the abovementioned conditions were assessed using a protocol given by Lowry et al. (1951) as modified by Herbert et al. (1971) by taking absorbance at 650 nm using a UV-Vis spectrophotometer (Hitachi U2900). From OD, subsequent protein values were calculated using BSA as standard. To estimate the growth rate of algae, 1 mL of algal suspension with a protein value of 100 μg mL−1 was further harvested from established cultures and inoculated in 100 mL of freshly prepared BG-11 medium having different concentrations of ZnO nanoparticles and its bulk counterpart. From these newly established cultures, protein contents were assessed regularly starting from 96 h, after every fifth day. IC50 values were also calculated to analyze the growth rates under the exposure conditions.

Biochemical parameters

All the biochemical analyses were performed on the 25th day of the experiment. Day 25 was chosen because in the growth curve, the highest protein value was detected on that day and after that, a decline was observed. Therefore, to assess the impact of treatment on the physiological condition of the algae, the 25th day was chosen for further assessment.

Estimation of chlorophyll and carotenoids

Extraction of pigment was done in methanol and their relative amounts were estimated using equations as per Mackinney (1941): 13.42 × A665 = μg chlorophyll mL−1 and 200× A420 = μg carotenoids mL−1.

Lipid peroxidation (MDA)

One milliliter of interacted cell suspensions was added to 2 mL of trichloroacetic acid (20%) and centrifuged for 45 min at 7000 rpm. The supernatant was added to 3 mL of 2-thiobarbituric acid (0.5%) and heated for 10 min in boiling water bath. After cooling, absorbance was measured at 532 nm as per Metzler et al. (2011).

Lactate dehydrogenase assay

To quantify membrane damage, lactate dehydrogenase (LDH) assay was performed (Wacker et al. 1956; Pakrashi et al. 2013). One milliliter of each algal cell suspension under various treatments was centrifuged at 7000 rpm for 10 min. About 100 μL of supernatant was collected and added to 100 μL of 30 mM sodium pyruvate followed by 2.8 mL of 0.2 M Tris-HCl. And just before measuring the decrease in absorbance, about 100 μL of 6.6 mM NADH was added. Ten readings at 340 nm were measured using UV-Vis spectrophotometer.

Superoxide dismutase activity

Algal cell suspensions were prepared by adding 2 mL (0.5 M) PBS buffer solution (pH 7.5) in 50 mg biomass harvested from treated algal cultures. Samples were centrifuged for 10 min at 4 °C at 13000 rpm. One hundred microliters of the supernatant was collected and mixed with reaction mixture prepared as per Yilancioglu et al. (2014). This was incubated for 10 min at 37 °C and absorbances were recorded at 560 nm.

Catalase activity

Catalase (CAT) enzyme helps in decomposing hydrogen peroxide into water and oxygen. To quantify the activity, 50 mg of interacted algal biomass was harvested and suspended in 2 mL (0.5 M) PBS buffer (pH 7.5). Samples were centrifuged at 12000 rpm at 4 °C for 30 min. One hundred microliters of supernatants was collected and mixed with reaction mixture as per details given by Roy et al. (2016). CAT activity was represented in terms of percent decrease with respect to the control.

Microscopic analysis

Comparative microscopic analyses of algal cells under treatments were done repeatedly using a compound microscope (Olympus CH20i). Furthermore, to study the algal morphology and particle localization, untreated and treated (1-mg L−1 concentration of ZnO treatments of both bulk and nano-forms) algal cells were analyzed through TEM (Tecnai, G-20 (FEI), USA) and SEM (EVO 18, Zeiss, Germany). For TEM analysis, blocks were prepared, ultrathin sectioning were done, and sections were loaded on copper grids, whereas for SEM, algal cell drops of each sample were coated, air-dried via gold sputtering, and subjected to analyses.

Statistical analysis

All the experiments were done in triplicates. Mean and standard deviation were calculated using MS Excel (office version 10.0) and values are shown as mean ± SD in Table 1. Correlation and regression equation were estimated using MS Excel. Statistically significant differences between control and treatment were analyzed using a one-way ANOVA with the help of Prism Software (version 3.02), as P values less than 0.5 were assumed as the significant differences.

