Exposure effects of iron oxide nanoparticles and iron salts in blackfish (Capoeta fusca): Acute toxicity, bioaccumulation, depuration, and tissue histopathology

Journal Pre-proof Exposure effects of iron oxide nanoparticles and iron salts in blackfish (Capoeta fusca): Acute toxicity, bioaccumulation, depuration, and tissue histopathology Mohammad Hossein Sayadi, Borhan Mansouri, Elham Shahri, Charles R. Tyler, Hossein Shekari, Javad Kharkan PII:

S0045-6535(20)30092-8

DOI:

https://doi.org/10.1016/j.chemosphere.2020.125900

Reference:

CHEM 125900

To appear in:

ECSN

Received Date: 12 November 2019 Revised Date:

4 January 2020

Accepted Date: 10 January 2020

Please cite this article as: Sayadi, M.H., Mansouri, B., Shahri, E., Tyler, C.R., Shekari, H., Kharkan, J., Exposure effects of iron oxide nanoparticles and iron salts in blackfish (Capoeta fusca): Acute toxicity, bioaccumulation, depuration, and tissue histopathology, Chemosphere (2020), doi: https:// doi.org/10.1016/j.chemosphere.2020.125900. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Credit Author Statement

Mohammad Hossein Sayadi, Elham Shahri, Javad Kharkan: Conceptualization, Methodology, Software. Borhan Mansouri and Charles R. Tyler: Data curation, WritingOriginal draft preparation. Javad Kharkan, Hossein Shekari, and Elham Shahri: Visualization, Investigation. Mohammad Hossein Sayadi: Supervision. Borhan Mansouri: Software, Validation: Borhan Mansouri, Charles R. Tyler, and Mohammad Hossein Sayadi: Writing- Reviewing and Editing,

Graphical abstract

Exposure effects of iron oxide nanoparticles and iron salts in blackfish (Capoeta fusca): Acute toxicity, bioaccumulation, depuration, and tissue histopathology Mohammad Hossein Sayadi1, Borhan Mansouri2, Elham Shahri1, Charles R. Tyler3, Hossein Shekari1, Javad Kharkan1 1- Department of Environmental Sciences, School of Natural Resources and Environment, University of Birjand, Birjand, Iran; E-mail: [email protected]; [email protected]; [email protected] 2- Substance Abuse Prevention Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran; E-mail: [email protected] 3- Biosciences, College of Life and Environmental Sciences, University of Exeter, Geoffrey Pope, Stocker Road, Exeter, Devon EX4 4QD, United Kingdom; E-mail: [email protected]

Abstract We assessed the toxicity of iron oxide nanoparticles compared with iron salts in the blackfish (Capoeta fusca). After an acute toxicity assessment, we conducted a chronic exposure to a sub-lethal concentration of Fe3O4 NPs, and iron salts (ferric nitrate (Fe(NO3)3), ferric chloride (FeCl3), ferrous sulfate (FeSO4)) to measure iron uptake over a period of 28 days and then subsequent clearance of the iron uptake in the exposed fish that were transferred to clean water for 28 days. Fe(NO3)3 was the most acutely toxic compound followed by FeCl3, FeSO4, and Fe3O4 NPs. Exposure to Fe3O4 NPs and iron salts induced histopathology anomalies in both gills and intestine that included aneurism, hyperplasia, oedema, fusion of lamellae, lamellar synechiae, and clear signs of necrosis (in the gills) and increases in the number of goblet cells, blood cell counts, and higher numbers of lymphocyte (in the intestine). Fe3O4 NPs showed a higher level of uptake in the body tissues compared with iron salts (p < 0.05) with levels of Fe in the gill > intestine > liver > kidney. Fe was shown to be eliminated most efficiently from the gills, followed by the kidney, then liver and finally the intestine. The highest tissue bioconcentration factors (BCF) occurred in the liver for FeCl3, Fe3O4 NPs, and FeSO4 and in the gills for Fe(NO3)3. We thus show differences in the patterns of tissue accumulation, clearance and toxicological responses for exposures to Fe3O4 NPs and iron salts in blackfish with implications for different susceptibilities for biological effects. Keywords: Acute toxicity, chronic toxicity, accumulation, iron oxide nanoparticles, histopathological changes

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1. Introduction Nanotechnology seeks to exploits the unique properties of compounds that operate at the nanoscale. Iron oxide nanoparticles (NPs) have attracted enormous interest in applications due to their relatively cheap price compared with other nanoparticles, abundance, unique magnetic properties, rapid reaction, and high catalytic abilities (Huber 2005; Zhu et al. 2012; Sharafeldin et al. 2017). Iron oxide NPs are used in very broad range of biomedical and bioengineering applications including in sensors, terabit magnetic storage, drug and gene transfer to cells, magnetic hyperthermia, for labeling cells and macromolecules, antivirals, cancer therapy, and magnetic resonance imaging (Ito et al. 2005; Luong et al. 2016; Sun et al., 2016; Kumar et al. 2019). Iron oxide NPs are also used for environmental applications, for example in soil remediation, treatment of groundwater and wastewater, removal of heavy metals and degradation organic components from water, photocatalytic processes, and biosensors (Huber 2005; Khedr et al., 2009; Saif et al. 2016; Buccolieri et al. 2017). The increasing use of iron oxide NPs for biomedical, bioengineering and environmental applications will inevitably result in their further release and increasing presence in terrestrial and aquatic ecosystems with potential health risks to humans and wildlife. Thus, studies seeking to understand the environmental hazard of iron oxide NPs in natural ecosystems are much needed. Nanoparticles and their salt species have been shown to behave differently in aquatic environments and can have different toxicological effects in aquatic animals. However, comprehensive ecotoxicological assessments on the effects of metal oxide NPs, and particularly for iron oxide NPs, in comparison to their iron salts are limited. Various studies have reported that waterborne iron oxide NPs and iron salt species may induce toxic effects on aquatic organisms, albeit for the most part at a concentration exceeding those found in the 2

