# Antagonistic Effects of Sublethal Concentrations of Certain Mixtures of Metal Oxide Nanoparticles and the Bulk (Al2O3, CuO, and SiO2) on Gill Histology in Clarias gariepinus

Authors: Amaeze Henry Nnamdi; Tam-Miette Dawarri Briggs; Oluwaseun Olusola Togunde; Henry Ebele Obanya
Journal: Journal of Nanotechnology
 (2019)

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## Abstract

Background. The effect of nanoparticles (NPs) on aquatic environments is poorly studied. Aim. This study evaluates the toxicity of joint effects of these different metal nanoparticles and their bulk in mixtures (Al2O3, CuO, and SiO2) on fish using histological biomarker. Materials and Methods. The bulk and nano sizes of three salts (Al2O3, CuO, and SiO2) were used. Nanosizes ranged from 25 nm to 100 nm. The juvenile fishes of Clarias gariepinus (mean Length: 12.3 ± 3.5 cm; mean weight: 18.52 ± 6.41 g) were used for the acute and chronic toxicity tests. They were exposed to 7 mg/L each of the bulk and nano sizes of the three metallic oxides either singly or in mixtures for 28 days. The basis for the sublethal concentration was that the 96 hr acute toxicity of the varied sizes of the three metallic oxides was nontoxic up to the concentrations of 100 mg/L with no significant mortality at the highest exposure concentrations. The gills were collected for histopathology. Results. Of the three metal oxide nanoparticles, SiO was the most toxic, with histopathological alteration index (HAI) of 20.0, followed by nano-CuO (HAI, 10.0) and nano-Al2O3 (HAI, 2.0). In single exposure, the gill alterations include high frequencies of erosion of gill lamella (EGL), hypertrophy (HPT), oedema (OD), and necrosis (N). Less damage was observed at the combination of the metal oxide nanoparticles of SiO + Al2O3, SiO + CuO and SiO + Al2O3 + CuO in equal (1 : 1—HAI, 2 and 6; 1 : 1 : 1—HAI, 6) and unequal ratios (1 : 2—HAI, 16 and 6; 2 : 1—HAI, 8 and 6). Similarly, all bulk combinations were also antagonistic except for the equal ratio of bulk CuO (HAI, 20) and bulk Al2O3 (HAI, 10) that gave additive effect with HAI of 32. Conclusion. The joint actions of nano Al2O3 and CuO with SiO produced a low toxic effect, unlike the high toxicity of their single trials; this also indicates that nano Al2O3 and CuO are antagonists. Similarly, among the bulk metal oxides (SiO, Al2O3, and CuO), CuO was the most toxic. Bulk SiO and Al2O3 are antagonistic on the effects of CuO on the fish gill. There is need to properly document the ecological implications of nanoparticles in the aquatic environment.


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## Body

## 1. Introduction

The rapid development of the nanotechnology industry in the last 20 years is yet to reach its potential [1]. This has been fuelled by the unusual properties that materials possess at the nanoscale; however, it is also that these properties are in part fuelling concerns regarding their potential toxicity and ecotoxicology. The engineering material applications of NPs can increase their concentrations in groundwater, soil, and surface water which presents the most significant exposure avenues, pathway, and fate for assessing environmental risks [2].Nanoparticles like silicon oxide have several applications in the preparation of nanocomposites to enhance thermal resistance and electrical and mechanical properties. Also, it is used in various fields of catalysts, pigment stabilization, electronics, and sensors. Nano-aluminium oxide is used as raw material in solid rocket propellant formulation and explosives. Nano-copper II oxide is used in biomedical applications such as antimicrobial and plasmonic materials as a component of reforming catalysts.Studies associated with the use of NPs are limited [3]. Miniaturization of materials is of great interest due to the difference in their physicochemical properties compared to the bulk materials. These properties include colour, solubility, conductivity, and catalytic activity of engineered nanomaterials [4]. In addition to their increased surface area to volume ratio, nanoparticles can serve as contaminant absorbents.Nanotoxicology explains the concepts of the toxicological basis of NPs on health and the environment [5]. Several studies have reported the harmful effects of NPs and their bulk salts on the biota, but presence of other contaminants and constituent mixture in environment has not been fully studied yet. The pollution of the aquatic ecosystems by NPs has been of global concern [6]. These pollutants including nanoparticles could increase the level of metals in natural water and seriously affect wetland habitats [7]. It was reported that the 95 hr LC10 of various nanoparticles for fish ranged from 100 μgL−1 to 1 mg·L−1, while the 95 hr LC50 of NPs reach the mg/L range [8]. Expected concentrations of NPs in surface waters range from μgL−1 to low mg·L−1 [8, 9].The use of fish histopathological alteration index as a tool for classification and categorization of stages of tissue changes relative to stress or chemical exposure has been reported by several ecotoxicologists and scientists [10–13]. This has been used to evaluate the effects of contaminants on the health of fish in the environment and to help establish a causal relation between exposure to toxic substances and the various biological responses [10]. The use of incidence and prevalence of fish diseases as associated with contaminants as indicators of environmental stress provides a definitive biological endpoint for the history of exposure [10, 13, 14].The fish gill is very sensitive to environmental changes and is easily affected by pollutants at low concentrations [15]. The gills have a large surface area and perform various vital functions such as respiration, osmoregulation, and excretion. Due to the fact that they are external structure in direct contact with the external environment, they are sensitive to chemical and physical changes of the aquatic environment [16]. The gills are the principal sites for gas exchange and other important functions such as ionic and osmotic regulation in addition to acid-base balance; histopathological changes in the structure of these organs involve respiratory disturbances and electrolyte imbalance [16].Mansouri et al. [15] have reported the most common histopathological anomalies in the gill of common carp such as hyperplasia, oedema, curvature, fusion, aneurism, and necrosis after 10 and 20 days exposure to TiO2 NPs (10.0 mg·L−1) and CuO NPs (2.5 and 5.0 mg·L−1) singly and in mixtures. The mixture effect was reported as synergetic. This study was similar to that of [17] that exposed Carassius auratus (goldfish) to mixtures of suspensions of 20 nm size Al2O3 and 50 nm size ZnO. Significant morphological alteration such as hyperplasia (with fusion of lamellae) was reported in the gills. Combined interactions within the mixtures could be antagonistic, synergetic, or additive depending on the specific properties and type of oxide NPs such as size and surface area [17]. However, there are limited studies on the joint effects of nanoparticles and their bulk metallic salts on the fish using histological biomarker.The aim of this study is to evaluate the toxicity of joint effect of different heavy metals in mixtures (Al2O3, CuO, and SiO2) on fish using histological biomarker.


