Effects of Iron-Oxide Nanoparticles (Fe3O4) Released From Synthesized Iron-based Thiourea Catalyst on the Growth, Cell Density, and Pigment Content of Chlorella Vulgaris

This study investigated the effects of Fe 3 O 4 nanoparticles released from synthesized Thiourea catalyst to Chlorella vulgaris as an essential primary producer in aquatic systems. A range of Fe 3 O 4 concentrations (0, 10, 100, 250, 500, 750, and 1000 mg L -1 ) was applied for the exposure test. Biological parameters of C. vulgaris, including cell density, cell viability, and pigment content were assessed. Bioconcentration factor and bioaccumulation were evaluated for contaminated microalgae. Non-carcinogenic risks were then assessed using target hazard quotient (THQ) for potential human consumptions. Findings showed that C. vulgaris cell numbers increased from 0 to 500 mg L -1 of Fe 3 O 4 . Chlorophyll a represented a time-dependent response, and greatest values were detected in 250 and 500 mg L -1 Fe 3 O 4 at 4.2 and 4 mg/g, respectively. Chlorophyll b content showed a time-related manner in exposure to Fe 3 O 4 with the highest values recorded at 250 mg L -1 after 96 h. Moreover, bioaccumulation displayed a dose-dependent response as bioaccumulated iron was in the largest amount at 15000 µg/g dw in 1000 mg L -1 , whereas the lowest one was in the control group at 1700 µg/g dw. The bioconcentration factor showed a concentration-relevant decrease in all iron treatments and 10 mg L -1 of Fe 3 O 4 represented the greatest BCF at 327.3611. Non-carcinogenic risks illustrated negligible hazard (THQ < 1) in a dose-response pattern and the largest EDI and THQ were calculated in 1000 mg L -1 at 7.4332E-07 (mg kg -1 day -1 ) and 1.06189E-09, respectively. In essence, iron is an essential trace element for biological aspects in aquatic systems, but in exceeding concentrations could impose toxicity effects in C. vulgaris populations.

The environmental concentration of iron-based materials has been increased in the biota, including soil, air, and water [10]. Literature have reported the fate, biotransformation, bioaccumulation, and toxicity of such elements in various ecosystems [11,12]. In particular, aquatic environments are the most important destinations for iron-based compounds through wastewater drainage and land lls in populated areas.
Fe 3 O 4 , in general, possess various behaviors when enter to the water systems, and undergo some processes, including aggregation, dissolution, redox reaction, and interaction with potential macromolecules [13]. These iron-based products can act as the best iron resource for microalgae, and in highly-contaminated systems lead to eutrophication and algal bloom [14]. Furthermore, iron-based materials in overloaded-conditions cause bioaccumulation and bioconcentration in the aquatic species, and thus, oxidative stress could result in through reactive oxygen species (ROS) production [15]. Aquatic ora and fauna are more sensitive to toxic materials compared with terrestrial species, and related compounds of iron can easily pass the cell membrane and cause severe damages to enzymes, proteins, and DNA integrity of organisms [16]. Lei et al. (2016) examined the toxicity of iron-based NPs to green algae in terms of fate, particle size, and oxidation effects [17]. Chlorella vulgaris (C. vulgaris) was used as an aquatic biological model to investigate iron-based oxidative stress [18]. The toxicity effects of iron oxide nanoparticles were assessed using Zebra sh (Danio rerio) in early life stages, that mortality, hatching delay, and malformation occurred in exposing to these nanomaterials [19]. The toxicity of nanoparticles to aquatic species has been investigated in numerous studies as Behzadi Tayemeh et al.
(2020) examined the toxic effects of silver nanoparticles and ions on C. vulgaris biological responses [20]. Sayadi et al. (2020) used black sh (Capoeta fusca) to study exposure effects of iron oxide nanoparticles and iron salts in causing toxicity, bioaccumulation, and tissue histopathology [21].
Microalga play a vital role in forming the primary production and energy base for all species inhabited in aquatic ecosystems [22,19]. Such valuable photosynthetic species ranged from microscopic to macro sizes and are the main cause of the food and oxygen production in both freshwater and marine systems.
Over the past decade, microalgae have gained signi cant attention among environmentalists due to their advantageous properties in aquatic ecological balance [23]. Many studies have been conducted concerning the toxic effects of metal contaminants on the biological aspects of microalgae, including growth rate, yield rate, pigment content, reproduction, and nutrient content [24]. Copper nanoparticles caused growth inhibition in Skeletonema costatum in exposing to microplastics [23]. Nutritional characteristics of microalgae examined through exposing to metals and metallic NPs, and pigments, biological macromolecules, and phenolic compounds reduced in the presence of these deleterious materials [26]. For this, microalgae have been always considered as ideal candidates and indicators in biological and ecological monitoring of the aquatic biota [27]. Moreover, phytoplanktons are being consumed as functional foods and reliable supplementary ingredients in human diet [26,27,29]. Having the richest source of unsaturated fatty acids, antioxidants, proteins, and pigments, microalgae have played a pivotal role in supplying aquatic-based bioactive compounds for consumers [30].
C. vulgaris is classi ed as unicellular green microalgae and known as an essential functional food in the world [19]. C. vulgaris is being sold in many countries like Japan, China, Germany, and India as the most important algal nutrient in human health and aquaculture activities [31]. This freshwater microalga, in addition, is known for its anticancer, anti-in ammatory, antioxidant, and antibacterial merits, which is widely used in pharmaceutical and food industries [32]. C. vulgaris has a great potential of biosorption in binding with toxic materials and metals dissolved in the water [33,34]. Having intracellular metal binding proteins such as metallothioneins, C. vulgaris has been identi ed as a viable microalga in alleviating the adverse effects of xenobiotics in aquatic ecosystems [35]. In addition, C. vulgaris has been an ideal biological model for primary producers to investigate the toxicological effects of metals and nanoparticles released into the aquatic environments. Therefore, this study intended to investigate the effect of iron-based nanoparticles released from synthesized Thiourea catalyst to C. vulgaris, as a pivotal primary producer in aquatic systems, in terms of bioaccumulation, bioconcentration, cell growth, pigment content, and risk assessment for potential human consumptions.

