Long-term Exposure of High Concentration Silver Nanoparticles Induced Toxicity, Fatality, Bioaccumulation, and Histological Alteration in Common Carp Fish (Cyprinus carpio)


 BackgroundCurrently, nanotechnology and nanoparticles have been quickly emerged and have gained the attention of scientists due to its massive applications in environmental sectors. However, these environmental applications of silver nanoparticles potentially cause serious effects on terrestrial and aquatic organisms. In the current study, freshwater fish C. carpio were exposed to blood mediated AgNPs for toxicity, mortality, bioaccumulation, and histological alterations. Silver nanoparticles were fabricated using animal blood serum and their toxic effect was studied against common carp fish at different concentrations level (0.03, 0.06, and 0.09 mg/L).ResultsThe findings have revealed a little effect of blood induced silver nanoparticles (B-AgNPs) on fish behavior at the highest concentration (0.09 mg/L). However, bioaccumulation of B-AgNPs was reported in different organs of fish. Maximum bioaccumulation of B-AgNPs was reported in the liver, followed by intestine, gills, and muscles. Furthermore, the findings have showed that the B-AgNPs bioaccumulation led to histopathological alterations including damage structure of gills tissue and caused necrosis. It is summarized that histopathological alteration in gills and intestine mostly occurred at the highest concentration of B-AgNPs (0.09 mg/L). ConclusionThis study provides evidence of the AgNPs influence on aquatic life; however, further systematic studies are crucial to access the effects of AgNPs on aquatic life.


Background
Nanotechnology (NT) and nanoscience is one of the remarkable area of science dealing with using of structure of nanoscale [1], and tremendously developed in the last decade [2]. Due to their wide range application, the NT is playing bene cial role in water treatment, food industry, house hold equipment, biomedical, and health care [3]. Nanoparticles (NPs) are a kind of nanomaterials with unique properties due to their small size and dispersal [4]. Among all the nanoparticles, silver nanoparticles (AgNPs) are well known for their commercialization worldwide [5,6], due to their sole biological activities [7], and utilizing widely in medicine [8]. Despite the biological activities and a wide range of applications of AgNPs, there is a lack of information regarding human health and environmental toxicity [9]. The extensive use of AgNPs in the world, release into the aquatic environment, which has elevated concern [10], for a high impact on aquatic life [11].
The rare knowledge of AgNPs regarding aquatic toxicity and their linkage with living components have raised concerns in the last decade [12]. It has been hypothesized and suggested the concentration of AgNPs in an aquatic ecosystem is in µg L − 1 or mg L − 1 [13], however, these concentrations are yet toxic to any environment [14]. It has been reported that alterable properties and small size of AgNPs are toxic and dangerous to the ecosystem [15,16]. However, silver is found in four different oxidation states (Ag, Ag + , Ag + 2 , and Ag + 3 ) in the aquatic environment [15,17]. Though, exact estimates of AgNPs toxicity are still hindered due to lack of acquaintance [12]. It has been accepted that the toxicity caused by AgNPs is due to the release of Ag + into the environment and is nally absorbed by living organisms [18]. Silver nanomaterials are the most producing nanoparticles and release extensively to both terrestrial and aquatic environments [19].
Common carp sh (Cyprinus carpio) is one of the most important sh species of freshwater [20], distributed in Asia and Europe [21]. It has been estimated that the production of common carp sh is about 4.32 million tons [20]. Fish, when exposed to different kinds of toxins, may lead to changes in biochemical, hematological parameters, and histological alteration [22]. Nowadays, NT is rapidly used as targeted speci c drugs to control sh diseases [23], because of its broad anti-bacterial activity [4] and anti-protozoal activity [24], but the exposure of AgNPs to sh led to the multiplicity of effects, even mortality [19], organ dysfunction, other lesions and gene mutation [8]. The small nanomaterials can easily penetrate inside the tissues such as the liver, gills, and brain of the sh [12]. Previous studies showed that this toxicity of AgNPs depends on the size and coating of NPs [25].
Among all aquatic organisms, sh is the most using aquatic specie for assessing environmental toxicity and toxic effects [12]. Therefore, this study was designed to evaluate the acute, chronic, and sublethal toxicity of three different concentrations of blood mediated AgNPs on common carp sh for a long exposure of twenty days' period. In vivo, the studies regarding toxicity and toxic effects of AgNPs is principally lacking; especially in aquatic systems where ecologically signi cant and diverse organisms are affected. In the current study, freshwater sh C. carpio were exposed to blood mediated AgNPs for toxicity, mortality, bioaccumulation, and histological alterations. The primary tissues exposed to various foreign pollutants in an aquatic sh are gills, muscles, intestine, and liver, hence these tissues were studied.

