Introduction

An increasing number of technologies are employed for manufacturing the engineered nanomaterials in many scientific and industrial sectors. Nowadays, nanoparticles (NPs) have applications in textiles, electronics, paints, cosmetics, and pharmaceuticals industries [1,2,3,4]. NPs are commonly used for their unique physico-chemical properties, especially metal oxide nanoparticles (MNPs) [5, 6]. Particularly noble metals such as silver, gold, platinum, and palladium have received much attention among scientists due to their high surface ratio and innovative applications [7,8,9]. In recent years, silver nanoparticles (AgNPs) have a new scope in functionalized surfactant and biocompatible [10, 11]. Ag has been used for medical purposes since the beginning of the last century [12,13,14].

Techniques have been developed and adapted as valuable tools to monitor pollution in aquatic environment for the last two decades [15, 16]. Usually, aquatic organisms are quickly and constantly exposed to toxic substances from natural or dissolved anthropogenic sources [17, 18]. The nanomaterials (AgNPs, ZnONPs, CuONPs, TiO2NPs, Fe3O4NPs, and NiNPs) contribute a great deal of aquatic pollution that poses threat to aquatic organisms [19,20,21,22,23,24,25]. Bioaccumulation and biomagnification capacity of nanomaterials (Ag, Cu, Zn, and CdCl2) have longer effects on aquatic species like planktonic crustacean (Daphnia magna) [26], ragworm (Nereis diversicolor) [24], mussel (Mytilus galloprovincialis) [27], fishes (Clarias gariepinus, Oreochromis niloticus, Oncorhynchus mykiss, Danio rerio, Carassius auratus) [28,29,30,31,32,33]. Increasing use of nanomaterials decreases the population of aquatic organisms mainly fishes. According to their intimate dependence on the environment and comparing with other species, fishes are more vulnerable to nanotoxic stress [34, 35]. Heavy metal/nanotoxicants accumulate in fish through biomagnifications and cause changes in their biological mechanisms [28, 36,37,38,39].

Fabrication of NPs can be achieved through different synthesis methods. Conventional (physical and chemical) approaches are the most widespread methods for the synthesis of NPs. However, in chemical approach, the use of toxic chemicals in the synthesis methods is inevitable. Since noble metal NPs are widely exposed to environment, there is a growing need to develop eco-friendly synthesis methods, which do not use toxic chemicals. Biogenic approach of developing AgNPs could reduce the environmental impact and generate minimal waste. Usually, AgNPs are applied in electronics and storage devices, biomedical, etc. [40, 41]. AgNPs are synthesized by several biological sources like bacteria, fungi, algae, and plants with the absence of hazardous materials [22, 42,43,44]. The biogenic AgNPs would be a least toxic and also an eco-friendly approach to inhibit the microbial contaminations in aquaculture environment. In addition, biogenic AgNPs are highly stable during large-scale production of fish feed and have significant antibacterial effects [45]. Biogenic synthesis of AgNPs is cost-effective and suitable for large-scale production in controlled environmental condition to their stability, shape, and size [46, 47]. In recent years, to promote environmental sustainability, nontoxic precursors are used in the development of nanomaterials [48]. Biogenic nanoparticles are the most admired inorganic NPs utilized as an efficient antibacterial, antifungal, antiviral, anti-inflammatory agents, and food additives [49]. Biogenic nanomaterials are nontoxic that increase growth and immune responses in various fishes like Catla catla, Labeo rohita, Cirrhinus mrigala, and Oreochromis niloticus and which plays a crucial role in aquaculture operations [50,51,52,53,54]. Several studies showed that the toxicity of conventional AgNPs depending on particle size, capping or coating agent, binding to DNA, residues of Ag+ ions, and release of reactive oxygen species (ROS) [55,56,57,58,59]. Through various pathways, AgNPs cause severe impacts on living organisms [60, 61]. The dispensable release of AgNPs in the aquatic ecosystem eventually may modify the physico-chemical and biological characteristics of aquatic system resulting in an environmental imbalance [35, 62,63,64].

Despite their number of applications, detailed information on the toxic effect and mechanical action of biogenic AgNPs on fish is limited. Therefore, the current study was to examine the toxicological impacts of biogenic AgNPs on freshwater common carp (Cyprinus carpio). This study further investigates the total accumulation, haematological parameters, and changes in antioxidant enzymes, oxidative stress genes, and histology in fish C. Carpio treated with sublethal concentrations of biogenic AgNPs.

Materials and Methods

Synthesis and Characterization of AgNPs

Using aqueous onion peel extract (Allium cepa L.), AgNPs were synthesized as described in our previous study [65]. Briefly, 5 g of A. cepa peel was cleaned and boiled in 100 ml of double distilled water (dd H2O) at 60 °C for 20 min in 250 ml of Erlenmeyer flask. Ten millilitres (10 ml) of resulting filtrate was mixed drop wise with 100 ml of (0.001 M) AgNO3 solution. The darkish brown colour indicates the formation of AgNPs, which were characterized using UV–Vis spectrophotometer, X-ray diffraction (XRD), fourier transform infrared (FT-IR), and transmission electron microscopy (TEM) with energy dispersive spectrum (EDS).

Animal Maintenance

Freshwater fish Cyprinus carpio was procured from Nathan Fish Farm, Thanjavur, Tamil Nadu, India. The mean body weight and length of the fish were 5.34 ± 0.53 g and 6.06 ± 0.69 cm, respectively. Fish were kept in 2000 l circular water tanks in the Aquarium Facility at Department of Animal Science, Bharathidasan University. The water was changed routinely and its temperature, pH, and dissolved oxygen (DO) were maintained at 29 ± 2 °C, 7.0 ± 0.1, and 6.5 ± 0.5 mg/l, respectively. Fish were allowed to acclimatize for 7 days and fed with commercial pellet feed at ad libitum.