Chemicals and instruments
All chemicals and reagents were purchased from Merck Company (Germany). Infrared (IR) spectra is applied to analyze the spectral experiment using spectrometer (Shimadzu FT-IR-8400S). Following the chemicals preparation, all rst-made products were examined using 1 H NMR (500 MHz) spectra and Bruker DRX-500 to identify the chemical characteristics. To solve the chemicals, an advanced spectrophotometer equipped with CDCl 3 (as the solvent) and tetramethylsilane (TMS) (as the internal standard) at ambient temperature was applied. Zeiss-Sigma (VP 500) was used to obtain SEM images and EDX spectra was recorded on an Oxford Instrumental® version. A vibrating magnetometer was used to identify the magnetic properties of the synthesized catalyst (MD Co., Iran, www.mdk-magnetic.com).
To analyze the thermogravimetric properties, thermal analyzer with a heating rate of 20°C min −1 was used over a temperature range of 25 to 1100°C under owing compressed nitrogen.

Catalyst preparation
To synthesize the catalyst, 5 mmol FeCl 3 .6H 2 O and 2.5 mmol FeCl 2 .4H 2 O were dissolved in 100 mL deionized water under vigorous stirring (800 rpm). Then, NH 4 OH solution (25%, w/w, 30 mL) was added to the prepared mixture at room temperature until adjusting the pH above 11. To form a black suspension, NH 4 OH solution was continuously added to maintain the pH value between 11 and 12. The resulting black dispersion was continuously stirred for 1 h at room temperature and then re uxed for 6 h. Following the methodology, ethanol (40 mL) was applied at 40°C for 1 h to purify Fe 3 O 4 nanoparticles for coating a layer of silica on the surface of the catalyst. Subsequently, tetraethylorthosilicate (TEOS, 10 mL) was charged to the reaction vessel, and the mixture was continuously stirred for 24 h. The silica-coated nanoparticles were collected by a magnet, followed by washing ve times with EtOH, diethylether and drying at 100°C in vacuum for 12 h. A sample of MNPs-SiO 2 (1 g) dispersed in a mixture of 50 mL of dry toluene containing (3-chloropropyl) trimethoxy-silane (1ml) as the effective linker with two different electrophile heads. The mixture was re uxed for 48 h. The nal product was separated by ltration, washed with toluene, and dried under vacuum for 24 h at 150°C. The prepared MNPs@SiO 2 @-Si-(CH 2 ) 3 -Cl (1g) and KI (1.66 g, 0.01 mol) were added to a solution of thiourea (0.76 g, 0.01 mol) and K 2 CO 3 (1.38 g) dissolved in acetonitrile (50 mL) transferred to a round-bottom ask and the mixture was stirred under re ux condition for 8 h. The obtained solid was then magnetically collected from the solution and washed abundantly with water/ethanol followed by drying at 80°C for 12 h.