Nanoparticles preparation
The Sheep's (Ovis aries) blood was collected from the slaughterhouse, Xiao Xihu Gonglin Road, Lanzhou (longitudes of 102°30" to 104°30" and latitudes of 35°5" to 38°), Gansu, China. The blood was collected in a close container in the presence of buffer solution, which keep maintain the proteins of blood serum. The blood was stored at room temperature for serum separation and then centrifuged three times at 12000 rpm for 30 minutes. The serum was separated and stored at 4℃ for further analysis. Pure silver nitrate (99.99%) solution (Sigma Aldrich Beijing, China) of 10 mL was mixed with 90 mL distilled water (10 mM AgNO 3 solution). Subsequently, 100 mL of serum was added to 100 mL of silver nitrate solution (10 mM) in a 500 mL Erlenmeyer ask and incubated for 24 hours at 37℃ at 150 rpm. The synthesized B-AgNPs were puri ed and separated through repeated centrifugation at 12,000 rpm for 20 minutes and stored for further use.

Characterization of Nanoparticles
The synthesis of B-AgNPs was indicated by the color change of the solution from reddish to brownish after incubation. However, UV-Vis spectrometry was used and plasmon bands were recorded at 422 nm that verify the B-AgNPs formation.
The obtained B-AgNPs were characterized for their shape and size. The phase purity and crystalline structure of the obtained B-AgNPs were analyzed by X-ray diffraction using Bruker D2 Phaser with graphite monochromator operated at 30kV and 15 mA. Transmission electron microscopy (TEM) and Scanning Electron Microscope (SEM) was used to characterize the detailed morphology, size, and distribution of synthesized B-AgNPs. The size of the synthesized B-AgNPs was measured by ImageJ software.

Fish species and their laboratory maintenance
The juvenile Common carp sh (C. carpio) (length: 12.3 ± 1.28 cm, weight: 9.59 ± 1.83 g) were brought from the Yantan local market, Chengguan district (36007°N 103082°E; Lanzhou, Gansu). These sh were allowed to adapt in tap water under a natural photoperiod of 14:10 hour light-dark period for one week. After acclimation, the healthy sh were selected for further studies, which were con rmed through physical appearance (skin luster, eyes, color, and behavior). All shes were kept at ambient temperature, pH, and dissolved oxygen (DO 2 ) given in ( Figure S1). Fish were fed with a commercial food diet two times a day (9:00 AM and 9:00 PM), manufactured by JK. Aquarium, China (algae powder, shrimp meal, wheat gluten, soya bean, crude protein, and fresh shmeal). The overall experiment was conducted in 10:14 dark and light cycles. In the whole experiment, 20% of water was changed daily for avoiding bacterial or fungal infection while 100% of water was changed once a week.

Experimental setup
The current study was conducted according to the requirements of the Organization for Economic Cooperation and Development [26] for long time exposure to B-AgNPs. There were four experimental groups as follows: A control group and three B-AgNPs (different concentrations) treatment groups. The sh in the water tanks were exposed to B-AgNPs 0.03 mg/L (LC 50 ), 0.06 mg/L (LC 50 ), and 0.09 mg/L (LC 50 ). Each group was comprising of twenty sh. Fish were not fed for 24 hours before starting the experiment. After every 48 hours, the sh were sampled from every group of studies. The collected sh were anesthetized and dissected for different organs (muscles, gills, intestine, and liver) collection. The collected organs were stored at -80 °C for bioaccumulation and histopathological alteration.

