The Toxicity Assessment of Iron Oxide (Fe3O4) Nanoparticles on Physical and Biochemical Quality of Rainbow Trout Spermatozoon

The aim of this study was to evaluate the in vitro effect of different doses (50, 100, 200, 400, and 800 mg/L) of Fe3O4 nanoparticles (NPs) at 4 °C for 24 h on the kinematics of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) spermatozoon. Firstly, Fe3O4 NPs were prepared at about 30 nm from Iron (III) chloride, Iron (II) chloride, and NH3 via a co-precipitation synthesis technique. Then, the prepared Fe3O4 NPs were characterized by different instrumental techniques for their chemical structure, purity, morphology, surface properties, and thermal behavior. The size, microstructure, and morphology of the prepared Fe3O4 NPs were studied by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) spectroscopy, and scanning electron microscopy (SEM) equipped with an energy-dispersive X-ray spectrometer (EDS). The thermal properties of the Fe3O4 NPs were determined with thermogravimetric analysis (TGA), differential thermal analysis (DTA), and differential scanning calorimeter (DSC) analysis techniques. According to our results, there were statistically significant (p < 0.05) decreases in the velocities of spermatozoon after treatment with 400 mg/L Fe3O4 NPs. The superoxide dismutase (SOD) and catalase (CAT) activities were significant (p < 0.05) decrease after 100 mg/L in after exposure to Fe3O4 NPs in 24 h. As the doses of Fe3O4 NPs increases, the level of malondialdehyde (MDA) and total glutathione (tGSH) significantly (p < 0.05) increased at doses of 400 and 800 mg/L.


Introduction
In today's technology, magnetic nanoparticles have an increasing importance [1][2][3]. In particular, Fe 3 O 4 is widely used in advanced technological applications such as magnetic imaging, drug release systems, enzyme immobilization matrices, catalyst support materials, cell separating molecules, hyperthermia, and reinforcement for some composites. Recently, many papers have reported on the implications and applications of Fe 3 O 4 nanoparticles (NPs), which are termed magnetite. Magnetite exhibits outstanding physicochemical properties due to the presence of both Fe(II) and Fe(III) in its structure. In particular, it behaves as a super paramagnetic when the particle size is reduced to a few nanometers [4]. Iron-based NPs have been used in many applications such as the treatment of chlorinated solvents and metals, the prevalent application for soil and groundwater remediation, biomedical applications (magnetic resonance imaging, drug delivery, and cell labeling), the treatment of water adsorption capacity, to improve surface modification, and in protective shells, solid supports, and the doping of a second metal [5]. For instance, Naha et al. showed that dextran-coated bismuth-Fe 3 O 4 NPs could be utilized as contrast agents for computed tomography and magnetic resonance imaging [6]. Similarly, several groups synthesized Fe 3 O 4 NPs-loaded polymeric microbubbles to create a bimodality platform; they also investigated the use of these particles as multimodal contrast agents [7,8]. In addition, it has been reported that Fe 3 O 4 NPs can be used to both control plaque-biofilms and prevent dental caries since they have an intrinsic enzyme mimetic activity similar to natural peroxidases [9,10]. As a result of these wide applications, Fe 3 O 4 NPs are also increasingly released in the environment. Furthermore, the potential ecotoxicological impacts in aquatic environments have aroused increasing attention. For example, over the last five years, there have been some studies about the ecotoxicology of iron-based NPs in aquatic animals such as an Indian major carp fish (Labeo rohita) [11], tilapia (Oreochromis niloticus) [12], Chinook salmon (Oncorhynchus tshawytscha) [13], zebrafish (Danio rerio) [14][15][16], Mytilus galloprovincialis [17], Daphnia magna [18], Artemia salina [19,20], and rotifer (Brachionus rotundiformis) [21].
In vitro methods allow the determination of the mechanisms and areas of action of pollutants by applying a wide range of exposure times and concentrations to compare the toxicity levels of environmental pollutants [22]. However, there are not many studies on in vitro toxicity of Fe 3 O 4 NPs in fish sperm. Therefore, in this study we aimed to determine the in vitro toxicity of Fe 3 O 4 NPs and its effects on the kinematics and oxidative stress markers of spermatozoon in rainbow trout (Oncorhynchus mykiss, Walbaum 1792). Thus, we aimed to test and understand the effects of this toxicity on the reproductive system of fish in aquatic ecology.