Characterization of synthesized Thiourea catalyst
To identify the characteristics of Synthesized catalyst, a range of optico-chemical techniques, including FT-IR, VSM, XRD, SEM, EDX, and TGA were applied. Results relating to the FT-IR spectra of magnetic NPs reaction was visualized using scanning electron microscopy (SEM) images as depicted in gure 1-VII. Based on the SEM analysis, the catalyst was made up of nanometer-sized iron particles shaping spherical morphology. Energy-dispersive X-ray spectroscopy (EDX) method was used to characterize the elemental composition of Fe 3 O 4 @SiO 2 @SiO 3 (CH 2 ) 3 NHCSNH 2 ( Fig. 1-VI). According to the EDX analysis, the well-dispersion of Fe 3 O 4 nanoparticles was obtained through the preparation process. Thermal Gravimetric Analysis (TGA) and Differential Thermal Analysis (DTA) were applied in three steps ( Fig. 1-V).
The rst rupture is associated with the loss of water 1% wt around 60°C, and the second and main loss in weight was recorded at 5% wt, 0.083 mol/100 g catalyst, associated with the elimination of thiourea and its propyl spacer between 200 to 300°C. The nal thermal destruction, approximately 1 % wt, was observed from 350 to 700 ˚C, corresponding loss of silica portion of nanoparticles.
The doublet peak was observed in the low eld relating to the H 1 , near electron donor group. In addition, the unit singlet peak was related to the H 3 and another doublet peak was for H 2 that it depended on the group connecting to the aromatic bond which is the same for electron donor group, but the peak H 2 had shifted to NO 2 as this group had responded and shifted to the high/low eld.

Pigment analysis
Pigments, including Chlorophyll a, Chlorophyll b, and carotenoid were considered as microalgae pigments and evaluated using a standard protocol prescribed by Silkina et al. (2015) with some modi cations [36].
Following the pigment analysis, a volume of 13 ml medium was centrifuged for obtaining cultivated algal cells using 20,000 rpm for 5 min at 4°C. The supernatant was rejected and then the maintained cells were preserved at -80°C until experiments. Pigments were extracted according to the method stated by Tsiola et al. (2017) who used 1.5 ml of 90 % ethanol added to the collected cells and then vortexed severely for 2 min and kept for 24 h in a dark condition at room temperature [37]. A microplate reader (BioTek, 800 TS Absorbance Reader; USA) was used to read the pigments in each treatment using 150 µl in triplicates at 470, 666, and 653 nm.

Bioaccumulation and bioconcentration factor
To measure intracellular Fe 3 O 4 concentrations, algal cells were rst collected from the water medium and then were thoroughly washed with cultured medium (without heavy metal ion) to remove the potential extracellular Fe 3 O 4 ions. Digestion was conducted using HNO3:HClO 4 at 80°C and 130°C for 1 and 3 h, respectively, based on the method prescribed by Qian et al. (2011) with some modi cations [28]. Digested material was diluted with de-ionized water, and Fe 3 O 4 concentrations were detected using ame atomic absorption spectrophotometer (Model 67OG, USA). Bioconcentration factor (BCF) was calculated using equation 1 as suggested by Kuppu et al. ( 2018).
Where CMDT is concentration of metal in dry tissue (mg kg −1 ), and CSMW is the concentration of same metal in water (mg L −1 ).

