# Impact of Green-Synthesized Zinc Oxide Nanoparticles (ZnO NPs) on the Biochemical and Histological Profiles of Cyprinus carpio

Hira Asad, Tanveer Ahmed, Huma Naz, Khalid Abbas, Fariha Latif, Saba Suleman, Rana Zeeshan Habib, Zainab Bashir, Muhammad Tayyeb Rizwan, Rashid Iqbal, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Hira Asad, Tanveer Ahmed, Huma Naz, Khalid Abbas, Fariha Latif, et al. Impact of Green-Synthesized Zinc Oxide Nanoparticles (ZnO NPs) on the Biochemical and Histological Profiles of Cyprinus carpio. Aquaculture Research, 2025, 2025 (1), 10.1155/are/5327177. hal-05189540

HAL Id: hal-05189540

https://imt-mines-ales.hal.science/hal-05189540v1

Submitted on 28 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
WILEY

Research Article

# Impact of Green-Synthesized Zinc Oxide Nanoparticles (ZnO NPs) on the Biochemical and Histological Profiles of *Cyprinus carpio*

Hira Asad<sup>ID,<fnref n="1" /></sup> Tanveer Ahmed<sup>ID,<fnref n="1" /></sup> Huma Naz<sup>ID,<fnref n="2" /></sup> Khalid Abbas<sup>ID,<fnref n="3" /></sup> Fariha Latif,<sup>4</sup> Saba Suleman,<sup><fnref n="1" /></sup> Rana Zeeshan Habib,<sup>5</sup> Zainab Bashir<sup>ID,<fnref n="6" /></sup>,<sup><fnref n="7" /></sup> Muhammad Tayyeb Rizwan,<sup>7</sup> Rashid Iqbal<sup>ID,<fnref n="8" />,<fnref n="9" /></sup> and Ayman A. Swelum<sup>ID <fnref n="10" /></sup>

Correspondence should be addressed to Tanveer Ahmed; tanvirahmeduf@gmail.com and Ayman A. Swelum; aswelum@ksu.edu.sa

Received 20 September 2024; Accepted 7 June 2025

Copyright © 2025 Hira Asad et al. Aquaculture Research published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

This study investigated the impact of varying concentrations, including 0.5 mg L⁻<fnref n="1" /> (T₁), 1 mg L⁻<fnref n="1" /> (T₂), and 2 mg L⁻<fnref n="1" /> (T₃) of zinc oxide nanoparticles (ZnO NPs) on hematological, biochemical, and histological indices of *Cyprinus carpio*. As the use of ZnO NPs grows, there are rising concerns about their potential environmental impacts, particularly on aquatic ecosystems. When released into water bodies, these NPs can be ingested by aquatic organisms, leading to bioaccumulation and potential toxicity. Therefore, understanding their effects on aquatic species, like *C. carpio*, is crucial. Significant alterations in the erythrocyte count, hemoglobin (Hb), and hematocrit (Ht) levels were found. Compared to the control group, the number of lymphocytes, EOSO, monocytes, and platelets in the Zn NPs-exposed groups dramatically increased. The biochemical analysis found considerable changes in blood total protein, albumin, globulin, and glucose levels. Histological examinations of the gills, intestines, and kidneys were conducted to assess structural or pathological changes. Treated fish showed progressive architectural distortions like necrosis (N), hyperplasia (HP), hypertrophy (HT), fusion of gill lamella (FGL), and proliferation of mucocytes (PM) in gills. Distension of the intercellular space (DIS), increased rate of the mucous cell (IMC), villous atrophy (VA), HP, and dysplasia (DP) were found in the intestines of ZnO NPs treated *C. carpio*. Tubular dilatation, tubular necrosis, severe congestion, hematopoietic tissue degradation, constricted lumen, glomerulopathy, and bowmans capsule dilution in kidneys of ZnO NPs treated *C. carpio* were noted. The findings from this study shed light on the biochemical and histological effects of ZnO NPs on *C. carpio*, contributing to a better understanding of the potential risks associated with NP exposure in aquatic environments. The results highlight the need to regulate the use of these NPs as well as their disposal in order to reduce the potential impact that they could have on aquatic ecosystems and public health.

**Keywords:** biochemical; common carp; histological parameters; ZnO NP

<footnote n="1"><sup>1</sup>Department of Life Sciences, Khawaja Fareed University of Engineering and Information Technology, Rahim Yar Khan, Pakistan</footnote>

<footnote n="2"><sup>2</sup>Department of Zoology, Cholistan Institute of Biological Sciences, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan</footnote>

<footnote n="3"><sup>3</sup>Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad, Pakistan</footnote>

<footnote n="4"><sup>4</sup>Institute of Zoology, Bahauddin Zakariya University, Multan, Pakistan</footnote>

<footnote n="5"><sup>5</sup>Polymères Composites Hybrides (PCH - IMT Mines Alès), IMT-MINES Ales, France</footnote>

<footnote n="6"><sup>6</sup>Department of Physics, University of Agriculture, Faisalabad, Pakistan</footnote>

<footnote n="7"><sup>7</sup>Government of Punjab Department of Fisheries, Lahore, Punjab, Pakistan</footnote>

<footnote n="8"><sup>8</sup>Department of Agronomy, Faculty of Agriculture and Environment, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan</footnote>

<footnote n="9"><sup>9</sup>Department of Life Sciences, Western Caspian University, Baku, Azerbaijan</footnote>

<footnote n="10"><sup>10</sup>Department of Animal Production, College of Food and Agriculture Sciences, King Saud University, Riyadh 11451, Saudi Arabia</footnote>
# 1. Introduction

One of the most popular food commodities traded globally is fish and fish products. The world's fastest-expanding industry for producing food is aquaculture. Global production of aquaculture has expanded dramatically in the last few decades. This is mostly due to several advancements that have increased production process control and competitiveness [1].

Common carp (*Cyprinus carpio*) belongs to the order Cypriniformes and the family Cyprinidae, which is regarded as the biggest family of freshwater fish. It rarely lives in brackish water and usually occupies freshwater habitats, including water bodies such as lakes, ponds, and rivers. Although it is extensively available in practically every nation on earth, Asia and several European nations are the biggest markets for it. The third most commonly introduced species globally is the *C. carpio*. Because of its exceptional capacity for environmental and dietary adaptation, it is being explored as a possible commercial aquaculture candidate in Asia and certain European nations [2].