Acute Toxicity Test

After the acclimatization period, fish were randomly divided into six groups in plastic troughs and each group contains 10 fish (Triplicate setup) for the determination of LC50. The acute study was conducted for 96 h according to OECD 203: Fish Acute Toxicity Test [66]. A series of AgNPs suspension (0, 2, 4, 6, 8, and 10 mg/l) (100 W, 53 kHz at 30 min, Misonix Ultrasonicator, USA) was exposed to each group separately. The NPs exposed to water were changed every 24 h to ensure and maintain the concentration of NPs. The experimental fish were fed with 10% of body weight of commercial feed. According to Finney [67], the LC50 values were obtained using probit analysis (SPSS ver. 16.0, IBM, Chicago, IL, USA).

Sublethal Toxicity

For sublethal toxicity, C. carpio fish were separated into four groups; each group consists of 90 fish (3 replicates). The first group was left as control. The suspension of 1/15th, 1/10th, and 1/5th of 96 h LC50 concentrations were prepared by dispersing (100 W, 53 kHz at 30 min, Misonix Ultrasonicator, USA) of AgNPs and exposed to remaining groups (2–4). The total experiment was carried out for 28 days. Optimal parameters were maintained similar to the acclimatization period. Renewal method was carried out daily with the same concentrations of NPs after 30 min of feeding.

Fish Sampling

AgNPs exposed fish were collected for sampling with an interval of every 7 days. Fish were anesthetized by hypothermia method (non-chemical method); then, the blood sample was withdrawn from the dorsal aorta, and the vital organs like the gill, liver, and muscle were dissected out and stored for further analysis at −80 °C.

Analysis of Bioaccumulation

The accumulation of Ag content in the gill, liver, and muscles of fish C. carpio was estimated using slightly modified method of Zhang [68]. Briefly, at the end of the exposure, fish tissue samples (0.5–1 g) were digested by the triple acid digestion method with the mixture of concentrated HNO3, HCl, and H2O2 in the ratio of 3:3:1. Finally, the total suspension (25 ml) was made with dd H2O and stored in polypropylene tubes. The bioaccumulation of Ag in the target tissues was estimated using ICP-OES (Manufacturer: PerkinElmer; Model: Optima 5300 DV, USA; wavelength range 165–782 nm; RF generator 40 MHz; Detection limit: upto ppb level using SCD detector). The obtained results were expressed in µg/g of the tissue analysed.

Haematological Parameters

The blood sample was drawn with 1 ml syringe (30-gauge) at dorsal aorta with an interval of 7 days and immediately transferred into collection tubes (containing EDTA) and subjected to analyse the haematological parameters. Haemoglobin, haematocrit, red blood cells, and white blood cells were evaluated in Mindray BC-2800Vet® automated haematology analyser. The mean cell volume (MCV), mean cell haemoglobin (MCH), and mean cell haemoglobin concentration (MCHC) were calculated by the method of Dacie and Lewis[69].

$$\mathrm{MCV }(\mathrm{cu}.\mathrm{ microns}) =\mathrm{ Hct}/\mathrm{RBC }(10^6/\mathrm{ml})$$
(1)
$$\mathrm{MCH }(\mathrm{pg}) = [\mathrm{Hb }(\mathrm{g}/\mathrm{l}) \times 10]/\mathrm{RBC }(10^6/\mathrm{ml})$$
(2)
$$\mathrm{MCHC }(\mathrm{g}/\mathrm{dl}) = [\mathrm{Hb }(\mathrm{g}/\mathrm{l}) \times 10]/\mathrm{Hct }\times 100$$
(3)

Measurement of Oxidative Biomarkers

For evaluating the oxidative stress damage, the gill, liver, and muscle tissues were homogenized with cold phosphate buffer (pH 7.4) and centrifuged at 11,200 × g for 10 min and the filtrate was stored at −80 °C until used. Lowry [70] method was adopted to estimate the total tissue protein with BSA as standard. Superoxide dismutase (SOD) activity was analysed by the method of Marklund and Marklund [71]. The measured activity was expressed as unit/mg protein. The activity of catalase (CAT) enzyme was measured by the method of Claiborne [72]. One unit of catalase was defined as 1 μMol of H2O2 consumed/mg protein/min. Rotruck et al. [73] method was followed to identify the glutathione peroxidase (GPx) activity and expressed as μMol consumed/mg protein/min. Habig et al. [74] was used to determine the glutathione-S-transferase (GST) activity and expressed as unit/mg protein. Glutathione (GSH) was analysed by the method of Moron et al. [75] and expressed as μg/mg protein.

Histoarchitecture Analysis

For the analysis of AgNPs toxicity in vivo, the histopathological examinations were done in selected tissues of C. carpio (gill, liver, and muscle) fixed in 10% formalin. The paraffin-embedded tissues were fixed and stained (haematoxylin and eosin). Morphological alterations were observed under a microscope attached with a camera (DM750, Leica Microsystems, Germany).

RNA Extraction and cDNA Synthesis

Total RNA was extracted from liver tissue (~50 mg) of experimental fish using RNAiso Plus Kit (Takara Clontech, India). The purity and concentration of RNA were checked by BioDrop µLITE spectrophotometer. Then, the cDNA were synthesized from the obtained RNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Total volume of reverse transcription reaction mixture contains 5 × reaction buffer (4 µl), 10 mM dNTP mix (2 µl), RiboLock RNase inhibitor (1 µl), and RevertAid H minus (1 µl) with 800 ng of RNA. Reverse transcription was performed by the following thermal conditions 25 °C for 5 min, 42 °C for 60 min, and 70 °C for 5 min (Eppendorf MasterCycler® Gradient, Hamburg, Germany). The synthesized cDNA was stored at −20 °C.