Health risk assessment of contaminated C. vulgaris
The estimated daily intake (EDI) and target hazard quotient (THQ) were used to evaluate the noncarcinogenic risk of

Statistical analysis
All of the analyses and comparisons were conducted using SPSS software version 19.0 (SPSS, Chicago IL, USA). Kolmogorov-Smirnov test was performed to assess the normality of data, and one-way ANOVA was used to determine possible differences between treatments. In all analyses, data were presented as mean ± SD, and differences were considered signi cant at P < 0.05. For data management and drawing ( ) ( ) graphs, Microsoft Excel (version 2016, windows 7) was applied to de ne and calculate the risk assessment analysis.

Cell density and algal growth
Cell density analysis is represented in gure 2. Based on the ndingS, cell density of C. vulgaris showed that the number of cells increased with the elevation of iron-based NPs from 0 to 100 mg L −1 while in higher concentrations (i.e. 250, 500, 750, and 1000) demonstrated a decrease, especially after 72 and 96 h exposure period. Results showed that cell density elevated with the increase of nanomaterial concentration from 0 to 500 mg L −1 after 0, 24, and 48 h exposure time (Fig. 2).

Pigment analysis
Results relating to the chlorophyll a content is depicted in gure 3. It is apparent that the content of chlorophyll a was in the lowest level after 24 h exposure time, whereas in a 96-h period the highest chlorophyll a was recorded in all treatments. Indeed, chlorophyll a content showed a time-dependent response to the concentration of iron nanoparticles. Moreover, in treatments exposed to 250 and 500 mg L −1 , chlorophyll a had 4.2 and 4 mg/g, respectively (Fig. 3). Chlorophyll b, in addition, demonstrated a time-relevant response to Fe 3 O 4 NPs as chlorophyll a content increased from 0 to 96 h after exposure period in all concentrations (Fig. 4). The greatest pigment content was detected at the end of the experiment (96 h) while the lowest one was observed in the rst day of the test. In terms of Fe 3 O 4 NPs exposure doses, algal populations exposed to 250 and 1000 mg L −1 illuminated the highest and lowest chlorophyll b content, respectively. Findings demonstrated that carotenoid underwent a concentrationdependent decrease in all tested groups (i.e. 0, 10, 100, 250, 500, 750, and 1000 mg L −1 ). Furthermore, a time-relevant manner was observed between Fe 3 O 4 NPs concentrations, and carotenoid content increased with the elevation of exposure time from 24 to 96 h in control, 10, and 100 mg L −1 iron nanomaterials. However, C. vulgaris populations exposed to 250, 500, 750, and 1000 mg L 1 showed the lowest carotenoid content in comparison with the lower concentrations and control group (Fig. 5).

Bioaccumulation and bioconcentration factor
Bioaccumulated Fe 3 O 4 in C. vulgaris populations exposed to different concentrations is represented in gure 6. Based on the outcomes, bioaccumulation factor exhibited a concentration-related pattern in the tested groups. The highest bioaccumulation recorded at 15072.9 µg/g dw in exposing to 1000 mg L −1 and the lowest one identi ed at 1592.32 µg/g dw in the control group where algal populations did not expose to Fe 3 O 4 concentration (Fig. 5) and Table 1). Findings regarding the bioconcentration factor (BCF) illustrated that 10 mg L −1 of Fe 3 O 4 had the greatest BCF at 327.3611, whereas other concentrations (0, 100, 250, 500, 750, and 1000) demonstrated BCF no more than 50 (Fig. 7).

Health risk assessment
Risk assessment analysis presented in Table 2. Outcomes suggested that estimated daily intake (EDI) increased with the elevation of Fe 3 O 4 concentration, and the highest EDI was calculated at 7.4332E-07 for C. vulgaris populations who were exposed to 1000 mg L −1 . Target hazard quotient (THQ) revealed a concentration-dependent response to iron concentrations, and the largest THQ was obtained in exposing to 1000 mg L −1 at 1.06189E-09. THQ in all contaminated populations of C. vulgaris to Fe 3 O 4 was below 1 (i.e. THQ < 1), and therefore, cultivated microalgae represented negligible hazard for algal consumers.