Nanotechnology is the study and manipulation of materials at dimensions of one to 100 nm, where unique phenomena enable novel applications [3]. It is a new and creative approach that has created new avenues for research on biological molecules, specifically delivering drugs, the manufacturing of proteins or cells, medical diagnosis, the development of nonviral gene treatment vectors, DNA transport, disease treatments, etc. [4]. Nanoparticles (NPs) have always been present, although their exposure increased significantly during the heyday of the Industrial Revolution. At the beginning of the 21st century, interest in the emerging fields of nanoscience and nanotechnology increased. Nanotechnology has a wide range of applications in aquaculture and can significantly alter this industry [5]. Detection and management of infections, treatment of water, pond cleaning, efficient administration of nutrients, and administration of medications are some of its current applications [6].

Zinc (Zn) is one of the most important minerals for all creatures, including fish. It is essential for the synthesis of transcription factors and proteins, which interact with Zn and are thought to be for certain tasks. Zn plays a pivotal role in several metabolic processes and is a particular cofactor for a number of enzymes. Nano Zn oxide (ZnO) may be substantially more hazardous than other particles made of metal oxide and may pose serious dangers to aquatic environments and organisms [7]. After exposure, NPs might aggregate and spread throughout the bodily tissues of fish, causing oxidative damage and histopathological alterations. Hematological and histological indicators, peroxidation of lipids as an indicator of damage from oxidative stress, and carp health are all adversely affected by feeding ZnO NPs [8, 9].

ZnO NPs have both beneficial and harmful effects on carp. Hematological analysis showed that fish exposed to ZnO NPs had dose-dependent decreases in hemoglobin (Hb), Hct, and number of red blood cells [10]. The mean corpuscular volumes (MCVs), mean corpuscular hemoglobin (MCH) levels, and white blood cell counts were all greater in fish treated with greater concentrations of ZnO NPs. Biochemical analysis

revealed that common carp exposed to ZnO NPs had significantly greater levels of sugar levels, overall protein, albumin, globulin, lipids, triglycerides, cholesterol, and creatinine. Rasheed et al. [11] detected alterations in blood biochemistry and impacts on the inner structure of the kidneys, intestine, and gills after 12–14 days of exposing the fish to ZnO NPs. Additionally, NPs were found in the kidney's glomerulus, red blood cells, and the outermost layer of all tissues. The detected alterations in serum and tissues may offer helpful data on environmental conditions and aquatic creature risk evaluations. When exposed to ZnO NPs, the target tissues may be the liver and gills. Recent studies reveal that ZnO NPs can harm freshwater fish when exposure times are too long or NP concentrations are too high.

An alternate and promising method for creating safer and more ecologically friendly NPs is the green synthesis of NPs utilizing plant extracts [12]. Plant extracts contain a variety of biomolecules that can serve as stabilizing and reducing agents in the green synthesis of NPs, including proteins, polysaccharides, amino acids, organic acids, and vitamins, as well as phytochemicals like polyphenols, flavonoids, terpenoids, alkaloids, tannins, and alcoholic compounds [13]. Green-synthesized ZnO NPs had less toxicological effects than chemically synthesized ZnO NPs on the activities of catalase, lipid peroxidation, glutathione S-transferase, and histopathology [14].

The objective of this study was to evaluate the efficiency of NPs made of ZnO as a tool for monitoring hematological and biochemical changes in *C. carpio*. Similarly, examining the potential histological alterations caused by NP exposure and establishing a link between NP concentrations and observed effects was also the objective of this study.

# 2. Materials and Methods

## 2.1. Fish Sampling and Acclimatization.
Experimental fish (*C. carpio*) fingerlings (10.5–15 cm in length and 6.5–10.5 g in weight) were collected for this study from the local public fish hatchery in Fazilpur, Punjab, Pakistan. Live fish samples were transferred to the Fisheries Research Lab, Department of Life Sciences, Khwaja Fareed University of Engineering and Information Technology, Rahim Yar Khan, Pakistan. Fish samples were acclimatized at laboratory conditions by keeping them in glass aquaria for 14 days. The fish were maintained on a regular photoperiod (12 h of light and 12 h of darkness) and fed commercial fish feed twice a day during the acclimation period.

## 2.2. Synthesis of ZnO NPs.
ZnO NPs were synthesized by following the methods of Rasheed et al. [11]. A 30 g of *Citrus limon* dried leaves in powder form were taken by adding 500 mL of double distilled water. To avoid much increase in temperature only 40–45°C was managed on a heating plate after sometimes extract was filtered, prepared, and preserved for further experimental work. A 30 mL extract of *C. limon* was added to a conical flask. Place on the hotplate and start stirring. During stirring, add NaOH solution dropwise until pH reaches to 13. After that, added 1 mL solution of 3 g zinc nitrate hexahydrate solution. After sometimes color change from dark brown to pale yellow confirmed the synthesis of colloidal
ZnO NPs. Stirring was continued to get the maximum yield of particles. The colloidal suspension of ZnO NPs was centrifuged at 4000 rpm. NPs were washed with 50%, 70%, and 90% absolute alcohol. Solution of dry powder ZnO NPs have prepared with a mass of 1.904 g in distilled water.

2.3. *Characterization of ZnO NPs.* The UV-visible (UV-Vis) absorption spectrum of the ZnO NPs was recorded in the wavelength range of 300–700 nm using XRD (Powder X-ray diffractometer Equinox 2000, Thermo Fisher Scientific, USA). FTIR of the synthesized ZnO NPs was performed (IR-Prestige-21, Shimadzu, Japan) in order to identify the different phytochemicals involved in the synthesis and stabilization of the NPs. The ZnO NPs' size, shape, and morphology were evaluated using SEM (SEM Inspect S-50, Thermo Fisher Scientific, USA). Energy-dispersive X-ray (EDX) analysis was used to identify the elemental components of the biosynthesized NPs.