Instrumentation and Reagents
The chemicals used for nanoparticle synthesis and toxicological studies were all of high purity. FeCl 3 ·4H 2 O, FeCl 2 ·4H 2 O, and NH 3 were obtained from a distributor of Merck Co. in Turkey. All chemicals used for toxicological studies were obtained from Sigma-Aldrich Co. (Saint Louis, MO, USA). All other chemicals were of the highest purity and commercially available and all solutions were prepared with distilled water.
Fourier transform infrared spectroscopy (FTIR) (Mattson 1000) was used to characterize the chemical structure of prepared Fe 3 O 4 NPs. The thermal properties of the prepared Fe 3 O 4 NPs were determined using TGA-50 (Shimadzu, Kyoto, Japan) and DTA-50 (Shimadzu, Kyoto, Japan) under a static air atmosphere and at a heating rate of 10 • C min −1 in the temperature range from 30 to 1000 • C. The DSC measurements of the Fe 3 O 4 NPs were performed on a DSC-60 (Shimadzu, Kyoto, Japan). All samples (about 5 mg) were placed in sealed aluminum pans before heating under nitrogen flow (20 mL/min) at a scanning rate of 10 • C/min. An Al 2 O 3 (5 mg)-filled aluminum crucible was used as a reference. The surface structure and morphological properties of the obtained Fe 3 O 4 NPs were investigated with SEM-EDX (LEO Evo-40 VPX). The Fe 3 O 4 NPs were characterized by XRD for the crystal structure and impurity. A Rigaku Rad B-Dmax II powder X-ray diffractometer was used for the XRD patterns of these samples. The 2θ values were taken from 2 • to 85 • with a step size of 0.04 • using Cu Kα radiation (λ value of 2.2897 Å).

Synthesis of Fe 3 O 4 NPs
In the study, FeCl 2 ·4H 2 O (2 g) was added to 50 mL of distilled water and mixed for 1 h. Then, FeCl 3 ·4H 2 O (5.45 g) was added to 50 mL of water in another flask, and stirred for 1 h. At the end of the 1-h mixing time, 50 mL of oleic acid was added to each of the solutions in each portion and mixed for 30 min. After that, the first and second portions of the solutions were taken into a 250 mL balloon and mixing was continued for 30 min. Then, 20 mL of 1.5 M NH 3 solution was added dropwise. The mixture was stirred at room temperature for 1 day with a mechanical stirrer and a light brownish material was obtained at the end of the reaction. The product was centrifuged and washed with ethanol four times.

Collection and Exposure of Sperm Samples
The rainbow trout males (1850 ± 110 g) were maintained in the hatchery station at a commercial fish farm, Malatya, Turkey. Sperm samples were obtained in January 2018. Stripping was performed by massage from the front to the back of the fish abdomen without anesthesia. Fresh sperm samples were diluted with immotile solution (IMS) and activated by motile solution (MS). Immotil solution (IMS) was prepared by NaCl, 103 mmol/L; KCl, 40 mmol/L; CaCl 2 , 1 mmol/L; MgSO 4 , 0.8 mmol/L; Hepes, 20 mmol/L; 1000 mL of distilled water; pH 7.8 as a stock solution [23]. Motil solution (MS) was prepared by CaCl 2 1 mM; Tris 20 mM, Glycine 30 mM, NaCl 125 mM; 1000 mL of distilled water; pH 9 [24,25].
The pooled sperm samples, taken from six individual fish, were exposed to Fe 3 O 4 NPs. The pooled sample was diluted with IMS to obtain a spermatozoon density of about 13 × 10 8 cells/mL.