Real-Time Quantitative PCR Analysis

RT-qPCR was performed by using a single-step real-time PCR machine (LightCycler® 96, Roche Life Science, USA) with 1X SYBR Green (Takara), 0.5 μl of each primer (Table 1), and 1 μl synthesized cDNA. The RT-qPCR conditions were initial denaturation (95 °C for 5 min), 45 cycles of 3 step amplification (95 °C for 10 s, 48/52 °C for 30 s, and 72 °C for 10 s). Table 1 shows the annealing temperatures of each primer. β-actin served as the internal control to normalize the RNA level. Three technical repeats and experimental replicates were performed for each gene. Threshold cycle (Ct) values were used to quantify the gene expression by 2−∆∆CT method [76].

Table 1 Primer sequences and annealing temperature for target genes and the housekeeping gene for real-time qPCR
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Statistical Analysis

The data obtained were showed as mean ± standard deviation (SD) for each group. The differences among groups were analysed statistically at p < 0.05 against the control group using one-way Analysis of variance (ANOVA) followed by Duncan’s multiple range (DMRT) as post hoc test (SPSS).

Results and Discussion

Characterization of Biologically Synthesized AgNPs

AgNPs optical property was studied by using UV–Vis spectroscopy (Fig. 1a). Herein, the synthesis of AgNPs may be due to the phytochemical constituents present in the peel of onion. It has been already reported that the onion wastes (Allium cepa L.) contain dietary fibre and bioactive compounds [77]. During synthesis, the formation of AgNPs was observed by the colour change due to the excitation of surface plasmon resonance (SPR) in the visible region ranging from 350 to 700 nm. The wavelength region has a typical SPR absorption band of synthesized AgNPs at a wavelength of maxima (λ max) at around 466 nm. The previous study biosynthesized AgNPs showed SPR around at 460 nm [78]. The results were an agreement with earlier study conducted in Dimocarpus Longan Lour. peel extract synthesized AgNPs [79].

Fig. 1
figure 1

Characterization of biologically synthesized AgNPs by using onion peel extract. a UV–vis spectrum. b XRD analysis. c FT-IR analysis. d and e TEM analysis with EDS spectrum

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The XRD pattern of biogenic AgNPs was indexed completely with the results supporting that the prepared material exhibited and confirmed by JCPDS card no: 03–0921 with a face-centred cubic (fcc) structure of silver (Fig. 1b). The planes of 1 1 1, 2 0 0, 2 2 0, and 3 1 1 were located the 2θ at 38.07°, 46.18°, 64.32°, and 77.35°, respectively, and confirmed the face-centred cubic (fcc) structure of AgNPs, which might have resulted from the bioactive compounds in the onion peel extract. The average particle size of the biogenic synthesized AgNPs was calculated at 33 nm with the Scherrer equation. D = Kʎ/βcosθ, whereas D is particle size, ʎ is X-ray wavelength (0.15426 nm), β is full width at half maximum (FWHM), and θ is Bragg’s angle. In comparison to the previous report [50], the AgNPs particles are slightly smaller, with an XRD modal diameter of 12 nm.

The AgNPs synthesized using brown skin of onion peel extract, which contains different phytochemical constituents such as dietary fibre, phenolics, and flavonoids [77]. These phytochemicals play an important role in the formation and stabilization of AgNPs. The FT-IR spectral peaks at 3429-and 1637 cm−1 assigned to the deformative vibration of water molecules (Fig. 1c). The weak peaks at 2922, 1259, 1546, and 1044 cm−1, corresponding to the stretching vibration of methyl [80], germinal methyl [81], amide I groups proteins [82], and C = O stretching [83]. Characteristics peaks at 801 and 545 cm−1 were attributed to Ag–O stretching vibration [84]. From this functional group analysis, which revealed the presence of flavonoids, phenolics, methyl, amide groups, and proteins was present in the biogenic AgNPs.

TEM micrograph with EDS was given in Fig. 1d. The size of the synthesized NPs was 8–50 nm and exhibited with the agglomerated spherical silver nanoparticles [85]. Ganesh Kumar et al. [86], reported, that the morphology of the NPs were depending on the concentration of reducing agents, which gives the structure to the NPs. The EDS analysis reveals a strong signal at 3 keV (Fig. 1e), which was generally exhibited by metallic Ag nanocrystals due to SPR, the peaks at 0.3 and 0.5 keV showed the un-reacted precursors of AgNO3 and biomolecules [87].