2.4. *Experimental Layout.* A total of 40 fish samples of *C. carpio* were shifted in four glass aquaria (10 fish fingerlings in each aquarium) after acclimatization. One group was control and three are experimental groups. Each aquarium contains 40 L of water with 2 x 1.5 x 1.25 length, height, and width.

2.5. *NP Exposure in Aquariums.* In the experimental group, three separate aquariums contain NP solutions with varying concentrations: 0.5 mg L⁻¹ (*T*₁), 1 mg L⁻¹ (*T*₂), and 2.0 mg L⁻¹ (*T*₃). In contrast, the control group's aquariums do not incorporate any NP solutions. During the 14 days trial, the fish in both the experimental and control groups were not subjected to any water changes. This standardized approach ensures that the only variable being tested is the presence and concentration of NPs in the aquariums, allowing for a more accurate assessment of their impact on the hematological, biochemical and histological changes.

2.6. *Blood Sampling.* Blood was drawn for hematological analysis in vials coated with EDTA from the caudal vein, immediately behind the caudal fins, using aseptic syringes. Additionally, blood is gathered in the gel. For separating serum for biochemical analysis, use clot yellow vacuum tubes. After 35 min of incubation, the serum separated on the upper side of the Gel Clot vacutainers and was easily transferred to an Eppendorf via a micropipette. The Eppendorf was then promptly kept at 5°C in the refrigerator for future processing.

2.7. *Extraction of Organs.* For the examination of histological changes, fish were given anesthesia with clove oil (60 mg L⁻¹) before being dissected. Gills, intestine, and kidneys were removed and stored in formalin solution.

2.8. *Histological Analysis.* The paraffin-embedding procedure was used to examine samples of kidney, liver, and gills under a light microscope by using the method of Bancroft et al. [15].

2.9. *Histomorphometry of Selected Organs.* All the selected organs were subjected to histomorphometry using prepared slides. Numerous histomorphometry measures, including shortening of the secondary lamella, epithelial lifting, vascular congestion, and nuclear fusion were investigated using methods of Bancroft et al. [15].

2.10. *Statistical Analysis.* The normality assumption of the data was verified by using Shapiro-Wilk test. Hematological and biochemical indices mean ± SE were calculated for both group of fishes. A one-way ANOVA was applied to measure the statistical difference at *p* ≤ 0.05 between the groups [16] using the software Statistix 8.1.

## 3. Results

### 3.1. Characterization of ZnO NPs

3.1.1. *UV-Vis Spectra.* The UV-Vis absorption spectrum of the ZnO NPs is recorded in the wavelength range of 300–700 nm using XRD (Powder X-ray diffractometer Equinox 2000, Thermo Fisher Scientific, USA). The spectrum has a maximum absorbance peak at 374 nm which indicates the synthesis of ZnO NPs (Figure 1).

3.1.2. *FTIR.* FTIR of the synthesized ZnO NPs was performed in order to identify the different phytochemicals involved in the synthesis and stabilization of the NPs. The characteristic peaks in the region between 400 and 600 cm⁻¹ have been assigned to metal-oxygen. The FTIR spectrum of synthesized ZnO NPs indicates characteristic peaks of ZnO at 457 and 550 cm⁻¹ that were assigned to ZnO stretching vibration. The broad peak at 3505 cm⁻¹ is attributed to the O–H stretching vibration of hydroxyl group in phenolic and alcohol compounds. The peak at 3332 cm⁻¹ indicates the N–H stretching vibration in the amine. The small peak at 2980 cm⁻¹ belongs to C–H symmetric stretching of alkanes. The narrow peak at 2308 cm⁻¹ is attributed to C≡N group. The peak at 1638 cm⁻¹ corresponds to C=C stretching in the aromatic ring in polyphenols. The band at 1120 cm⁻¹ is attributed to C–O stretching vibration (phenolics, carbohydrate ring, flavonoids). The narrow peak at 1015 cm⁻¹ is assigned to the C–N stretches in amino acids. The peak at 905 cm⁻¹ and 720 cm⁻¹ is attributed to nitrate and C–H out-of-plane bending of the aromatic ring. The FTIR results confirm the formation of ZnO NPs and various phytochemicals (polyphenols, flavonoids, alkaloids, and pectin) present in the aqueous plant extract are involved in both the formation and stabilization of synthesized ZnO NPs (Figure 2).

3.1.3. *SEM Analysis.* The ZnO NPs' size, shape, and morphology were evaluated using SEM. The ZnO NPs had a nearly spherical appearance and exhibited agglomeration (Figure 3).

3.1.4. *EDX Analysis.* EDX was used to identify the elemental components of the biosynthesized NPs. The ZnO elemental analysis shows that the sample contains both Zn and O, indicating the purity of the synthesis. Zn and O have weight % values of 74.34% and 25.66%, respectively, and atomic % values of 41.48% and 58,52%, respectively (Figure 4).

3.2. *Changes in Hematological Parameters.* Hematological parameters include Hb concentration, hematocrit (Ht), erythrocyte count (RBC), MCV, mean corpuscular hemoglobin concentration (MCHC), and MCH. By using a microscope to analyze blood smears, more details about the red blood cell system can be discovered. For example, the percentage of immature red blood cells, or erythroblasts, indicates
<figure><img src="image_3.png" /><figcaption>FIGURE 1: UV-Vis spectra of synthesized ZnO NPs.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>FIGURE 2: FTIR spectrum of synthesized ZnO NPs.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>FIGURE 3: SEM images of ZnO NPs.</figcaption></figure>

erythropoietic activity, and the percentage of different red blood cell anomalies, such as cellular and nuclear deformities, indicates cytotoxicity and genotoxicity. The noticed alterations brought on by toxicity may differ and indicate a rise or fall in the levels of all or some RBC's parameters (Table 1).

### 3.3. *Changes in Blood Biochemical Parameters.*
Considerable changes in blood total protein, albumin, globulin, and the level of glucose were found in the biochemical analysis (Figure 5).