Determination of Biochemical Oxidative Markers
For sperm samples preparation for a biochemical assay, each sample was sonified with an ultrasonifier (Bandel in Sonopuls HD 2070) in phosphate buffer solution (PBS) following a previously published protocol [26]. Afterward, the homogenates were centrifuged at 10,000 rpm for 10 min at 4 • C and the supernatants were separated for further analysis. For the measurement of antioxidant enzymes activity, the catalase (CAT) activity was measured by following the reduction of hydrogen peroxide (H 2 O 2 ) at 240 nm at room temperature [27]. The CAT activity was then calculated according to the rate of the change in absorbance and expressed in U/mg protein. One unit of CAT represents the amount of enzyme that decomposes 1 µmol of H 2 O 2 per minute.
Superoxide dismutase (SOD) activity was determined using the xanthine oxidase/cytochrome C method [28]. One unit of SOD activity is the amount of enzyme required to cause a half-maximal inhibition of cytochrome C reduction. Results were expressed in U/mg protein.
Lipid peroxidation was measured by using the thiobarbituric acid (TBA) solution for malondialdehyde (MDA). The solution was added to the semen samples and the reaction mixture was incubated at 100 • C for 30 min. The samples were cooled and centrifuged at 14,000 rpm for 10 min. Then the resulting supernatant was separated and the absorbance of the supernatant was taken at 440 nm at room temperature using an ELISA microplate reader (Biotek, Winooski, VT, USA). The level of MDA was expressed as nmol MDA/mg protein.
Protein was estimated according to the method reported by Bradford using bovine serum albumin (BSA) as a standard [30].

Determination of Spermatozoon Kinematics
After 24 h, the samples were examined under an Olympus CX31 microscope with a 200× magnification lens and a Sony CCD camera with 30 fbs. Spermatozoon velocity parameters such as VSL: straight line velocity (µm/s), VCL: curvilinear velocity (µm/s), and VAP: angular path velocity (µm/s), as well as movement style parameters such as LIN: linearity (%), the ratio of net distance moved to total path distance, BCF: beat cross frequency turning points of the spermatozoon head (Hz) and ALH: amplitude of lateral displacement of the spermatozoon head (µm) [31] were carried out by the computer-assisted sperm analysis systems, BASA-Sperm Aqua, produced by Merk Biotechnology Ltd. Co. in Turkey.

Statistics
Descriptive analysis (Means ± SE, p < 0.05) in Univariate Variance (two way-ANOVA) and Multiple Variance Analysis (MANOVA) with the Duncan test were used between groups after the homogeneity of each group was tested through the Test of Homogeneity of Variance in the SPSS 17 program. The graphics were created by Graph Pad Prism 5.

Characterization of Fe 3 O 4 NPs
The FTIR spectrum for Fe 3 O 4 NPs structures is given in Figure 1A. In this figure, the hydrogen bond tension of the -OH groups on the surface of the nanoparticles is seen between 3300 and 3600 cm −1 . Moreover, the peak at 600 cm −1 is due to the Fe-O stretching vibration. All these peaks demonstrate the desired Fe 3 O 4 NPs chemical structure [32]. The X-ray spectrum of the synthesized Fe 3 O 4 NPs was also monitored, and is shown in Figure 1B. In this spectrum, 220 peaks at 29.3 • , 311 peaks at 35.1 • , and 400 peaks at 43.4 • can be seen [33]. In addition, 422 peaks at 54.3 • , 511 peaks at 56.7 • , and 440 peaks at 62.7 • confirm the Fe 3 O 4 NPs structure [34]. The obtained Fe 3 O 4 structures were obtained as pure and clean, as seen in the X-ray spectrum. Therefore, when we look at the TGA thermogram, no mass loss is observed. Even with heating up to 800 • C, there is an absence of mass change. A similar interpretation is seen the DTA thermogram. All thermograms show a classic and routine Fe 3 O 4 NPs slope decline [35]. The given DSC thermogram confirms the results of other thermal analyses. Moreover, it can be seen that the obtained nanoparticle structure has a high purity according to the DSC thermogram ( Figure 2).

Statistics
Descriptive analysis (Means ± SE, p < 0.05) in Univariate Variance (two way-ANOVA) and Multiple Variance Analysis (MANOVA) with the Duncan test were used between groups after the homogeneity of each group was tested through the Test of Homogeneity of Variance in the SPSS 17 program. The graphics were created by Graph Pad Prism 5.