Acute Toxicity of AgNPs

The toxicity of different NPs has been widely studied and used in the fields of biomedical and pharmaceuticals. Due to the increasing production and application of NPs, there is a growing likelihood of occupational and possibility of environmental exposure [88]. NPs in the aquatic ecosystem encounter various changes, such as dissolution, aggregation, oxidation, and bioavailability that cause toxicity to the aquatic species [89]. Nowadays, traditional and biological NPs exhibit attractive characteristics and can trigger several risk factors [90]. The determined LC50 values were used to fix the sublethal concentrations to investigate the chronic effect of biogenic synthesized nanomaterials. No mortality was noted in the control groups; however, the biogenic nanomaterial-treated fish groups showed mortality in the increasing concentrations and 100% mortality was recorded at the maximum tested concentrations (10 mg/l) at 96 h. The 96 h acute exposure of AgNPs synthesized from A. cepa was toxic to C. carpio at the LC50 of 2.76 mg/l. In the earlier study, exposure of AgNPs synthesized using blood serum of sheep showed LC50 at 0.61 mg/l to common carp, which is higher toxicity concentration than the present study [91]. Liaqat et al. [92] analysed 96 h LC50 concentration of AgNPs synthesized using Halymenia porphyraeformis in C. carpio was found to be 0.331 mg/l, which is more toxic than the LC50 value shown in this present investigation. On the other hand, commercially available colloidal AgNPs showed highest mortality at the 48 h LC50 concentration of 0.5 mg/l [93]. Ramachandran et al. [94] reported that the 96 h LC50 of biologically synthesized AgNPs were toxic to adult zebrafish at 24.5 µg/l. A study conducted by Krishnaraj et al. [95] has shown the highest toxicity even on the lowest concentration of AgNPs synthesized using aqueous extract of Malva crispa leaves at 142.2 µg/l to adult zebrafish. Similarly, another study has revealed that fish exposed to AgNPs synthesized using Psidium guajava in zebrafish were found to be 400 µg/l [96]. Aquatic toxicity evaluation may provide insights to the relative sensitivity of different species to AgNPs, which may also provide suitable data on the adverse effect of NPs on aquatic environment, as these species hold important positions in aquatic ecosystems. The toxicity difference in the different species reveals the sensitivity of species distributions. This study indicates that the toxic nature of NPs was not only dependent on the species difference but also depends on the concentration and physio-chemical properties of NPs [97,98,99,100,101].

Sublethal Toxicity Analysis

Following the LC50 determination, 28 days chronic toxicity was analysed. The sublethal concentrations of 1/15th, 1/10th, and 1/5th of the LC50 (2.36 mg/l) of AgNPs (0.184, 0.276, and 0.552 mg/l) were used. No mortality was observed in control and AgNPs treated groups during the experiment.

Bioaccumulation Assay

ICP-OES is a powerful multi-element analyser used to quantify the toxic metals in fish and other aquatic organisms even at subparts per billion because of its high sensitivity [102,103,104]. Total accumulation level of Ag at the end of 28th day in gill, liver, and muscle of C. carpio fish exposed to biogenic AgNPs was shown in Table 2. The biotransformation in the present study observed that the accumulation in all organs was dose-dependent. According to Iversen et al. [105] report, the accumulation of NPs was based on the composition, size, surface charge, and surface coating. Generally, metal uptake is depending upon water which pumped through gills. Significantly accumulated Ag content was observed in the gill, liver, and muscle when comparing with control fish. In this study, the accumulation was observed in the following order of gill > liver > muscle in AgNPs exposed C. carpio fish. Generally, gills are in direct contact with aquatic environment and are physiologically complex and vulnerable structures that makes them easily target organ for waterborne toxicants [106]. In addition, AgNPs can accumulate and bind with gills by transportation process, which affects the ability of fish respiration to hypoxic (low oxygen) conditions, and passes through blood stream to liver tissue leading to oxidative stress. Generally, fish have various transportation systems to maintain their required mineral levels for a wide range of metabolic pathways, either by regulating the absorption of minerals through their diet [1]. Results of another study confirmed the highest accumulation of Zn was observed in gills after exposure of ZnONPs [107, 108]. These findings are comparable with previous studies that have shown the bioaccumulation of Ag in various vital organs of O. mykiss [109, 110]. The study of possible uptake of TiO2NPs showed that the gill, intestine, and brain have higher level of Ti content, and the least amount of Ti was accumulated in the muscle of the goldfish (C. auratus) [33]. Liver is known to be one of the vital organs in fish and have detoxification process when exposed to contamination via excretion. Thus, a lower content of Ag elimination in the liver of common carp may be due to the binding of the metallothionein-like proteins produced in these tissues and its eventual detoxification and storage in them; hence, suggesting that Ag is excreted gradually and slowly in the liver tissues in comparison to the gills [111]. The accumulation of NPs in fish could occur due to the exposure of NPs for longer times [112]. This was most pronounced in fish treated with higher concentrations of AgNPs [113, 114]. The influence of different metals induces toxicity and it might have been caused by the structural variabilities of the particles, potential adsorption by tissues, and physiological and toxicological responses by organisms. Moreover, the accumulation of nanomaterials in common carp was depending on the target organ, concentration, and duration of exposure [106].

Table 2 Total accumulation of Ag (µg/g) in the gill, liver, and muscle of C. carpio exposed to different sublethal concentrations of AgNPs
Full size table

Haematological Analysis

Haemoglobin (Hb) and haematocrit (Hct) content in control and AgNPs treated fish were illustrated in Fig. 2a, b. Hb and Hct content in the AgNPs treated fish was significantly decreased (p < 0.05) on all sampling days except the 14th day, which showed the higher content of Hb at 0.184 mg/l compared to control. Exposure of AgNPs on O. niloticus significantly decreased the values of erythrocyte count, Hct and Hb compared to the control groups [115]. Reduced values of haematological parameters were observed by Dhanapakiam and Ramasamy [116] after exposing Cu for 30 days in C. carpio were due to the destruction of erythrocytes. Similar to the present findings, Alkaladi et al. [117] and Rather et al. [52] found a decreased value of Hct in fish exposed to varying sublethal levels of ZnONPs and AgNPs, respectively. Similar observations were also reported in C. catla treated with zinc and cadmium [118], arsenic [119], and O. niloticus treated with cadmium [28]. A decline in the Hct level suggests anaemia or destruction of the erythrocyte membrane, resulting in haemodilution [120,121,122].