### 3.4. Histological Studies

3.4.1. *Histology of Gills.* The gills of the *C. carpio* consisted of mucous cells, filament, pillar cells, epithelial cells, secondary lamellae, and water cannulas. The circulatory system, stratified epithelium, and cartilaginous skeleton made up the primary gill lamellae. In this study, treated group shows progressive architectural distortions like necrosis (N), hyperplasia (HP), hypertrophy (HT), fusion of gill lamella (FGL), and proliferation of mucocytes (PM) as shown in Table 2 and Figure 6.

3.4.2. *Histology of Intestine.* The histopathological alterations in the intestinal tissue of *C. carpio* exposed to varying
<figure><img src="image_6.png" /><figcaption>FIGURE 4: EDX spectra of ZnO NPs.</figcaption></figure>

<table><caption>TABLE 1: Response of hematological indices of *C. carpio* kept under different concentrations of ZnO NPs.</caption><thead><tr><th>Parameters</th><th>Control</th><th>T<sub>1</sub></th><th>T<sub>2</sub></th><th>T<sub>3</sub></th></tr></thead><tbody><tr><td>Hemoglobin (g/dL)</td><td>29.44 ± 0.08<sup>A</sup></td><td>29.68 ± 0.09<sup>A</sup></td><td>26.66 ± 0.1<sup>B</sup></td><td>24.69 ± 0.06<sup>C</sup></td></tr><tr><td>Erythrocyte (10<sup>6</sup>/mm<sup>3</sup>)</td><td>1.94 ± 0.01<sup>A</sup></td><td>1.95 ± 0.01<sup>A</sup></td><td>1.76 ± 0.01<sup>B</sup></td><td>1.39 ± 0.01<sup>C</sup></td></tr><tr><td>Hematocrit (%)</td><td>25.63 ± 0.13<sup>A</sup></td><td>25.64 ± 0.12<sup>A</sup></td><td>23.5 ± 0.07<sup>B</sup></td><td>20.73 ± 0.17<sup>C</sup></td></tr><tr><td>Mean corpuscular volume (fL)</td><td>158.57 ± 0.27<sup>C</sup></td><td>158.52 ± 0.29<sup>C</sup></td><td>165.81 ± 0.27<sup>B</sup></td><td>178.82 ± 0.21<sup>A</sup></td></tr><tr><td>MCH</td><td>35.04 ± 0.18<sup>A</sup></td><td>35.09 ± 0.07<sup>A</sup></td><td>30.48 ± 0.11<sup>B</sup></td><td>26.55 ± 0.11<sup>C</sup></td></tr><tr><td>Mean corpuscular hemoglobin concentration (g/dL)</td><td>230.19 ± 0.19<sup>A</sup></td><td>229.86 ± 0.19<sup>A</sup></td><td>226.23 ± 0.21<sup>B</sup></td><td>220.12 ± 0.12<sup>C</sup></td></tr><tr><td>Platelets</td><td>12.46 ± 0.09<sup>C</sup></td><td>12.54 ± 0.09<sup>C</sup></td><td>15.67 ± 0.18<sup>B</sup></td><td>21.36 ± 0.07<sup>A</sup></td></tr><tr><td>Leukocyte counts (10<sup>3</sup>/mm<sup>3</sup>)</td><td>43.12 ± 0.19<sup>A</sup></td><td>42.97 ± 0.22<sup>A</sup></td><td>39.48 ± 0.12<sup>B</sup></td><td>36.43 ± 0.06<sup>C</sup></td></tr><tr><td>Neutrophil (%)</td><td>6.58 ± 0.1<sup>C</sup></td><td>6.41 ± 0.08<sup>C</sup></td><td>8.65 ± 0.09<sup>B</sup></td><td>10.57 ± 0.09<sup>A</sup></td></tr><tr><td>Lymphocyte (%)</td><td>73.86 ± 0.13<sup>C</sup></td><td>73.91 ± 0.13<sup>C</sup></td><td>80.15 ± 0.2<sup>B</sup></td><td>82.9 ± 0.18<sup>A</sup></td></tr><tr><td>EOS</td><td>0.49 ± 0.02<sup>C</sup></td><td>0.51 ± 0.02<sup>C</sup></td><td>2.41 ± 0.01<sup>B</sup></td><td>3.76 ± 0.01<sup>A</sup></td></tr><tr><td>Monocyte (%)</td><td>7.37 ± 0.02<sup>C</sup></td><td>7.33 ± 0.02<sup>C</sup></td><td>13.24 ± 0.04<sup>B</sup></td><td>15.75 ± 0.05<sup>A</sup></td></tr></tbody></table>

Note: Significant differences at *p*<0.05. Superscript letters means followed by the same letter, within a row, are not significantly different (*p*<0.05). Abbreviations: EOS, eosinophils; MCH, mean corpuscular hemoglobin.

concentrations of ZnO NPs and fish under control are noted. The findings revealed that the most typical alterations of the intestine are distension of the intercellular space (DIS), increased rate of mucous cell (IMC), villous atrophy (VA), HP, and dysplasia (DP) as shown in the Table 3 and Figure 7.

### 3.4.3. Histology of Kidneys.
The kidneys of fish are engaged in several excretory processes, such as homeostasis, excretion, and osmoregulation. Histological analysis of the kidneys exposed to ZnO NPs showed modest histopathological alterations. When treated with different concentrations, tubular dilatation, tubular necrosis, severe congestion, hematopoietic tissue degradation, constricted lumen, glomerulopathy, and bowmans capsule dilution (Table 4; Figure 8).