natural environment. These effects include oxidative stress and disturbances of liver enzyme function in tilapia (Ates et al. 2016), induced DNA damage and mutagenic effects in guppy (Qualhato et al. 2017), induced hematological, ionoregulatory and gill Na+/K+_ATPase activity of an Indian major carp (Remya et al. 2015), and suppression of population growth of marine microalgae species (Demir et al. 2015). Exposures to iron salts have also been shown to reduce mucus cell number in rainbow trout gills (1mM, Leguen et al. 2011), enhance activity of transaminases in blood plasma of common carp (87 mg L-1; Slaninova et al. 2014), cause filament epithelium proliferation, induce respiratory dysfunction in roho labeo (at 145.18 mg L-1 Singh et al. 2019), reduce growth and development in boreal toad (Bufo boreas) tadpoles, and reduce growth in mountain whitefish (Prosopium williamsoni) and reduce reproductive output in California blackworm (Cadmus et al. 2018). The solubility of nano-Fe3O4 particles in water is extremely low and therefore they tend to attach to other particles and/or to aquatic organisms (Brunner et al., 2006; Hu et al. 2012). Studies have shown a high accumulation of nano-Fe3O4 in zebrafish but equally high rates of elimination also (Zhang et al., 2015). Chronic exposure effects of Fe3O4 NPs in fish (e.g. Nile tilapia, O. niloticus) have included immunotoxic effects (Ates et al., 2016). Most of the reported effects of Fe3O4 NPs have been attributed to increased oxidative stress, likely through, the release of free iron salts from the particles, and disruption of iron homeostasis (Kornberg et al. 2017). Therefore, in this study we set out to assess the toxicity of iron oxide NPs compared with various salts of iron in blackfish (Capoeta fusca) for acute and chronic exposures, making assessments on the uptake, depuration, and toxicity (including via tissue histopathology) for exposures specially to Fe3O4 NPs, ferric nitrate (Fe(NO3)3), ferric chloride (FeCl3), and ferrous sulfate (FeSO4). The toxicological measurements were assessed against the measured iron content for the different exposure materials and exposures for 3

chronic toxicological assessments were set at one-tenth of the lethal concentration for each compound. 2. Materials and methods 2.1. Fe3O4 NPs characterization and Fe salts Iron oxide nanoparticles (Fe3O4 NPs) with purity 98% were obtained from 3302 Twig Leaf Lane, Houston, TX 77084, USA, (product code of 1317-61-9). Iron salts consisting of chloride (FeCl2.4H2O), iron nitrate (FeN3O9.9 H2O), and iron sulfate (FeSO4.7H2O) were obtained from Merck (Merck, Germany). Fe3O4 NPs had a particle size of 20-30 nm, with spherical morphology, specific surface area (SSA) of 40-60 m2/g, bulk density of 0.84 g/cm3, and true density of 4.8-5.1 g/cm3. The Fe3O4 NPs were subjected to systematic characterization using powder X-ray diffraction (XRD; PHILIPS, Netherland), Field Emission Scanning Electron Microscopy (FESEM; TE-SCAN, Czech Republic), and Transmission Electron Microscopy (TEM; Hitachi, Japan) techniques (Fig. 1). 2.2. Test Organism and Experimental Conditions Blackfish (Capoeta fusca) were collected from qanats (underground channels used to transport water from an aquifer or water well to the ground surface for irrigation and potable water) in South Khorasan province (Eastern Iran) and transferred to the laboratory. Fish were maintained for 4 weeks in 200 L tanks prior to any experimentation in order to acclimatize them to the laboratory conditions. The tap water parameters during the exposure period were as follows: dissolved oxygen 7.1 ± 0.2 mg L-1, pH - 7.4 ± 0.4, temperature - 23.4 ± 1.5 ºC, electrical conductivity - 1441 ± 32 µS cm-1, and water hardness - 371 ± 15 mg L-1. Blackfish was exposed to Fe3O4 NPs and different iron salts (FeCl3, Fe(NO3)3, FeSO4) to first assess their acute toxicity (LC50) and subsequently, their chronic toxicity, during which time

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assessments were made on compound uptake, tissue effects (via histopathology) and compound clearance. 2.3. Acute toxicity