Discussion
Iron is one of the most abundant metal in the earth's crust (34.6 %) and would be naturally available in the environment [42]. This vital trace element plays a crucial role in producing materials in the photosynthetic organisms as well as cell division processes. There are a variety of iron-based compounds in the environment, but the most common forms are Fe 2 O 3 , Fe 3 O 4 , FeSO 4 , FeCl 3 , and Fe 2 NO 3 .
Iron in certain concentrations is essential for living-beings in the environment, but at higher concentrations, it can be toxic. In microalgae population, iron involves in metabolism pathways, including photosynthesize, pigment, DNA synthesis, nitrogen xation, and respiration. Furthermore, iron-based materials are constructed through a wide range of human-made activities which result in iron pollution in the biota. Synthesized catalysts are designed to functionalize chemical reactions via reducing the cost and increasing the speed of processes. For this, many industries have applied organocatalysts containing metals as a reliable and viable base. The ever-increasing application of such catalysts has resulted in environmental pollution and aquatic toxicity for many organisms. Literature reported that wastewater from industrial developments contains 10 to 120 mg L −1 iron [43]. Iron content of wastewater and sediment were estimated to be 50 to 3500 µg L −1 and 4300 to 7000 mg kg −1 in a lake located in Western Australia. Copper smelter companies are one of the most important sources of iron-based materials that cause 882 mg kg −1 iron in the sediment. Our ndings revealed that the bioaccumulation of Fe 3 O 4 NPs in C. vulgaris has a concentration-dependent manner in response to iron-based catalyst used in this study.
Fe3O4 accumulated increasingly from 3273.61 ± 244.298 µg/g dw in populations exposed to 10 mg L −1 to 15072.9 ± 1928.64 µg/g dw detected in 1000 mg L −1 concentration. To support this, C. vulgaris has a great potential of absorption and bioaccumulation of environmental metals and is known as an accumulator used in toxicological studies. This bioindicator possesses a hard and thick cell wall containing a considerable amount of ber that results in absorbing metal ions released into the aquatic biota. Metals such as iron are more desire to absorb and accumulate in the body of aquatic species, rather than being dissolved in the water. Trace elements could be adsorbed by the sh body and its organs in aquatic systems [44]. Another study was conducted using four species of marine algae to evaluate their bioaccumulated iron and its risk for human consumption. Results showed that Gracilariopsis sp. and Sargassum sp. contained larger amount of iron at 1960 and 1570 µg/g dw, respectively, compared to other species. BCF demonstrated that there is a concentration-relevant decrease among populations exposed to Fe 3 O 4 , and the highest value was calculated for 10 mg L −1 treatment at 327.3611, whereas algae exposed to 1000 mg L −1 showed the lowest BCF at 15.07287 after 96 h of exposure. BCF has indirect relationship with the metal concentration exposed to the water medium, but with the increase of bioaccumulated Fe 3 O 4 in algal body, this factor elevates. In general, microalgae are classi ed as good metal accumulators in aquatic systems, and BCF > 1000 suggested that the studied species has a great ability to bioconcentrate metals. Researchers claimed that marine diatom Phaeodactylum tricornutum could actively adsorb Cd (II) ions to remove such toxic materials from the water. They stated P. tricornutum showed BCF is a stronger cadmium bioconcentrator as its BCF was above 1000 in most tested concentrations [45]. In the present study, there was no BCF higher than 327.3611 (10 mg L −1 ) and most tested concentrations displayed BCF below 50. To support this, a complete biosorption can occur by microalgae cells in case they expose to lower Fe 3 O 4 concentrations, and in turn, higher BCF could be obtained. With the increase of Fe 3 O 4 concentration in the water, binding sites in the cell wall of the C. vulgaris are rapidly saturated, which inhibit the binding of more metal ions [45]. Moreover, in highly metal-contaminated environments, intracellular mechanisms in C. vulgaris regulate the gradient concentration between the water and organism [46].