## 2. Materials and Methods

### 2.1. Experimental Chemicals and Preparation of Test Solutions

The bulk and nano sizes of three salts (Al2O3, CuO, and SiO2) were procured from Sigma Aldrich. They were stored at room temperatures in the laboratory before use. Nano-Al2O3 appeared as grey coloured powder, density of 2.70 g/cm3, and size of 40 nm with 99.9% purity. Nano-CuO has red to yellow powder, density of 8.94 g/ml at 25°C with size <50 nm. SiO2 is a brown yellow powder, density of 2.33 g/ml at 25°C with size <100 nm.Stock suspensions of the uncoated powder were made using dechlorinated tap water to obtain various concentrations of the test solution. Respective grams of the metallic oxides were dissolved in 1 L of the dechlorinated tap water to obtain appropriate mixtures to obtain stock solutions (Figure1). Literature search was used to obtain the test concentrations. The concentrations of the test compounds were prepared as single solutions and mixtures of 1 : 1, 1 : 2, 2 : 1, and 1 : 1 : 1 both for bulk and nanoscale oxides.Figure 1
(a) Histological section of gill of fish for control experiment showing primary gill lamella (PGL), blunt secondary gill lamella (SSGL), and blood vessel (BV) (H&E stains, ×400). (b) Histological section of gill of fish exposed to nanosize SiO showing total destruction of primary gill lamella (PGL) and secondary gill lamellae (SGL), eroded epithelium (ERE), and area of severe necrosis (N) and oedema (OD) (H&E stains, ×100).
(a)
(b)


### 2.2. Experimental Organisms: Collection and Acclimatization

The African sharp tooth Catfish/African Catfish,Clarias gariepinus, was used for this study. It was selected on the basis of ease of culture and known responses to pollutants from existing literatures. The fishes were procured from a fish farm in Akoka, Lagos, and transported in open drums to the Department of Zoology, Laboratory Annex at the Biological Garden. The fishes were transported into large holding tanks (100 L capacity) in the laboratory which were half filled with dechlorinated tap water left open for 24 hours. The juvenile fishes (mean length: 12.3 ± 3.5 cm; mean weight: 18.52 ± 6.41 g) were employed for the sublethal/chronic toxicity tests. Only catfishes of the same batches were used for the experiment.The fishes were acclimatized in the lab using the 100 L capacity holding tanks at a stocking density of 20 fishes per litre for the fingerlings and 5 fishes per litter for the juveniles. The acclimatization lasted for 7 days after which the bioassay commenced. The acclimatization was conducted under standard laboratory conditions (temp, 26.0 ± 3.0°C; humidity, 75 ± 6%; photoperiod, light: dark, 12 : 12 hours). The bioassay media (dechlorinated tap water) was also of suitable quality (pH: 6.8; D.O, 8.5 mg/L; Salinity, 0.0 ppt).


### 2.3. Laboratory Bioassay

#### 2.3.1. Acute Toxicity Assay

The acute toxicity test/range finding exercise was conducted over 96 hours with varying concentrations in order to achieve mortality. The bioassay containers were made of plastics (6 m × 4  cm × 4 cm). During the acute toxicity test, the fingerlings were stocked at 10 fishes per litre of water, made up by equivalent concentrations of the stock solution of the bulk and nanoscale heavy metals acting singly. Each setup was in duplicate, i.e., 20 fingerlings per concentrations.The fishes were exposed to concentrations of 1 mg/L, 10 mg/L, 100 mg/L, 1000 mg/L, and 3000 mg/L as required based on the response of the test organism. The 96-hour acute toxicity of the nano and the bulk metals was found to be nontoxic up to the concentration of 100 mg/L, and no significant mortality was found at the highest exposure concentration.


#### 2.3.2. Chronic Toxicity Assay

The juvenile catfishes were employed in the chronic toxicity tests in which they were exposed to 7 mg/L each of the bulk and nano sizes of the three metallic oxides either singly or in mixtures for 28 days. The chronic toxicity assays were conducted in larger plastics (11  cm × 9  cm × 7.5 cm) using 5 L of water because of the larger sizes of the fishes compared to the acute toxicity test. At the end of the exposure period, the fishes were immobilized by spinal puncture and dissected to collect the gills and preserved for histopathological investigation.


#### 2.3.3. Histopathological Examination

The gills were fixed in 10% formalin, dehydrated in graded ethanol [18], cleared in xylene, embedded in paraffin wax, and sectioned at 5 μm on a rotary microtome. Slides were stained using the haematoxylin and eosin technique for light microscopy [18]. The histopathological changes were evaluated according to [18] and [10], which includes the calculation of the histopathological alteration index (HAI) for each fish using the following formula:(1)HAI=1∑I+∑II+∑III.Because I, II, and III correspond to the number of stages of change, the mean HAI was scored on six-point scale: 0 = normal tissue; 2.0 = mild damage to the tissue; 4.0 = moderate damage to the tissue; 6.0 = partially severe damage to the tissue; 8.0 = severe damage to the tissue; 10 = irreparable damage to the tissue. These stages of change and scoring system or scale are given in Table1.Table 1
Stages of change and histological alterations of the gill.
Alteration score
Score description
Histological alteration
0
Normal tissue
No lesion or any alteration (NT)
2.0
Mild damage
Mild thickening of gill lamella (GL1)Epithelial hyperplasia (EH)Hypertrophy (HPT)
4.0
Moderate damage
Moderate thickening of gill lamella (GL2)Oedema (OD)
6.0
Partially severe damage
Eroded outer operculum (ERO)Epithelial lifting (EPL)Partial fusion of secondary lamella (FSGL1)Erosion of gill lamella (EGL)Shortening of secondary lamella (SSGL)Stunted gill lamella (SGL)Blunt secondary lamella (BSGL)Uncontrolled proliferation of epithelial cells (PEC)
8.0
Severe damage
Severe thickening of gill lamella (GL3)Complete fusion of secondary lamella (FSGL1)Aneurysm (ANS)
10.0
Irreparable damage
Necrosis (N)Total damage to the lamella (TDL)