The acute toxicity test for Fe3O4 NPs and iron salts (FeCl3, Fe(NO3)3, FeSO4) on blackfish (total length: 4.5 ± 0.3 cm; weight: 2.3 ± 0.2 g) was carried out according to the OECD guidelines No. 203 (LC50; OECD 2019). The iron compounds were sonicated for three periods of 30 min (100W, 50KHz, Ultrasonic bath, PARSONIC 7500s, IRAN), and stock solution of 1000 mg L-1 prepared in distilled deionized water. The test concentrations of Fe3O4 NPs were 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg L-1 and for iron salts (FeCl3, Fe(NO3)3, FeSO4), 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg L-1. Ten fish were exposed to each compound concentration for 96 h. Mortalities were monitored during the test period and these mortalities quantified after 24, 48, 72 and 96 h. Exposures were performed in triplicate. 2.4. Chronic Exposure Study: Uptake, clearance, and tissue effects

Using the information derived from the lethality test, blackfish were exposed to the Fe3O4 NPs and different iron salts at a concentration of one-tenth of their 96h LC50. Blackfish were divided into five groups of 25, each placed within glass aquaria (including one control group) and exposed to Fe3O4 NPs and the different iron salts for a period of 28 d to measure iron uptake and then allowed to undergo depuration in clean water for a further 28 d. To maintain the required concentrations of test compounds in the aquaria, during the experiment, 50% of the aquarium water containing the desired concentrations of the iron compound was replaced every 48 h. Also, at these times fish waste was removed from the aquaria. Fish exposed to the different treatments were sampled at 7, 14 and 28 d. The remaining fish were then placed into tanks with clean water (i.e. not test compound) to assess for body clearance of Fe during at 7, 5

14 and 28 d. At the end of each exposure period, gill, intestine, liver, and kidney tissues were dissected from the blackfish and underwent acid digestion, conducted using a mixture of nitric acid (HNO3, 65%, Merck, Germany) and perchloric acid (HClO4, 70%, Merck, Germany) in a ratio of 2 to 1. To each tissue, 5 ml of nitric acid was added and allowed to digest overnight. 2.5 ml of perchloric acid was then added to each sample, which was then placed inside a water bath (TW12, Julabo Co., Germany) for 4-6 h at 100 °C to allow the solutions to clear. After filtration of the samples (nitrocellulose filter 0.45 m), the concentration of Fe on each tissue was measured by graphite furnace atomic absorption spectrometer (ContrAA 700, Analytik Jena AG, Germany). The standard CHEM_LAB iron solutions with Code Number 21.1791706.50 were used to calibrate the device. The limit of detection (LOD) and relative standard deviation (%RSD) for Fe were 0.01 mg L-1 and < 3%, respectively. For assessing the effects of the iron compound exposures on body tissues, histopathology was conducted on the gill and intestine tissues after 28 d exposure. For this, gills and intestine were dissected out and immediately placed in 10% formalin for 24 h, then transferred to 70% ethanol. Dehydration steps were performed using a sequential alcohol series of 90, 95 and 100% and cleared in xylene, infiltrated with liquid paraffin at 60 ºC. The tissue samples were then embedded with paraffin blocks and sectioned at 5 µm on a rotary microtome (MicroTec, Rotary microtome, CUT 4050). The sections cut were then stained with Haematoxylin and Eosin. Photograph taken from slides using the light microscope (Nikon, eclips-E200) for determining the histopathological effects. A semiquantitative scoring system of the histopathological lesions’ was applied to three randomly selected fish from each group and inspecting four randomly selected areas on those slides for each organ studied. 2.5. Toxicokinetics modeling 6

The uptake and depuration kinetics of Fe in the tissues of blackfish were described using a first-order one-compartment model. Both versions assumed that the background concentration in the blackfish is a fixed value C0 measured in the organism at time zero, and is not a component of depuration in the kinetics models (OECD 2012; Pavlaki et al. 2017).

For the uptake phase the model used reads: Qt = C +

k × Ce × 1 − e ×  Equation 1 k

And for the depuration phase: Qt = C +

k × C × e ×  − e ×  Equation 2 k

Where Q(t) is the Fe concentration in fish (µg g-1) at sampling time t; C0 is the background concentration in the organism in µg Fe g-1 dry body weight at time 0; k1 is the uptake rate constant (per d); k2 is the depuration rate constant (per d); Ce is the exposure concentration in the medium in µg Fe L−1; tc is the time when the fish where transferred to fresh uncontaminated medium; These constants allow assessment of the bioconcentration factor (BCF) because this equation considers that Q(t) reaches a steady state. This way, t continues to increase until ek *t 2 =0;

in these, the bioconcentration factor (BCF) and the time that organisms required to