Lethal concentration (LC 50 )
The mortality and acute toxicity were carried out according to the Organization for Economic Cooperation and Development [26]. The mortality rates were recorded every 24 hours for 20 days.

Tissue silver contents
The collected organs were washed and properly dried. Dried organs were kept inside the mu e furnace at 450℃ for 2 hours for digestion. Further 10 mL HCL of 6 mM was added to 1 g of ash. After the complete absorption of ash in HCL, the solution was allowed for ltration following the protocol of [12].

Analytical detection of AgNPs in tissue
The ltered solution was analytically analyzed by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-OES) (Agilent Technologies, 5100) for the bioaccumulation of AgNPs in different tissues of Common carp sh.

Tissue preparation for light microscopy
Tissues were observed for histopathological alteration under a light microscope. The dissected tissues were xed in a 9% formaldehyde solution. After the xation, the tissues were dehydrated in gradient alcohol solutions and implanted with para n wax. The tissue of 4 μm thickness was prepared by a rotary microtome and placed on a glass slide and stained with eosin and hematoxylin.

Biomarker assay
Tissue extracts for antioxidant enzymes measurements were obtained from gill and liver by homogenizing the tissues using 0.1 M sodium phosphate buffer, pH 6.5 containing 20% glycerol, I mM EDTA, and 1.4 mM dithioerythritol. The glutathione S-transferase (GST) activity was conducted using 1-chloro-2,4-dinitrobenzene substrate. Glutathione reductase activity (GR) was conducted using homogenizing the tissues in 0.1 M K-phosphate, 5 mM ascorbate. Catalase assay (CAT) mixture of 3 ml consisted of 0.05 ml tissue extract, 1.6 ml phosphate buffer (100 mM buffer, pH 7.0), 0.5 ml H 2 O 2 , and 0.95 ml distilled water.

Statistical analysis
In the overall experiment, the mortality of sh and treatment concentrations were analyzed through Probit (LC 50 ). All the experimental data were tested for normality and presented as means with standard deviation. The data were tested for time (days) and treatment effects by Two-Way analysis (ANOVA) followed by post-hoc Duncan. P-value < 0.05 was considered statistically signi cant.

Characterization of B-AgNPs
In this study, stable, spherical, small size, and well disperse AgNPs were obtained by using animal blood. The synthesis of B-AgNPs was initially con rmed from the color change of the solution, however, the maximum absorption of UV-Vis spectrometry at 422 nm (Figure 1a) further con rmed the B-AgNPs synthesis. The X-ray diffraction analysis showed peaks 111, 200, 220, and 311 that indicate crystalline structure if the obtained B-AgNPs (Figure 1b). The other peaks lower than 30, is due to residue of organic components present in the blood serum. The size of the obtained B-AgNPs was a small-sized range from 20 -50 nm (Figure 2a). Moreover, the SEM analysis showed spherical and well-dispersed AgNPs at 2.0 μm (Figure 2b).

Toxicity of B-AgNPs against sh
The obtained B-AgNPs were applied in vitro for its mortality and toxicity in sh fauna. C. carpio was very prone to B-AgNPs absorption and caused damage at the tissue level, but with very little mortality. In the present study, the behavior of sh was studied and the sh group (0.09 mg/L) have shown very less abnormal behavior while exposed to B-AgNPs given in Table 1. Therefore, B-AgNPs could be an appropriate indicator for further use in any sheries department. Mortality potential or lethal effect of any applicable materials is the rst parameter of any application study. The sh mortality in the lethal concentration of B-AgNPs was dose-dependent. e.g. the highest mortality was found at the highest dose (0.09 mg/L) given in (Table 2, Fig 3). There was no mortality found in the other three groups (control, 0.03 mg/L, 0.06 mg/L) of the study. In the overall experiment, the mortality was observed on days 2, 6, 16, and 20 and no mortality has occurred in other days of the experiment. Median lethal concentration (LC 50 ) values for 2, 6, 16, and 20 days are given in Table 2.  Figure 3. The mortality of C. carpio during chronic exposure to B-AgNPs (0.09 mg/L). The sh were exposed to different concentrations of B-AgNPs for 20 days. Note: No mortality has been seen at 0.03 mg/L and 0.06 mg/L.