Table 1 Biochemical parameter analysis of Coelastrella terrestris under different treatment levels
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Results and discussion

Nanoparticle characterization

Transmission electron microscopy analysis revealed polymorphic shape and size (≤ 100 nm) of ZnO nanoparticles (Fig. 1a). The transmittance peaks at 518.48, 480.38, and 424 wavenumber cm−1 of ZnO nanoparticles were comparable with the standard graph of ZnO (Fig. 1b). Sonication was essential as particles have tendency to aggregate in aqueous form (Angel et al. 2013). Zeta potential of suspended ZnO nanoparticles was observed to be − 24.6 mV in stock solution prepared within deionized water (Fig. 2b). The dynamic light scattering study showed the hydrodynamic average size of ZnO nanoparticles within the deionized water was 252 nm in diameter with PDI value of 0.164 (Fig. 2a) and showed a slow and clear tendency to aggregate further as the size is much greater than the actual size of ZnO nanoparticles analyzed through TEM. The reason behind the aggregation is the increase in ionic concentration which subsequently decreases the repulsive forces between the ZnO nanoparticles. Hence, in aqueous medium, hydrodynamic sizes usually greater than the actual particle sizes were found (Bian et al. 2011).

Fig. 1
figure 1

Zinc oxide nanoparticle characterization: a TEM micrograph and b FTIR spectrum

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Fig. 2
figure 2

a Hydrodynamic size nanoparticles measured via dynamic light scattering of zinc oxide nanoparticle stock solution in deionized water. b Zeta potential of suspended nanoparticles

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Algal growth kinetics

Growth of Coelastrella terrestris was found adversely affected under the ZnO nanoparticles and its bulk counterpart treatments. However, analyzing the growth pattern through protein content, it was observed that the nano-form of ZnO is more toxic to the Coelastrella terrestris than its bulk form. Toxicity in both cases, nano-form and bulk form, was found to be dose-dependent. It is clearly evident that the increase in dosage concentration leads to the increase in toxicity similar to the previously reported study (Schiavo et al. 2016). To assess the growth rate, estimation of protein is one of the important and useful parameters (Turhani et al. 2005). Protein as a parameter has also been found useful in assessing the toxicity induced by heavy metal and nanoparticles in few earlier published reports (Rai et al. 1992; Harish et al. 2008; Gong et al. 2011). Further, ZnO NPs were found to exhibit a strong adsorption capability for proteins; therefore, estimating the protein is critical to understand the toxicity level (Horie et al. 2009). Earlier studies also concluded the toxicological responses because of Zn+ ion dissolution which depends on the adsorption potential of proteins resultant into deprived growth rate in algae (Manzo et al. 2013; Suman et al. 2015). On the 25th day, when highest protein level was marked by control, a 27% reduction in growth rate under bulk (y = 50.635x, R2 = 0.173) and a 54% reduction in growth rate under nano-form (y = 34.673x, R2 = − 0.101) at 1 mg L−1 of treatment level were observed in terms of protein level. In other treatment concentrations also, reduction in soluble protein under nano-form was higher compared with the bulk was observed (Fig. 3). Even at the lowest concentration (0.1 mg L−1), a significant reduction (25%) in protein content was observed in the case of nano-form (y = 26.302x + 166.58; R2 = 0.1846), whereas in the bulk, the reduction was almost negligible (y = 47.095x + 149.63; R2 = 0.3557). A remarkable difference under the nano- and bulk treatments was also observed at 0.5 mg L−1 where a 35% reduction (y = 17.764x + 144.3, R2 = 0.1226) in nano-form and a 27% reduction (y = 36.385x + 130.43, R2 = 0.262) in bulk were detected. Reduced level of proteins clearly showed that the nano-form of particles is toxic in comparison with the bulk. Moreover, IC50 values were also calculated in both of the cases. IC50 calculated on the 25th day revealed that the nano-form is more toxic (IC50 = 0.255 mg L−1) in comparison with the bulk form (IC50 = 0.455 mg L−1). It means that lower concentration of nano-form is required to induce a 50% growth inhibition and higher dose of bulk is required to induce the same level of inhibition. Results clearly revealed that the nano-form of ZnO nanoparticles is more toxic than the bulk form. IC50 growth rate parameter is more appropriate scientifically and provides better interpretations between the comparative studies (Bergtold and Dohmen 2011).

