Introduction

Pollution of water bodies through metal discharges from industries greatly affects the ecosystem. Because metals are highly persistent pollutants in the aquatic ecosystem, they can cause alteration of growth, development, morphology, physiological, and biochemical metabolism in aquatic organism (Assche and Clijsters 1990; Bidar et al. 2007; Ajitha et al. 2019). Metals enter the aquatic system and primarily act on small organisms, algae, which are ubiquitously distributed throughout the aquatic environment; and such metal contaminants are widely distributed out to and among various organisms due to unavoidable presence in the aquatic food chain system (Liu et al. 2008). Among various animal taxa, extensive studies on the adverse effects of metal contamination in animals including aquatic vertebrates and invertebrates are available; however, relatively little attention has been given on the importance of primary producers, microalgae.

Microalgae are known to be sensitive to the alterations in the environment and often used as biological indicators for assessing the toxic effects of metals (Chouteau et al. 2004; Durrieu et al. 2011; Kumar et al. 2015), as they are extensively prevalent in lakes and seas (Chen et al. 2012) (Table 1). Among microalgae, Chlorella vulgaris is a well-known photosynthetic freshwater microalga and generally used for toxicity tests due to its high sensitivity to xenobiotics (Ajitha et al. 2019). In fact, metals having characteristics of non-biodegradability, biomagnification, and high toxicity are of great threat to the aquatic ecosystem, resulting in a significant reduction in algal diversity and productivity, which all contribute to changes in algal composition (Harding and Whitton 1976; Foster 1982; Shehata and Whitton 1982; Takamura et al. 1989; Gupta and Chandra 1994; Bajguz 2000; Mallick 2004). Furthermore, consequences of metal stress in algae include detrimental effects on growth, cell division, photosynthesis, and destruction of primary metabolites (Pokora and Tukaj 2010; Tukaj and Tukaj 2010; Wang et al. 2011).

Table 1 Review on the metal exposure performed to the microalgae and the endpoints and responses assessed
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Among various metal pollutants present in aquatic ecosystem, Zn is considered to be an essential microelement; however, at higher concentration, Zn is strongly phytotoxic and leads to the obstruction of algal growth, while the non-essential element Hg becomes highly toxic in metallic, ionic, and organic forms which is deleterious to aquatic fauna and flora (Ouyang et al. 2012; Dinesh Kumar et al. 2014). Also, Hg pollution is of great concern due to its high toxicity and resistance to biodegradability, and potential for bioaccumulation through trophic chains. Growth inhibition of Chlorella by Hg has been widely acknowledged (Hutchinson and Stokes 1975; Gipps and Biro 1978). Moreover, studies have evaluated Hg toxicity in aquatic organisms, highly focusing on the bioaccumulation and trophic transfer as well as lethal and sub-lethal toxicity to fish (Boening 2000). In addition, few studies have explored Hg toxicity to aquatic plants and larval stages of insects (Azevedo-Pereira and Soares 2010; Dirilgen 2011). Several findings revealed that Hg causes a significant reduction of plant growth and biomass (Godbold 1991; Israr et al. 2006; Cargnelutti et al. 2006; Zhou et al. 2007) and generates oxidative stress by the generation of reactive oxygen species (ROS) (Cargnelutti et al. 2006; Zhou et al. 2007).

To date, many toxicity tests have been performed based on individual toxicity; however, due to the potential combined effects, toxicity exerted by the combinations of various metals is likely more serious and threatening (Zeb et al. 2017). Also, literature on the assessment of the combined toxicity of metals (Mochida et al. 2006; Su et al. 2012; Qu et al. 2013) particularly on single and/or synergistic effects of metals in the microalga C. vulgaris have been reported (Rai et al. 1981a; Franklin et al. 2002; Qian et al. 2009, Qian et al.2011). For example, the combined nitrogen limitation and cadmium stresses have led to significant inhibition of growth and cell density of C. vulgaris (Chia et al. 2015). However, a detailed recent study illustrates the additive and synergistic effects on C. vulgaris in response to six metals such as Ni, Fe, Zn, lead (Pb), cadmium (Cd), and chromium (Cr) (Mo et al. 2019). Exposure of the microalga C. pyrenoidosa to copper and cadmium, individually and in combination, resulted in growth inhibition (Nugroho et al. 2017). Also, single, combined, and second exposure effect of Cu2+ and chlortetracycline (CTC) on the microalgae C. pyrenoidosa and Microcystis aeruginosa demonstrated variation in toxicity due to differences in recovery potential among the two species (Lu et al. 2015). Moreover, the action of binary mixtures of cetyltrimethyl ammonium chloride (CTAC) and aromatic hydrocarbon showed synergetic and antagonistic effects on C. vulgaris (Ge et al. 2010). Similarly, single and combined effects of cadmium and 4-n-nonylphenol (4-n-NP) on growth inhibition and oxidative stress in the microalga C. sorokiniana were have been reported (Wang et al. 2018) (Table 2).