Bioaccumulation of B-AgNPs in tissue
The result of bioaccumulation of B-AgNPs in different organs has been given in (Fig 4). Overall the B-AgNPs were mostly bioaccumulated in the liver, followed by intestine, gills, and muscles (p < 0.05). In all organs, the bioaccumulation of B-AgNPs was dose-dependent e.g. the bioaccumulation of B-AgNPs was increased while the dose was increased.
Comparatively the treatment groups consisted of different concentrations of B-AgNPs. The results revealed that the highest bioaccumulation of B-AgNPs was seen at the highest concentration of B-AgNPs (0.09 mg/L) while the lowest bioaccumulation was seen at the lowest concentration B-AgNPs (0.03 mg/L). The overall absorption of B-AgNPs in different organs is given in (Fig S2) while the daily basis bioaccumulation of B-AgNPs in different organs of (Cyprinus carpio) is given in (S3 = Gills, S4 = Intestine, S5 = Muscle, and S6 = Liver). The liver was the most bioaccumulated organ (Fig 4a).  (Table 3)

Histological investigation
The histological investigation showed that exposure of sh fauna to B-AgNPs led to an alteration in tissue level in targeted organs (gills, intestine). Tissue alteration in the sense of damage, atrophy, shortening of secondary lamella, degeneration, and necrosis at different concentrations of B-AgNPs have been observed. The gill tissue structure was damaged and led to atrophy and necrosis while exposed to 0.03 mg/L of AgNPs shown in ( Figure 5). (Figure 6) shows that necrosis has occurred, which led to vacuolation and the tissues are arranged disorderly. The shedding and degeneration have started in the tissues of gills at 0.06 mg/L concentration of B-AgNPs. At the highest concentration of B-AgNPs (0.09 mg/L), the reduction or shortening of lamella was observed as shown in (Figure 7). Not only the gills but the AgNPs also caused histological alterations in intestinal tissues. In the current study, some intestinal villi mucosal epithelial cells are shaded and a small number of epithelial cells on the top are missing. (Figure 8) shows that there is degeneration, shedding, and necrosis have been observed. The sh exposed to 0.03 mg/L B-AgNPs concentration shows that there is shedding and small number of epithelial cells on the top of intestinal villi are missing. The sh exposed to 0.06 mg/L concentration led to necrosis and degeneration in mucosal cell. The exposure of sh to 0.09 mg/L led to necrosis and cell lysis in the villi mucosal epithelial cells as shown in (Figure 8c). So, it was summarized that lesions, degeneration, necrosis, and shedding even cell lysis were formed mostly at the highest concentration of B-AgNPs exposure given in Table S1 and S2.