## 2.1. Experimental Chemicals and Preparation of Test Solutions

The bulk and nano sizes of three salts (Al2O3, CuO, and SiO2) were procured from Sigma Aldrich. They were stored at room temperatures in the laboratory before use. Nano-Al2O3 appeared as grey coloured powder, density of 2.70 g/cm3, and size of 40 nm with 99.9% purity. Nano-CuO has red to yellow powder, density of 8.94 g/ml at 25°C with size <50 nm. SiO2 is a brown yellow powder, density of 2.33 g/ml at 25°C with size <100 nm.Stock suspensions of the uncoated powder were made using dechlorinated tap water to obtain various concentrations of the test solution. Respective grams of the metallic oxides were dissolved in 1 L of the dechlorinated tap water to obtain appropriate mixtures to obtain stock solutions (Figure1). Literature search was used to obtain the test concentrations. The concentrations of the test compounds were prepared as single solutions and mixtures of 1 : 1, 1 : 2, 2 : 1, and 1 : 1 : 1 both for bulk and nanoscale oxides.Figure 1
(a) Histological section of gill of fish for control experiment showing primary gill lamella (PGL), blunt secondary gill lamella (SSGL), and blood vessel (BV) (H&E stains, ×400). (b) Histological section of gill of fish exposed to nanosize SiO showing total destruction of primary gill lamella (PGL) and secondary gill lamellae (SGL), eroded epithelium (ERE), and area of severe necrosis (N) and oedema (OD) (H&E stains, ×100).
(a)
(b)


## 2.2. Experimental Organisms: Collection and Acclimatization

The African sharp tooth Catfish/African Catfish,Clarias gariepinus, was used for this study. It was selected on the basis of ease of culture and known responses to pollutants from existing literatures. The fishes were procured from a fish farm in Akoka, Lagos, and transported in open drums to the Department of Zoology, Laboratory Annex at the Biological Garden. The fishes were transported into large holding tanks (100 L capacity) in the laboratory which were half filled with dechlorinated tap water left open for 24 hours. The juvenile fishes (mean length: 12.3 ± 3.5 cm; mean weight: 18.52 ± 6.41 g) were employed for the sublethal/chronic toxicity tests. Only catfishes of the same batches were used for the experiment.The fishes were acclimatized in the lab using the 100 L capacity holding tanks at a stocking density of 20 fishes per litre for the fingerlings and 5 fishes per litter for the juveniles. The acclimatization lasted for 7 days after which the bioassay commenced. The acclimatization was conducted under standard laboratory conditions (temp, 26.0 ± 3.0°C; humidity, 75 ± 6%; photoperiod, light: dark, 12 : 12 hours). The bioassay media (dechlorinated tap water) was also of suitable quality (pH: 6.8; D.O, 8.5 mg/L; Salinity, 0.0 ppt).


## 2.3. Laboratory Bioassay

### 2.3.1. Acute Toxicity Assay

The acute toxicity test/range finding exercise was conducted over 96 hours with varying concentrations in order to achieve mortality. The bioassay containers were made of plastics (6 m × 4  cm × 4 cm). During the acute toxicity test, the fingerlings were stocked at 10 fishes per litre of water, made up by equivalent concentrations of the stock solution of the bulk and nanoscale heavy metals acting singly. Each setup was in duplicate, i.e., 20 fingerlings per concentrations.The fishes were exposed to concentrations of 1 mg/L, 10 mg/L, 100 mg/L, 1000 mg/L, and 3000 mg/L as required based on the response of the test organism. The 96-hour acute toxicity of the nano and the bulk metals was found to be nontoxic up to the concentration of 100 mg/L, and no significant mortality was found at the highest exposure concentration.


### 2.3.2. Chronic Toxicity Assay

The juvenile catfishes were employed in the chronic toxicity tests in which they were exposed to 7 mg/L each of the bulk and nano sizes of the three metallic oxides either singly or in mixtures for 28 days. The chronic toxicity assays were conducted in larger plastics (11  cm × 9  cm × 7.5 cm) using 5 L of water because of the larger sizes of the fishes compared to the acute toxicity test. At the end of the exposure period, the fishes were immobilized by spinal puncture and dissected to collect the gills and preserved for histopathological investigation.


### 2.3.3. Histopathological Examination

The gills were fixed in 10% formalin, dehydrated in graded ethanol [18], cleared in xylene, embedded in paraffin wax, and sectioned at 5 μm on a rotary microtome. Slides were stained using the haematoxylin and eosin technique for light microscopy [18]. The histopathological changes were evaluated according to [18] and [10], which includes the calculation of the histopathological alteration index (HAI) for each fish using the following formula:(1)HAI=1∑I+∑II+∑III.Because I, II, and III correspond to the number of stages of change, the mean HAI was scored on six-point scale: 0 = normal tissue; 2.0 = mild damage to the tissue; 4.0 = moderate damage to the tissue; 6.0 = partially severe damage to the tissue; 8.0 = severe damage to the tissue; 10 = irreparable damage to the tissue. These stages of change and scoring system or scale are given in Table1.Table 1
Stages of change and histological alterations of the gill.
Alteration score
Score description
Histological alteration
0
Normal tissue
No lesion or any alteration (NT)
2.0
Mild damage
Mild thickening of gill lamella (GL1)Epithelial hyperplasia (EH)Hypertrophy (HPT)
4.0
Moderate damage
Moderate thickening of gill lamella (GL2)Oedema (OD)
6.0
Partially severe damage
Eroded outer operculum (ERO)Epithelial lifting (EPL)Partial fusion of secondary lamella (FSGL1)Erosion of gill lamella (EGL)Shortening of secondary lamella (SSGL)Stunted gill lamella (SGL)Blunt secondary lamella (BSGL)Uncontrolled proliferation of epithelial cells (PEC)
8.0
Severe damage
Severe thickening of gill lamella (GL3)Complete fusion of secondary lamella (FSGL1)Aneurysm (ANS)
10.0
Irreparable damage
Necrosis (N)Total damage to the lamella (TDL)


## 2.3.1. Acute Toxicity Assay

The acute toxicity test/range finding exercise was conducted over 96 hours with varying concentrations in order to achieve mortality. The bioassay containers were made of plastics (6 m × 4  cm × 4 cm). During the acute toxicity test, the fingerlings were stocked at 10 fishes per litre of water, made up by equivalent concentrations of the stock solution of the bulk and nanoscale heavy metals acting singly. Each setup was in duplicate, i.e., 20 fingerlings per concentrations.The fishes were exposed to concentrations of 1 mg/L, 10 mg/L, 100 mg/L, 1000 mg/L, and 3000 mg/L as required based on the response of the test organism. The 96-hour acute toxicity of the nano and the bulk metals was found to be nontoxic up to the concentration of 100 mg/L, and no significant mortality was found at the highest exposure concentration.


## 2.3.2. Chronic Toxicity Assay

The juvenile catfishes were employed in the chronic toxicity tests in which they were exposed to 7 mg/L each of the bulk and nano sizes of the three metallic oxides either singly or in mixtures for 28 days. The chronic toxicity assays were conducted in larger plastics (11  cm × 9  cm × 7.5 cm) using 5 L of water because of the larger sizes of the fishes compared to the acute toxicity test. At the end of the exposure period, the fishes were immobilized by spinal puncture and dissected to collect the gills and preserved for histopathological investigation.


