Toxicity of ZnFe-SO4 layered double hydroxide in Tetradesmus obliquus and evaluation of some physiological responses of the microalgae for stress management

Layered double hydroxides (LDHs), regarding their physical and structural properties, have different and wide applications industry and their increasing use may raise ecological and human health concerns. However, the potential toxicity mechanisms of LDHs in different organisms are still unclear. In the present work, after synthesizing of ZnFe-SO4 LDH and studying of its characterization by XRD, FT-IR, SEM, EDX-mapping, TEM and Raman, its toxicity in Tetradesmus obliquus was evaluated. According to experimental results, the growth of the algae and content of photosynthetic pigments were significantly decreased after treatment with 100 mg/L of ZnFe-SO4 LDH. The high dose exposure to the LDH also inhibited the activity of SOD and POD enzymes, possibly due to the LDH- catalyzed reactive oxygen species production. In addition, lipid peroxidation and the content of phenolic compounds, as no-enzymatic antioxidants were increased by enhancement of the LDH concentration. The rise of phenol, flavonoids and MDA contents could be regarded as some manifestations and responses to the toxic effects of the contaminant in the algae cells. The results provided a better understanding of the undesirable effects and toxicity of LDHs in aquatic organisms.

of pollutants, microalgae such as species of Scenedesmacease family are critical biomarkers for environmental changes and have been of much interest 11 .Tetradesmus (Scenedesmus) obliquus (Turpin) M.J. Wynne is a member of the family that has gained attention for the production of biocomponents that were used in wastewater treatment and for other environmental purposes 12 .The influential role of the microalgae has been well confirmed in the remediation of phosphorus, sulfate, phosphate, ammonium, phenolic compounds, heavy metals and some medicinal compounds such as paracetamol and salicylic acid [13][14][15] .The microalgae was also used for biofuel production, aquaculture, development of biosensors and applied as bioreactor like some cyanobacteria [16][17][18] .T. obliquus showed excellent sensitivity to pesticides, heavy metals and some toxic substances like formaldehyde and can be used for detecting pollutants in aqueous media 11 .However, despite the mentioned extensive researches about the microalgae, no study have focused on the potential mechanism of LDHs toxicity to T. obliquus.
The present work aimed to determine the toxicological impacts of ZnFe-SO 4 LDH on T. obliquus, as well as to screen some biochemical and physiological responses of the algae to the existence of LDH in its environment.Toward this goal, the ZnFe-SO 4 LDH was synthesized and characterized by SEM (Scanning electron microscopy), EDX (Energy dispersive X-ray) mapping, TEM (Transmission electron microscopy), XRD (X-ray diffraction) and FT-IR (Fourier transform infrared).Next, after the treatment of the algae with ZnFe-SO 4 LDH, the toxicological effects of the contaminants on some algal physiological parameters, including growth, pigments content and enzymatic and non-enzymatic antioxidants, were determined.