Table 2 Synergistic effect of heavy metals and their endpoint assays on various algae
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Based on the previous study on metal composition in treated electroplating industrial effluent (Ajitha et al. 2019), we selected Zn (upper limit 167.25 mg/L) and Hg (upper limit 104.2 mg/L) as the testing metal elements for this study. Single and combined effects of Zn and Hg were analyzed based on both acute and chronic exposure periods, 48 h and 7 days, respectively. This study aims to better understand how Zn, Hg, and Zn+Hg combination affects the biological process in C. vulgaris. To corroborate, we investigated the effects of Zn and Hg and their combination on the accumulation of oxidative radicals, the impairments on physiological parameters (pigments and protein), and the counter-response of antioxidant defense mechanisms (CAT and SOD) in the oxidative stress-induced microalga C. vulgaris. Besides, the morpho-variability and aberrations in C. vulgais during chronic exposure to metals at different concentrations were observed. Overall, even though without any affirmed mechanism of action of synergism or the combinatorial effects of Zn and Hg on C. vulgaris, this study provides insight into the response of C. vulgaris in response to Zn, Hg, and Zn+Hg and helps to predict the biological effects, which paves way for the directions for identifying the molecular effects of metal synergism in C. vulgaris.

Materials and methods

The microalga C. vulgaris was obtained from Central Marine Fisheries Research Institute, Kochi, Kerala in India and maintained axenically at the National Centre for Aquatic Animal Health (NCAAH), Cochin University of Science and Technology (CUSAT), Kerala, India, until it was used for this study (Ajitha et al. 2019). C. vulgaris was maintained in aerated Bold’s basal medium (BBM) (Bischoff and Bold 1963) under 16:8 h light and dark cycle at 25±2°C, 45 mmol m−2 s−1 photon flux intensity. All investigations were carried out following the guidelines provided by the Institutional Biosafety Committee (IBSC) at NCAAH, CUSAT, Kerala in India.

To obtain the elemental Zn and Hg, ZnCl2 and HgCl2 with 99.9% purity were selected. Stock solutions of ZnCl2 (2.4 mM contain 167.25mg/L Zn) and HgCl2 (0.076 mM contain 104.2 mg/L Hg) were prepared with Milli-Q water and considered as 100%. The upper limits of Zn (167.25 mg/L) and Hg (104.2 mg/L) were selected based on the previous findings (Ajitha et al. 2019) and considered as 100%. Various concentrations of Zn and Hg used for the study are given in Tables 4 and 5. To assess single and combined effects of Zn and Hg during acute and chronic toxicity tests, C. vulgaris cells were exposed to metal solutions prepared in BBM media in different concentrations (2.5 to 80%). Control was maintained by using the same BBM medium without metals. Experimental cell cultures were initiated at 0.6 × 106 cells/mL. Cell number was determined using a hemocytometer (Improved Neubauer, Rohem, India), as described in Ajitha et al. (2019). Chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Field emission scanning electron microscopic (FESEM) analysis was performed using C. vulgaris samples with distinct concentrations of Zn and Hg (2.5, 20, and 80%). Microalgal cultures were harvested and washed in 1x phosphate-buffered saline (PBS) for 2–3 times followed by centrifugation at 12,400×g. To obtain the cell pellets, 1 mL 2.5% glutaraldehyde was added and kept for overnight at 4°C. After 12 h, cells were harvested and washed in 1x PBS for 2–3 times. One milliliter of 2% osmium tetroxide was added and incubated for 4 h at 4°C. Cells were harvested and washed with 1x PBS followed by the dehydration with acetone and air dry (Grantt 2008). Nova NanoSEM 450UoK scanning electron microscope (Nova NanoSEM, Los Angeles, USA) was used to observe the microalgal cells.

C. vulgaris samples treated with various metal concentrations (2.5 to 80% for both Zn and Hg) were prepared for the assessment of photosynthetic pigments. Two-milliliter microalgal sample was centrifuged at 2200×g for 5 min and the pellets were suspended in 2 mL methanol, incubated for 30 min at 45°C. The supernatant was discarded and absorbance was taken at 665.2, 652.4, and 470 nm (Lichtenthaler 1987).

For the evaluation of protein content, six dilutions of metal samples along with the control were tested in C. vulgaris cultures. Followed by the standard protocol (Barbarino and Lourenço 2005), protein was extracted. To quantify the total protein contents, Bradford assay (Bradford 1976) was carried out with the precipitated proteins via bovine serum albumin (BSA) as the standard.

