In this study, stable, spherical, small size, and well-dispersed AgNPs were obtained by using animal blood. The synthesis of B-AgNPs was initially confirmed from the color change of the solution, however, the maximum absorption of UV–Vis spectrometry at 422 nm (Fig. 1a) further confirmed the B-AgNPs synthesis. The X-ray diffraction analysis showed peaks 111, 200, 220, and 311 that indicate the crystalline structure of the obtained B-AgNPs (Fig. 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 to 50 nm (Fig. 2a). Moreover, the SEM analysis showed spherical and well-dispersed AgNPs at 2.0 μm (Fig. 2b).
Fig. 1The confirmation of B-AgNPs by UV–Vis spectrometry at 422 nm (a). The X-ray diffraction pattern of blood-mediated silver nanoparticles (b)
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Fig. 2The scanning electron microscopy result of blood-induced silver nanoparticles (a). The transmission electron microscope result of blood-mediated nanoparticles (b)
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The obtained B-AgNPs were applied in vitro for their mortality and toxicity in fish 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 fish was studied and the fish group (0.09 mg/L) has shown very less abnormal behavior while exposed to B-AgNPs as given in Table 1. Therefore, B-AgNPs could be an appropriate indicator for further use in any fisheries department. The mortality potential or lethal effect of any application materials is the first parameter of any application study. The fish 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, 13, and 19 while no mortality has occurred on other days of the experiment. Median lethal concentration (LC50) values for 2, 6, 13, and 19 days are given in Table 2.
Table 1 The behavior study of fish during the exposure period to different concentrations of B-AgNPsFull size table
Table 2 Median lethal concentration LC50 of B-AgNPs to common carp fish in 2, 6, 13, and 19 daysFull size table
Fig. 3The mortality of C. carpio during chronic exposure to B-AgNPs (0.09 mg/L). The fish were exposed to different concentrations of B-AgNPs for 20 days. No mortality has seen at 0.03 mg/L and 0.06 mg/L
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The result of bioaccumulation of B-AgNPs in different organs is given in Fig. 4. Overall the B-AgNPs were mostly bioaccumulated in the liver, followed by the 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 Aditional file (Table S3 = gills, S4 = intestine, S5 = muscle, and S6 = liver). The liver was the most bioaccumulated organ (Fig. 4a). The highest absorption of B-AgNPs was observed in a group (0.09 mg/L), i.e., 272.09 mg in the 20 days exposure. 299.15 mg B-AgNPs were absorbed in the intestine for 20 days exposure as shown in Fig. 4b. Gills have absorbed 225.99 mg of B-AgNPs for 20 days (Fig. 4c) while the lowest absorption of B-AgNPs was observed in the muscles of fish, i.e., 76.64 mg (p < 0.05) shown in Fig. 4d. However, the highest level of absorption of B-AgNPs was seen in the liver and intestine (p < 0.05). The bioaccumulation of B-AgNPs reported in this study was compared with previous studies (Table 3).
Fig. 4The absorption and bioaccumulation of B-AgNPs in the gills (a) in the intestine (b) in the liver (c) and the muscles (d) of common carp fish (C. carpio) after the exposure to 0.03, 0.06, and 0.09 mg/L for 20 days (p < 0.05). There was no accumulation of AgNPs in control groups
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Table 3 Comparison of the present study with previous studies, showing the results at the end of the experimentFull size table
The histological investigation showed that exposure of fish fauna to B-AgNPs led to an alteration in tissue level in targeted organs (gills, intestine). Tissue alterations 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 Fig. 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 Fig. 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 fish exposed to 0.03 mg/L B-AgNPs concentration shows that there is shedding and a small number of epithelial cells on the top of intestinal villi are missing. The fish exposed to 0.06 mg/L concentration led to necrosis and degeneration in the mucosal cell. The exposure of fish 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 Additional file 1: Table S1 and S2.