Characterization
To examine the surface morphology and elemental composition of ZnFe-SO 4 LDH, FESEM and EDX analyses were performed, respectively.The uniformness of the nanomaterials properties is crucial 19 .Figure 1a,b, displays the formation of nanosheets (average thickness of about 26 nm).The successful synthesis of ZnFe-SO 4 LDH exhibited characteristic hexagonal plate-like LDH sheets and was predominately smooth.The results of ZnFe-SO 4 LDH were in a close agreement with the corresponding literature data 20,21 .Furthermore, for ZnFe-SO 4 LDH to check the possible impurity elements from the synthesis processes, EDX-map measurements were performed 22 .The results of the EDX-mapping microanalysis are indicated in Fig. 1c-i.The presence of Zn, Fe, S, and O in its spectra confirmed the successful synthesis of ZnFe-SO 4 LDH without other impurities.Due to the ZnFe-SO 4 LDH being synthesized with a ratio of 3:1, the presence of Zn, Fe, S, and O was 36.84%,5.9%, 8.7%, and 48.57% in the LDH structure, respectively.Also, the elemental mapping images revealed that Zn, Fe, S, and O were homogeneously and uniformly distributed throughout ZnFe-SO 4 LDH parallel with the amounts used in the synthesis.
The morphology of the as-synthesized samples was measured by TEM images through different magnifications (Fig. 2).The TEM mode is well-suited for crystal orientation analysis and periodic structure analysis of nanomaterials.In addition, diffraction patterns obtained from TEM mode can provide valuable insights into the crystal structure of the sample.The TEM images showed that the prepared plate-like ZnFe LDH included crystallites which were approximately hexagonal.The structure indicated the layered essence of the LDH.
The functional groups and the molecular structure of ZnFe-SO 4 LDH were determined via FTIR analysis (Fig. 3b).The -OH stretch vibrations with hydrogen bonding and surface water molecules have been detected with the broad, strong bands within the range of 3448 1/cm.The interlayer water bending modes could be identified by the bands in the range of 1645 1/cm.The bands located at 1128 1/cm were related to sulfate ions.The peaks showed stretching and vibration modes of M-O and O-M-O in the hydrotalcite around 400-800 1/cm and 400-600 1/cm, respectively, where M denotes the cations of Zn 2+ and Fe 3+24 .
Raman spectroscopy provides identification of crystalline phases present in materials from the number of observed bands and their wave numbers which are related to their space group and the bonds force constants, respectively.Usually, the Raman spectrum of a nanomaterial remains sufficiently similar to the corresponding single-crystal one to allow univocal identification.The interlayer anions of LDH structures are characterized by the weak band at 900-1000 cm −1 in the Raman spectrum (Fig. 3c).The lattice vibration modes of M-O and O-M-O, could be identified by the bands in the low-frequency area.The existence of the peak at about 600 cm −1 was related to the Fe-O-Fe stretching vibrations.Also, the peak at 300 cm −1 and 450 cm −1 could be assigned to Zn-O bonds and Zn-O-Fe bonds, respectively 25 .