eliminate half the amount of Fe (DT50) were calculated as: DT# = BCF =

ln2 Equation 3 %

% Equation 4 %

2.6. Data analysis

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SPSS software version 20 was used to analyze the data. The significance level was 0.05. The LC50 values (concentration that causes 50% of mortality) lethal concentration for 50% (LC50) values were calculated for each chemical exposure using the Probit analysis program with the software SPSS Statistics Package (version 20). To investigate the effect of exposure period on the concentration of Fe3O4 NPs and iron salts in blackfish, a one-way analysis of variance (One-way ANOVA) with Tukey-Kramer post hoc test was used. A two-way analysis of variance (two-way ANOVA) was applied to assess for the effects of tissues and times of exposure on bioconcentration and depuration of Fe3O4 NPs and iron salts. The severity of histopathological changes in each tissue type was determined semi-quantitatively using a grading system with four effect levels designated as (-) no histological damage, (+) mild histopathological damage, (++) moderate histopathological damage, and (+++) severe histopathological alteration (Jerome et al. 2017). 3. Results 3.1. Fe3O4 NPs characterization The Fe3O4 NPs characteristics, as assessed via XRD, TEM and SEM are shown in Fig. 1 (ac). The XRD pattern of Fe3O4 NPs at 30.25◦, 35.56◦, 43.18◦, 53.49◦, 57.21◦ and 62.64◦, corresponded to the (220), (311), (400), (422), (511), and (440) reflection phases, respectively (Fig. 1a). These peaks were indexed as the magnetite phase which is in accordance with JCPDS No. 19-0629. The size of Fe3O4 NPs calculated from XRD spectral data was 22.37nm. According to the SEM images, the size distribution of Fe3O4 NPs was in the nanometer range and they were generally spherical in shape (Fig. 1b). Indications of clumping of the Fe3O4 nanoparticles in the SEM image likely occurred due to hightemperature calcination before imaging. The TEM image of Fe3O4 NPs (Fig. 1c) showed a uniform dispersion of similarly sized particles 8

3.2. Acute toxicity No mortalities were observed during the experimental period in the control fish. The acute toxicity of Fe3O4 NPs and iron salts in blackfish at 24, 48, 72 and 96 h are shown in Table 1. The LC50-96 h for Fe3O4 NPs, Fe(NO3)3, (FeCl3), and FeSO4 were 32.3, 1.6, 3.3, and 5.2 mg L-1, respectively. Thus, according to our results, Fe(NO3)3 had the highest toxicity compared with the other iron structures (Fe3O4 NPs and iron salts). The toxicity of iron salts was considerably greater than that for Fe3O4 NPs (iron salts LC50= 1.6 to 5.2 mg L-1, compared with an LC50= 32.3 mg L-1 for Fe3O4 NPs). The differences in these relative toxicities likely relate to the amount of bioavailable iron. 3.3. Body tissue bioaccumulation and clearance of Fe3O4 NPs and iron salts The accumulation and depuration trend of Fe3O4 NPs and iron salts in different tissues of blackfish are presented in Fig. 4. Both time and tissue had significant effects on the accumulation and elimination of Fe salts (Table 2, p < 0.01). After 28 d of exposure, there was a significant difference in the amount of iron accumulated between different treatment groups compared to controls (p < 0.01; Table 2). For exposure to the nanomaterial (Fe3O4 NPs), the gills were seen to accumulate the highest tissue concentrations of iron, followed by the intestine, liver, and then kidney. Clearance for Fe3O4 NPs occurred most quickly in the gills, followed by the kidney, liver, and intestine. Accumulation of FeCl3 and FeSO4 were highest in the intestine followed by the gill, liver, and kidney. The lowest accumulation of iron for the compounds tested occurred for exposure to Fe(NO3)3 in the kidney (Table 2). The kinetics parameters for Fe ion activity obtained by a first-order one-compartment model are shown in Table 3. The uptake rate constant k1 and depuration rate constant k2 were higher in the intestine tissue compared to other tissues (except for liver in the FeSO4 treatment in k1) and differed between the four groups of Fe3O4 NPs and iron salts. As no 9

steady-state was reached in any of the treatments, bioconcentration factors (BCF) were calculated from the ratio of k1 and k2 values. Accordingly, the maximum BCF in FeCl3, Fe3O4 NPs, and FeSO4 groups occurred in the liver, and for the Fe(NO3)3 group, the gills. The lowest BCF occurred for FeCl3, Fe(NO3)3 and FeSO4 in the kidney and for Fe3O4 NPs in the intestine. The estimated time needed for the blackfish to eliminate half of the sequester Fe (DT50) was estimated in the range between 15 d for Fe3O4 NPs up to 93 d for Fe(NO3)3. 3.4. Tissue histopathology A series of histopathological alterations were seen in the gills and intestine in fish exposure to Fe3O4 NPs and iron salts. Effects seen on the gills exposed to the iron NPs and salts are shown in Fig. 2. The gills of control fish indicated a few small histopathological abnormalities that included clubbed tips and slight curvature in some of the secondary lamellae that might relate to a degree of stress. However, the gills of fish in the other treatment groups showed some significant alterations due to the iron exposures including aneurism, hyperplasia, oedema, the fusion of lamellae, lamellar synechiae, and clear signs of necrosis (Fig. 2). The frequency of necrosis in secondary lamellae was highest in the Fe(NO3)3 treated blackfish. The frequency of fusion of the secondary lamellae and epithelial hyperplasia abnormalities was highest in the FeCl3 and Fe3O4 NPs treated fish compared with fish treated with the other salts (Table 4). Lamellar synechiae and curvature were the major histopathological alterations seen in the gill epithelium in FeCl3 and FeSO4 treatment groups compared to all other groups (Table 4). In the intestine after 28 d of exposure, some histopathological lesions were seen for exposures to Fe3O4 NPs and the iron salts treatments which included increases in the number (and swelling) of goblet cells, higher blood cell counts higher numbers of lymphocyte and expansion of the villi structure (Fig. 3). The frequency of these abnormalities was higher in 10