Fig. 5Histopathological alteration of the gills of common carp fish after the exposure to 0.03 mg/L concentrations of B-AgNPs. (↑) The atrophy of gill lamella, necrosis, and cloudy of the lamella. The tissues were stained using eosin and magnified with × 400
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Fig. 6Histopathological alteration of the gills of common carp fish after the exposure to 0.06 mg/L concentrations of B-AgNPs. (↑) Vacuolation of gill lamella, (↑) epithelial cell of gill shedding, and (↑) necrosis. The tissues were stained using eosin and magnified with × 400
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Fig. 7Histopathological alteration of the gills of common carp fish after the exposure to 0.09 mg/L concentrations of B-AgNPs. (↑) Atrophy and shortening of the lamella. (↑) The interlayer of lamellae fused. The tissues were stained using eosin and magnified with × 400
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Fig. 8a The histopathological alterations of common carp fish intestine after the exposure to B-AgNPs at 0.03 mg/L concentrations. (↑) Degeneration, necrosis, and loss of epithelium cells on the top of villi in the small intestine, (↑) the increased number of lymphocytes. b The histopathological alterations of common carp fish intestine after the exposure to B-AgNPs at 0.06 mg/L. (↑) Necrosis, and shedding, lamina propria disintegrate in mucosal cells (↑) lymphocytes infiltration. c The histopathological alterations of common carp fish intestine after the exposure to B-AgNPs at 0.09 mg/L. (↑) Degeneration, necrosis, and cell lysis in villi of the intestine, (↑) increased lymphocytes. The tissues were stained using eosin and magnified with × 400
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Antioxidant enzyme activities are summarized in Table 4. The different concentrations of B-AgNPs led to significant changes in the hepatic and gill enzymatic activities. With a reduction in the fish group exposed to (0.03 mg/L and 0.06 mg/L) and an increase in the highest concentration of B-AgNPs (p < 0.05). At the highest concentration (0.09 mg/L) of B-AgNPs, a rise in GST and decrease were noticed in GR in the case of the liver. But in the gills, GST and GR both have been decreased as compared to control. The CAT increased at 0.03 mg/L while decreased in both 0.06 mg/L and 0.09 mg/L in case of the gills and liver. The GR decreased at all the concentrations of B-AgNPs comparing to the control group gills while in the liver the GR has increased at 0.03 mg/L and 0.06 mg/L but has decreased at 0.09 mg/L.
Table 4 Enzymatic assay of glutathione S-transferase (GST), glutathione reductase (GR), and catalase (CAT) in the tissues of liver and gills of C. carpio after exposure to 0.03 mg/L, 0.06 mg/L, and 0.09 mg/L for long-time exposure of B-AgNPsFull size table
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 fish (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 gills, 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 the intestine, gills, and muscles. During the overall experiment, no abnormal behaviors were noticed. For instance, the fish properly moved toward the food. The food was not rejected by fish. The feces excretion was normal. No irregular operculum movement was found, but the fish 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]. The high surface-to-volume ratio in metallic nanoparticles increases the probability of releasing ion form nanoparticles [2]. According to [34], the toxicity of AgNPs in fish fauna is mostly because of Ag ions instead of nanoparticles.
To investigate the consequences of B-AgNPs, mortality (LC50) was measured in the current study. The results revealed that LC50 was dose-dependent. There was no mortality at 0.03, 0.06 mg/L concentration of B-AgNPs while 5/20 fish 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 fish died at 0.03 mg/L after 96 h of exposure to AgNPs [2], but in our study, there was no mortality at the lowest concentration (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 for the first time using animal blood serum and tested for the bioaccumulation in fish fauna. The results for the bioaccumulation revealed that the B-AgNPs were mostly accumulated in the liver, followed by the intestine, gills, and muscle (Fig. 4a–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 on the fish toxicity of AgNPs. Based on the above results, it can be assumed or hypothesized that fish liver might play an important role in the metabolism of silver. Previously studies described that the silver first increase in blood when a fish is exposed to AgNPs, then the liver absorbs the silver, therefore bioaccumulation of silver NPs highly took place [37, 38]. In the current study, the fish were fed two times a day, so it can be assumed that while ingesting food particles, they also ingested silver nanoparticles, therefore the intestine is the second most bioaccumulated organ followed by 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 finger-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, gills were the third most B-AgNPs bioaccumulated organ. This might be because of two reasons: (1) gills are highly exposed to the external environment; (2) due to large surface area, the gills have the 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 sublethal concentrations of blood-induced silver nanoparticles, different lesions and alterations were observed in the intestine and gills (Figs. 5, 6, 7, 8). In detail, it has been observed that there was necrosis, degeneration, and cell lysis in the gills and intestine of fish at different concentrations of B-AgNPs given in Additional file 1: Tables S1 and S2. These kinds of changes in gills are the indications of a 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 gills primarily [43]. In the current study by comparison with other previously conducted studies. It has been observed that our study showed less toxicity in the sense of mortality, bioaccumulation, and histological alterations. Therefore, blood serum-mediated silver nanoparticles might be an alternative to the other biological synthesized AgNPs.
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