Fig. 2
figure 2

Analysis of haematological parameters. a Hb. b Hct. c RBC. d WBC. e MCV. f MCH. g MCHC on AgNPs exposed C. carpio fish. Each value represents the mean ± SD; n = 3. Different letters above the bars show a significant difference among the groups at p < 0.05

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The erythrocyte count of control and fish exposed to sublethal concentrations of AgNPs were illustrated in Fig. 2c. The RBC in the AgNPs treated fish was decreased significantly (p < 0.05) when comparing with control. This phenomenon was more evident in fish treated with higher sublethal concentrations of AgNPs. Reduction in RBC may also be due to a decline in haematopoiesis [123, 124]. Exposure of NPs causes respiratory dysfunction through gill damage and eventually alters the RBC values [125]. Farmen et al. [126] have reported that the RBC count and haemoglobin values significantly decreased, resulting in macrocytic anaemia in Atlantic salmon exposed to AgNPs. A similar reduction of RBC was reported in Oreochromis mossambicus treated with Cu [127], O. mykiss treated with CuNPs [128], L. rohita treated with waterborne Fe2O3 NPs [129], and ZnONPs treated O. niloticus [117].

In the present investigation, significant increase (p < 0.05) in white blood cells (WBC) count was observed in the AgNPs -treated fish than control (Fig. 2d). In the higher sublethal concentration (0.276 and 0.552 mg/l), the WBC count was decreased significantly, whereas the lower concentration (0.184 mg/l) showed a maximum increase. Due to the nano-toxicity, the WBCs respond immediately to the medium alteration. This shows that the fish can build a defensive mechanism to withstand nanotoxic stress. The increase in WBC may be due to induced proliferation as a result of the nano-toxicity of pluripotential hematopoietic cells, which, in effect and may result from decreased circulation between differentiated cells [130, 131]. In this study, increased WBC counts suggest the stress of the fish caused by nano-toxicants, which may have caused hypoxia and gill damages.

The MCV, MCH, and MCHC levels of AgNPs treated fish were illustrated in Fig. 2e–g, respectively. RBC index level in the treated fish was significantly altered (p < 0.05) throughout the experiment. This was most pronounced in fish exposed to the sublethal concentrations of AgNPs. The present results confirmed the alteration of haematological indices as a non-specific immune response to NPs toxicity. Similar results were found in C. mrigala exposed to AgNPs [52] and O. niloticus exposed to ZnONPs [117]. Exposure of nanomaterials may disrupt the iron absorption in intestine or hematopoietic tissues and increases the destruction rate of RBC due to osmoregulatory dysfunction [132].

Oxidative Biomarkers

The activity of antioxidant enzyme SOD in the gills of AgNPs was shown in Fig. 3a. AgNPs significantly altered (p < 0.05) the SOD levels in the gill tissues of AgNPs exposed groups comparing control. In contrast, 7th day treatment showed a similar response except for 0.184 mg/l of AgNPs treated fish gill. On 14thth day of AgNPs exposure significantly enhanced the enzyme activity at 0.552 mg/l compared with control and other tested concentrations. On 21stst day exposure, there was a significant decrease in AgNPs exposed groups. On 28th day, levels of SOD were decreased in 0.276 and 0.552 mg/l. The liver and muscle SOD activity were increased significantly throughout the study period compared with the control (Fig. 3b and c). C. carpio exposed to 0.552 mg/l AgNPs showed a significant increase in muscle SOD activity up to 21st day and significantly decreased on 28th day. Lower and intermediate concentrations of AgNPs induced a significant alteration. These results indicated the generation of O•−2 in the tissues of C. carpio and demonstrated that AgNPs induced oxidative stress after chronic exposure. In another study, similar findings were reported by Pirsaheb et al. [133], a significant decrease in activity of SOD was observed in gills of C. carpio exposed to metal-doped TiO2NPs and the mechanism behind this, is the metallic nature of NPs and the presence of transition metals encourages the production of ROS leading to oxidative stress. For maintaining cell homeostasis and preventing oxidative stress, SOD catalyses superoxide anion radical dismutation by forming less-reactive molecular oxygen [134]. A similar response was observed in O. mossambicus exposed to AgNPs [135] and juvenile C. auratus exposed to fullerene C60 [136].

Fig. 3
figure 3

Antioxidant enzyme activity of C. carpio exposed to sublethal concentrations of AgNPs. a Gill SOD. b Liver SOD. c Muscle SOD. d Gill CAT. e Liver CAT. f Muscle CAT. g Gill GPx. h Liver GPx. i Muscle GPx. j Gill GST. k Liver GST. l Muscle GST. m Gill GSH. n Liver GSH. o Muscle GSH. Each value represents the mean ± SD; n = 3. Different letters above the bars show a significant difference among the groups at p < 0.05

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CAT activity in the gill was found to be increased in AgNPs treated groups comparing the control, whereas the gill CAT activity was decreased significantly on 14th day (0.552 mg/l of AgNPs) (Fig. 3d). In this present study, the liver and muscle of AgNPs treated fish showed significantly decreased CAT activity throughout the study period compared with control (Fig. 3e and f). Excessive productions of ROS from SOD catalytic activity inhibits the CAT function [137,138,139,140]. Several studies have also demonstrated a biphasic tendency in CAT activity in fish after treating with increasing levels of TiO2 in O. niloticus and C. Carpio [141,142,143].