the Fe(NO3)3 treated fish when compared with all other treatment groups (Table 4). Similarly to that for gills, the highest levels of histopathological alterations were found in the intestine for exposed to iron salts than iron oxide NPs (Table 4). 4. Discussion 4.1. Acute toxicity The acute toxicity of the Fe3O4 NPs and iron salts differed for the different exposure times. In the first 24 h of exposure, FeCl3 was most toxic to the blackfish, but at 96 h the most toxic compound was Fe(NO3)3. Some studies have reported that ferrous (Fe2+) compounds are more toxic to aquatic animals than colloidal forms of ferric (Fe3+) compounds because they have greater binding affinity to gill surfaces and in turn bioavailability, and intracellular Fe2+ can accelerate the formation of highly reactive free radicals (Teien et al. 2008; Farina et al. 2012; Singh et al. 2019). In some other fish species, studies have shown that Fe(III) has relatively higher toxicity because of a high tendency to precipitate on gill surfaces (Lappivaara et al. 1999; Lappivaara and Marttinen 2006). Previous studies have shown 96 h LC 50 values for iron to range between 72.20 mg L-1 and 164.65 mg L-1 in three fish species of fish Catla catla, Labeo rohita and Cirrhina mrigala (Javed and Abdullah, 2006). In Daphnia magna a much higher 96-h LC50 ( 2.3×10-4 mg/ml) has been reported for Fe3O4 NPs chloride (García et al. 2011) compared with that we report here for blackfish. Collectively, these findings illustrate the 96 h LC50 values for the different iron-based compounds vary between fish species. Our observations on behavioral changes in blackfish that included the gradual loss of equilibrium and increased surfacing activity for exposures to Fe3O4 NPs or iron salts compared with controls are similar to the findings of Saravanan et al. (2015). 4.2. Bioaccumulation of Fe3O4 NPs and iron salts 11

Our findings indicate that of the iron-based compounds studied, Fe3O4 NPs were most readily accumulated in the body of the blackfish and Fe(NO3)3 showed the lowest level of accumulation. Although Fe3O4 NPs showed greater uptake into the blackfish compared with Fe(NO3)3, Fe(NO3)3 induced a greater level of tissue damage compared with Fe(NO3)3 (i.e. Fe(NO3)3 was more overtly toxic). Fe3O4 NPs are likely more permeable across biological membranes due to their smaller size compared with Fe salts, as has been reported in Ates et al. (2016). Zn NPs also have been shown to pass across biological membranes more readily than their bulk counterparts in both common carp (Hao et al., 2013) and tilapia (Kaya et al., 2015). According to the findings of this study, gill tissue and intestine accumulated the greatest amounts of total Fe after 28 d exposure and this was greatest for exposure to Fe3O4 NP. Little study has been done previously comparing the effects of iron NPs with iron salts, but respiratory effects due to iron NP accumulation in the gill have been shown previously in both carp (Remya et al., 2015) and rainbow trout (Özgür et al., 2018). Previous studies have also illustrated that direct adsorption/adhesion of Fe3O4 NP aggregates onto the surface of the exposed aquatic organisms, including fish, can delay hatching, inhibit Na+/K+-ATPase activity (albeit at the very high exposure level of 25 mg L-1), disturb ion balance, damage cell membranes, and deplete oxygen exchange and cause hypoxia (Cheng et al., 2011; He et al., 2011; Saravanan et al., 2015). Zhu et al. (2012) reported that adsorption/adhesion of Fe3O4 NP aggregates on the surface of the exposed organisms can enhance the release of free ions resulting in greater biological accumulation. The accumulation of Fe3O4 NPs in blackfish tissues increased over time compared with that for the iron salts, and this was especially the case in the gill tissue and the intestine. The enhanced Fe3O4 NPs uptake in the gills is likely, in part, to be due to the tissue’s direct 12

contact with the surrounding water and also the tendency for particles to bind with the mucus in gills. In our study, of the tissues assessed we found the gill and liver tissues showed the highest BCFs for all the different iron groups. Considering the dynamics of the uptake of Fe3O4 NPs, studies in Ceriodaphnia dubia, have shown that the maximal uptake occurred after a 6h exposure, but in that study, the exposure concentrations were far higher compared with our study on the blackfish (Hu et al., 2012). Furthermore, the conditions of the exposure solution differed and this can have a major bearing on NP bioavailability and uptake (Tervonen et al., 2010; Hu et al. 2012; Skjolding et al. 2014). 4.3. Clearance of Fe3O4 NPs and iron salts Clearance of the different iron compounds taken up varied across the different tissues. Gill tissues showed the fastest elimination rates compared with other tissues, and these findings support several other studies in freshwater fish on rates of NPs/metals clearance (Kalay and Canli 2000; Mansouri et al., 2012, 2016). The rapid excretion of Fe from the gill likely relates to their direct contact with the surrounding water whereby compounds can move easily out into the water column down a concentration gradient when the fish are placed into the clean water (Kalay and Canli, 2000). The slowest rates of elimination of iron nanoparticles and iron salts (assessed after 28 d of depuration) occurred in the intestinal and liver tissues. The lower rates of components excretion from the liver are likely due to the fact that some of the metal uptaken is temporarily bound up with storage proteins and also has to be passed to other organs for excretion (Kalay and Canli, 2000). Studies by Zhang et al. (2015) report that accumulated iron oxide nanoparticles in zebrafish can be eliminated efficiently when fish were moved to nanoparticle-free water at rates of between 86% and 100% after 24 d. Interestingly, in that work, the author’s reported that iron oxide nanomaterials absorbed via the gastrointestinal tract may be stored for more than 12 d. Accordingly, it is important when 13