Estimation of ROS was done using dihydroxyrhodamine123 (DHR123) dye. Various metal concentrations (2.5, 20, and 80%) were prepared along with the control at 48 h and 7 days. The cell pellets were stained with DHR123 at a final concentration of 5μg/L for 1h. Cells were centrifuged and washed twice in fresh BBM medium. The cultures were resuspended in fresh BBM medium and observed under a confocal microscope (Nikon A1R, Tokyo, Japan) to check the ROS generation in single cells (Sathasivam et al. 2016).

Antioxidant enzyme assays were performed by following our previous paper (Ajitha et al. 2019). Briefly, C. vulgaris cultures in response to various concentrations of metals (i.e., zinc and mercury) ranging from 2.5 to 80% were kept for 48h and 7 days. C. vulgaris cells (100 mg) were homogenized in 0.5 M PBS (pH7.5), 1 mM ethylene-diamine-tetraacetic-acid (EDTA), and a pinch of polyvinyl polypyrrolidone. The homogenate was centrifuged at 12,400×g at 4 °C for 30 min. Enzyme extraction was carried out at 0–4°C and the supernatants were stored as aliquot for enzyme estimation. Catalase activity was examined following the standard protocol (Chance and Maehly 1955). Reaction mixture was prepared with 2.5 mL 10 mM PBS, 0.5 mL H2O2, and 0.2 mL enzyme extract. Reduction in absorbance at 230 nm was analyzed in a spectrophotometer (Hitachi U-3900, Tokyo, Japan) and the specific activity was expressed in terms of changes in absorbance/min/extinction coefficient/mg protein. Superoxide dismutase activity was determined by standard protocol (Das et al. 2000). Reaction mixture of 1.5 mL aliquot comprised of 0.3 mL each of 50 mM PBS (pH7.4), 20 mM methionine, 1% (v/v) Triton X-100, 10 mM hydroxylamine hydrochloride, and 50 μM EDTA. To this aliquot, 200 μL, the supernatant was added followed by the pre-incubation at 37°C for 5 min. Eighty microliters of 50 μM riboflavin was added to the tubes and the mixture was placed below a light source for 10 min. One milliliter of Griess reagent was added to each tube and the absorbance of the color formed was measured at 543nm against buffer taken as blank. Each test was performed in triplicates.

One-way analysis of variance (ANOVA) was done to confirm the validity of the data using SPSS® software (version21; SPSS Inc., Chicago, IL, USA) followed by Tukey’s post hoc test (Tukey’s, P<0.05 and P<0.01), which shows statistically significant differences in all treatments.

Results and discussion

In the present study, single and combined effects of Zn and Hg were assessed through morphological changes, photosynthetic pigment content, total protein content, ROS generation, and antioxidant enzyme (CAT and SOD) activities in C. vulgaris. SEM provided direct observation of microalgal cells in which high magnification and resolving power facilitates the improved examination of morphology and surface attachment. Under the chronic exposure of different concentrations of Zn, C. vulgaris cells featured various physical transformations in cell size and structure compared to the control. C. vulgaris cells of the control showed typical size for the species (Fig. 1A and Suppl. Fig. 1A). Morphological variations and aberrations in C. vulgaris cells were visible from 2.5% concentration onwards compared to the control. Cell wall showed signs of shrinkage and structural damages illustrated in Fig. 1 Suppl. Fig. 1, and the deformations were severe under the higher concentration (80%). Under Hg exposure, microalgal cells in response to 2.5, 20, and 80% concentrations of Hg also showed structural alterations compared to the control (Fig. 1B), nearly similar to Zn exposed cells. However, in the combined toxicity test, the adverse effects were marginally higher compared to the single metal toxicity, especially at the highest concentration (Fig. 1C). Indeed, ruptured and shrank cells in the test groups demonstrated the severity of metal-induced toxicity on C. vulgaris and further suggests that the combined effects of the two metals are likely to exert higher toxicity within the cells, ultimately leading to cell structural deformities at a higher extent.

Fig. 1
figure 1

Scanning electron micrograph of Chlorella vulgaris treated with Zn, Hg, and Zn+Hg shows morpho-variation and aberrations. Cell wall damages are observed at 2.5, 20, and 80% heavy metals present in concentrations tested (Scale bar = 3 μm)