## 4. Discussion

This study investigated the impact of varying concentrations including of ZnO NPs on hematological, biochemical, and histological indices of *C. carpio*. Nanomaterials, such as ZnO NPs, are increasingly being utilized in various fields, including

medicine, agriculture, and environmental management. NPs have always been present, although their exposure increased significantly during the heyday of the industrial revolution. NPs have a wide range of applications in aquaculture including detection and management of infections, treatment of water, pond cleaning, efficient administration of nutrients, and administration of medications. ZnO NPs have both beneficial and harmful effects on carp. Zn plays a pivotal role in several metabolic processes and is a particular cofactor for a number of enzymes. Nano ZnO may be substantially more hazardous than other particles made of metal oxide and may pose serious dangers to aquatic environments and organisms [7]. After exposure, NPs might aggregate and spread throughout the bodily tissues of fish, causing oxidative damage, and histopathological alterations. Hematological and histological indicators, peroxidation of lipids as an indicator of damage from oxidative stress, and carp health are all adversely affected by feeding ZnO NPs [8, 9]. The inferences of this study have shown that ZnO NPs can alter the hematology and biochemical parameters of *C. carpio*. NPs' toxicity level is dependent on several variables,
<figure><img src="image_7.png" /><figcaption>FIGURE 5: Response of biochemical Indices of *C. carpio* kept under different concentrations of ZnO NPs.</figcaption></figure>

<table><caption>TABLE 2: Histological changes of gills when treated with different concentrations of ZnO NPs.</caption><thead><tr><th>Parameters</th><th>Control</th><th>T<sub>1</sub></th><th>T<sub>2</sub></th><th>T<sub>3</sub></th></tr></thead><tbody><tr><td>Necrosis (N)</td><td>—</td><td>+</td><td>++</td><td>+++</td></tr><tr><td>Hyperplasia (HP)</td><td>—</td><td>+</td><td>+</td><td>++</td></tr><tr><td>Hypertrophy (HT)</td><td>—</td><td>+</td><td>++</td><td>+++</td></tr><tr><td>Fusing of gill lamellae (FGL)</td><td>—</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Proliferation of mucocytes (PM)</td><td>—</td><td>+</td><td>+</td><td>++</td></tr></tbody></table>

Note: (–) Not detected, (+) Minor, (++) Moderate, (+++) Major.

such as their size and shape, length of exposure, concentration, accumulation, area of surface, and charge. Aquatic creatures exposed to NPs develop a variety of harmful features, such as causing cytotoxicity, genotoxicity and epigeneticity [17]. ZnO NPs have caused considerable concern to researchers because of their effects on water as well as on aquatic organisms [18, 19].

**4.1. Hematological Indices.** Hematological indices such as the count of red blood cells (RBCs), the concentration of Hb, and the levels of Ht have been shown to fluctuate. Exposure to ZnO NPs has been associated with decrease Hb concentration, Hct, and number of red blood cells, all of which may point to an erythropoiesis activation and stress due to lack of food. This could be a means of protection against the negative effects of oxidative stress on the NPs. Fish exposed to ZnO NPs for longer periods of time or at higher concentrations may exhibit hematological system damage shown by a decrease in RBC count, Hb, and Hct levels. Our results show an increase in

the number of leukocytes and monocytes in groups with 1 and 2 mg L⁻¹ ZnO NPs is due to phagocytosis. Moreover, alterations in parameters like HCT, MCV, MCH, and MCHC suggest changes in the size and Hb content of RBCs. Additionally, disturbances in biochemical parameters such as alterations in serum enzymes, electrolyte imbalance, and oxidative stress markers might indicate the impact of ZnO NPs on the physiological balance of *C. carpio*. These findings emphasize the potential hematological and biochemical implications of exposure to ZnO NPs in this fish species. Changes in total protein, albumin, and levels of glucose have been observed, suggesting disturbances in glucose homeostasis and protein metabolism. The release of Zn ions from ZnO NPs may potentially be responsible for the negative effects seen since an excess of Zn can disrupt a biological system's ability to function normally. According to recent findings fish that consume ZnO NPs may experience hematological and metabolic abnormalities as a result of the oxidative stress, cellular disruption, and inflammatory reactions they induce [11]. Similar outcomes were reported for hematological indicators in *Heteroclarias* by Kori-Siakpere and Ubagu [20] when fish were treated with different concentrations of ZnO NPs. After receiving injections through the peritoneum or intravenous delivery of ZnO NPs, *C. carpio* exhibited a significant decrease in RBCs, Ht, and Hb levels in comparison to the control group. As erythropoietin tissue is subjected to stress from toxicants, a reduced HCT fixation also demonstrates cell shrinkage [21]. The levels of RBCs, Hb, and HCT had dramatically changed in *Oreochromis niloticus* [22]. In fish treated with toxicants, the levels of Hb are reduced because toxicants may have an inhibitory impact on the catalyst's profile, which produces the
<figure><img src="image_8.png" /><figcaption>FIGURE 6: Microscopic (400× magnification) examination in gills of *C. carpio* exposed to different concentration of ZnO NPs showed the proliferation of mucocytes (PM), necrosis (N), fusion of gills lamella (FGL) and hyperplasia (HP), hypertrophy (HT).</figcaption></figure>

<table><caption>TABLE 3: Histological changes of intestine when treated with different concentrations of ZnO NPs.</caption><thead><tr><th>Intestine</th><th>Control</th><th>T<sub>1</sub></th><th>T<sub>2</sub></th><th>T<sub>3</sub></th></tr></thead><tbody><tr><td>Distension of the intercellular space (DIS)</td><td>—</td><td>+</td><td>++</td><td>+++</td></tr><tr><td>Increased rate of mucous cell (IMC)</td><td>—</td><td>+</td><td>+</td><td>++</td></tr><tr><td>Villous atrophy (VA)</td><td>—</td><td>+</td><td>++</td><td>+++</td></tr><tr><td>Hyperplasia: (HP)</td><td>—</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Dysplasia (DP)</td><td>—</td><td>+</td><td>+</td><td>++</td></tr></tbody></table>

Note: (–) Not detected, (+) Minor, (++) Moderate, (+++) Major.

mixture of Hb [23]. The lower levels of HCT and Hb due to the structural bending of RBCs under the influence of the metal were reported by Venkatachalam and Natarajan [24] in *Labeo rohita*. According to the study conducted by Rajput et al. [25], it was disclosed that the gill and liver histology and erythrocyte count have been changed in *O. niloticus* samples treated with ZnO NPs. In the experiment performed by Khan et al. [26], *O. mossambicus* was treated with ZnO NPs and reported that biomarkers most often affected include the RBCs, WBCs, MVCs, MCHs, HB, platelets, and neutrophils.