## 2.3.3. Histopathological Examination

The gills were fixed in 10% formalin, dehydrated in graded ethanol [18], cleared in xylene, embedded in paraffin wax, and sectioned at 5 μm on a rotary microtome. Slides were stained using the haematoxylin and eosin technique for light microscopy [18]. The histopathological changes were evaluated according to [18] and [10], which includes the calculation of the histopathological alteration index (HAI) for each fish using the following formula:(1)HAI=1∑I+∑II+∑III.Because I, II, and III correspond to the number of stages of change, the mean HAI was scored on six-point scale: 0 = normal tissue; 2.0 = mild damage to the tissue; 4.0 = moderate damage to the tissue; 6.0 = partially severe damage to the tissue; 8.0 = severe damage to the tissue; 10 = irreparable damage to the tissue. These stages of change and scoring system or scale are given in Table1.Table 1
Stages of change and histological alterations of the gill.
Alteration score
Score description
Histological alteration
0
Normal tissue
No lesion or any alteration (NT)
2.0
Mild damage
Mild thickening of gill lamella (GL1)Epithelial hyperplasia (EH)Hypertrophy (HPT)
4.0
Moderate damage
Moderate thickening of gill lamella (GL2)Oedema (OD)
6.0
Partially severe damage
Eroded outer operculum (ERO)Epithelial lifting (EPL)Partial fusion of secondary lamella (FSGL1)Erosion of gill lamella (EGL)Shortening of secondary lamella (SSGL)Stunted gill lamella (SGL)Blunt secondary lamella (BSGL)Uncontrolled proliferation of epithelial cells (PEC)
8.0
Severe damage
Severe thickening of gill lamella (GL3)Complete fusion of secondary lamella (FSGL1)Aneurysm (ANS)
10.0
Irreparable damage
Necrosis (N)Total damage to the lamella (TDL)


## 3. Results and Discussion

### 3.1. Histopathological Effects and Joint Action of Single and Combined Exposures ofClarias gariepinus on 28-Day Sublethal Concentrations of Nanometallic Salts