Characterization of Fe3O4 NPs
The FTIR spectrum for Fe3O4 NPs structures is given in Figure 1A. In this figure, the hydrogen bond tension of the -OH groups on the surface of the nanoparticles is seen between 3300 and 3600 cm −1 . Moreover, the peak at 600 cm −1 is due to the Fe-O stretching vibration. All these peaks demonstrate the desired Fe3O4 NPs chemical structure [32]. The X-ray spectrum of the synthesized Fe3O4 NPs was also monitored, and is shown in Figure 1B. In this spectrum, 220 peaks at 29.3°, 311 peaks at 35.1°, and 400 peaks at 43.4° can be seen [33]. In addition, 422 peaks at 54.3°, 511 peaks at 56.7°, and 440 peaks at 62.7° confirm the Fe3O4 NPs structure [34]. The obtained Fe3O4 structures were obtained as pure and clean, as seen in the X-ray spectrum. Therefore, when we look at the TGA thermogram, no mass loss is observed. Even with heating up to 800 °C, there is an absence of mass change. A similar interpretation is seen the DTA thermogram. All thermograms show a classic and routine Fe3O4 NPs slope decline [35]. The given DSC thermogram confirms the results of other thermal analyses. Moreover, it can be seen that the obtained nanoparticle structure has a high purity according to the DSC thermogram (   Figure 3A. In these images, the NPs are very homogeneous and appear to be around 30 nm. Furthermore, electron diffraction spectroscopy revealed the Fe3O4 NPs structure. EDX images of Fe3O4 structures are given in Figure 3B. The basic values of O and Fe are clearly seen in this structure. For O and Fe, diffraction peaks are shown at 0.705 and 6.398 keV, respectively. This proves that the nanoparticles have a magnetic ferrite nanostructure.

Spermatozoon Kinematics
The in vitro kinematics of rainbow trout spermatozoon exposed for 24 h to different concentrations (50, 100, 200, 400, and 800 mg/L) of Fe3O4 NPs are shown in Figure 4A-F. The VSL value decreased with increasing Fe3O4 NP doses. In particular, the decrease was found to be significant (p < 0.05) after exposure to 400 mg/L of Fe3O4 NPs (19.91 ± 1.93 μm/s) in Figure 4A. The VCL value of the control group was determined to be 130.84 ± 1.64 μm/s. The lowest VCL value was observed at the dose of 800 mg/L, being 97.88 ± 2.71 μm/s, and this decrease was also significant (p < 0.05) in Figure 4B. The control group had a VAP value of 70.76 ± 8.22 μm/s, which was the highest VAP value in all the groups. The changes in the VAP value were observed to be statistically significant (p < 0.05) at the doses of 400 mg/L and 800 mg/L of Fe3O4 NPs in Figure 4C.