### 4.2. Biochemical Parameters.
Considerable changes (decrease) in blood total protein, albumin, globulin, and the level of glucose were found in the biochemical analysis in this study of fish kept in ZnO NPs compared to the control group of fish. Decreased levels may indicate liver dysfunction or protein synthesis impairment. Glucose level fall in this study may be due to stress, indicating an activation of the fish's stress response. Total serum protein, albumin, and globulin are markers of

good health and resilience to illness and stress because of their roles in immunological response [27]. Levels of albumin and globulin in fishes are influenced by both intrinsic and extrinsic factors, including species, size, gender, maturity, habitat, nutrition, and weather. The defense and clotting systems were linked to the controlled proteins and pathways, which may have been a reaction to the fish administered with NPs made of ZnO experiencing a disruption in homeostasis [9]. Rashidian et al. [28] observed reduced levels of total protein in *C. carpio* after exposure to ZnO NPs. Similarly, in *O. niloticus* under subchronic exposure, the ZnO NPs results showed alterations in the serum biochemistry and histology [29].

### 4.3. Histological Alteration.
The impacts of ZnO NPs on *C. carpio* indicate changes in the intestine, kidney, and gills when treated with varying concentrations. At higher concentrations (*T*<sub>3</sub>) in gills, these NPs exhibited hemorrhage and cup shape of secondary gill filament. Gills, being crucial for respiration, show progressive architectural distortions like N, HP, HT, FGL, PM due to exposure to ZnO NPs, particularly at higher concentrations (*T*<sub>3</sub>). This occurred because ZnO NPs demonstrated that the hypertrophy pavement epithelial cells on the gill lamella and the chloride had collapsed of their apical region structure. When the concentrations were higher, these symptoms were worse. Histological changes such as HP of epithelial cells, epithelial lifting, and lamellar fusion can increase the area in which toxins come into touch with the gill vascular system, impairing the fish's ability to breathe and their overall health [30]. Hao et al. [31] selected juveniles of *C. carpio* in their study and found that ZnO NPs exhibit greater cellular reactivity as well as bioaccumulation. Due to the special characteristics of gills, such as their vast surface area, distinct histological structure, and direct interaction with the aquatic environment, as
<figure><img src="image_9.png" /><figcaption>FIGURE 7: Microscopic examination in intestine (400× magnification) of *C. carpio* exposed to different concentrations of ZnO NPs showing distension of the intercellular space (DIS), increased rate of mucous cell (IMC), villous atrophy (VA), hyperplasia: (HP), dysplasia (DP).</figcaption></figure>

<table><caption>TABLE 4: Histological changes of kidneys when treated with different concentrations of ZnO NPs.</caption><thead><tr><th>Kidney</th><th>Control</th><th>T<sub>1</sub></th><th>T<sub>2</sub></th><th>T<sub>3</sub></th></tr></thead><tbody><tr><td>Tubular dilatation</td><td>—</td><td>+</td><td>++</td><td>+++</td></tr><tr><td>Tubular necrosis</td><td>—</td><td>+</td><td>+</td><td>++</td></tr><tr><td>Severe congestion</td><td>—</td><td>+</td><td>++</td><td>+++</td></tr><tr><td>Hematopoietic tissue degradation</td><td>—</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Constricted lumen</td><td>—</td><td>+</td><td>+</td><td>++</td></tr><tr><td>Glomerulopathy</td><td>—</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Bowmans capsule dilution</td><td>—</td><td>+</td><td>+</td><td>+</td></tr></tbody></table>

Note: (–) Not detected, (+) Minor, (++) Moderate, (+++) Major.

well as being crucial to the maintenance of the acid–base balance and osmoregulation; for these reasons, it is regarded as the primary target organ for aquatic toxicants. Similar findings were reported by Sukhsangchan et al. [32] in Asian Seabass (*Lates calcarifer*). There are currently very few studies that describe the histological alterations that NPs generate in fish gills [33]. Similarly, Khan et al. [26] found disrupted gill lamellae, the cartilaginous core failure brought structural changes, the secondary gill lamellae reduced size, blood clots formed in the gill tissues of *O. mossambicus* treated with ZnO NPs.

Our results revealed significant alterations in the histological structure of the intestine, leading to alterations of the intestine are DIS, IMC, VA, HP, and DP. Wang et al. [34] found similar results about increased villus width, length, and surface area of the intestine. Similarly, in the kidney, ZnO NP exposure might cause nephrotoxicity and affect renal functions of the *C. carpio* when treated with ZnO NPs in this study. Our observations indicated that the liver and kidneys may be the most sensitive to ZnO NP exposure through the gastrointestinal route Zn buildup was not observed in the examined internal structures of treated fish after prolonged exposure to ZnO NPs.

After a period of recuperation, exposure appears to impact liver function by influencing alterations in liver enzyme activity, while it also causes minor histological changes in the kidney. The lysosome's black specks of varying sizes, the nucleus membrane, and the nucleus's shape were all altered by changes to the liver's ultrastructure. There were some slight changes in the size and number of lysosomes in the kidney's blood arteries. Furthermore, RBCs, the glomerulus of the kidney, and the cytoplasm of every tissue were discovered to contain NPs. The identified changes in serum and tissues could provide beneficial information on environmental parameters and aquatic animal risk assessments [10]. Compared with inferences from previous studies, our findings summarized that exposure of aquatic animals to ZnO NPs may induce histological changes in crucial organs, such as the hepatic, renal, and intestine; suppress biochemical parameters in the serum; and impair hematological components [35–37]. Different concentrations of ZnO NPs negatively affected the growth of Asian seabass and reduced basic immunological defense as compared to the control group [32].

The results of this finding may be used to inform regulatory choices about the use of NPs made from ZnO in several sectors, that frequently employ these NPs, such as the textile, food packaging, and cosmetics industries.

## 5. Conclusion

In conclusion, this comprehensive study illuminates the effects of ZnO NPs on the hematological, biochemical, and histological parameters of *C. carpio*. The results emphasize how crucial it is to control both the use and disposal of these NPs to lessen the potential harm they could do to aquatic ecosystems. The findings underscore the necessity for further exploration and monitoring of NP-induced toxicity in aquatic habitats, highlighting the significance of understanding and mitigating
<figure><img src="image_10.png" /><figcaption>FIGURE 8: Microscopic examination in kidneys (400× magnification) of *C. carpio* exposed to different concentrations of ZnO NPs showing tubular dilatation (TD), tubular necrosis (TN), severe congestion (SC), hematopoietic tissue degradation (HTD), constricted lumen (CL), glomerulopathy, Bowman's capsule dilution (BCD).</figcaption></figure>

the potential ecological implications of NP exposure on aquatic organisms. If we are to fully comprehend the prolonged effects that ZnO NPs have on fish health and ecology, more research is necessary.

