Synthesis of spinel ferrites nanoparticles and investigating their effect on the growth of microalgae Picochlorum sp.

Abstract Spinel ferrites nanoparticles (SNPs) have been extensively studied, synthesized and used in many applications including wastewater treatment, biosensors and as photocatalysts. The studies of the effect of these SNPs at cellular and molecular levels and their influence in the environment are scarce. Thus, in the current study, different metal ferrite SNPs were synthesized via sol-gel-combustion route and were characterized by X-ray Diffraction (XRD), Scanning Electron Microscope (SEM) with Energy Dispersive X-ray (EDX) and Vibrating Sample Magnetometry (VSM) analysis techniques. The effect of these SNPs on the growth of the microalgae Picochlorum sp. was then investigated. The results showed that the synthetic method used to prepare the metal ferrite SNPs leads to highly crystalline SNPs with average crystallites size between 33–36 nm except for ZnFe2O4 NPs which have crystallite size of 52 nm. The results of XRD and EDS confirmed the formation of SNPs. VSM analysis of CoFe2O4 showed the highest magnetic energy (U) and saturation magnetization (Ms) of 30870 erg/g and 60 emu/g, respectively. Although, the microalgae culture of Picochlorum sp. treated with different SNPs showed significant difference in viable cells concentration at 48 h and 72 h of incubation compared to control samples, the growth pattern of both treated and untreated samples were seen similar. This could indicate that SNPs may reduce the growth of the microalgae but will not cause severe inhibition when used at the proper concentration. This promotes their potential use for many applications.