Spermatozoon Kinematics
The in vitro kinematics of rainbow trout spermatozoon exposed for 24 h to different concentrations (50, 100, 200, 400, and 800 mg/L) of Fe 3 O 4 NPs are shown in Figure 4A-F. The VSL value decreased with increasing Fe 3 O 4 NP doses. In particular, the decrease was found to be significant (p < 0.05) after exposure to 400 mg/L of Fe 3 O 4 NPs (19.91 ± 1.93 µm/s) in Figure 4A. The VCL value of the control group was determined to be 130.84 ± 1.64 µm/s. The lowest VCL value was observed at the dose of 800 mg/L, being 97.88 ± 2.71 µm/s, and this decrease was also significant (p < 0.05) in Figure 4B. The control group had a VAP value of 70.76 ± 8.22 µm/s, which was the highest VAP value in all the groups. The changes in the VAP value were observed to be statistically significant (p < 0.05) at the doses of 400 mg/L and 800 mg/L of Fe 3 O 4 NPs in Figure 4C. The LIN value decreased with increasing Fe3O4 NPs doses, and this reduction was significant (p < 0.05) at both 400 mg/L and 800 mg/L doses in Figure 4D. The BCF value was observed to be significant (p < 0.05) at the dose of 400 mg/L of Fe3O4 NPs and up. The highest BCF value was 9.93 ± 0.76 Hz at the 800 mg/L dose in Figure 4E. The ALH value decreased with increasing Fe3O4 NPs doses, and this reduction was statistically significant (p < 0.05) after the 200 mg/L dose. The lowest ALH value was observed to be 17.82 ± 2.14 μm, at the 800 mg/L dose of Fe3O4 NPs in Figure 4F.  The LIN value decreased with increasing Fe 3 O 4 NPs doses, and this reduction was significant (p < 0.05) at both 400 mg/L and 800 mg/L doses in Figure 4D. The BCF value was observed to be significant (p < 0.05) at the dose of 400 mg/L of Fe 3 O 4 NPs and up. The highest BCF value was Toxics 2018, 6, 62 8 of 13 9.93 ± 0.76 Hz at the 800 mg/L dose in Figure 4E. The ALH value decreased with increasing Fe 3 O 4 NPs doses, and this reduction was statistically significant (p < 0.05) after the 200 mg/L dose. The lowest ALH value was observed to be 17.82 ± 2.14 µm, at the 800 mg/L dose of Fe 3 O 4 NPs in Figure 4F.

Oxidative Stress Biomarkers and the Effective Concentration (EC50)
The oxidative stress biomarkers (tGSH, CAT, SOD, and MDA) were determined after spermatozoon were exposed to Fe 3 O 4 NPs (50, 100, 200, 400, and 800 mg/L) for 24 h, and the results were compared with the control group. The effect of Fe 3 O 4 NPs on the biochemical parameters (tGSH, CAT, SOD, and MDA) are presented in Figure 5A-D.

Oxidative Stress Biomarkers and the Effective Concentration (EC50)
The oxidative stress biomarkers (tGSH, CAT, SOD, and MDA) were determined after spermatozoon were exposed to Fe3O4 NPs (50, 100, 200, 400, and 800 mg/L) for 24 h, and the results were compared with the control group. The effect of Fe3O4 NPs on the biochemical parameters (tGSH, CAT, SOD, and MDA) are presented in Figure 5A-D.
According to results, there was significant (p < 0.05) increase in tGSH levels after 400 mg/L dose ( Figure 5A). On the other hand, as the dose of Fe3O4 NPs increased, the CAT activity decreased compared to the control value. This decrease was significant (p < 0.05) after the 50 mg/L dose of Fe3O4 NPs ( Figure 5B). It was observed a significant (p < 0.05) decrease in SOD activity between the control and groups after a dose of 100 mg/L ( Figure 5C). The level of MDA increased, but no significant (p ˃ 0.05) changes in the MDA levels were observed until the 200 mg/L dose. Only at doses of 400 and 800 mg/L significantly (p < 0.05) increased to the levels of MDA when compared to the control ( Figure  5D). The EC50 is the dose at which 50% of the maximum effect is produced, or the concentration of toxicant at which the toxicant is 50% effective. However, the effective concentration (EC50) against According to results, there was significant (p < 0.05) increase in tGSH levels after 400 mg/L dose ( Figure 5A). On the other hand, as the dose of Fe 3 O 4 NPs increased, the CAT activity decreased compared to the control value. This decrease was significant (p < 0.05) after the 50 mg/L dose of Fe 3 O 4 NPs ( Figure 5B). It was observed a significant (p < 0.05) decrease in SOD activity between the control and groups after a dose of 100 mg/L ( Figure 5C). The level of MDA increased, but no significant (p > 0.05) changes in the MDA levels were observed until the 200 mg/L dose. Only at doses of 400 and 800 mg/L significantly (p < 0.05) increased to the levels of MDA when compared to the control ( Figure 5D).
The EC50 is the dose at which 50% of the maximum effect is produced, or the concentration of toxicant at which the toxicant is 50% effective. However, the effective concentration (EC50) against Fe 3 O 4 NPs exposure was calculated in the values of VSL, VCL, and VAP of spermatozoon in rainbow trout ( Figure 6A-C).