Comparing with control, significant increase (p < 0.05) in gill GPx activity was observed throughout the experiment period except on 14thth day (Fig. 3g). GPx activity in the liver decreased significantly in all the concentrations of AgNPs after 7th day exposure compared with control fish (Fig. 3h). AgNPs induced a biphasic trend elevation of GPx in the muscle on all sampling days compared with its control (Fig. 3i). A maximum elevation of muscle GPx was observed on 28th day experiment in (0.552 mg/l) of AgNPs. Reduced intracellular GSH levels or toxicity of nanomaterials on enzyme correlate with the reduction or induction of GPx activity. Likewise, Wu and Zhou [144] found significant alternations in GPx activity of fish Oryzias latipes exposed to AgNPs. Elimination of hydroperoxides by GPx through the reduction of GSH alters the function [145]. In response to CuONPs exposure, GPx activity was stimulated in the liver and gill of O. niloticus to counteract hydroperoxides [31].

The GST activity in the gill was significantly increased on all the sampling days except on 7th day of treatment, and significantly decreased activity was observed on 14th day experiment (0.552 mg/l) (Fig. 3j). Induced activity of GST is an adaptive mechanism for neutralizing the impacts of nanomaterials. Lee et al. [146] observed increased activity of GST in tissues of C. carpio treated with various concentrations of AgNPs and suggested that GST enzyme is induced to protect the fish against the NPs toxicity. The increase in GST activity in gills could be a good marker for compensatory tissue response against to toxicant exposure. The antioxidant enzyme activity of GST in the liver tissues exposed to AgNPs was shown in Fig. 3k. AgNPs exposure significantly decreased the liver GST activity throughout the experiment period except for the 7th day compared with its control. Significantly decreased (p < 0.05) muscle GST activity was observed throughout the experiment (Fig. 3l). Stimulated activity of GST in Brycon amazonicus fish indicates its significance against ROS [147]. Similar induced activity of GST was reported by Chae et al. [148] after studying the effects of AgNPs in Japanese medaka (Oryzias latipes). Increased activity of GST reveals ROS generation due to the existence of nanomaterials in fish [149]. The altered antioxidant defense mechanism exhibits imbalanced oxidant and antioxidant levels in the tissues [150, 151].

A maximum elevation of gill GSH was observed on 14th day (0.276 mg/l) and a minimum of decreased activity was found on the same day at 0.552 mg/l concentration (Fig. 3m). The changes of liver GSH activities in the AgNPs exposed groups were shown in Fig. 3n. It was found that GSH activity was varied with the exposure concentration and different times in this study. The change in the levels of GSH is inter-related with the activity of GST. According to Reddy et al. [152], increased glutathione reductase activity is a result of pro-oxidant system which forms reduced GSH by recycling oxidized GSH. Increased activity of GSH level was noted in fish (Lateolabrax japonicus) after treated with benzo[a]pyrene by Jifa et al. [153]. In muscle, the AgNPs treatment significantly decreased the elevation of GSH activity throughout the experiment period compared with the control group (Fig. 3o). AgNPs exposure depleted the glutathione activity in cells associated with increasing ROS levels [154, 155]. The ability of NPs can induce the toxicity to fish by several factors such as their concentration, the exposure period, accessibility to the target site, and distribution in organism’s tissues and kind of species [156].

Histological Observations

Histopathological analysis of fish tissues has been used as a tool that is essential for evaluating the quality of aquatic water bodies [157,158,159]. Gills are vital organs used for many physiological functions [160]. Typical gills of the untreated fish indicated a neat architecture of primary and secondary lamellae, mucous cells, pillar cells, epithelial cells, and central venous sinus, which could be differentiated easily (Fig. 4a–d). The AgNPs treated (0.184 and 0.276 mg/l) fish gill showed lamellar blood sinus constricts, leukocyte infiltration of epithelium and fusion of lamellae were noticed (Fig. 4e–l), whereas 0.552 mg/l of AgNPs treated fish showed severely damaged primary and secondary lamellae, complete fusion of lamellae, epithelial necrosis, epithelial desquamation, aneurysm, epithelial lifting, hyperplasia, thickening of primary lamellae, and curved lamellae (Fig. 4m–p). Damages in the gill are a chain-like process induced by toxicants, leading to respiratory disease [161]. Histopathological changes in fish gills when exposed to a toxicant lead to toxicity by direct and indirect. Necrosis and degeneration of lamellae were directly affected by toxicants, whereas indirect mode is based on the epithelial lifting, hyperplasia [162]. Alterations, such as epithelial hyperplasia, fusion of lamellae, shortening of lamellae, and aneurysm, can be considered as adaptive responses of fish when exposed to toxicants [163]. In addition, the aneurism in the gills represents the blood-filled and swelling blood vessel may lead to disturbances in blood flow in the gills, increase risk of disagreement and result in severe haemorrhage and bleeding or death [164]. Lamellar fusion, hypertrophy, hyperplasia, and thickening of lamellae are considered as defense responses, widely studied in fish, and can prevent the toxicants from the bloodstream. In the present study, the severity of histo-morphological changes in the gill of common carp was concentration dependent, so that the fish treated with higher concentration of AgNPs showed more intense damages. Most important observed damages were fusion of secondary lamellae, shortening of lamellae, hyperplasia, and aneurism. These histo-morphological differences were defense mechanisms against toxic substances to protect from further damages [108].