Discussion
The toxicity of AgNPs in the aquatic ecosystem is affected by the capability of uptake of aquatic organisms. We herein measured and evaluated the long-time exposure toxicity of newly synthesized B-AgNPs using animal blood in Common carp sh (C. carpio). In the current study, the focus was on mortality, bioaccumulation in different organs, abnormal behavior, and the histopathological study of B-AgNPs. During the study period, the bioaccumulation of B-AgNPs in gill, liver, intestine, and muscles was observed. The results of the current study revealed that the B-AgNPs were mostly bioaccumulated in the liver, followed by intestine, gill, and muscles. During the overall experiment, no abnormal behaviors were noticed. For instance, the sh properly moved toward the food. The food was not rejected by sh. The feces excretion was normal. No irregular operculum movement was found but the sh movement toward the surface was normal. Moreover, the B-AgNPs also damage and cause necrosis, even cell lysis in intestinal villi.
It has been proved that the AgNPs are not much stable and able to be dissolved in aqueous solutions so, it releases Ag ions progressively [32]. Though the toxic effects of the silver in nano form have been widely reported the toxicity of AgNPs is seems to be primarily due to the Ag + release [2,33]. In fact, the high surface to the volume ratio in metallic nanoparticles increases the probability of releasing ion form nanoparticles [2]. According to [34], the toxicity of AgNPs in sh fauna is mostly because of Ag ions instead of nanoparticles.
To investigate the consequences of B-AgNPs, on the mortality (LC 50 ) were accomplished in the current study. The results revealed that LC 50 was dose-dependent. There was no mortality at 0.03, 0.06 mg/L concentration of B-AgNPs while 5/20 sh in the same group at the variant time died at the highest dose (0.09 mg/L) of B-AgNPs. The highest mortality (2/20) was noted on the 16th day of the experiment. In comparison, our results revealed the lowest mortality at the highest concentration of B-AgNPs. A study conducted by [27] revealed the mortality in every concentration even by the lowest concentration (0.01 mg/L) of AgNPs. Another conducted study exhibited that 7/21 sh died at 0.03 mg/L after 96 hours of exposure to AgNPs [2], but in our study, there was mortality at 0.03 mg/L till the end of the experiment.
It has been assumed that biological synthesized AgNPs are less toxic as compared to chemically synthesized nanoparticles [35]. In the current study, the AgNPs were synthesized newly by using animal blood and tested for the bioaccumulation in sh fauna. The results for the bioaccumulation revealed that the B-AgNPs were mostly accumulated in the liver, followed by the intestine, gill, and muscle (Fig. 4a, b, c, d). Similar results have been achieved by [36], in which the AgNPs were accumulated mostly in the liver and intestine. No doubt, there are few studies conducted in the sh toxicity of AgNPs. Based on the above results, it can be assumed or hypothesized that sh liver might play an important role in the metabolism of silver. Previously studies described that the silver rst increase in blood when a sh is exposed to AgNPs then, the liver absorbs the silver, so bioaccumulation of silver occurred [37,38]. In the current study, the sh were fed two times a day, so it can be assumed that while ingestion food particles, they also ingested silver nanoparticles, therefore intestine is the second most bioaccumulated organ after the liver (Fig. 4a). Another previously conducted study also revealed that the intestine was the second most accumulated after the liver [39,40]. The small nger-like projections (villi) present in the intestine increase the surface area for the absorption of any particles. Therefore, [41] concluded that the increased bioaccumulation of AgNPs perhaps due to the absorption from the liver by the intestine. In the present study, gill was the third most B-AgNPs bioaccumulated organ. This might be because of two reasons, 1: Gill are very exposed to the external environment, 2: due to large surface area, the gill has capability to absorb more and more especially small sized materials. [36] concluded that small sized AgNPs were mostly absorbed as compared to large sized AgNPs.
After the long period of exposure of C. carpio to sub-lethal concentrations of blood induced silver nanoparticles, the different lesions and alterations were observed in the intestine and gill (Fig. 5, 6, 7, and 8). In detail, it has been observed that there was necrosis, degeneration, and cell lysis in gill and intestine of sh at different concentrations of B-AgNPs given in (Table S1 and S2). These kinds of changes in gill are the indications of defense mechanism, subsequently, they increase the gap between the external environment and bloodstream, and prevent the penetration of pollution [42]. In the current study, during exposure to AgNPs, the tissue damaging mostly depended on the concentration of B-AgNPs. At the highest concentration (0.09 mg/L) caused severe damage as compared to the lowest concentration (0.03 mg/L). Among all NPs, silver is the most powerful toxicant that affects the gill primarily [43]. So, in the current study by comparison with some other conducted studies. It has been observed that our study showed less toxicity in the sense of mortality, bioaccumulation, and histological alterations.

Conclusion
The current study aimed to investigate the toxicity and toxic effects of B-AgNPs. According to the results of the present study, it might be said that after the long-time exposure, the B-AgNPs were bioaccumulated in the targeted organs while the liver was the main target for bioaccumulation. Furthermore, the bioaccumulation of B-AgNPs have led to histological alterations in gill and intestine. The surface capping, morphology, and size of AgNPs are the major di culties while studying the nano-ecotoxicity. These properties of AgNPs are the main factors for causing toxicity in sh fauna. Therefore, it is concluded that the focus and attention should be given to all these factors while synthesizing the green AgNPs. This might help in the future to reduce the nano-toxicity in biodiversity.