The interaction of NPs with other contaminants is dependent on the properties of the NPs such as size, composition, morphology, porosity, aggregation/disaggregation, and aggregate structure. The devastating effects of NPs and bulk are mainly due to the dispersion, persistence, and bioaccumulation and biomagnification potentials in addition to their toxicity in the biological tissues [19].The fish gill is the primary target organ affected by NPs. Jayaseelan et al. and Griffitt et al. [20, 21] had reported physiological alterations such as dysfunction in osmoregulation, respiratory gas exchange, and body fluid permeability balance. Due to the large superficial area of the epithelium per volume ratio, the organ is more susceptible to effects of contaminants [22]. Histological and biochemical analysis in previous studies revealed the gills to be the primary target organ [10, 11, 21]. The physiological alteration could become visible as histological alteration as revealed by Griffitt et al. [21], who reported that NPs produced hypertrophy of epithelial cells in the gills [21].In this study, the most histopathological lesions in fish gills such as hyperplasia, oedema, curvature, shortening and fusion of gill lamellae, aneurism, and necrosis are described and quantified using an index as used in several studies [10–13]. Tables 2 and 4–7 and Figures 1–12 show the histopathological effects and joint action of single and combined exposures of Clarias gariepinus to 28-day sublethal concentrations of nanometallic salts. Single exposure to nano-SiO, nano-Al2O3 and nano-CuO produced varying effects after 28 days. Nano-SiO was the most toxic, with histopathological alteration index (HAI) of 20.0, followed by nano-CuO (HAI, 10.0) and nano-Al2O3 (HAI, 2.0). The gill alterations include high frequencies of erosion of gill lamella (EGL), hypertrophy (HPT), oedema (OD), and necrosis (N).Table 2
A 28-day single and joint exposure ofClarias gariepinus to nanosize metals.
Conc. ratio
Nanometals
Assumed HAI
Toxic level
Actual HAI
Toxic level
Type of joint effect
Gill pathology
Singly
SiOAl2O3CuO
20.02.010.0
HighLowModerate
NANANA
NANANA
NANANA
EGL, hypertrophyOedemaNecrosis
1 : 1
SiO + Al2O3SiO + CuO
22.030.0
HighHigh
2.06.0
LowLow
AntagonisticAntagonistic
Hypertrophy, SSGL
2 : 1
SiO + Al2O3SiO + CuO
22.030.0
HighHigh
8.06.0
LowLow
AntagonisticAntagonistic
EH, SSGL
1 : 2
SiO + Al2O3SiO + CuO
22.030.0
HighHigh
16.06.0
ModerateLow
AntagonisticAntagonistic
PEC, EPL, necrosis
1 : 1 : 1
SiO + Al2O3+ CuO
30 or 22
High
6.0
Low
Antagonistic
EGL, SSGL
Toxic level: HAI:X < 10, low; 10 ≥ X < 20, moderate; X ≥ 20, high.Figure 2
(a) Histological section of gill exposed to bulk SiO. Deformities such as oedema of secondary gill lamella (OD), hypertrophy (HPT), and epithelial hyperplasia are observed (HPS). Primary gill lamella is also observed (H&E stains, ×400). (b) Histological section of gill exposed to bulk CuO. Total destruction of both primary and secondary lamella is seen. Areas of eroded epithelium (ERE), aneurysm (ANS), necrosis (N), and blood vessel (BV) are also observed (H&E stains, ×100).
(a)
(b)Figure 3
(a) Histological section of gill exposed to nano-CuO showing oedema of primary gill lamella (OD), epithelial hyperplasia (HPS), eroded epithelium (ERE), and the blood vessel (BV) (H&E stains, ×100). (b) Histological section of gill exposed to bulk Al2O3 showing primary gill lamella (PGL), shortened secondary gill lamella (SSGL), hypertrophy (HPT), and some areas of mild necrosis (N) (H&E stains, ×400).
(a)
(b)Figure 4
(a) Histological section of gill exposed to nano-Al2O3 showing primary gill lamella (PGL), curved secondary gill lamella (CSGL), epithelial hyperplasia (HPS), epithelial lifting (EPL), and blood vessel (BV) (H&E stains, ×400). (b) Histological section of gill exposed to bulk SiO/CuO (1 : 1) showing secondary gill lamella (SGL), hypertrophy (HPT), and epithelial lifting (EPL) (H&E stains, ×400).
(a)
(b)Figure 5
(a) Histological section of gill exposed to nano-SiO/CuO (1 : 1) showing primary gill lamella (PGL) shortened secondary gill lamella (SSGL), blood vessel, oedema of primary lamella, and eroded epithelium (H&E stains, ×100). (b) Histological section of gill exposed to nano-SiO/Al2O3 (1 : 1) showing primary gill lamella (PGL), eroded secondary gill lamella (ESGL), and hypertrophy (HPT) (H&E stains, ×100).
(a)
(b)Figure 6
(a) Histological section of gill exposed to bulk SiO/Al2O3 (1 : 1) showing primary gill lamella (PGL), eroded secondary gill lamella (ESGL), and hypertrophy (HPT). Shortened secondary gill lamella (SSGL) and area of necrosis (N) are also observed (H&E stains, ×100). (b) Histological section of gill exposed to bulk Al2O3/CuO (1 : 1) showing primary gill lamella (PGL), total destruction of secondary gill lamella, oedema (OD), aneurysm (ANS), and necrosis (N) (H&E stains, ×100).
(a)
(b)Figure 7
(a) Histological section of gill exposed to nano-SiO/Al2O3 (2 : 1) showing secondary gill lamella (SGL), blood vessel, and epithelial hyperplasia (HPS) (H&E stains, ×400). (b) Histological section of gill exposed to bulk SiO/Al2O3 (2 : 1) showing primary gill lamella, shortened secondary gill lamella (SSGL), and blood vessel (BV) (H&E stains, ×400).
(a)
(b)Figure 8
(a) Histological section of gill exposed to nano-SiO/CuO (2 : 1) showing fused secondary gill lamella (FSGL), secondary gill lamella (SGL), and epithelial hyperplasia (HPS) (H&E stains, ×400). (b) Histological section of gill exposed to bulk SiO/CuO (2 : 1) showing hypertrophy (HPT), blood vessel (BV), and epithelial hyperplasia (HPS) (H&E stains, ×400).
(a)
(b)Figure 9
(a) Histological section of gill exposed to bulk Al2O3/CuO (2 : 1) showing oedema (OD) and epithelial hyperplasia (HPS) (H&E stains, ×400). (b) Histological section of gill exposed to bulk Al2O3/CuO (1 : 2) showing primary gill lamella (PGL), epithelial lifting (EPL), and hypertrophy (HPT) (H&E stains, ×400).
(a)
(b)Figure 10
(a) Histological section of gill exposed to nano-Al2O3/CuO (1 : 2) showing primary gill lamella (PGL), curved secondary gill lamella (CSGL), and shortened secondary gill lamella (SSGL) (H&E stains, ×400). (b) Histological section of gill exposed to nano-SiO/CuO (1 : 2) showing secondary gill lamella (SGL), inflammation of lamella (INF), and blood vessel (H&E stains, ×400).
(a)
(b)Figure 11
(a) Histological section of gill exposed to bulk SiO/CuO (1 : 2) showing epithelial hyperplasia (HPS), necrosis (N), and hypertrophy (HPT) (H&E stains, ×400). (b) Histological section of gill exposed to bulk SiO/Al2O3 (1 : 2) showing mild necrosis (N) and secondary gill lamella (SGL). No serious damage is observed (H&E stains, ×400).
(a)
(b)Figure 12
(a) Histological section of gill exposed to nano-SiO/Al2O3 (1 : 2) showing curved secondary gill lamella (CSGL), epithelial lifting (EPL), and hypertrophy (HPT) (H&E stains, ×400). (b) Histological section of gill exposed to nano-SiO/Al2O3/CuO showing total destruction of gill architecture. Blood vessel is seen (H&E stains, ×400).
(a)
(b)The histopathological alteration index (HAI) showed that the gills are affected by nanoparticles of these metal oxides, and similar damages have been reported in other fish species exposed to Cu NPs [22, 23], TiO2 NPs [24], and other NPs such as cobalt (III) oxide (Co2O3) nanoparticle [24] and colloidal silver nanoparticle [25].If the effect of the joint exposures of nano-SiO with another nanometallic salt of equal ratio (1 : 1) were to be the addition of individual effect, then the HAI of SiO + Al2O3 and SiO + CuO should be 22.0 and 30.0, respectively. The actual combined effects of SiO + Al2O3 and SiO + CuO were HAI: 2.0 and 6.0, respectively. The gill alterations include low frequencies of hypertrophy (HPT) and shortening of secondary gill lamella (SSGL). This implies that joint actions of Al2O3 and CuO with SiO produced a low toxic effect, unlike the high toxicity of their single trials; this also indicates that Al2O3 and CuO are antagonists.Increasing the concentration of SiO in the mixtures (SiO + Al2O3 and SiO + CuO) in ratio 2 to 1 makes no difference in the toxic level. The actual combined effects of SiO + Al2O3 and SiO + CuO were HAI: 8.0 and 6.0, respectively. The gill alterations include low frequencies of gill epithelial hyperplasia (EH) and shortening of secondary gill lamella (SSGL).Increasing the concentrations of Al2O3 and CuO in the mixtures (SiO + Al2O3 and SiO + CuO) in ratio 1 to 2 still had no change in the toxic level for CuO with HAI of 6.0, but there was moderate toxicity for Al2O3 with HAI of 16.0. The gill alterations include moderate frequencies of uncontrolled proliferation of epithelial cells (PEC), gill epithelial lifting (EPL), and necrosis (N).Mixtures of the three nanometallic salts (SiO, Al2O3, and CuO) in ratio 1 : 1 : 1 produced low toxic effect (HAI, 6.0) compared to the addition of the individual effect, assumed to be HAI: 30 or 22. The gills have low frequencies of epithelial lifting (EPL) and shortening of secondary gill lamella (SSGL).


### 3.2. Histopathological Effects and Joint Action of Single and Combined Exposures ofClarias gariepinus on 28-Day Sublethal Concentrations of Bulk Metallic Salts