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In fact, Hg and Zn compounds induced morphological transformations and oxidative stress in microalgal cells including Chlorella sp. (Nuzzi 1972; Gold et al. 2003; Li et al. 2006; Morin and Coste 2006; Tripathi and Gaur 2006). Similarly, in the diatoms Thalassiosira pseudonana (Sunda 1975) and Skeletonema costatum (Morel et al. 1978), morphological aberrations were reported in response to Cu2+. Furthermore, a photosynthetic protist Euglena gracilis exposed to chromium showed rigorous morphological and biochemical alterations (Rocchetta et al. 2006), while morphological changes due to metal intoxication were widely reported in Chlorophyceae (Rosko and Rachlin 1977), Chrysophyceae (Davies 1974), Bacillariophyceae (Morel et al. 1978; Nuzzi 1972; Sunda 1975), and ciliates (Tingle et al. 1973). In contrast, few studies in the past have shown ameliorating effects of iron (Fe) against the toxicities of other heavy metals in various algae, including Micrasterias (Volland et al. 2011; Volland et al. 2012; Andosch et al. 2012) which showed significant improvements of cell morphogenesis, photosynthesis, cell division rates, and the structures of chloroplasts (Volland et al. 2014). Also, it has been shown that Fe and Zn assist in ameliorating the toxicity of heavy metal such as chromium (Cr) (Mallick et al. 2010; Branzini et al. 2012) possibly induced by the presence of competition for carrier up-take into the cell (di Toppi and Gabbrielli 1999; Shanker et al. 2005). However, due to possible species-specific differences in metal toxicities among algae, further molecular analyses on the metal-uptake potential are required to fully understand the differences in metal toxicities. Aside from species-specific differences in metal-uptake and subsequent induction of toxicity, other factor such as biosorption potential of C. vulgaris may account for the physiological malformation due to Zn and Hg-induced toxicity. Indeed, biosorption in aquatic plants cells is crucial against toxicity as they are involved in removal of toxic elements (Mehta et al. 2002; Michalak and Chojnacka 2010) and threshold concentration for different metals has been reported (Wan Maznah et al. 2012), suggesting species-and metal-specific differences in biosorption potential, contributing to concentration-dependent increase in cell deformities in C. vulgaris under the two metal exposures. Taken together, it is evident that the observed morphological variations in C. vulgaris samples were likely due to metal toxicity (Table 3).

Table 3 Review on morpho-variations and aberrations in the metal exposed microalgae
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The main photosynthetic pigments are comprised of chlorophyll-α, -β, and carotenoids (Chl-α, Chl-β, and Car) (Yang et al. 2020). Chlorophyll is essential in photosynthesis which enables microalgae and cyanobacteria to generate energy from light absorption, Chl-α, specifically (Takamura et al. 1990; Van Baalen and O’Donnell 1978). The results presented in Fig. 2A and Tables 4 and 5 clearly demonstrate a dose-dependent toxic effect of Hg and Zn on pigment contents of C. vulgaris and combined effect of Zn and Hg induced more stress on pigments compared to the single-dose experiment and correspondingly the pigments were decreased. This pattern was observed in both acute (48 h) and chronic (7 days) experiments. However, a random reduction of pigment was observed in chronic toxicity test (Fig. 2A). C. vulgaris cultures exposed to different concentrations of Zn during 48 h and 7 days showed a reduction in Chl-α, Chl-β, and Car contents respectively (Fig. 2A, Suppl. Fig. 2). Above 2.5% concentration, diminution of pigments was noticed compared to the control in both acute and chronic studies. Interestingly, both single and combined toxicity of metals induced higher contents of Chl-β compared to that of Chl-α in all concentration treatment. Single effects of Zn on C. vulgaris cultures from lower to higher concentration showed a concentration-dependent significant reduction (P<0.05) in pigment content and the percentage reductions were 47.64 to 90.93% and 67.09 to 94.17% at lower (2.5) and higher (80) concentrations during acute and chronic exposure (Tables 6 and 7).

Fig. 2
figure 2

Effect of Zn, Hg, and Zn+Hg from 48-h and 7-day experiments on photosynthetic pigment production (A) and protein content (B) in Chlorella vulgaris cells. All the values are mean of triplicates ± SD. Different letters represent significant differences (P < 0.05) in response to different concentrations after Tukey’s post hoc analysis

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Table 4 Effect of Zn, Hg, and Zn+Hg on Chlorella vulgaris after 48h post-exposure
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Table 5 Effect of Zn, Hg, and Zn+Hg on Chlorella vulgaris after 7th day post-exposure
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Table 6 Percentage increase (+)/decrease(−) from control of Zn, Hg, and Zn+Hg on Chlorella vulgaris after 48h post-exposure
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Table 7 Percentage increase (+)/decrease (−) from control of Zn, Hg, and Zn+Hg on Chlorella vulgaris after 7th day post-exposure
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Previously, photosynthetic pigments were found to be diminished under excess concentrations of Zn in microalgal cultures (De Filippis and Pallaghy 1976; Rai et al. 1981b). Similarly, Chl-α concentration was reduced in the green microalga Pseudokirchneriella subcapitata in response to Zn exposure (Soto et al. 2011). Also, high concentrations of Zn reduced total chlorophyll content, ATPase activity and carotenoid/chlorophyll ratio, and cell division and mobility in the green microalgae Scenedesmus obliquus and S. quadricauda (Omar 2002).