Fig. 3
figure 3

Effect of zinc oxide nanoparticles and its bulk counterpart on Coelastrella terrestris growth kinetics measured via protein content

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As the nano-form of particles possesses greater surface area to volume ratio which might be one of the key factors in attributing toxicity to the algae, smaller size leads to the stronger interaction with the algal cell and alters the cell wall thickness (Kim et al. 2016). In fact, intrusion of nanoparticles is much easier in the case of newly formed algal cells in comparison with the mature ones (Dash et al. 2012) which might be the reason of retarded growth rate response in the case of nanoparticle treatments. Inherent pores of algal cell also suggested allowing nanoparticle internalization. It was reported that more pores must be induced after nanoparticle exposure. It produces oxidative stress and leads to disruption of the cell (Navarro et al. 2008; Xia et al. 2015; Taylor et al. 2016; Tripathi et al. 2017). The size of the particle plays a prominent role in toxicological pavements (Franklin et al. 2007). Earlier studies done on ZnO nanoparticles reported that different particle shapes and sizes of the same metal oxide result into a different toxicity level (Peng et al. 2011; Samei et al. 2019). Likewise, Manzo et al. (2013) revealed that the bulk ZnO particles were less toxic than its nanoparticles for Dunaliella tertiolecta. In the present study, the lowest concentration (0.1 mg L−1) of bulk treatment was not observed to be harmful to the algae, whereas the same concentration in nano-form was found generating a negative impact on algal growth. However, at the 1-mg L−1 concentration, both were detected affecting the growth adversely but the toxicity was found more in the case of nanoparticles in comparison with the bulk. Similar results were observed in the case of two marine algae, Tetraselmis suecica and Phaeodactylum tricornutum, while assessing the ZnO nanoparticles and bulk form toxicity (Li et al. 2017). The surface of algal cells was observed to be occupied by ZnO nanoparticles (Fig. S1), which could have obstructed the nutrient exchange between the growth medium and algal cell (Bhattacharya et al. 2010). A similar phenomenon was observed by Metzler et al. (2011) in assessing the effect of titanium dioxide nanoparticle on algal growth. Moreover, aggregates entrapping algal cells must have reduced the availability of light which could have affected the cell growth activity (Fig. S1). Similarly, Huang et al. (2005) also concluded that the inhibition of algal cell growth is due to the adsorption of titanium dioxide nanoparticles on algal cell surface. Interestingly, with the increase in dosage concentration, we observed delays in the growth pattern at the onset of exponential phase in both bulk and nano-forms of ZnO. Merdzan et al. (2014) reported ZnO nanoparticle aggregation in correlation with the dissolution phenomenon and concluded that both the processes could simultaneously contribute to produce toxicological impact. Moreover, earlier dissolution was found to damage the cell wall and destructive cell organelles, which was also apparent in the present study after microscopic analysis, which will be discussed later. Moreover, the toxicological attributes were also because of the time of the exposure. As the duration of exposure was extended, aging of particles also occurred which also influences the toxicity in the case of nanoparticles (Schiavo et al. 2018). The overall impact of ZnO nano-form and bulk form is found negative on the protein content of algal cells which in terms of growth was suppressive.

Biochemical parameters

Toxicity assessment by evaluating biological endpoints like chlorophyll, enzyme activities, and peroxidation reveals the oxidative stress of the algae and helps in correlating the mechanism, imparting the toxicity. Therefore, it is important to determine the biochemical parameters (Chen et al. 2019).

Chlorophyll and carotenoid content

Significant deduction in chlorophyll content was observed after the exposure of ZnO nano- and bulk counterpart at 0.5- and 1-mg L−1 concentrations (Table 1). At the lowest concentration of 0.1 mg L−1, no significant changes in chlorophyll were detected in both nano- and bulk samples. Chlorophyll content was found more sensitive under treatments as it was earlier detected in stress conditions reported by Houimli et al. (2010) whereas contrastingly carotenoid content was not found much reduced. This supports the earlier studies reporting that metal stress in algae responded in carotenoid synthesis to quench oxidative stress (Nikookar et al. 2005; Wang et al. 2010).

Lipid peroxidation (MDA)

Oxidative stress could be detected by the quantification of lipid peroxidation, as the membrane lipid damage indicates the stress conditions (Sayeed et al. 2003). A dose-dependent increase in the lipid peroxidation was observed in all concentrations exposed, except in bulk at the 0.1-mg L−1 concentration. A similar dose-dependent response was reported in the earlier study conducted by Wang et al. (2011). In nano-form, increase in lipid peroxidation was also reported by Chen et al. (2012). Peroxidation levels were found much higher in the case of nano-treatments in comparison with its bulk counterpart as a biomarker of oxidative stress indicating that nano-form is more toxic.