Introduction
Spinel ferrite nanoparticles (SNPs) have general formula of MFe 2 O 4 , where M is a metal cation with (2þ) charge such as Mn, Fe, Cu, Ni and Zn.These NPs have unique magnetic and electrical properties and thus have been utilized in many applications such as biomedical (Ahamed et al., 2011), biosensors (Sandu, Presmanes, Alphonse, & Tailhades, 2006), high frequency components (Praveena, Chen, Liu, Sadhana, & Murthy, 2016), photocatalytic activity (Sharma, Bansal, & Singhal, 2015;Valero-Luna, Palomares-Sanch ez, & Ru ız, 2016;Yean-Ling, Steven, Hwai-Chyuan, & Wen-Tong, 2016) and degradation of organic Azo dyes (Oladipo, Ifebajo, & Gazi, 2019) and removal of tetracycline antibiotic from aqueous solutions (Ifebajo, Oladipo, & Gazi, 2019).The SNPs have been synthesized using various methods including microemulsion (Ganure, Dhale, Katkar, & Lohar, 2017), hydrothermal (Wang, 2006), solvothermal (Li et al., 2015;Ni, Lin, Xiaoling, Xinqing, & Hongliang, 2015), co-precipitation (Pereira et al., 2012), sol-gel (Jasso-Ter an et al., 2017) mechanical milling (Marinca, Chicinas ¸, Isnard, & Neamt ¸u, 2016) and chemical combustion (P erez, Gomez-Polo, Larumbe, P erez-Landazabal, & Sagredo, 2012).Due to the increase in the production of designed nanoparticles, human exposure to SNPs and their potential for adverse health effects have been of great concern.Thus, there are number of publications focused on studying the toxicity and the effects of these SNPs on different human cells and living organisms.For example, NiFe 2 O 4 SPs caused a decrease in cell viability in some types of human cells (Ahamed, Akhtar, Alhadlaq, Khan, & Alrokayan, 2015;Ahamed et al., 2011) and their cytotoxicity increases as the size of the particle increases (Yin, Too, & Chow, 2005).ZnFe 2 O 4 SPs induced cytotoxicity and oxidative stress in some human cells in the dosage range of 10-40 mgmL À1 (Alhadlaq, Akhtar, & Ahamed, 2015).On the other hand, CoFe 2 O 4 SPs showed no significant toxicity effect on human cancer line cells at the concentration of 0.095-0.95mgmL À1 and cell viability increases in parallel with the concentration of the NPs (Kim, Kim, Kim, Shim, & Lee, 2007;Pa sukonien _ e et al., 2014).Considering the effect of SNPs on aquatic organisms, Sebastian, Vijayalakshmy, Lakshmi, Saramma, and Mohammed (2014) observed a decrease in the content of chlorophyll of Chlorella pyrenoidosa algae on exposure to high concentration (> 0.5 mM) of ZnFe 2 O 4 SNPs.L opez-Moreno et al. ( 2016) studied the effect of CoFe 2 O 4 on the growth of Lycopersicon lycopersicum (tomato plants) and observed that when the concentration of NPs was increased, root growth and the uptake of Fe, Co, Mg and Ca in the plant tissues slightly increased.It has been found that the size of Fe 3 O 4 SNPs had a great influence on the growth of Picochlorum sp. and increasing particle size lowered the toxicity and the negative effect was only observed in the early growth stages (Hazeem et al., 2015).
In the present paper, various metal ferrite nanoparticles were synthesized via sol-gel combustion method and the structures of these SNPs were characterized by XRD, SEM with (EDX) which is used to identify the elements in the sample quantitatively.The magnetization properties of the nanoparticles were followed by VSM.The effect of these SNPs on the growth of the marine microalgae Picochlorum sp. were investigated.The effect of SNPs was tested using 10 mgL À1 during exponential growth phase.The concentration was selected according to recent publications using different NPs and their effect on microalgae, in which the overall findings indicated that the NPs generally at low concentrations do not have a negative impact on the growth of microalgae.For examples, Li et al. (2006) found that Cu þ2 and Zn þ2 salts were not toxic to the marine microalgae Pavlova viridis (3012) at low concentrations (0 and 3 mgL À1 ) and Zn þ2 salt was used at concertation's ranged between 0 and 32.5 mgL À1 .Additionally, ZnFe 2 O 4 was found to enhance algal growth at low concentration (0.5 mM (0.12 mgL À1 )) using the microalgae Chlorella pyrenoides and at higher concentration (1 to 2 mM (0.24-0.48 mgL À1 )), the growth was found to be hindered (Sebastian et al., 2014).Ahmad, Yao, Zhou, and Liu (2015) treated the microalgae Chlorella vulgaris with CoFe 2 O 4 at concentrations ranged between 6.3 to 100 mM (1.48 to 23.5 mgL À1 ) and found that the NPs didn't generate a negative effect on microalgae at low concentration 6.3 mM (1.48 mgL À1 ).Hazeem et al. (2015) found that the magnetic Fe 3 O 4 NPs inhibited cell growth of the microalgae Picochlorum sp. using 20 nm NPs, whereas the 40 nm Fe 3 O 4 NPs and the bulk material of Fe 3 O 4 were found to promote algal growth.Furthermore, they revealed that the 20 nm Fe 3 O 4 NPs did not show significant difference between the treated and untreated samples, in the lag and decline phases at different concentrations (50-1600 mgL À1 ).Therefore, the authors of the current study and according to previous published work and literature search decided to evaluate the effect of the synthesized SNPs at low concentration for comparison with existing literature.

Method and materials
All chemicals and solvents were of analytical grade and they were purchased from Sigma-Aldrich and used without further purification.

Chemical synthesis of spinel ferrites nanoparticles (sol-gel combustion method)
The SNPs were synthesized using sol-gel combustion method reported by P erez et al. ( 2012).An equimolar mixture of M(NO 3 ) 2 .XH 2 O, where M ¼ Cr, Co, Cu, Ni, Zn, and Fe(NO 3 ) 3 .9H 2 O salts and citric acid (1 g, 0.0025 mol) in 150 mL distilled water was stirred for  Then the mixture was heated at 135˚C until formation of gel and then was burned to obtain the dark brown to black powder.The powder product was kept for characterization.

Characterization of SNPs
The crystal structure, phase composition and average particle size of the newly prepared spinel ferrites nanoparticles were determined using Rigaku-Ultima IV x ray diffractometer equipped with Cu-radiation (k¼1.542A˚) in the range of 20 to 80 with an accelerating voltage of 40 kV and counting rate of 1 /min.The morphology and the chemical composition of the samples were identified using EVO LS 10 Scanning Electron Microscope (SEM) with EDX.The magnetic properties measurements were carried out at room temperature using PMC MicroMag 3900 model VSM in the field range of þ10 kOe to À10k Oe.SNPs at a concentration of 10 mgL À1 .Each treatment was run in triplicates and the incubation test was conducted under controlled laboratory conditions (18 C, 100 mmol photons/m 2 .secand 12:12 h light: dark cycle).Viable cell concertation was measured using Muse TM cell analyzer (Millipore, USA) at 24, 48 and 72 h after incubation to elucidate the effects of SNPs on the growth of microalgae.The data is presented as mean and standard deviation (SD).Sigma Plot 13 was used to plot the graph between viable cell concentration and time.Oneway ANOVA was used to determine whether there exists any significant difference between control and treated samples at any time.