Discussion
The goal of the present study was to investigate the toxic impacts of different concentrations of Fe3O4 NPs on the spermatozoon of rainbow trout, Oncorhynchus mykiss. Although the in vitro spermatozoon toxicity has attracted the attention of many researchers, there are limited studies on the toxicity of nanoparticles for the risk assessment of nanomaterials, especially concerning fish reproductive systems.
The literature has shown that iron-based NPs have toxic effects on aquatic organisms. For example, Ates and his colleagues studied the chronic (60 days) effects of alpha and gamma Fe2O3 structures (0.1, 0.5, and 1.0 mg/L) on tilapia (Oreochromis niloticus). They focused on the effects of particle morphology on accumulation, elimination, hematology, and immune responses. According to their results, while the spleen had the largest accumulation, the intestine, kidney, liver, gills, brains and muscle tissues followed close behind. They determined no significant changes in hemoglobin, hematocrit, red blood cell, and white blood cell counts. While the level of serum glucose (GLU) decreased, the levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and lactate dehydrogenase (LDH) increased. Finally, they determined that Fe2O3 NPs can induce differential uptake, assimilation, and immunotoxic effects on O. Niloticus under chronic exposure [12].
Another study focused on the effects of different doses (0.01, 0.1, and 1.0 mg/mL) of metal oxide NPs (SnO2, CeO2, and Fe3O4) on mortality and behavioral and biochemical responses of Artemia salina