Hg-exposed cells showed decreased photosynthetic pigments in both acute and chronic tests (from 2.5 to 80%), compared to the control. Under both acute and chronic exposure, the reduction of pigment contents was in a concentration-dependent manner, compared to the control (i.e., gradual reduction of pigment contents). In a single metal exposed experiment, marginal reduction in pigment contents was noticed in Hg-treated cells than Zn which varied at approximately 3–4% variation on toxic endpoints (Tables 6 and 7).

Hg toxicity has been reported to cause perturbation in various biological functions. For example, growth reduction by Hg in Chlorella was extensively acknowledged (Gipps and Biro 1978; Hutchinson and Stokes 1975), and the photosynthetic capacity of Chlorella was affected at a concentration of 2.5 × 10−5 M HgCl2 (Greenfield 1942).0.1 mg/L Hg, which is highly toxic than Cu or Pb, completely inhibited cell division in Chlorella (Hannan and Patouillet 1972). In previous findings, Hg showed a detrimental effect on various microalgae, showing the decreased photosynthetic pigments as the characteristic of Hg-exposed microalgae (Rai et al. 1981b). Based on previous findings and the results obtained in this study (Tables 4, 5, 6, and 7), it is suggestive that Hg has higher potential to cause significant toxicity in C. vulgaris compared to Zn.

Under chronic exposure, combined toxicity tests with Zn and Hg illustrated the inhibitory effects that were similar to those of the single-exposure experiments with Zn and Hg (Fig. 2A; right panel). In diverse concentrations of combinations of Zn and Hg, gradual decrement of pigment content was shown in C. vulgaris cells during 48 h and 7 days, indicating that this is likely due to dose-dependent inhibitory effect of Zn and Hg. Combination of Zn and Hg provides a significantly higher impact (P<0.05) on microalgal cells than that of single-exposed effect. Due to the metal-induced effects of Zn and Hg in the combined test, the pigment contents were reduced to >90%, compared to control and the marginal difference between the single and combined one was ~4–23% (P<0.05), suggestive of combined metal effect toxicities (Tables 4, 5, 6, and 7).

The main indication of metal toxicity has been prevalently found with the reduction of chlorophyll contents, which is likely associated with oxidative stress. Indeed previous findings from Euglena (De Filippis et al. 1981), metal-exposed higher plants (Clijsters et al. 1999), and the lichen Xanthoria parietina in response to environmentally relevant concentrations of hexavalent chromium (di Toppi et al. 2004), all showed similar outcomes on the species due to metal-induced oxidative stress. Similar to the present study, except the metal concentration used, a concentration-dependent reduction of photosynthetic pigments in response to Zn and Hg was reported earlier in C. vulgaris (Rai et al. 1991). In the pearl millet Pennisetum typhoideum, chlorophyll synthesis was suppressed in response to Hg and Pb (Prasad and Prasad 1987). Diminution in chlorophyll pigments was reported in the microalgae C. kessleri and Coelastrum sphaericum and the delphacid planthopper Stenocranus acutus in response to high concentrations of copper (Schiariti et al. 2004). The green microalga Chlamydomonas reinhardtii showed chlorophyll pigment reduction in response to Cd and Cu (Prasad et al. 1998). The microalga C. protothecoides in response to various concentrations (30–300 μΜ) of the herbicide SANDOZ 9785(4-chloro-5-[dimethylamino]-2-phenyl-3[2H]pyridazinone) caused a reduction in the Chl-α/Chl-β ratio (Samuel and Bose 1987). Also, Cd and Pb reduced the Chl-α/Chl-β ratio in wheat seedlings (Öncel et al. 2000). Pigment reduction was reported in C. vulgaris in response to Cr (Rai et al. 2013). Overall, combined toxicities of metals indeed induced significant loss of chlorophyll contents, possibly by uncontrolled accumulation of metal ions within the cell (Shakya et al. 2007). In addition to the accumulation of heavy metals within a cell, reduction of chlorophyll contents could possibly be due to the inhibition of chlorophyll biosynthesis from heavy metal interference in magnesium in the porphyrin ring of the chlorophyll molecule (Kupper et al. 1998; Kupper et al. 2002). Moreover, loss of chlorophyll pigments is one of the bio-indication for heavy-metal induced injury in plant cells (Muradoglu et al. 2015), suggesting that single and combined toxicity of metals (Zn and Hg) could cause detrimental effect in C. vulgaris. In addition, acute exposure (i.e., 96 h) of Zn and Cu decreased pigment content (Kebeish et al. 2014; Kumar et al. 2016; Zeraatkar et al. 2016) and photosynthetic rates in C. vulgaris (Saavedra et al. 2018).