ZnFe-SO 4 LDH effect on algal cell growth and pigments, content
Nanomaterials would inhibit algae's growth by affecting gene expression, metabolism, photosynthesis and nitrogen fixation.The inhibition of algae growth may vary with the type, concentration, exposure time of nanomaterials and testing organisms 9 .The growth profiles of the algal cells grown under treatment with 10 and 20 mg/L of ZnFe-SO 4 LDH for six days were similar (Fig. 4a), and these concentrations seldom imposed a significant effect on the cell growth.Treatment of concentrations higher than 20 mg/L (50 and 100 mg/L ZnFe-SO 4 LDH) reduced the final cell density to near 40% and 48.2%, respectively.In general, the higher exposure concentration www.nature.com/scientificreports/ of LDH would induce inhibition of the microalgae growth.The toxicity of some other nanomaterials, such as carbon nanotubes, Fe 2 O 3 nanoparticles and MgO nanoparticles was also reported for various green algae.For instance, MgO nanoparticle was found to be toxic to S. obliquus and Pseudokirchneriella subcapitata even at low (0.8 mg/L) and high concentrations (100 mg/L), respectively 26,27 .Different sensitivity of algal species to various concentrations of nanomaterials was also observed for iron-based nanoparticles.The toxic effects of Fe 2 O 3 -NP on Nanochloropsis sp. and Isochrysis sp.appeared at low concentrations 28 ; meanwhile, in Chlorella vulgaris, the high concentration of nanoparticles caused toxicity effects 29 .These various responses of algal species to different concentrations of the contaminants might be a species-specific behavior shared by virtually all members of a given species 27 .
After exposure of the algae cell to different concentrations of ZnFe-SO 4 LDH, the content of photosynthetic pigments was determined.Treatment of the algal cells by 10, 20 and 50 mg/L of the pollutant led to the enhancement of Chl a, b, as well as total carotenoids contents, as compared to the control experiment (Fig. 4b).The increase of Chl a and b content was statistically significant only for 50 mg/L of the pollutant (p < 0.05).Treatment of 100 mg/L of the contaminant reduced Chl a and b, as compared to the control sample.Chlorophyll content is one of the important indicators confirming the tension situations in photosynthetic organisms and its depletion can lead to the reduction of the photosynthetic rate when followed by the decrement in the growth rate of the given organism 30 .In contrast to Chl changes, the total carotenoids were raised at all concentrations of ZnFe-SO 4 LDH (Fig. 4b), but these increases were not statistically significant (p > 0.05).According to the literature, the carotenoid content was also enhanced in C. vulgaris cells after treatment with some nanoparticles such as ZnO and Fe 2 O 3 during heavy metals stress 31,32 .Moreover, it was shown that despite Ni negatively affects on chlorophyll contents, it increases carotenoids content in Dunaliella sp 33 .An increase in the amount of some antioxidant compounds, including carotenoids, is one of the events that can occur following undesirable environmental conditions, such as the existence of pollutants in the environment 34 .Carotenoids protect the integrity of the cell membranes and increase their stability by inhibition of lipid peroxidation 35,36 .
Figure 4c also illustrates light microscopic images of the microalgae in control samples and after exposure with 100 mg/L ZnFe-SO 4 LDH.The adsorption of nanomaterials to cell surface or their entrance to algal cells and destruction of the subcellular structures were reported as adverse effects of nanomaterials in algae 9 .In the present study, the treated cells showed varying degree of vacuolization and increased cavitations, as compared to the images of the control cells (Fig. 4c).However, the exact effects of the LDH on cell organelles is not clear and needs further investigation.Accordingly, SOD activity was significantly enhanced after treatment by 50 mg/L ZnFe-SO 4 LDH, resulting in the 2.2-fold induction of the SOD activity, as compared to the corresponding control (Fig. 5a).Augmentation and induction of SOD activity were reported after treatments of different concentrations of nonomaterials such as carbon nanotubes, Fe 2 O 3 nanoparticles and ZnO nanoparticles in S. obliquus and C. vulgaris 27,32 .The induction of SOD activity was also reported in different microalgae species in response to heavy metal stress.A concentrationdependent increase in SOD activity has been revealed with Cu, Pb and Cd in C. vulgaris and with Cu and Zn in Pavlova viridis and S. vacuolatus [37][38][39] .However, the enzyme activity was reduced significantly after six days of treatment with 100 mg/L ZnFe-SO 4 LDH.In biological systems, SOD, as a front line of defense mechanisms, is involved in combatting reactive oxygen species (ROS) produced during different stress conditions 40 .Thus, an increase in SOD activity has been correlated with the mechanisms responsible for tolerating and coping the stress conditions 41 .POD is one of the most important enzymes for ROS capture in cell of microalgae under heavy metal stress and works in close synchrony with SOD to prevent production of more ROS during different stresses 42 .Treatment of 10 and 20 mg/L of the LDH led to increase of POD activity, but contrary to the induction of the SOD activity after treatment with 50 mg/L ZnFe-SO 4 LDH, the treatment of 50 mg/L along with 100 mg/L lowered the POD activity (Fig. 5b).Increased POD activity has been shown in different microalgae such as C. vulgaris, S. obliquus and Dunaliella sp.under exposures to Cu, Cd, Ni and Pb 15,33,37 .In contrast to such induction of antioxidant enzymes activities, their inhibition following the treatment of living organisms with high concentrations of contaminants has been confirmed in other studies 40 .
Due to the structural properties of phenolic compounds, such as aromatic rings in their chemical structures, they can act as radical scavengers 43 .According to Fig. 5c, some concentration-dependent increase was observed in the amounts of phenols and flavonoids.The phenol content in the microalgae was elevated up to 13.3 and 15.6 mg GA/g FW after treatment with 50 and 100 mg/L ZnFe-SO 4 LDH, respectively (compared to its content in the control sample, 8.2 mg GA/g FW).Furthermore, a significant enhancement in the flavonoids content of the algae was observed after its treatment with 50 and 100 mg/L ZnFe-SO 4 LDH (Fig. 5d).Although, unlike plants, there is not much information about the type and content of phenolic compounds in algae and about these compounds involvement in cellular responses to ROS generated during different stresses 44 .It was found that the phenolic profile of diatom Phaeodactylum tricornutum was dependent upon the addition of copper and iron to the culture media and the production of relevant amounts of phenolic compounds acted as protective mechanism against toxicity of Cu and Fe 45 .The accumulations of gentisic acid, (+) catechin and (−) epicatechin, the most prominent phenolic compounds, in Dunaniella tertiolecta have also been previously reported after the algae treatment with 790 nmol/L Cu 46 .In addition to the phenolic compounds, the augmentation in flavonoids content was reported after the treatment of C. sorokiniana and S. acuminatus by Zn 47 .