Tables3 and 4–7 and Figures 1–12 show the histopathological effects and joint action of single and combined exposures of Clarias gariepinus to 28-day sublethal concentrations of bulk metallic salts. Single exposure to bulk SiO, bulk Al2O3, and bulk CuO produced varying effects after 28 days. Bulk CuO was the most toxic, with histopathological alteration index (HAI) of 20.0, followed by bulk Al2O3 (HAI, 10.0) and bulk SiO (HAI, 6.0). The gill alterations include high frequencies of erosion of gill lamella (EGL), hypertrophy (HPT), oedema (OD), stunted gill lamella (SGL), aneurysm (ANS), and necrosis (N).Table 3
A 28-day single and joint exposure ofClarias gariepinus to bulk size metals.
Conc. ratio
Bulk metals
Assumed HAI
Toxic level
Actual HAI
Toxic level
Type of joint effect
Pathology
Singly
CuOSiOAl2O3
20.06.010.0
HighLowModerate
NANANA
NANANA
NANANA
Hypertrophy, EGL, OD, SGL, ANS, necrosis
1 : 1
CuO + SiOCuO + Al2O3
26.030.0
HighHigh
14.032.0
ModerateHigh
AntagonisticAdditive
Hypertrophy, EGL, OD, EPL, ANS, N, TDL
2 : 1
CuO + SiOCuO + Al2O3
26.030.0
HighHigh
6.06.0
LowLow
AntagonisticAntagonistic
EH, necrosisHypertrophy
1 : 2
CuO + SiOCuO + Al2O3
26.030.0
HighHigh
14.08.0
ModerateLow
AntagonisticAntagonistic
EH, oedema
1 : 1 : 1
CuO + SiO + Al2O3
30 or 26
High
12.0
Low
Antagonistic
EGL, SSGL
Toxic level: HAI:X < 10, low; 10 ≥ X < 20, moderate; X ≥ 20, high.Major alterations in fish under acute and chronic exposure to NPs and their bulk are changes in the morphology of the lamellar epithelium. The epithelium layer of secondary lamella becomes oedema with reduced surface area, resulting in asphyxiation. This change might also indicate acute inflammation due to the failure of the epithelial sodium pump [26]. The epithelial oedema and fusion affect toxicokinetics and distribution of pollutants in the gill epithelium [27].Furthermore, changes in the structure of the lamellar epithelium cause changes in the volume of gas and ion exchange [28, 29]. Similar studies by Ostaszewska et al. and Johari et al. [30–32] reported these histological changes such as swelling of goblet cell, villus deformation, hyperplasia, inflammation, necrosis, and vacuoles in the intestinal tissues.If the effect of the joint exposures of bulk CuO with another bulk metallic salt of equal ratio (1 : 1) were to be the addition of individual effect, then the HAI of CuO + SiO and CuO + Al2O3 should be 26.0 and 30.0, respectively. The actual combined effects of CuO + SiO and CuO + Al2O3 were HAI: 14.0 and 32.0, respectively. The gill alterations for CuO + SiO include moderate frequencies of hypertrophy (HPT), erosion of gill lamella (EGL), and gill epithelial lifting (EPL). The gill alterations for CuO + Al2O3 include moderate frequencies of oedema (OD), aneurysm (ANS), total damage to gill lamella (TDL), and necrosis (N). This implies that joint actions of SiO and Al2O3 with CuO produced a low toxic effect, unlike the high toxicity of their single trials; this also indicates that bulk SiO and bulk Al2O3 are antagonists.Increasing the concentration of CuO in the mixtures (CuO + SiO and CuO + Al2O3) in ratio 2 to 1 makes no difference in the toxic level. The actual combined effects of CuO + SiO and CuO + Al2O3 were HAI: 6.0 and 6.0, respectively. The gill alterations include low frequencies of gill epithelial hyperplasia (EH), hypertrophy, and necrosis (N).Increasing the concentrations of SiO and Al2O3 in the mixtures (CuO + SiO and CuO + Al2O3) in ratio 1 to 2 makes no difference in the toxic level for Al2O3. The actual combined effects of CuO + Al2O3 were HAI: 8.0. There was moderate toxicity with SiO with HAI of 14.0. The gill alterations include moderate frequencies of epithelial hyperplasia (EH) and oedema (OD).Mixtures of the three bulk metallic salts (SiO, Al2O3, and CuO) in ratio 1 : 1 : 1 produced low toxic effect (HAI, 12.0) compared to the addition of the individual effect, assumed to be HAI: 26 or 30. The gills have low frequencies of epithelial lifting (EPL) and shortening of secondary gill lamella (SSGL).Aneurysm is one common alteration in this study. This is the swelling of the blood vessel in the gill tissues, which could disturb blood flow [32, 33]. The histological responses in the gills of fish are mostly caused by circulatory disturbances, as aneurism, regressive and progressive changes, and hyperplasia [34, 35].


## 3.1. Histopathological Effects and Joint Action of Single and Combined Exposures ofClarias gariepinus on 28-Day Sublethal Concentrations of Nanometallic Salts