Fig. 4
figure 4

Light microscopic structural analysis of gill tissue of freshwater fish C. carpio exposed to AgNPs. (× 40, H & E). a, b, c, and d are control gills of 7th, 14th, 21stst, and 28th days, respectively, showing MC — mucus cells, PL — primary lamellae, SL — secondary lamellae, EC — epithelial cells, PC — pillar cells, and red star mark — central venous sinus. The other photo plates (e-p) showing the alterations of AgNPs treated gill tissue structures such as CF — complete fusion, ED — epithelial desquamation, EN – epithelial necrosis, FL — fusion of lamellae, NSC — naked supporting cartilage, H/HP — hyperplasia, SSL — shortening of secondary lamellae, FCL — fully collapsed lamellae, LI — leukocyte infiltration of epithelium, LBSC — lamellar blood sinus constricts, A — aneurism, SPL — splitting of primary lamellae, DSL — disintegrating secondary lamellae, FC — complete fusion of lamellae, CL — curved lamellae, EL — epithelial lifting, black star mark — thickening of primary lamellae, D — debris, and CCSL — completely collapsed secondary lamellae. Scale bars are 50 μm

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Exposure of fish to nanomaterials contributes to enter into the digestive tract and respiratory organs then distributed to all over the body through blood circulation and deposited eventually in the liver, which is considered as an essential metabolic organ that detoxifies toxic matters [165,166,167,168,169]. Histological examination of the control liver showed the normal hepatocytes with sinusoids and nucleus (Fig. 5a–d). Our results showed that waterborne AgNPs (0.184 mg/l) induced blood congestion, karyolysed hepatocytes, infiltrating erythrocytes in the liver (Fig. 5e–h). Moreover, cloudy degradation of hepatocytes, kupffer cells, karyolysis, necrotic pancreatic tissue, and nuclear alteration were noticed in the liver of 0.276 mg/l AgNPs treated fish (Fig. 5i–l). However, 0.552 mg/l AgNPs treated liver has different structural characters like karyolysis, kupffer cells, dilated sinusoids, necrosis, cytoplasmic vacuolation, and aggregation of melanoacrgophages (Fig. 5m–p). An induced proliferation of endoplasmic membranes may cause alterations in the liver, biotransformation, and metabolism of intracellular enzymes [160, 170, 171]. Among the changes, erythrocyte infiltration is an initial immunological fish defense system because of damages caused by toxicants [172]. Similar alterations were reported by Rodrigues et al. [173] in O. mykiss and Cunha and de Brito-Gitirana [174] in D. rerio.

Fig. 5
figure 5

Light microscopic structural analysis of liver tissue of freshwater fish C. carpio exposed to AgNPs. (× 40, H & E).a, b, c, and d are control gills of 7th, 14th, 21stst and 28th days, respectively, showing H — hepatocytes, N — nucleus, and S — sinusoids. The other photo plates (e–p) showing the alterations in the liver tissue structures such as HC — hepatocyte congestion, CDH — cloudy degradation of hepatocytes, KC — kupffer cells, KL/K — karyolysis, V — cytoplasmic vacuolation, BC — blood congestion, PN — pancreatic nucleus, KH — karyolysed hepatocytes, PNZ — pancreatic necrotic zone, NPT — necrotic pancreatic tissue, CBV — congestion of blood vessels, IE — infiltrating erythrocytes, NA — nuclear alteration, KAC — karyolysed aciner cell, MA — melano-macrophage aggregates, DS — dilated sinusoids, N — necrosis, and EN — enlarged nucleus. Scale bars are 50 μm

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In this study, the standard histological structure of the muscle shows an elegant assembly of bundles of muscle fibres, sarcolemma, nucleus, and connective tissue. The fibres appeared irregular with nuclei as black dots in section. The entire muscle mass was covered in a dense collagenous tissue sheath called epimysium. As the name implies, they also had many cross striations (Fig. 6a–d). Fish exposed to AgNPs (0.184 mg/l) revealed several tissue alterations, namely, degradation of muscle bundle, pyknotic nuclei, and fragmented fibre (Fig. 6e–h). Several alterations in muscle cytoplasm and degeneration of muscle bundle may be related to oxidative stress in the target tissue caused by generated ROS through nanomaterials exposure [175, 176]. However, vacuolar degradation in muscle, necrosis in muscle fibre, bend muscle fibre, necrotic zone, and degeneration endomysium were also observed in 0.279 mg/l AgNPs treated fish (Fig. 6i–l). Similar changes were detected in 0.552 mg/l AgNPs treated fish muscle with increased eosinophilia of the cytoplasm, intracellular space, lesion of striated muscle, shrinkage of muscle fibre, and tissue debris (Fig. 6m–p). Histological changes might have occurred as a toxicity response to the increased accumulation of toxicants in the muscle tissues. Maharajan et al. [177] reported cytoplasmic degeneration, damaged epithelium, hydropic swelling of hepatocytes, blood congestion, nuclear pyknosis, cytoplasmic vacuolation, and nuclear degeneration, accumulation of dark granules, and cellular necrosis were the responses of copper exposure to Asian sea bass (Lates calcarifer).