LDH release

Lactate dehydrogenase leakage is one of the parameters to measure cytotoxicity induced by nanoparticles (Yang et al. 2009). LDH release was found to be enhanced with the dosage concentration under nanoparticle treatment, whereas, at lowest concentration (0.1 mg L−1) of bulk, no such release was detected (Table 1). However, at the same concentration, ZnO nano-form was observed to be more cytotoxic to the algae than its bulk counterpart via LDH analysis. At 1 mg L−1 of nano-form concentration, substantial membrane damage was notified indicating the toxicity as reported earlier (Zhang et al. 2012).

SOD activity

Enzymatic activity was reflective even at lower concentrations of treatment. The maximum activity detected in nano- and bulk form was 31.62 ± 0.3 and 27.44 ± 0.3 unit mg−1prot at 1 mg L−1, respectively. Enzymatic activity observed was directly proportional to dose concentration. Superoxide dismutase (SOD) activity clearly showed stressful physiological condition in algae after the exposure of ZnO in nano- and bulk forms as reported earlier (Suman et al. 2015).

CAT activity

Catalytic activity was found increasing in linear manner with respect to the dosage concentrations. However, under nano-treatments, catalase activity was more profound than the bulk treatments. At the 1-mg L−1 ZnO nanoparticle exposure, the value 4.77 ± 0.1 unit mg−1prot was obtained which is the highest among all, whereas at the same concentration, 3.32 ± 0.1 1 unit mg−1prot was observed in bulk form. Only under the 0.1 mg L−1 bulk, no significant activity of catalase was noticed.

Microscopic analysis

Preliminary screening of algal cells done by compound microscope embarked a difference between the cultures grown under nano- and bulk treatments and provided us supportive evidences (Fig. 4). In comparison with the control, particles of ZnO along with media particles were found aggregated under both of the cases which must have hindered the nutrient availability required by the algal cells to grow. Peculiar morphological proofs and lack of motility of Coelastrella terrestris allowed an easier aggregation of ZnO around the cells. Another observation was that with the increase of dosage concentrations in both of the cases, aggregation was found to increase. This reduced the availability of light to the algal cells which have obstructed the growth (Aruoja et al. 2009; Gong et al. 2011).

Fig. 4
figure 4

Compound microscopy images of Coelastrella terrestris under control and under different treatment concentrations of zinc oxide nanoparticles and its respective bulk form after 600 h: a control or untreated algal cells; b–d zinc oxide nanoparticle–treated algal cells with 0.1-, 0.5-, and 1-mg L−1 concentrations, respectively; e–g zinc oxide bulk–treated algal cells with 0.1-, 0.5-, and 1-mg L−1 concentrations, respectively; h algal cultures under treatment along with control (1 control; 2, 3, and 4 represent 0.1-, 0.5-, and 1-mg L−1 zinc oxide nanoparticle treatments, respectively; 5, 6, and 7 represent 0.1-, 0.5-, and 1-mg L−1 zinc oxide bulk treatments, respectively)

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Similar results were evident by SEM analysis (Fig. 5). One more trend was observed that in the case of nano-form, surface adsorptions of ZnO nanoparticles were observed (Fig. S1) as reported earlier (Li et al. 2017). It could be the reason why nano-form is more toxic than bulk as it covers larger surface area of algal cell via forming large aggregates and stops the exchange between the cell and the media and light. Moreover, aggregation increases the direct interaction of ZnO nano- and bulk forms with the algal cell surface which could be the reason of cytological damages manifested after the TEM analysis under both treatments (Xia et al. 2015). TEM results revealed that cell organelles after the exposure collapsed and fragmented (Fig. 5). Cell wall was found ruptured; cytoplasm was shrunken. Overall analysis showed that the long-term exposure of ZnO nanoparticles induces cytological abnormalities to which the growth of the algae was affected.

Fig. 5
figure 5

Scanning electron microscopy: aCoelastrella terrestris control cells, b algal cells treated with zinc oxide nanoparticles, c algal cells treated with zinc oxide bulk counterpart (arrows representing detrimental effects of treatments). Transmission electron microscopy: dCoelastrella terrestris control cells, e algal cells treated with zinc oxide nanoparticles, f algal cells treated with zinc oxide bulk counterpart; both micrographs were taken after 25 days of exposure treated with 1 mg L−1

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Conclusion

Our results revealed that physiochemical nature of the particles plays an important role with respect to the development phase of the algae. This study clearly revealed that the growth rate of algae was suppressed. The nano-form of ZnO was found remarkably toxic in comparison with its bulk counterpart. Toxicity was found directly proportional to the dosage concentration under both nano- and bulk treatments, and aggregation was found to play a vital role in attributing toxicity. To develop a better understanding regarding the nature of aggregation with respect to the media and exposure time, more investigation needs to be done.