C. vulgaris cultures in response to different concentrations of Zn demonstrated a concentration-dependent reduction in protein content. At 2.5% concentration, protein content was high, supporting with similar findings (Mishra et al. 2006), where the rising of protein content at lower concentration is likely due to the increase of stress proteins including antioxidant enzymes to maximize the defense against toxicity or could be the indication of the maximum defense threshold. Total amino acid content was increased at less concentration of Zn but was decreased at higher Zn concentration (Omar 2002). The reduction of protein content was due to increased proteolytic activity.

Significant dose-dependent reduction (P<0.05) in the protein content was observed in both acute and chronic studies. Interestingly, in all concentration, the protein content in the combined metal exposed cells was higher than the single-dose Zn-exposed cells, suggesting an elevated level of stress protein accumulation in the algal cells. This pattern was observed in both acute and chronic toxicity studies (Fig. 2B and Tables 4, 5, 6, and 7).

C. vulgaris cells in response to Hg for 48 h and 7 days showed a similar trend as shown in Zn-exposed microalgal cells. Significant enhancement (P<0.05) of protein content at a lower concentration of Hg indicates the generation of stress proteins through which they eradicate their stress but requires further studies to confirm it.

In combined toxicity tests, C. vulgaris cultures in response to different concentrations of Zn and Hg showed similar changes, but at a higher level, to those of single-exposed cultures with Zn and Hg single treatment. Due to the combined effect of Zn and Hg, the metal-induced effects on C. vulgaris cells were higher than the individual exposed effect.

A percentage reduction of protein content in Zn during acute exposure at 20% concentration compared to control was found to be 20.2 ± 2.6, and in Hg, it was around 17 ± 1.1 while in the combined test (Zn+Hg), the value was significantly reduced to 15 ± 1.1 (Table 6) which is highly significant P<0.01 and P<0.05, respectively, compared to that of the individual tests. In chronic exposure, a significantly higher percentage reduction in protein content was observed for both individual and combined tests in a concentration-dependent manner (Table 7).

Induction of protein content at 2.5% concentration was further supported by earlier findings such as an increase of protein content at lower doses (Osman et al. 2004) and it would be one of the mechanisms either eliminating toxic effects or increasing the cellular respiration leading to utilization of carbohydrate in favor of protein accumulation. In microalgae, the toxicity of metals leads the binding to sulfhydryl groups in proteins or the disruption of an essential element and/or interruption of protein structure (Tripathi and Gaur 2006). Elevation in oxidative stress-induced protein degradation demonstrates a correlation between protein reduction and proteolytic activity in response to oxidative stress (Romero-Puertas et al. 2002). Indeed, in agreement to other previously reported studies, the results obtained in C. vulgaris in response to Zn and Hg, under both single and combined as well as acute and chronic exposure, have demonstrated significant increase at the lowest concentration tested, most likely attributing to a wide array of metabolic processes including expression of stress-related proteins for defense against environmental stressors (Xu et al.2008). Taken together, increase in the protein content under a low concentration of both single and combined effect of metals (i.e., Zn and Hg), possibly suggests that C. vulgaris can cope with the metal-induced stress only up to certain concentrations; however, the threshold concentration would most likely be in a species-specific manner.

To analyze whether metals induce oxidative stress, single and combined toxicity tests in C. vulgaris cultures in response to 2.5, 20, and 80% concentrations of Zn and Hg at 48 h and 7 days were performed. In this investigation, ROS generation was found to be increasing significantly (P<0.05) in both metal exposures, in both single and combined effects (Figs. 3 and 4; Tables 4, 5, 6, and 7) in concentration-dependent manner. In the individual toxicity test with Zn, during acute exposure at 2.5% concentration, 5-fold increase in ROS production was observed compared to the control, and in Hg, ~ 6-fold increase was observed; whereas in the combined test (Zn+Hg), 7-fold difference was observed (Table 6). All the values were statistically significant at P<0.01 and P<0.05. While compared to the acute test significantly higher percentage increase in ROS production was noticed in the chronic test (Table 7). ROS generation by the various concentrations of metal compounds suggests induction of oxidative stress in C. vulgaris, which further verifies the toxic nature of the metals in Chlorella.