considering bioconcentration of iron-based compounds to ensure accurate reporting on the target tissues studied, the length of exposure, and the concentration of the compounds to which the fish have been exposed, as these factors can all have a major bearing on the outcome of the dynamics and amount of these compounds that are uptaken and retained in the organism. 4.4. Tissue histopathology Tissue histopathology is widely used to assess for signs of stress in organisms exposed to toxicants. Gills are especially sensitive to the effects of pollutant exposure, in part because of their direct contact with pollutants in the environmental (Ostaszewska et al. 2016; Capaldo et al. 2019). The main histopathological effects after 28 d of exposure to Fe3O4 NPs and iron salts were aneurisms, hyperplasia, secondary lamellae fusion, lamellar synechiae, and necrosis. Similar histological changes in gills were reported by Singh et al. (2019), in a study with the freshwater fish Labeo rohita exposed to iron (ferrous) at three sub-lethal test concentrations for 96 h. Curvature as well as dilated and clubbed tips in the lamellar epithelium are often seen as an early effect histopathological marker for the effect of toxicants on aquatic species more generally (Mansouri et al. 2016). Lamellar synechiae were seen to be higher in the FeSO4 and FeCl3 treatment groups than in the other groups, a finding supported in a similar study by Nero et al. (2006) where pollutant effects on proliferation of chloride and epithelial cells led to synechiae in gill lamellae. In our study, hyperplasia of the gill epithelium and secondary lamellae fusion was the most frequent histopathological anomaly in Fe3O4 NPs and FeCl3 treatment groups. Gill hyperplasia and secondary lamellae fusion appear to be a protective mechanism adopted to limit the respiratory surface in contact with the pollutants and reduce the pollutant absorption rate (Capaldo et al. 2019; Beegam et al. 2019). These effects, however, may lead to 14

respiratory dysfunctions. Previous studies have shown that the high levels of NPs (both copper and iron oxide NPs) in the gill tissue of fish inhibit the Na+/K+_ATPase activity resulting in osmoregulatory failure; in the teleost, gill Na+/K+_ATPase plays an important role in the maintenance of electrolytes between extra and intracellular milieus (Shaw et al. 2012; Remya et al. 2015). Severe gill lesions seen in the blackfish due to iron overload may relate to hypoxia-like conditions created. Hypoxic condition stimulates the formation and releases into the circulation of more erythrocytes from hemopoietic tissue through adrenergic stimulation (Chen et al. 2011; Ostaszewska et al. 2016; Beegam et al. 2019). Wu et al. (2012) reported that the waterborne Fe (II) and long-term exposure to 0.1 to 2.0 mg L-1 Fe (II) induced respiratory dysfunction and gill anomalies in juvenile turbot. Increases seen in the number of goblet cells, swelling of those goblet cells, higher numbers of blood cells, and expansion at of the villi at their surface were the most common intestinal anomalies seen in black fish exposed to both Fe3O4 NPs and iron salts. Goblet cells secrete mucin that has an important role in the absorption of iron from the environment (Bury et al. 2001). In this cascade, iron-bound with mucin then passes through the mucous layer and enters the enterocytes via divalent metal transporters (Cooper et al. 2007). Our findings on the goblet cells concur with observations by others on the effects of other metals and NPs on fish intestine (Al-Bairuty et al. 2013; Osborne et al. 2015). In a similar study, Li et al. (2009) reported that nano-iron caused swelling of epithelial cells of the intestine of medaka (Oryzias latipes) after 2 weeks of exposure. Similar effects were seen also in medaka (Oryzias latipes) after 14 d of exposure to sublethal concentrations of iron NP, and where both inflammation and thinner intestinal walls were reported (Chen et al., 2011). The enlargement of goblet cells and their increase in number are indicative of defense/repair mechanisms associated with the severe pathological changes (Suganthi et al. 2015). 15

5. Conclusion Our findings illustrate differences in body tissue accumulation, effects and depuration of Fe3O4 NPs and iron salts in blackfish and show various iron salts have higher acute toxicity compared with Fe3O4 NP. Exposure to both Fe3O4 NP and iron salts disrupted tissue structure (and likely function) for sub-lethal exposures, albeit at levels that exceed those found in all but perhaps the most contaminated environments. We further illustrate that the gill and intestinal tissues accumulated the highest amount of Fe and thus may be amongst the most vulnerable body tissues for exposure effects.