The interaction of NPs with other contaminants is dependent on the properties of the NPs such as size, composition, morphology, porosity, aggregation/disaggregation, and aggregate structure. The devastating effects of NPs and bulk are mainly due to the dispersion, persistence, and bioaccumulation and biomagnification potentials in addition to their toxicity in the biological tissues [19].The fish gill is the primary target organ affected by NPs. Jayaseelan et al. and Griffitt et al. [20, 21] had reported physiological alterations such as dysfunction in osmoregulation, respiratory gas exchange, and body fluid permeability balance. Due to the large superficial area of the epithelium per volume ratio, the organ is more susceptible to effects of contaminants [22]. Histological and biochemical analysis in previous studies revealed the gills to be the primary target organ [10, 11, 21]. The physiological alteration could become visible as histological alteration as revealed by Griffitt et al. [21], who reported that NPs produced hypertrophy of epithelial cells in the gills [21].In this study, the most histopathological lesions in fish gills such as hyperplasia, oedema, curvature, shortening and fusion of gill lamellae, aneurism, and necrosis are described and quantified using an index as used in several studies [10–13]. Tables 2 and 4–7 and Figures 1–12 show the histopathological effects and joint action of single and combined exposures of Clarias gariepinus to 28-day sublethal concentrations of nanometallic salts. Single exposure to nano-SiO, nano-Al2O3 and nano-CuO produced varying effects after 28 days. Nano-SiO was the most toxic, with histopathological alteration index (HAI) of 20.0, followed by nano-CuO (HAI, 10.0) and nano-Al2O3 (HAI, 2.0). The gill alterations include high frequencies of erosion of gill lamella (EGL), hypertrophy (HPT), oedema (OD), and necrosis (N).Table 2
A 28-day single and joint exposure ofClarias gariepinus to nanosize metals.
Conc. ratio
Nanometals
Assumed HAI
Toxic level
Actual HAI
Toxic level
Type of joint effect
Gill pathology
Singly
SiOAl2O3CuO
20.02.010.0
HighLowModerate
NANANA
NANANA
NANANA
EGL, hypertrophyOedemaNecrosis
1 : 1
SiO + Al2O3SiO + CuO
22.030.0
HighHigh
2.06.0
LowLow
AntagonisticAntagonistic
Hypertrophy, SSGL
2 : 1
SiO + Al2O3SiO + CuO
22.030.0
HighHigh
8.06.0
LowLow
AntagonisticAntagonistic
EH, SSGL
1 : 2
SiO + Al2O3SiO + CuO
22.030.0
HighHigh
16.06.0
ModerateLow
AntagonisticAntagonistic
PEC, EPL, necrosis
1 : 1 : 1
SiO + Al2O3+ CuO
30 or 22
High
6.0
Low
Antagonistic
EGL, SSGL
Toxic level: HAI:X < 10, low; 10 ≥ X < 20, moderate; X ≥ 20, high.Figure 2
(a) Histological section of gill exposed to bulk SiO. Deformities such as oedema of secondary gill lamella (OD), hypertrophy (HPT), and epithelial hyperplasia are observed (HPS). Primary gill lamella is also observed (H&E stains, ×400). (b) Histological section of gill exposed to bulk CuO. Total destruction of both primary and secondary lamella is seen. Areas of eroded epithelium (ERE), aneurysm (ANS), necrosis (N), and blood vessel (BV) are also observed (H&E stains, ×100).
(a)
(b)Figure 3
(a) Histological section of gill exposed to nano-CuO showing oedema of primary gill lamella (OD), epithelial hyperplasia (HPS), eroded epithelium (ERE), and the blood vessel (BV) (H&E stains, ×100). (b) Histological section of gill exposed to bulk Al2O3 showing primary gill lamella (PGL), shortened secondary gill lamella (SSGL), hypertrophy (HPT), and some areas of mild necrosis (N) (H&E stains, ×400).
(a)
(b)Figure 4
(a) Histological section of gill exposed to nano-Al2O3 showing primary gill lamella (PGL), curved secondary gill lamella (CSGL), epithelial hyperplasia (HPS), epithelial lifting (EPL), and blood vessel (BV) (H&E stains, ×400). (b) Histological section of gill exposed to bulk SiO/CuO (1 : 1) showing secondary gill lamella (SGL), hypertrophy (HPT), and epithelial lifting (EPL) (H&E stains, ×400).
(a)
(b)Figure 5
(a) Histological section of gill exposed to nano-SiO/CuO (1 : 1) showing primary gill lamella (PGL) shortened secondary gill lamella (SSGL), blood vessel, oedema of primary lamella, and eroded epithelium (H&E stains, ×100). (b) Histological section of gill exposed to nano-SiO/Al2O3 (1 : 1) showing primary gill lamella (PGL), eroded secondary gill lamella (ESGL), and hypertrophy (HPT) (H&E stains, ×100).
(a)
(b)Figure 6
(a) Histological section of gill exposed to bulk SiO/Al2O3 (1 : 1) showing primary gill lamella (PGL), eroded secondary gill lamella (ESGL), and hypertrophy (HPT). Shortened secondary gill lamella (SSGL) and area of necrosis (N) are also observed (H&E stains, ×100). (b) Histological section of gill exposed to bulk Al2O3/CuO (1 : 1) showing primary gill lamella (PGL), total destruction of secondary gill lamella, oedema (OD), aneurysm (ANS), and necrosis (N) (H&E stains, ×100).
(a)
(b)Figure 7
(a) Histological section of gill exposed to nano-SiO/Al2O3 (2 : 1) showing secondary gill lamella (SGL), blood vessel, and epithelial hyperplasia (HPS) (H&E stains, ×400). (b) Histological section of gill exposed to bulk SiO/Al2O3 (2 : 1) showing primary gill lamella, shortened secondary gill lamella (SSGL), and blood vessel (BV) (H&E stains, ×400).
(a)
(b)Figure 8
(a) Histological section of gill exposed to nano-SiO/CuO (2 : 1) showing fused secondary gill lamella (FSGL), secondary gill lamella (SGL), and epithelial hyperplasia (HPS) (H&E stains, ×400). (b) Histological section of gill exposed to bulk SiO/CuO (2 : 1) showing hypertrophy (HPT), blood vessel (BV), and epithelial hyperplasia (HPS) (H&E stains, ×400).
(a)
(b)Figure 9
(a) Histological section of gill exposed to bulk Al2O3/CuO (2 : 1) showing oedema (OD) and epithelial hyperplasia (HPS) (H&E stains, ×400). (b) Histological section of gill exposed to bulk Al2O3/CuO (1 : 2) showing primary gill lamella (PGL), epithelial lifting (EPL), and hypertrophy (HPT) (H&E stains, ×400).
(a)
(b)Figure 10
(a) Histological section of gill exposed to nano-Al2O3/CuO (1 : 2) showing primary gill lamella (PGL), curved secondary gill lamella (CSGL), and shortened secondary gill lamella (SSGL) (H&E stains, ×400). (b) Histological section of gill exposed to nano-SiO/CuO (1 : 2) showing secondary gill lamella (SGL), inflammation of lamella (INF), and blood vessel (H&E stains, ×400).
(a)
(b)Figure 11
(a) Histological section of gill exposed to bulk SiO/CuO (1 : 2) showing epithelial hyperplasia (HPS), necrosis (N), and hypertrophy (HPT) (H&E stains, ×400). (b) Histological section of gill exposed to bulk SiO/Al2O3 (1 : 2) showing mild necrosis (N) and secondary gill lamella (SGL). No serious damage is observed (H&E stains, ×400).
(a)
(b)Figure 12
(a) Histological section of gill exposed to nano-SiO/Al2O3 (1 : 2) showing curved secondary gill lamella (CSGL), epithelial lifting (EPL), and hypertrophy (HPT) (H&E stains, ×400). (b) Histological section of gill exposed to nano-SiO/Al2O3/CuO showing total destruction of gill architecture. Blood vessel is seen (H&E stains, ×400).
(a)
(b)The histopathological alteration index (HAI) showed that the gills are affected by nanoparticles of these metal oxides, and similar damages have been reported in other fish species exposed to Cu NPs [22, 23], TiO2 NPs [24], and other NPs such as cobalt (III) oxide (Co2O3) nanoparticle [24] and colloidal silver nanoparticle [25].If the effect of the joint exposures of nano-SiO with another nanometallic salt of equal ratio (1 : 1) were to be the addition of individual effect, then the HAI of SiO + Al2O3 and SiO + CuO should be 22.0 and 30.0, respectively. The actual combined effects of SiO + Al2O3 and SiO + CuO were HAI: 2.0 and 6.0, respectively. The gill alterations include low frequencies of hypertrophy (HPT) and shortening of secondary gill lamella (SSGL). This implies that joint actions of Al2O3 and CuO with SiO produced a low toxic effect, unlike the high toxicity of their single trials; this also indicates that Al2O3 and CuO are antagonists.Increasing the concentration of SiO in the mixtures (SiO + Al2O3 and SiO + CuO) in ratio 2 to 1 makes no difference in the toxic level. The actual combined effects of SiO + Al2O3 and SiO + CuO were HAI: 8.0 and 6.0, respectively. The gill alterations include low frequencies of gill epithelial hyperplasia (EH) and shortening of secondary gill lamella (SSGL).Increasing the concentrations of Al2O3 and CuO in the mixtures (SiO + Al2O3 and SiO + CuO) in ratio 1 to 2 still had no change in the toxic level for CuO with HAI of 6.0, but there was moderate toxicity for Al2O3 with HAI of 16.0. The gill alterations include moderate frequencies of uncontrolled proliferation of epithelial cells (PEC), gill epithelial lifting (EPL), and necrosis (N).Mixtures of the three nanometallic salts (SiO, Al2O3, and CuO) in ratio 1 : 1 : 1 produced low toxic effect (HAI, 6.0) compared to the addition of the individual effect, assumed to be HAI: 30 or 22. The gills have low frequencies of epithelial lifting (EPL) and shortening of secondary gill lamella (SSGL).