Fig. 3
figure 3

Generation of reactive oxygen species in response to Zn, Hg, and combination (Zn+Hg) during acute and chronic exposure. Different letters represent significant differences (P < 0.05) in response to different concentrations after Tukey’s post hoc analysis. Data are the mean ± SD of triplicates

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Fig. 4
figure 4

Fluorescent confocal microscopy images demonstrate Zn-, Hg-, and Zn+Hg-induced reactive oxygen species (ROS) generation in Chlorella vulgaris. (A) Non-treated cells (control), cells treated with 2.5, 20, and 80% concentrations of Zn for 48 h and 7 days; (B) Non-treated cells (control), cells exposed to different concentrations of Hg (2.5, 20, and 80%) incubated for 48 h and 7 days; Non-treated cells (control), cells exposed to different concentrations of Zn+Hg (2.5, 20, and 80%) incubated for 48 h and 7 days. Red fluorescence is the auto-fluorescence of C. vulgaris cells, green fluorescence originated during the reaction of ROS with DHR123. (Scale bar = 5 μm)

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Previously, ROS generation was observed in various algae in response to metals and xenobiotics. For example, exposure of CuO on the green microalga C. reinhardtii induced oxidative stress (Melegari et al. 2013) and exposure to polychlorinated biphenyl (PCB) in the dinoflagellate Lingulodinium polyedrum (da Leitao et al. 2003) led to significant induction of oxidative stress. Also, ROS generation was noticed in the dinoflagellate Prorocentrum minimum in response to CuCl2 and PCB (Ponmani et al. 2015), while the accumulation of the intracellular ROS was reported in the microalgae C. vulgaris and P. subcapitatain response to Cu (Knauert and Knauer 2008). In agreement with our results, the similar tendency in ROS generation has been reported in the microalgal cells of Anabaena sp. in response to Zn2+, reaching the maximum peak at a concentration higher than 0.7 mg/L Zn2+. In fact, heavy metals promote oxidative damage in two ways, by increasing the cellular concentrations of ROS (Winterbourn 1982) and/or by reducing the cellular antioxidant potential (Sies 1999). The adverse effects of ROS accumulation on cellular levels are highly associated with protein oxidation, lipids, and nucleic acids, which ultimately lead to alterations in cell structure and mutagenesis (Halliwell and Gutteridge 1999; Pinto et al. 2003). Furthermore, negative effects of metal and/or xenobiotic-induced ROS generation and consequent oxidative stress pose a higher threat in photosynthetic organisms compared to animals, as the common biological source of oxygen is acquired through intense electron flux within the microenvironment, which is already filled with elevated oxygen levels and metal ion concentrations, making photosynthetic organisms highly susceptible to oxidative stress (Pinto et al. 2003). Taken together, the single and combined effect of metal (Zn and Hg) induced a significant increase in ROS levels in C. vulgaris, which may be closely associated with concentration-dependent cell morphological deformity; however, further studies on morphological alteration in association to intracellular ROS levels are required to fully elucidate this phenomenon due to the presence of ambiguity in mechanisms of heavy metal toxicity.

Microalgae have diverse antioxidant enzymes to mitigate the increased generation of ROS caused by metals. For ROS scavenging, antioxidant enzymes perform a vital role (Kang et al. 1999; Sharma et al. 2012). Antioxidant enzymes are well-known biomarkers of protection in response to oxidative stress and generation of antioxidant enzymes is regarded to be one of the ways to avoid or overcome the metal-induced cell destruction (Wu and Lee 2008). CAT, SOD, glutathione reductase (GR), and glutathione peroxidase (GPx) are meant to safeguard cells and tissues from oxidative damages and to counteract the toxicity of ROS (Ensibi et al. 2013). CAT is the key enzyme for the conversion of H2O2 to H2O and O2. CAT involved in the mechanism to shield the cells against the damage caused by ROS to cellular components including nucleic acids, lipids, and proteins (Imlay 2002).

The initial key enzyme for ROS scavenging is considered to be SOD in plants and other organisms, playing an important role in active O2 metabolism and altering superoxide radicals (O2-) to H2O2 at a rapid rate. SOD, among other antioxidant enzymes, detoxifies superoxide anions (Beyer et al. 1991; Mellado et al. 2012) and considered to be a renowned biomarker of defense in response to oxidative stress (Assche and Clijsters 1990; Chongpraditnun et al. 1992). In this study, both single and combined exposure to Zn and Hg in C. vulgaris resulted in significant elevation at initial concentration, followed by gradual reduction under acute and chronic exposures (Fig. 5A and Tables 4, 5, 6, and 7). A bell-shaped concentration-response pattern was noticed in the SOD activity. In the individual toxicity tests of Zn and Hg, the percentage reduction in SOD activity during acute exposure at 20% concentration was found to be 16.7± 0.4 and 6.5 ± 0.1, while in the combined test, the value was significantly reduced to 3.7 ± 0.1 due to the combined effect of Zn and Hg (Table 6). All the values were statistically significant at P<0.01 and P<0.05. A concentration-dependent significant percentage reduction was noticed in both acute and chronic tests with Zn, Hg, and Zn+Hg (Tables 6 and 7).