**Data Availability Statement**

The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Ethics Statement**

This study was performed by following institutional guidelines and has been approved by the appropriate institutional bioeth- ical committee.

**Conflicts of Interest**

The authors declare no conflicts of interest.

**Author Contributions**

Tanveer Ahmed conceptualized the study. Hira Asad conducted the study. Muhammad Tayyeb Rizwan assisted in fish sampling. Saba Suleman assisted during the trial and data collection. Zai- nab Bashir assisted in the formulation of ZnO NP. Huma Naz and Fariha Latif analyzed the histology results. Hira Asad and Tanveer Ahmed wrote the article. Khalid Abbas, Rana Zeeshan Habib, Rashid Iqbal, and Ayman A. Swelum analyzed the data and technically reviewed the article.

**Funding**

The authors extend their appreciation to the Ongoing Research Funding Program (ORF-2025-971), King Saud University, Riyadh, Saudi Arabia, for funding this research.

**Acknowledgments**

The authors extend their appreciation to the Ongoing Research Funding Program (ORF-2025-971), King Saud University, Riyadh, Saudi Arabia, for funding this research. ChatGPT and Grammarly were used in the preparation of some parts (a few lines of Introduction and Discussion) of this manuscript.

**References**

[1] S. Afewerki, F. Asche, B. Misund, T. Thorvaldsen, and R. Tveteras, "Innovation in the Norwegian Aquaculture Industry," *Reviews in Aquaculture* 15, no. 2 (2023): 759-771.

[2] M. Ottinger, K. Clauss, and C. Kuenzer, "Aquaculture: Relevance, Distribution, Impacts and Spatial Assessments—A Review," *Ocean & Coastal Management* 119 (2016): 244-266.

[3] B. Bhushan, "Introduction to Nanotechnology," in *Springer Handbook of Nanotechnology*, (Springer, 2017): 1-19.

[4] J. E. Hulla, S. C. Sahu, and A. W. Hayes, "Nanotechnology: History and Future," *Human & Experimental Toxicology* 34, no. 12 (2015): 1318-1321.

[5] C. Fajardo, G. Martinez-Rodriguez, J. Blasco, J. M. Mancera, B. Thomas, and M. De Donato, "Nanotechnology in Aquaculture: Applications, Perspectives and Regulatory Challenges," *Aquaculture and Fisheries* 7, no. 2 (2022): 185-200.

[6] B. J. Shaw and R. D. Handy, "Physiological Effects of Nanoparticles on Fish: A Comparison of Nanometals Versus Metal Ions," *Environment International* 37, no. 6 (2011): 1083-1097.

[7] R. E. Ibrahim, G. E. Elshopakey, A. A. Abdelwarith, et al., "Chitosan Neem Nanocapsule Enhances Immunity and Disease Resistance in Nile Tilapia (*Oreochromis niloticus*)," *Heliyon* 9, no. 9 (2023): e19354.

[8] M. M. Rahman, "Role of Common Carp (*Cyprinus carpio*) in Aquaculture Production Systems," *Frontiers in Life Science* 8, no. 4 (2015): 399-410.

[9] L. Chupani, H. Niksirat, J. Velíšek, et al., "Chronic Dietary Toxicity of Zinc Oxide Nanoparticles in Common Carp
(Cyprinus carpio L.): Tissue Accumulation and Physiological Responses," *Ecotoxicology and Environmental Safety* 147 (2018): 110–116.

[10] J.-W. Lee, J.-E. Kim, Y.-J. Shin, et al., "Serum and Ultrastructure Responses of Common Carp (*Cyprinus carpio* L.) During Long-Term Exposure to Zinc Oxide Nanoparticles," *Ecotoxicology and Environmental Safety* 104 (2014): 9–17.

[11] A. Rasheed, K. J. Iqbal, A. Safdar, et al., "Toxicological Effects of Zinc Oxide Nanoparticles on Hemato-Biochemical Profile of Common Carp (*Cyprinus carpio*)," *Journal of King Saud University - Science* 35, no. 7 (2023): 102835.

[12] R. G. Saratale, G. D. Saratale, H. S. Shin, et al., "New Insights on the Green Synthesis of Metallic Nanoparticles Using Plant and Waste Biomaterials: Current Knowledge, Their Agricultural and Environmental Applications," *Environmental Science and Pollution Research International* 25, no. 11 (2018): 10164–10183.

[13] S. Iravani, "Green Synthesis of Metal Nanoparticles Using Plants," *Green Chemistry* 13, no. 10 (2011): 2638–2650.

[14] S. Rasool, M. Faheem, U. Hanif, et al., "Toxicological Effects of the Chemical and Green ZnO NPs on, *Cyprinus carpio*, L. Observed Under Light and Scanning Electron Microscopy," *Microscopy Research and Techniques* 85, no. 3 (2022): 848–860.

[15] J. D. Bancroft, A. D. Floyd, and S. K. Suvarna, *Bancroft's Theory and Practice of Histological Techniques* (Elsevier Health Science, New York (NY) USA, 7th edition, 2013).

[16] R. G. D. Steel, J. H. Torrie, and D. A. Dickey, *Principles and Procedures of Statistics* (McGraw Hill Book Co., Singapore, 3rd edition, 1996).

[17] A. Sibiya, J. Jeyavani, P. Santhanam, E. Preetham, R. Freitas, and B. Vaseeharan, "Comparative Evaluation on the Toxic Effect of Silver (Ag) and Zinc Oxide (ZnO) Nanoparticles on Different Trophic Levels in Aquatic Ecosystems: A Review," *Journal of Applied Toxicology* 42, no. 12 (2022): 1890–1900.

[18] W. Bai, Z. Zhang, W. Tian, et al., "Toxicity of Zinc Oxide Nanoparticles to Zebrafish Embryo: A Physicochemical Study of Toxicity Mechanism," *Journal of Nanoparticle Research* 12, no. 5 (2010): 1645–1654.