Results and discussion
The structures of the prepared SNPs were identified by XRD and SEM with EDX.The results of XRD analysis are shown in The diffraction patterns of all the powders confirmed the formation of the corresponding spinel metal ferrite (MFe 2 O 4 ), except for CrFe 2 O 4 which is unstable and thus the weight percentage was difficult to estimate (Table 1) and the peak pattern most probably corresponded to FeCr 2 O 4 .In addition, the diffraction peaks of all the samples were sharp which indicate highly crystalline nature of the prepared samples.Some traces of metal oxides phases are present in the prepared samples in which the determination of the charge of distribution has been of general interest (Afsin & Roberts, 1994).SEM was used to determine the external morphology, shape and chemical composition of a crystalline material.Figure 2 2.
Vibrating Sample Magnetometer (VSM) is used to measure the magnetic properties of magnetic materials.The magnetic properties of the prepared samples were measured at room temperature between þ10 kOe À10 kOe.The M-H relationships of the studied ferrites were demonstrated in Figure 4.In order to compare magnetic energy of the studied ferrites, magnetic energy U (erg/g) was used.CoFe 2 O 4 NPs has high hysteresis loss (Table 1), which means it is hard metal ferrite.The hard ferrites are usually characterized by high coercivity and magnetic energy.CoFe 2 O 4 SPs exhibited ferromagnetic behavior with coercivity (Hc) and saturation magnetization (Ms) of about 1029 Oe and 60 emu/g, respectively.These results agree with the data reported by Matsuda, Nakanishi, Kaneko, and Osaka (2015).NiFe 2 O 4 is soft metal ferrite and exhibited saturation magnetization and coercivity of 47 emu/g and 120 Oe, respectively, which are in conformity with the literature values (P erez et al., 2012).ZnFe 2 O 4 had the lowest magnetic energy with the saturation magnetization of 44 emu/g (Alhadlaq et al., 2015).It was found that coercivity values (Hc) of the prepared SNPs increased as the crystallite size decreased.For example, the coercivity values are found about 1029, 119 and 40 Oe for CoFe 2 O 4 , NiFe 2 O 4 and ZnFe 2 O 4 with the corresponding crystallite sizes of 33, 35 and 52, respectively.Such experimental result was known previously for different magnetic materials and alloys (Adler & Pfeiffer, 1974;Bertotti, Fiorillo, & Pasquale, 1991;Degauque, Astie, Porteseil, & Vergne, 1982;Landgraf, Da Silveira, & Rodrigues, 2011), which shows the influence of grain size (GS) upon the magnetic properties of one group of compounds.
The effect of the synthesized SNPs on the growth of Picochlorum sp. is depicted in Figure 5.Control samples showed an increase in viable cell concentration during 72 h of incubation time from 14.3 Â 10 3 cells/mL (±2.8) to 32.23 Â 10 3 cells/mL (±3.2).There was no significant difference between the control and treated samples at 24 h; however, a significant difference (p < 0.05) was observed between the control and treated samples at 48 and 72 h.
The microalgae treated with different SNPs showed similar trend of growth, but the number of viable cells were lower than the control samples, which implicated that the SNPs could reduce the growth of microalgae.Complete inhibition of cell growth was not recorded with any of the tested SNPs which make them potential materials for many environmental, industrial and medical applications.
The results of this study agree with the previous literature.For instance, the effect of zinc ferrite NPs on the growth of the freshwater Chlorella pyrenoidosa was investigated and it was found that the effect changes depending on the concentration of the NPs.Algal growth can be promoted when proper concentrations of NPs are used (Sebastian et al., 2014).L opez-Moreno et al. ( 2016) demonstrated that Lycopersicon lycopericum (tomato plants) did not exhibit any negative effect when exposed to cobalt ferrite NPs (CoFe 2 O 4 ).Tomato plants were able to tolerate the NPs concentration up to 1000 mgL À1 without noticeable toxicity.Hazeem et al. (2015) found that magnetic Fe 3 O 4 NPs promoted algal growth during late growth stages (stationary   and decline phases).Kim et al. (2007) revealed that CoFe 2 O 4 was not toxic and the tissues injected (ICR mice cells) showed no notable changes in comparison to the control samples.Ahmad et al. (2015) found that CoFe 2 O 4 nanobeads at 6.3 mM did not show any significant inhibition to microalgae growth or chlorophyll a content using Chlorella vulgaris, though, concentrations above 12.5 mM caused oxidative stress and mechanical damage to the algae.Contrary to the current study, Ahamed et al. (2011) found that nickel ferrite NPs induced dosedependent cytotoxicity in human lung epithelial (A549 cells) through ROS generation and oxidative stress.Additionally, Ahamed et al. (2015) found that nickel ferrite also induces dose-dependent cytotoxicity in liver HepG2 and breast MCF-7 cell lines through many assays including oxidative stress.Matsuda et al. (2015) found that CoFe 2 O 4 exhibited higher coercivity than Fe 3 O 4 NPs of the same diameter (10 nm).This higher coercivity led to more heating efficiency of NPs under an AC magnetic field and consequently caused cell death of human breast cancer MCF-7 cells (Matsuda et al., 2015).
In some other studies, some dissimilarities were noticed.Pa sukonien _ e et al. (2014) have observed different responses in different cell lines (human pancreatic and ovarian cancer cells) on the treatment with superparamagnetic cobalt ferrite NPs (Co-SPOINS).They found that both cell lines accumulated Co-SPOINS, but they displayed different responses.A2780 cells (ovarian cancer cell line) found to be more sensitive to exposure to the NPs.However, the general conclusion of their study indicated that Co-SPOINs were not toxic when a safe concentration is used.A summary of the different responses of different cell lines to SNPs is illustrated in Table 3.