According to outcomes emerged to this study, cell density of C. vulgaris populations increased in lower concentrations of Fe 3 O 4 at 10, 100, and 250 mg L −1 after 72 and 96 h exposure period, and the highest cell growth was observed in populations exposed to 100 mg L −1 iron. However, in higher concentrations (i.e. 250, 500, 750, and 1000 mg L −1 ) cell density was inhibited. It is investigated that the cell density in C. vulgaris populations exposed to silver nanoparticles (AgNPs) and silver ions (AgNO 3 ) during a 72-h exposure time increased rstly with the elevation of metal nanoparticles and ions, but in higher exposure concentrations and times, it remained stable due to inhibitory effects of such materials [20]. Aquatic microalgae possess limitations to use micronutrients and absorb trace elements in their inhabitants, and in higher concentrations these essential elements act as growth inhibitors. These ndings mirrored those reported by Naorbe and Serrano (2018)  This may mean that microalgae such as C. vulgaris has their own growth regulatory mechanisms that act as inhibitory factors to adjust their populations based on the environmental conditions. The suppression of C. vulgaris growth in exposing to cadmium, lead, and copper stress was studied, and results concerned that the growth and chemical compositions decreased during the rst 48 h of exposure time, and copper was more effective inhibitory factor than lead and cadmium to prevent cell growth [49]. Increasing on iron bioavailability above 200 µM could mitigate the growth rate of C. vulgaris populations exposed to Fe and increased the amount of lipid radical content in the intracellular space [18].
Pigment analysis, in this study, showed that chlorophyll a content increased over the exposure period (i.e.  [39]. Pigment content of microalgae can be used as a reliable biomarker in investigating the health status of the organism, and in whole, community and ecosystem. Literature reported that the most important reason regarding the pigment reduction in microalgae is due to adaptation to unsuitable environmental conditions through changing the pigments to supply organic nitrogen [51]. According to our results, chlorophyll b content showed a similar pattern as chlorophyll a, and a signi cant increase was observed during 96-h exposure time. The highest and lowest chlorophyll b were identi ed at 250 and 1000 mg L −1 , respectively. Carotenoid content of the studied microalgae showed a concentration-related decrease from 0 to 1000 mg L −1 . Literatures have proven that the loss of pigment content during the exposure period to metals is mainly because of damage in chloroplast ribosome as well as inhibition in the electron transport chain in the donor center of the C. vulgaris cells [52]. In toxic concentration, Fe 3 O 4 interfere in the pigment production process and alter the essential enzymes and proteins accounted for pigment synthesis [41]. It is highly possible that with the ever-increasing applications of iron-based catalysts in chemical reactions used for various industries, the environmental concentration of Fe 3 O 4 increase in the aquatic biota, including freshwater rivers, marine environments, lakes, and wetlands. The importance of microalgae as functional foods and supplementary ingredients in human diet has been proven in many studies [54][55][56][57]. Having a diverse range of unsaturated fatty acids, antioxidants, omega-3, proteins, and pigments, C. vulgaris has engrossed many attentions among nutrition scientists and aquaculture companies. However, the health status of the C. vulgaris, in terms of bioaccumulated metals, is vital for those who consider such valuable ingredients in their daily diets. Because of this, carcinogenic and non-carcinogenic risk assessment tests are applied to examine the potential risk of edible microalgae for consumers. In this study, estimated daily intake (EDI) and target hazard quotient Although iron is a vital micronutrient for algal viability and reproduction, inhibitory and toxicity effects may occur in its exceeding concentrations above 500 mg L −1 . As a strong accumulator in aquatic systems, C. vulgaris, could desire to absorb released Fe 3 O 4 ions, that led to increasing bioaccumulation with the elevation of iron concentrations. C. vulgaris possesses a great capacity to bioconcentrate iron nanoparticles, and in this regard, this species plays an essential role in biomagni cation through food web in aquatic environments. EDI and THQ revealed that non-carcinogenic risk of Fe 3 O 4 was at negligible risk (THQ < 1) for people. Taken together, although iron-based organocatalysts used in industrial applications are green and safe, in over-used conditions act as inhibitor for primary producers like C. vulgaris and threaten the health status of consumers as aquatic functional food.

Declarations Declaration of interests
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.   Cell density of C. vulgaris exposed to different concentrations of Fe3O4 in a 96-h period.

Figure 3
Chlorophyll a content of C. vulgaris exposed to different concentrations of Fe3O4 in a 96-h period.
Page 21/23 Figure 4 Chlorophyll b content of C. vulgaris exposed to different concentrations of Fe3O4 in a 96-h period.

Figure 5
Carotenoid content of C. vulgaris exposed to different concentrations of Fe3O4 NPs in a 96-h period.  Bioconcentration factor of Fe3O4 in C. vulgaris during a 96-h exposure period.