[19] E. Sánchez-López, D. Gomes, G. Esteruelas, et al., "Metal-Based Nanoparticles as Antimicrobial Agents: An Overview," *Nanomaterials* 10, no. 2 (2020): 292.

[20] O. Kori-Siakpere and E. O. Ubogu, "Sublethal Haematological Effects of Zinc on the Freshwater Fish, Heteroclarias Sp. (Osteichthyes: Clariidae)," *African Journal of Biotechnology* 7, no. 12 (2008): 2068–2073.

[21] M. Saravanan, K. P. Kumar, and M. Ramesh, "Haematological and Biochemical Responses of Freshwater Teleost Fish *Cyprinus carpio* (Actinopterygii: Cypriniformes) During Acute and Chronic Sublethal Exposure to Lindane," *Pesticide Biochemistry and Physiology* 100, no. 3 (2011): 206–211.

[22] A. A. Abdel-Khalek, M. Kadry, A. Hamed, and M. A. Marie, "Ecotoxicological Impacts of Zinc Metal in Comparison to Its Nanoparticles in Nile Tilapia; *Oreochromis niloticus*," *The Journal of Basic & Applied Zoology* 72 (2015): 113–125.

[23] D. Pamila, P. S. Subbaiyan, and M. Ramaswamy, "Toxic Effects of Chromium and Cobalt on *Sarotherodon mossambicus* (Peters)," *Indian Journal of Environmental Health* 33, no. 2 (1991): 218–224.

[24] T. Venkatachalam and A. V. Natarajan, "Haematological Investigation on Freshwater Teleost *Labeo rohita* (Ham.) Following Aquatic Toxicities of Cr (III) and Cr (VI),"

*International Journal of Research in Biosciences* 3, no. 3 (2014): 1–14.

[25] V. D. Rajput, T. M. Minkina, A. Behal, et al., "Effects of Zinc-Oxide Nanoparticles on Soil, Plants, Animals and Soil Organisms: A Review," *Environmental Nanotechnology, Monitoring & Management* 9 (2018): 76–84.

[26] G. B. Khan, N. Akhtar, M. F. Khan, Z. Ullah, S. Tabassum, and Z. Tedesse, "Toxicological Impact of Zinc Nanoparticles on Tilapia Fish (*Oreochromis mossambicus*)," *Saudi Journal of Biological Sciences* 29, no. 2 (2022): 1221–1226.

[27] M. A. O. Dawood and S. Koshio, "Recent Advances in the Role of Probiotics and Prebiotics in Carp Aquaculture: A Review," *Aquaculture* 454 (2016): 243–251.

[28] G. Rashidian, C. C. Lazado, H. H. Mahboub, et al., "Chemically and Green Synthesized ZnO Nanoparticles Alter Key Immunological Molecules in Common Carp (*Cyprinus carpio*) Skin Mucus," *International Journal of Molecular Sciences* 22, no. 6 (2021): 3270.

[29] K. Shahzad, M. N. Khan, F. Jabeen, et al., "Toxicity of Zinc Oxide Nanoparticles (ZnO-NPs) in Tilapia (*Oreochromis mossambicus*): Tissue Accumulation, Oxidative Stress, Histopathology and Genotoxicity," *International Journal of Environmental Sciences and Technology* 16 (2019): 1973–1984.

[30] S. Subashkumar and M. Selvanayagam, "First Report on: Acute Toxicity and Gill Histopathology of Fresh Water Fish *Cyprinus carpio* Exposed to Zinc Oxide (ZnO) Nanoparticles," *International Journal of Scientific and Research Publications* 4, no. 3 (2014): 1–4.

[31] L. Hao, L. Chen, J. Hao, and N. Zhong, "Bioaccumulation and Sub-Acute Toxicity of Zinc Oxide Nanoparticles in Juvenile Carp (*Cyprinus carpio*): A Comparative Study With its Bulk Counterparts," *Ecotoxicology and Environmental Safety* 91 (2013): 52–60.

[32] R. Sukhsangchan, J. Phaksopa, A. Uchuwittayakul, C.-C. Chou, and P. Srisapoome, "Effects of Zinc Oxide Nanoparticles (ZnO NPs) on Growth, Immune Responses and Histopathological Alterations in Asian Seabass (*Lates calcarifer*, Bloch 1790) Under Low-Salinity Conditions," *Animals* 14, no. 18 (2024): 2737.

[33] G. Federici, B. J. Shaw, and R. D. Handy, "Toxicity of Titanium Dioxide Nanoparticles to Rainbow Trout (*Oncorhynchus mykiss*): Gill Injury, Oxidative Stress, and Other Physiological Effects," *Aquatic Toxicology* 84, no. 4 (2007): 415–430.

[34] L. Wang, C. Hu, and L. Shao, "The Antimicrobial Activity of Nanoparticles: Present Situation and Prospects for the Future," *International Journal of Nanomedicine* 12 (2017): 1227–1249.

[35] H. Kaya, F. Aydin, M. Gürkan, et al., "Comparative Toxicity Study Between Small and Large Size Zinc Oxide Nanoparticles in Tilapia (*Oreochromis niloticus*): Organ Pathologies, Osmoregulatory Responses and Immunological Parameters," *Chemosphere* 144 (2016): 571–582.

[36] H. Kaya, M. Duysak, M. Akbulut, et al., "Effects of Sub-Chronic Exposure to Zinc Nanoparticles on Tissue Accumulation, Serum Biochemistry, and Histopathological Changes in Tilapia (*Oreochromis niloticus*)," *Environmental Toxicology* 32, no. 4 (2017): 1213–1225.

[37] A. H. Mandal, S. Ghosh, D. Adhurjya, et al., "Exploring the Impact of Zinc Oxide Nanoparticles on Fish and Fish-Food Organisms: A Review," *Aquaculture Reports* 36 (2024): 102038.

[38] Q. Sun, J. Li, and T. Le, "Zinc Oxide Nanoparticle as a Novel Class of Antifungal Agents: Current Advances and Future Perspectives," *Journal of Agricultural and Food Chemistry* 66, no. 43 (2018): 11209–11220.