Conclusion
Various metal ferrite SNPs of high crystalline structures were synthesized in high purity via sol-gel-combustion method.The structures of the SNP samples were confirmed by XRD and SEM techniques.The average crystallite size of the prepared samples was determined between 33-36 nm, except for ZnFe 2 O 4 which was about 52 nm.The highest saturated magnetization was about 60 emu/g for CoFe 2 O 4 .There were no significant differences in cell growth on exposure to different spinel nanoparticles at 24 h of incubation.However, a significant difference between control samples and treated samples were noticed at 48 and 72 h.The growth pattern of the treated samples was similar to the control samples but with lower viable cell concentration, indicating that the SNPs may reduce the growth of microalgae at certain concentration, but do not induce complete inhibition.SNPs of different doses, different organisms and cell lines could be investigated to draw a more detailed picture on the effect of SNPs and their potential uses in many environmental, industrial and medical applications and to understand their ultimate impact on environment.
represents the SEM images of the prepared SNPs.The images revealed irregular shapes except for ZnFe 2 O 4 NPs which have non uniform spherical shape.Energy dispersive X-ray (EDX) results confirmed that the ratio of the transition metal atoms in each system is consistent with the nominal stoichiometry.The atomic ratio of Co/Fe, Ni/Fe, Cu/Fe and Zn/Fe for CoFe 2 O 4 , NiFe 2 O 4 , Zn Fe 2 O 4 and CuFe 2 O 4 was approximately 1:2.An ERX of ZnFe 2 O 4 NPs is shown in Figure 3 and Table

Figure 5 .
Figure 5. Changes in viable cell concentration in control and treated samples at 24, 48 and 72 h.(SNPs were used at 10 mgL À1 , pH 8).

Figure 4 .
Figure 4. Magnetization versus applied field of the MFe 2 O 4 synthesized NPs.

Table 1 .
Crystal structure and the magnetic parameters of the prepared nanoparticles.
Table 1 and Figure 1.XRD is usually used to analyze the crystal structure, phase equilibrium, and crystallite size.Debye-Scherrer equation was used for calculating particle size (D ¼ 0.94k/(b cos h), where k is the wavelength of the xray and b is the line broadening at half maximum intensity.The average crystallite sizes of FeCr 2 O 4 , CoFe 2 O 4 , NiFe 2 O 4 , CuFe 2 O 4 and ZnFe 2 O 4 were determined as 34, 33, 36, 35 and 52 nm, respectively.