## 3.2. Histopathological Effects and Joint Action of Single and Combined Exposures ofClarias gariepinus on 28-Day Sublethal Concentrations of Bulk Metallic Salts

Tables3 and 4–7 and Figures 1–12 show the histopathological effects and joint action of single and combined exposures of Clarias gariepinus to 28-day sublethal concentrations of bulk metallic salts. Single exposure to bulk SiO, bulk Al2O3, and bulk CuO produced varying effects after 28 days. Bulk CuO was the most toxic, with histopathological alteration index (HAI) of 20.0, followed by bulk Al2O3 (HAI, 10.0) and bulk SiO (HAI, 6.0). The gill alterations include high frequencies of erosion of gill lamella (EGL), hypertrophy (HPT), oedema (OD), stunted gill lamella (SGL), aneurysm (ANS), and necrosis (N).Table 3
A 28-day single and joint exposure ofClarias gariepinus to bulk size metals.
Conc. ratio
Bulk metals
Assumed HAI
Toxic level
Actual HAI
Toxic level
Type of joint effect
Pathology
Singly
CuOSiOAl2O3
20.06.010.0
HighLowModerate
NANANA
NANANA
NANANA
Hypertrophy, EGL, OD, SGL, ANS, necrosis
1 : 1
CuO + SiOCuO + Al2O3
26.030.0
HighHigh
14.032.0
ModerateHigh
AntagonisticAdditive
Hypertrophy, EGL, OD, EPL, ANS, N, TDL
2 : 1
CuO + SiOCuO + Al2O3
26.030.0
HighHigh
6.06.0
LowLow
AntagonisticAntagonistic
EH, necrosisHypertrophy
1 : 2
CuO + SiOCuO + Al2O3
26.030.0
HighHigh
14.08.0
ModerateLow
AntagonisticAntagonistic
EH, oedema
1 : 1 : 1
CuO + SiO + Al2O3
30 or 26
High
12.0
Low
Antagonistic
EGL, SSGL
Toxic level: HAI:X < 10, low; 10 ≥ X < 20, moderate; X ≥ 20, high.Major alterations in fish under acute and chronic exposure to NPs and their bulk are changes in the morphology of the lamellar epithelium. The epithelium layer of secondary lamella becomes oedema with reduced surface area, resulting in asphyxiation. This change might also indicate acute inflammation due to the failure of the epithelial sodium pump [26]. The epithelial oedema and fusion affect toxicokinetics and distribution of pollutants in the gill epithelium [27].Furthermore, changes in the structure of the lamellar epithelium cause changes in the volume of gas and ion exchange [28, 29]. Similar studies by Ostaszewska et al. and Johari et al. [30–32] reported these histological changes such as swelling of goblet cell, villus deformation, hyperplasia, inflammation, necrosis, and vacuoles in the intestinal tissues.If the effect of the joint exposures of bulk CuO with another bulk metallic salt of equal ratio (1 : 1) were to be the addition of individual effect, then the HAI of CuO + SiO and CuO + Al2O3 should be 26.0 and 30.0, respectively. The actual combined effects of CuO + SiO and CuO + Al2O3 were HAI: 14.0 and 32.0, respectively. The gill alterations for CuO + SiO include moderate frequencies of hypertrophy (HPT), erosion of gill lamella (EGL), and gill epithelial lifting (EPL). The gill alterations for CuO + Al2O3 include moderate frequencies of oedema (OD), aneurysm (ANS), total damage to gill lamella (TDL), and necrosis (N). This implies that joint actions of SiO and Al2O3 with CuO produced a low toxic effect, unlike the high toxicity of their single trials; this also indicates that bulk SiO and bulk Al2O3 are antagonists.Increasing the concentration of CuO in the mixtures (CuO + SiO and CuO + Al2O3) in ratio 2 to 1 makes no difference in the toxic level. The actual combined effects of CuO + SiO and CuO + Al2O3 were HAI: 6.0 and 6.0, respectively. The gill alterations include low frequencies of gill epithelial hyperplasia (EH), hypertrophy, and necrosis (N).Increasing the concentrations of SiO and Al2O3 in the mixtures (CuO + SiO and CuO + Al2O3) in ratio 1 to 2 makes no difference in the toxic level for Al2O3. The actual combined effects of CuO + Al2O3 were HAI: 8.0. There was moderate toxicity with SiO with HAI of 14.0. The gill alterations include moderate frequencies of epithelial hyperplasia (EH) and oedema (OD).Mixtures of the three bulk metallic salts (SiO, Al2O3, and CuO) in ratio 1 : 1 : 1 produced low toxic effect (HAI, 12.0) compared to the addition of the individual effect, assumed to be HAI: 26 or 30. The gills have low frequencies of epithelial lifting (EPL) and shortening of secondary gill lamella (SSGL).Aneurysm is one common alteration in this study. This is the swelling of the blood vessel in the gill tissues, which could disturb blood flow [32, 33]. The histological responses in the gills of fish are mostly caused by circulatory disturbances, as aneurism, regressive and progressive changes, and hyperplasia [34, 35].


## 4. Conclusion

The joint actions of Al2O3 and CuO with SiO produced a low toxic effect, unlike the high toxicity of their single trials; this also indicates that Al2O3 and CuO are antagonists. Similarly, among the bulk metal oxides (SiO, Al2O3, and CuO), CuO was the most toxic. The joint actions of SiO and Al2O3 with CuO produced a low toxic effect, unlike the high toxicity of their single exposures. Bulk SiO and bulk Al2O3 are antagonistic on the effects of CuO on the fish gill. There is a need to properly document the ecological implications of nanoparticles in the aquatic environment.



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*Source: 7686597-2019-06-12.xml*