Fig. 6
figure 6

Light microscopic structural analysis of muscle tissue of freshwater fish C. carpio exposed to AgNPs. (× 40, H & E). a, b, c, and d are control gills of 7th, 14th, 21st and 28thth days, respectively, showing MF — muscle fibre, S — sarcolemma, CT — connective tissue, N — nucleus, MB — muscle bundle, SM — striated muscle. The other photo plates (e–p) showing the alterations in the liver tissue structures such as MFN — muscle fibre necrosis, FF — fragmented fibre, DMB — degradation of muscle bundle, PN — pyknotic nuclei, VDM — vacuolar degradation in muscle, N — necrosis, NMF — necrotic muscle fibre, NZ — necrotic zone, CD — cellular debris, BMF — bend muscle fibre, DMT — degradation of muscle tissue, DMF — degradation of muscle fibre, IS — intracellular space, DE — degenerating endomysium, BMF — bend muscle fibre, ICE & P — increased eosinophilia in the cytoplasm and pyknotic nuclei, SMF — shrinkage of muscle fibre, and LSM — lesion of striated muscle. Scale bars are 50 μm

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Expression of Oxidative Stress Response Genes

It is well established that the induction of oxidative damage is an underlying mechanism of nanomaterials induced toxicity in the biological systems. The release of free radicals changes various signalling pathways causing apoptosis. The present study investigated the changes in the transcript level of genes (Cu–Zn SOD, CAT, GPx1a, GST-α, CYP1A, and Nrf-2) after the exposure of AgNPs, by adopting RTqPCR method. The expression of Cu–Zn SOD was found to be downregulated in fish exposed to AgNPs (Fig. 7a). Cu–Zn SOD is an important antioxidant defense system of all living organisms [178]. AgNPs significantly decreased the Cu–Zn SOD gene expression levels after 28 days of exposure. When the oxidant insult is produced, tissues can counteract and activate the Cu–Zn SOD gene expression more smoothly. Transcript levels of the CAT gene were downregulated in all the treatment conditions of AgNPs exposure (Fig. 7b). In the present study, AgNPs exposure downregulated the Cu–Zn SOD and CAT gene expression, which might be partially disturbed by other signalling factors in fish liver tissue [179].

Fig. 7
figure 7

Effect of AgNPs exposure on expression of genes involved in oxidative stress in the liver of C. carpio after 28 days exposure. a Cu–Zn SOD. b CAT. c GPx1a. d GST-α. e CYP1A. f Nrf-2. Each value represents the mean ± SD; n = 3. Different letters above the bars show a significant difference among the groups at p < 0.05

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The AgNPs exposure shows a similar response between control and higher concentration in GPx1a mRNA transcript level (Fig. 7c). The tissue-specific response might partially explain the expression of GPx1a under oxidative stress conditions. Increased level of GPx1a mRNA in the liver indicates its mechanism for compensating the damages caused by nanomaterials [180]. GPx1a is a unique enzyme which catalyses membrane phospholipid hydroperoxides [181]. Therefore, it is speculated that the liver requires increased GPx1a de novo synthesis to metabolize AgNPs. However, higher level of ROS resulting from the AgNPs exposure inactivates or decreases GPx1a activity.

The transcript levels of GST-α were downregulated in AgNPs exposed fish (Fig. 7d). GST-α plays a crucial role in detoxifying various types of toxicants and protects the DNA damage from ROS-mediated oxidative stress. Lee et al. [146] observed a similar response in C. carpio when exposed to 200 µg/l of AgNPs. GST-α gene expression might be a valuable tool for evaluating oxidative stress [182, 183]. Downregulation of GST-α indicates the failure of the defence system to counteract the increased ROS generation.

The expression of CYP1A was found to be downregulated in fish exposed to AgNPs (Fig. 7e). CYP1A enzyme controls the biotransformation of several toxic substances. Nanomaterial-related elevations of the CYP1A gene may be used as biomarkers for the metal detoxification process. Mostly detoxification is occurred in the microsomal mono-oxygenase enzyme system and dependent on the heme protein, which located in liver and other organelles of fish [184]. According to Oliva et al. [185] report, the activity of CYP1A varies in different metal contaminations.

Various cell-signalling pathways regulate antioxidant enzyme gene transcription mechanism [186]. The Nrf-2 signalling is an important expression factor for antioxidant enzyme genes in higher and lower vertebrates [187, 188]. Overlapping expression of Nrf-2 was observed in fish following exposure to AgNPs (Fig. 7f); transcript levels of Nrf-2 in fish treated with 0.184 and 0.552 mg/l concentration of AgNPs were downregulated, whereas 0.276 mg/l exposure showed enhanced Nrf-2 expression in the liver. Biogenic AgNPs stimulated the H2O2 production, which increased the expression of Nrf-2 mRNA [189]. This phase 2 detoxifying enzyme removes potential materials by converting them to harmless compounds and then eliminated them from the body [190]. There are mismatch alterations between antioxidant enzymes and antioxidant gene expression levels that might be the effect of time-lag after transcription and translation. When the antioxidant mechanism could not reduce or eliminate the excessive production of ROS, it can increase the risk of oxidative stress. These impacts may degrade the enzymes or reduce the activity [191, 192].

Conclusion

Recent reports clearly stated that biogenic nanomaterials are less toxic than chemically prepared nanomaterials. However, the present study demonstrated that biogenic AgNPs showed toxicity to freshwater common carp C. Carpio in a concentration dependent manner. Bioaccumulation analysis showed a maximum accumulation of Ag in the gills compared with other organs, indicating the dose-dependent activity and availability of target tissues. A significant alteration was noticed in haematological parameters during sublethal exposure periods. Phytogenic AgNPs potentially caused oxidative stress by either stimulating or inhibiting the SOD, CAT, GPx, GST, and GSH activities of antioxidant defence systems. Histological observations reveal the damages of C. carpio tissues caused by AgNPs released into the aquatic ecosystems and may pose a risk to aquatic life. The expression levels of the antioxidant defence system of C. carpio were downregulated. It is known that the induced activities of Cu–Zn SOD, CAT, GPx1a, GST-α, CYP1A, and Nrf-2, serve as a protective mechanism to overcome the free radicals. The present work indicated that the toxicity evaluation tools might be considered as potential biomarkers for assessing the health status of fish and the data obtained in this regard provide substantial information to sustain the quality of the aquatic bodies.