Fig. 5
figure 5

Effects of diverse concentrations of Zn, Hg, and Zn+Hg for 48-h and 7-day exposure on superoxide dismutase activity (A) and catalase activity (B) in Chlorella vulgaris cells. Different letters represent significant differences (P < 0.05) in response to different concentrations after Tukey’s post hoc analysis. Data are the mean ± SD of triplicates

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In plant cells, SOD activity increased as a result of different kind of chemical compounds and physical stresses (Mittler 2002). Induction of superoxide anion content was also shown in metals-exposed macroalgae (Çelekli et al. 2016; Wu et al. 2014). Increased SOD activity was observed in the cyanobacterium Spirulina platensis in response to Zn, Pb, Cu over a concentration gradient of 0.05–0.20 mg/L (Choudhary et al. 2007). Also, the combined effects of Cu and Cd increased the SOD activity in C. vulgaris (Qian et al. 2011) and the second exposure effect of Cu2+ and CTC showed increased SOD activity on the microalgae C. pyrenoidosa and M. aeruginosa (Lu et al. 2015). Furthermore, single and combined effects of Cd and 4-n-NP on the microalga C. sorokiniana for 48 h, 72 h, and 96 h showed induction of SOD activity and reduced during the exposure time increased (Wang et al. 2018). Overall, initial induction of SOD activity under acute and chronic exposures to single and combined treatment of Zn and Hg in C. vulgaris may suggest that both mitochondrial and chloroplast electron transport systems may be affected by heavy metal-induced oxidative stress (Pinto et al. 2003). Also, an initial increase in SOD activity relative to a reduction over time may suggest disruption of oxidative balance which possibly depends on the severity of the stress and metal properties.

When C. vulgaris cultures were exposed to various concentrations of Zn and Hg under acute and chronic periods (48 h and 7 days, respectively), a significant increase (P<0.05) in CAT activity was observed as shown in Fig. 5B and Tables 4, 5, 6, and 7. In the single toxicity tests, during acute exposure, the percentage increase in CAT activity compared to control at 80% concentration in Zn and Hg were 941 ± 2 (9-fold) and 1056 ± 2 (10-fold), respectively; whereas in the combined test (Zn+Hg), increase in CAT activity was significantly higher (P<0.01 and P<0.05) (1212 ± 1.4 [12-fold]) compared to the single exposure tests (Table 6). While compared to the acute exposure, percentage increase in CAT activity during chronic exposure was high in both individual and combined test (Table 7).

In the marine microalga Pavlova viridis, the antioxidant enzymatic activities were increased at the highest concentrations in response to Zn and Cu (Li et al. 2006). Similar to that of the findings in the toxicity test of C. vulgaris cells with Zn, significant increasing trend (P<0.05) in CAT activity was observed for both individual toxicity and combined toxicity (Fig. 5B). A dose-dependent increase in the antioxidant activity was reported in C. vulgaris in response to Cu, Pb, and Cd (Bajguz 2010; Cheng et al. 2016). Antioxidant enzymes interact together to prevail over metal impacts in the marine microalgae Acanthophora spicifera and Chaetomorpha antennina and the seaweed Ulva reticulate (Babu et al. 2014). CAT demonstrates an essential function in the microalga P. subcapitata at greater toxicant concentrations (Soto et al. 2011). Activation of CAT activity occurred in the freshwater cyanobacterium Anabaena doliolum in response to Cu (Mallick and Rai 1999). Production of antioxidant enzymes such as CAT, GR, and GPx in C. vulgaris in response to Cd exposure illustrates that these antioxidant enzymes perform together to reduce the toxic effects of metals (Cheng et al. 2016). Indeed, increase in CAT activity is referred to be an adaptation method developed by plants (Reddy et al. 2005). Single toxicity tests of Cu and Cd on C. vulgaris showed a slight increase in CAT activity (Qian et al. 2011). Besides, single and combined effects of Cd and 4-n-NP on the microalga C. sorokiniana demonstrated initial stimulation in CAT activity which then decreased over time (Wang et al. 2018). In this study, however, metal-induced (Zn and Hg) elevation in ROS was not successfully scavenged by the antioxidant CAT, despite significant increase in CAT activity in C. vulgaris, which may suggest incapability of CAT in the clearance of Zn and Hg-induced ROS, possibly due to concentration-specific modulations of SOD activities, which may not have fully catalyzed superoxide into oxygen in response to the two metals

In summary, Zn and Hg established a significant impact on the C. vulgaris cells. The observation from this study clearly documents a higher level of dose-dependent toxic effect of Hg and Zn in combination than in single, which suggests synergistic effects. These effects were observed in both acute and chronic toxicity studies through significant flection in photosynthetic pigment content, total protein content, ROS production, antioxidant enzymes (SOD and CAT) along with morphological aberrations. However, elaborative study with large data sets including the genomics and proteomics data are required to find out the synergistic effects of metals at the molecular level in C. vulgaris.