Discussion
The goal of the present study was to investigate the toxic impacts of different concentrations of Fe 3 O 4 NPs on the spermatozoon of rainbow trout, Oncorhynchus mykiss. Although the in vitro spermatozoon toxicity has attracted the attention of many researchers, there are limited studies on the toxicity of nanoparticles for the risk assessment of nanomaterials, especially concerning fish reproductive systems.
The literature has shown that iron-based NPs have toxic effects on aquatic organisms. For example, Ates and his colleagues studied the chronic (60 days) effects of alpha and gamma Fe 2 O 3 structures (0.1, 0.5, and 1.0 mg/L) on tilapia (Oreochromis niloticus). They focused on the effects of particle morphology on accumulation, elimination, hematology, and immune responses. According to their results, while the spleen had the largest accumulation, the intestine, kidney, liver, gills, brains and muscle tissues followed close behind. They determined no significant changes in hemoglobin, hematocrit, red blood cell, and white blood cell counts. While the level of serum glucose (GLU) decreased, the levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and lactate dehydrogenase (LDH) increased. Finally, they determined that Fe 2 O 3 NPs can induce differential uptake, assimilation, and immunotoxic effects on O. Niloticus under chronic exposure [12].
Another study focused on the effects of different doses (0.01, 0.1, and 1.0 mg/mL) of metal oxide NPs (SnO 2 , CeO 2 , and Fe 3 O 4 ) on mortality and behavioral and biochemical responses of Artemia salina larvae. After 48 h of exposure, they determined that although these nanoparticles did not induce any mortality of the larvae, they caused changes in behavioral and biochemical responses. So, while the cholinesterase activities were found to be significantly decreased in the larva exposed to SnO 2 NPs, they were significantly increased with CeO 2 and Fe 3 O 4 NPs. The glutathione-S-transferase (GST) activities decreased after exposure to all of the nanoparticles. The catalase activities gradually decreased in the SnO 2 NPs, while there were no changes in CeO 2 NPs. However, the catalase activities were significantly stimulated after exposure to all of the doses of , catalase (CAT) and glutathione peroxidase (GSH)) were substantially increased following exposure to Fe 3 O 4 NPs. They concluded with their study that Fe 3 O 4 NPs have the potential to affect aquatic organisms when released into marine ecosystems [19].
Some researchers studied zebra fish sperm kinematics, fertilization success, and vitality by activated media with perfluorooctane sulfonate (PFOS) (0.09, 0.9, and 9 mg/L). They determined that PFOS decreased the percentage of motile sperm, the curvilinear velocity (VCL), and the mean angular displacement (MAD) of spermatozoa, but there was no effect on the straight line velocity (VSL) or the angular path velocity (VAP). Also, they observed a significant decrease in fertilization success of spermatozoon at doses of 0.9 mg/L PFOS or greater [36]. Linhartova et al. (2015) studied about the effects of different in vitro doses (0.5, 1.75, 2.5, 5, and 10 µg/L) of Tetrabrombisphenol A on DNA integrity and oxidative stress in Sterlet (Acipenser ruthenus) spermatozoon over 2 h. They found that the spermatozoon velocity and percent motile sperm were significantly decreased and the DNA were damaged after exposure to about a 2.5 µg/L dose. Also, while the thiobarbituric acid reactive substances (TBARS) level was significantly increased after 5 µg/L, CP and SOD activity were significantly increased after a 0.5 µg/L dose [37].
In our study, it was determined that the VSL and VCL values of spermatozoon were statistically significantly (p < 0.05) decreased after 24-h exposure to 400 mg/L of Fe 3 O 4 NPs. Moreover, the VAP value was significantly decreased at the dose of 800 mg/L. So, Fe 3 O 4 NPs decreased all velocities of rainbow trout spermatozoon. Our results are supported and paralleled by the findings of other researchers [36][37][38].
Gokduman et al. investigated the dose, treatment, and time-dependent toxicity of superparamagnetic Fe 3 O 4 NPs (10 nm, 0-400 µg/mL) on primary rat hepatocytes. The obtained results suggested that the response of ROS increased with increasing concentrations of Fe 3 O 4 NPs [39,40].
Biochemical parameters are important to determine of toxicities of Fe 3 O 4 NPs in rainbow trout sperm spermatozoon. In our study, CAT and SOD activities decreased from 100 to 800 mg/L doses of Fe 3 O 4 NPs. This inhibition in the enzyme activities was probably due to the overproduction of ROS, induced by Fe 3 O 4 NPs. This is similar to the findings of Afifi et al. [41], in which the SOD and CAT activities were reduced in the brain of fish exposed to Ag NPs at a concentration of 4 mg/L. Similarly, CAT and SOD activities were reduced in the liver of ZnO NP-treated fish [42,43]. A significant increase in the tGSH level was observed after exposure to Fe 3 O 4 NPs over 24 h. The level of MDA increased at doses of 400 and 800 mg/L of Fe 3 O 4 NPs. The increase of MDA could be explained by the depletion of the antioxidant system. The same results have been reported previously in many studies. Adebayo et al. [44] and Afifi et al. [41] demonstrated the increasing effect of CeO 2 and Ag NPs on the MDA level. Therefore, we decided that the balance between the oxidative stress and antioxidant system was broken and that oxidative damage in spermatozoon was caused by Fe 3 O 4 NPs exposure, especially at doses of 400 and 800 mg/L. In this respect, the results are also similar to situations such as reproductive disorders and the deterioration of biological activities in rainbow trout spermatozoon. In addition, our results are supported by the data of other researchers [12,19,20,45,46].

Conclusions
According to our data, the kinematics (i.e., the velocities and movement styles) of rainbow trout spermatozoon were negatively affected by exposure to Fe 3 O 4 NPs (50, 100, 200, 400 and 800 mg/L), for 24 h. The kinematics of spermatozoon exhibited significant (p < 0.05) decreases, which came into effect after 400 mg/L for VSL, VAP, and LIN, 50 mg/L for VCL, and 200 mg/L for BCF and ALH, as compared to the control group. However, Fe 3 O 4 NPs have devastating effects on the antioxidant system after 50 mg/L for CAT and SOD, and 400 mg/L for MDA and tGSH, as compared to the control group. The effective concentration (EC50) against exposure to Fe 3 O 4 NPs was calculated to be 494.09 mg/L for VSL, 297.29 mg/L for VCL, and 285.94 mg/L for VAP of spermatozoon in rainbow trout. Finally, we concluded that the balance between the oxidative stress and antioxidant system was broken and that oxidative damage was caused in spermatozoon fur to Fe 3 O 4 NPs exposure, especially at concentrations of 200 mg/L and greater, in aquatic environments.