ZnFe-SO 4 LDH effect on the MDA content
One of the main destructive effects of ROS is removing hydrogen from unsaturated chain of fatty acids and causing their additional peroxidation at the cell membrane.During lipid peroxidation, different types of cytotoxic compounds such as MDA are produced.Therefore, MDA content is directly correlated with ROS accumulation and subsequent oxidative damage 9 .Treatment of microalgae with 20, 50 and 100 mg/L ZnFe-SO 4 LDH led to  a statistically significant rise of the MDA content up to 15.9, 20.6 and 25.9 nM/gFW, respectively (Fig. 6).The obtained result could be a sign of lipid peroxidation during the treatment of the algae with high concentrations of the pollutants.MDA has been extensively studied in different microalgae under various stress conditions and similar effects were detected after exposure of microalgae with different pollutants, such as other nanomaterials, heavy metals and antibiotics.For instance, MDA content was raised in S. obliquus and Raphidocelis subcapitata after exposure to high concentrations of carbon nanotubes and clarithromycin, respectively 27,48 .Moreover, exposure of S. obliquus and Euglena gracilis to Pb and Cr at their EC 50 concentrations increased the MDA content respectively 15,49 .

LDH synthesis
ZnFe-SO 4 LDH was synthesized via the co-precipitation method.FeSO 4 .7H 2 O (0.001 mol) and ZnSO 4 .7H 2 O (0.003 mol) were dissolved in 40 mL distilled water.The solution was titrated slowly with NaOH solution (2 mol/L) until the pH reached 8 at room temperature and under Ar atmosphere.After 24 h stirring, the precipitates were centrifuged and dried overnight in an oven at 60 °C3 .

Algae cultivation and treatments
T. obliquus was obtained from the Artemia and Aquaculture Research Institute, Urmia University.As mentioned in the text, T. obliquus was obtained from the Artemia and Aquaculture Research Institute, Urmia University.Identification of the microalgae, was already done by Asalpishe et al. 50.Briefly, using pairs of primers designed by Baldwin 51 , two segments of ITS (Internal transcribed spacer) (ITS1 and ITS 2) were amplified by PCR.For obtaining the precise sequence of PCR products, Sanger sequencing was applied.The sequence of the amplified region was submitted into NCBI with accession number OR393092.The microalgae was cultivated in 100 mL liquid mineral Bold's Basal Medium (BBM) using 250 mL flasks 52 .The flasks were at 22 ± 2 °C, illuminated with cool white fluorescent tubes at a continuous light intensity of 60 μE/m 2 /s (24 h photoperiod) and aerated with filtered air continuously.The microalgae were harvested every seven days.The flasks containing specific numbers of T. obliquus and culture medium were exposed to 10, 20, 50 and 100 mg/L of ZnFe-SO 4 LDH, during the experiment.

Measurement of the growth rate and pigments content
In order to perform the microalgae growth assay, pre-cultures of T. obliquus at the exponential phase were inoculated into the fresh culture medium containing different concentrations of ZnFe-SO 4 LDH (initial cell density: 10 6 cells/mL).The treatment lasted for seven days and the algal growth was monitored daily using a hemocytometer with Neubauer improved rulings (Boeca, Germany), after fixation of the microalgae cells with Lugol's solution.
The 2 mL micro-algae culture was centrifuged and after decanting the supernatant, the residue sediment was mixed with acetone 100% and then incubated for 24 h without light.Finally, the contents of Chl a, b, as well as total carotenoid were determined spectrophotometrically using the equations described by Lichtenthaler 53 .