Funding This study was supported by the Research Council of the University of Birjand (Grant Number: 24156/1398). Conflict of interest The authors declare no conflict of interest. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Acknowledgment The authors of this study gratefully acknowledge the Research Council of the University of Birjand for financial help. The contribution of the Department of Environmental Sciences, University of Birjand is sincerely appreciated. Abbreviations Nanoparticles (NPs) Iron oxide nanoparticles (Fe3O4 NPs) Lethal concentration 50 (LC50) Ferric nitrate (Fe(NO3)3) Ferric chloride (FeCl3) Ferrous sulfate (FeSO4) References Abdel-Khalek, A. A., Kadry, M. A., Badran, S. R., Marie, M. A. S., 2015. Comparative toxicity of copper oxide bulk and nano particles in Nile tilapia; Oreochromis niloticus: biochemical and oxidative stress. The Journal of Basic & Applied Zoology, 72, 43-57. 16

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20

Figures and Tables

b

c

Fig. 1 The XRD pattern (a), SEM (b), and TEM (c) images of Fe3O4 NPs

21

a

b F

Hp

d

c Oe

Hp

DCt

An

Cu LS F

e

f

LS N Fig. 2 Gill morphology in black fish (C. fusca) exposed chronically (28 days) to various iron-based compounds; Controls (a), b-f iron-based compound exposure groups. Other treatment groups b: Fe3O4 NPs, c: FeCl3, d-e: FeSO4, and f: Fe(NO3)3) all show injuries including aneurism (An), dilated and clubbed tips (DCt), hyperplasia (Hp), oedema (Oe), curvature (Cu), fusion of lamellae (F), lamellar synechiae (LS), and necrosis (N) 22

a

b

INBC V

c

d

D

INGC

EVS

e

f INBC SG

EVS Fig. 3 Intestine histopathology of black fish (C. fusca) exposed to Fe3O4 NPs and various iron salts (x400); Control (a), and exposures to b: Fe3O4 NPs, c: FeCl3, d: FeSO4, and e-f: Fe(NO3)3). Iron exposure treatments showed injuries including Degeneration of villi structure (D), a higher number of goblet cells (INGC), swelling of goblet cells (SG), higher numbers of blood cells (INBC), higher

23

Fig. 4 Accumulation and depuration of Fe (µg g-1) in the selected tissues (gill, intestine, kidney, and liver) of black fish (C. fusca) exposed to Fe3O4 NPs and various iron salts (Fe(NO3)3, FeCl3, FeSO4). N=6 (numbers of fish for each treatment groups)

Table 1 Lethal concentration (LC50) of Fe3O4 NPs and various iron salts in blackfish (C. fusca) NPs/ salts Concentration (mg L-1) 24 h 48 h 72 h 96 h Fe3O4 NPs

1000

107.1

81.2

32.3

Fe(NO3)3

57.5

8.3

3.3

1.6

FeCl3

30.9

8.7

5.6

3.3

FeSO4

42.6

10.0

7.9

5.2

24

25

Table 2 Bioaccumulation and depuration of Fe (µg g-1) in tissues of blackfish (C. fusca) Bioaccumulation Tissues