Antioxidants assays
To evaluate the antioxidant responses, T. obliquus cells were treated with different concentrations of ZnFe-SO 4 LDH suspensions in the nutrient solution.The harvested cells from 100 mL culture medium were homogenized in a buffer solution containing 0.2% polyvinylpyrrolidone (PVP) and centrifuged at 4 °C.The resulting supernatants were applied to assess the activities of antioxidant enzymes.Bradford assay was then applied for protein content measurement 54 .
The SOD activity was acquired by determining the inhibition of the photochemical reduction rate of nitroblue tetrazolium (NBT) through the algal extract.The reaction buffer contained 67 mmol/L potassium phosphate buffer solution (pH 7.8), 1.5 mmol/L NBT, 0.12 mmol/L riboflavin and a suitable aliquot of enzyme extract.The reaction mixture was illuminated for 15 min at light intensity of 5000 Lux.The absorbance was measured at 560 nm and the enzyme activity was indicated by U/mg protein 55 .To determine POD activity, a reaction mixture was used composed of guaiacol (ɛ: 26.6 mM −1 cm −1 ), H 2 O 2, citrate-phosphate-borate buffer and a suitable amount of enzyme extract 56 .The increase in absorbance at 470 nm during polymerization of guaiacol to tetraguaiacol was recorded for 3 min.
The content of the total phenols was calculated by a spectrometric method described by Singleton 57 .A proper amount of the algae extract was mixed with 600 μL of distilled water; then, 50 μL of the Folin-Ciocalteu's reagent was added to the mixture.After 7 min, 150 μL Na 2 CO 3 (20%) was added and the total volume of the mixture was 1 mL by distilled water.After 2-h incubation of the samples at the ambient temperature, the absorbance was determined at 760 nm.Their contents were expressed as mg of gallic acid equivalent (GAE) per g of the algae fresh weight (FW).To determine the flavonoids content, the aluminum chloride colorimetric method 58 by quercetin as a reference compound was used.To put it briefly, 600 μL of the algae methanolic extract ( www.nature.com/scientificreports/ was mixed with 2% AlCl 3 methanol solution.After the 60 min incubation at room temperature, the absorbance of the mixture was determined at 420 nm and expressed as mg quercetin equivalent (QE) g −1 FW.

Malondialdehyde (MDA) content
To determine malondialdehyde (MDA) accumulation, 0.01 g of fresh algae samples was ground in 1 mL of 0.1% (w/v) trichloroacetic acid (TCA), at 4 °C; then, a suitable amount of 0.5% (w/v) thiobarbituric acid (TBA) in 20% (w/v) TCA was added and heated at 95 °C for 30 min.The reaction mixture was refrigerated rapidly and centrifuged at 10,000g for 10 min (4 °C).The supernatant absorbance was determined at 532 nm and 600 nm and MDA content was expressed as nmol MDA g −1 FW (ɛ = 155 mM −1 cm −1 ) according to the method developed by Heath and Packer 59 .

Statistical analysis
One-way ANOVA followed by Tukey-Kramer, multiple comparisons test, was used for statistical data analysis (data with three replicates; using GraphPad Software, Inc. USA).The results were described as mean ± standard deviation (SD).

Conclusion
The toxicity of ZnFe-SO 4 LDH to a typical microalgae T. obliquus was determined.The growth inhibition and reduction of the chlorophyll contents were found when the microalgae was exposed to high concentration of the LDH.The LDH could also cause lipid peroxidation and induce the antioxidant defense system of T. obliquus to protect the microalgae from the undesirable toxic effects of the pollutant.The contents of total phenols and flavonoids were increased in response to treatment of high concentration of the LDH and possibly due to the induced ROS production during the high dose exposure to the pollutant.Furthermore, the activities of SOD and POD enzymes were inhibited after the treatment of T. obliquus with 100 mg/L of ZnFe-SO 4 LDH.The results of the present study will be helpful for the risk assessments of LDHs and provide a new insight into the toxicological effects of LDH in algae.

Figure 4 .
Figure 4.The effect of different concentration of ZnFe-SO 4 LDH (mg/L) on (a) growth and (b) contents of chlorophyll a, b, a + b and total carotenoids of T. obliquus (Mean ± SD, n = 3, different letters in each column showed statistically significant difference (p < 0.05)) and (c) Light microscopic images of T. obliquus cells in control condition (left) and after treatment with 100 mg/L ZnFe-SO 4 LDH (The scale bars are 10 μm; yellow arrows show the vacuolization of the cells).

Figure 5 .
Figure 5. (a) SOD and (b) POD activities and the content of (c) total phenol and (d) flavonoids in control microalgae and the microalgae exposed to different concentration of ZnFe-SO 4 LDH (Mean ± SD, n = 3, different letters in each part of the figure showed statistically significant difference (p < 0.05)).

Figure 6 .
Figure 6.Contents of MDA (nM/ g FW) in control T. obliquus and the microalgae exposed to different concentrations of ZnFe-SO 4 LDH [Mean ± SD, n = 3, different letters in each column showed statistically significant difference (p < 0.05)].