Time

Control

Fe3O4 NPs

FeSO4

0.03±0.0

1.19±0.8

0.48±0.1

0.48±0.1

0.72±0.2a

14

0.03±0.0a

3.50±0.6b

0.94±0.0b

1.10±0.2b

1.33±0.4b

0.03±0.0a

0.97±0.7a

0.31±0.0a

0.33±0.0a

0.47±0.0a

28

0.02±0.0a

6.93±1.6c

1.43±0.5c

1.81±0.6c

1.95±0.2c

0.04±0.0a

0.25±0.0a

0.26±0.0a

0.24±0.1a

0.42±0.0a

0.14

0.001

0.03

0.01

0.002

0.16

0.29

0.12

0.12

0.09

7

0.04±0.0a

0.67±0.2a

0.46±0.1a

0.48±0.1a

0.57±0.3a

0.03±0.0a

2.88±0.4a

0.75±0.2a

1.06±0.4a

1.07±0.4a

14

0.02±0.0a

2.15±0.7b

0.99±0.4b

1.38±0.4b

1.62±0.3b

0.03±0.0a

1.21±0.5b

0.50±0.2a

0.76±0.2a

0.60±0.2a

28

0.03±0.0a

4.02±1.6c

1.37±0.0c

2.05±0.6c

2.23±0.2c

0.03±0.0a

0.37±0.1c

0.39±0.0a

0.63±0.1a

0.48±0.2a

0.10

0.02

0.01

0.01

0.002

0.94

0.001

0.17

0.23

0.13

7

0.02±0.0a

0.41±0.0a

0.29±0.0a

0.23±0.1a

0.16±0.0a

0.03±0.0a

1.74±0.8a

0.88±0.3a

0.62±0.1a

0.63±0.1a

14

0.02±0.0a

1.21±0.6b

0.72±0.2b

0.87±0.2b

1.01±0.4b

0.03±0.0a

0.89±0.3b

0.72±0.0a

0.48±0.1a

0.41±0.0a

28

0.03±0.0a

3.01±0.7c

1.14±0.2c

1.26±0.2c

1.90±0.7c

0.03±0.0a

0.26±0.0b

0.54±0.2a

0.24±0.1b

0.33±0.0b

0.06

0.004

0.01

0.006

0.01

1.00

0.04

0.25

0.03

0.03

7

0.02±0.0a

1.70±0.8a

0.37±0.1a

1.12±0.4a

0.89±0.7a

0.03±0.0a

0.84±0.2a

0.96±0.1a

0.86±0.4a

0.81±0.4a

14

0.03±0.0a

3.40±0.7b

0.97±0.4b

1.63±0.0b

1.42±0.4b

0.04±0.0a

0.82±0.5a

0.75±0.3b

0.47±0.1a

0.39±0.0a

28

0.02±0.0a

3.61±1.3b

1.19±0.4b

1.75±0.2b

1.95±0.2a

0.02±0.0a

0.37±0.0b

0.63±0.2b

0.44±0.0a

0.34±0.1a

p-value times

0.85

0.01

0.08

0.09

0.11

0.63

0.04

0.04

0.37

0.17

p-value tissues

0.20

0.001

0.34

0.01

0.13

0.65

0.01

0.001

0.001

0.10

p-value times

0.34

0.001

0.001

0.001

0.001

0.92

0.001

0.01

0.01

0.001

p-value tissues × times

0.15

0.01

0.99

0.35

0.81

0.36

0.04

0.99

0.087

0.89

Liver

26

a

a

FeSO4

0.32±0.1

p-value times

a

FeCl3

0.38±0.0

Kidney

a

Fe(NO3)3

0.48±0.1

p-value times

a

Fe3O4 NPs

0.29±0.1

Intestine

a

FeCl3

0.04±0.0

p-value times

a

Fe(NO3)3

7

Gill

a

Depuration Control

a

Table 3 Bioaccumulation and depuration kinetics of Fe in the tissues of blackfish. K1: uptake kinetic constant in L/Kg/day, k2: elimination kinetic constant in 1/ day, T1/2: half-life in days, BCF: bioconcentration factor. CI: confidence intervals in brackets

Gills

Intestine

Fe3O4 NPs

Fe(NO3)3

FeCl3

FeSO4

Fe3O4 NPs

Fe(NO3)3

FeCl3

FeSO4

0.29±0.0

0.479±0.1

0.379±0.1

0.319±0.0

0.667±0.1

0.458±0.0

0.479±0.0

0.569±0.1

0.018±0.0

0.007±0.0

0.007±0.0

0.011±0.0

0.044±0.0

0.012±0.0

0.016±0.0

0.017±0.0

T1/2

37.7

93.7

93.3

62.7

15.6

59.3

42.13

41.8

BCF

15.8

15.0

15.5

23.0

64.8

39.1

21.7

25.4

K1 K2

Kidney

Liver

Fe3O4 NPs

Fe(NO3)3

FeCl3

FeSO4

Fe3O4 NPs

Fe(NO3)3

FeCl3

FeSO4

K1

0.415±0.0

0.296±0.0

0.232±0.0

0.163±0.0

0.405±0.0

0.376±0.1

1.1±0.1

0.886±0.0

K2

0.027±0.0

0.016±0.0

0.01±0.0

0.009±0.0

0.018±0.0

0.015±0.0

0.013±0.0

0.012±0.0

T1/2

25.8

50.9

72.5

71.5

39.3

47.0

52.3

55.6

BCF

51.1

29.0

24.2

85.2

28.8

34.3

16.8

71.0

27

Table 4 Semiquantitative evaluation of lesions recorded in the gill and intestine of blackfish (C. fusca) exposed to Fe3O4 NPs and various iron salts Histopathological changes Tissues Gill

An*

Cu

DCt

F

Hp

LS

N

Control

-**

+

+

-

-

-

-

Fe3O4 NPs

+

+

+

+++

+++

+

-

Fe(NO3)3

+

+

++

++

++

+

+++

FeCl3

+

++

++

+++

+++

++

-

FeSO4

+

++

++

++

++

++

+

D

EVS

INBC

INGC

INL

SG

V

Control

-

-

-

-

-

-

-

Fe3O4 NPs

+

+

+

+

+

+

++

Fe(NO3)3

++

++

++

++

++

+++

+

FeCl3

+

++

+

+

+

-

+

FeSO4

++

++

++

++

+

++

+

Intestine

* aneurism (An), Curvature (Cu), dilated and clubbed tips (DCt), lamellar fusion (F), hyperplasia (Hp), lamellar synechiae (LS), and necrosis (N) in gill; Degeneration (D), expansion at villi structure (EVS), increase in the number of blood cells (INBC), increase in the number of goblet cells (INGC), increase in the number of lymphocyte (INL), necrosis and erosion (NE), and swelling of goblet cells (SG) in intestine. **None (−), mild (+), moderate (++) and severe (+++)

28

Highlights 1. The Fe(NO3)3 (LC50: 1.62 mg L-1) was more toxic than FeCl3 (LC50: 3.38 mg L-1), FeSO4 (LC50: 5.24 mg L-1), and Fe3O4 (LC50: 32.35 mg L-1) for a period of 96h. 2. Histopathological anomalies were recorded in the tissues of blackfish. 3. Iron uptake in different tissues of blackfish were decreased in the sequence gill > intestine > liver > kidney. 4. The highest and lowest levels of Fe accumulation in different tissues were related to Fe3O4 NPs and Fe(NO3)3 respectively.