Antagonistic Effects of Sublethal Concentrations of Certain...

Research ArticleAntagonistic Effects of Sublethal Concentrations of CertainMixtures of Metal Oxide Nanoparticles and the Bulk(Al2O3 CuO and SiO2) on Gill Histology in Clarias gariepinus

Amaeze Henry Nnamdi Tam-Miette Dawarri Briggs Oluwaseun Olusola Togundeand Henry Ebele Obanya

Ecotoxicology Unit Department of Zoology University of Lagos Lagos Nigeria

Correspondence should be addressed to Henry Ebele Obanya henryobanyarocketmailcom

Received 28 November 2018 Revised 6 February 2019 Accepted 20 May 2019 Published 12 June 2019

Academic Editor Paresh Chandra Ray

Copyright copy 2019AmaezeHenryNnamdi et al+is is an open access article distributed under the Creative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Background +e effect of nanoparticles (NPs) on aquatic environments is poorly studied Aim +is study evaluates the toxicity ofjoint effects of these different metal nanoparticles and their bulk in mixtures (Al2O3 CuO and SiO2) on fish using histologicalbiomarker Materials and Methods +e bulk and nano sizes of three salts (Al2O3 CuO and SiO2) were used Nanosizes rangedfrom 25 nm to 100 nm +e juvenile fishes of Clarias gariepinus (mean Length 123plusmn 35 cm mean weight 1852plusmn 641 g) wereused for the acute and chronic toxicity tests +ey were exposed to 7mgL each of the bulk and nano sizes of the three metallicoxides either singly or in mixtures for 28 days +e basis for the sublethal concentration was that the 96 hr acute toxicity of thevaried sizes of the three metallic oxides was nontoxic up to the concentrations of 100mgL with no significant mortality at thehighest exposure concentrations +e gills were collected for histopathology Results Of the three metal oxide nanoparticles SiOwas the most toxic with histopathological alteration index (HAI) of 200 followed by nano-CuO (HAI 100) and nano-Al2O3(HAI 20) 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+Al2O3SiO+CuO and SiO+Al2O3 +CuO in equal (1 1mdashHAI 2 and 6 1 1 1mdashHAI 6) and unequal ratios (1 2mdashHAI 16 and 6 2 1mdashHAI 8 and 6) Similarly all bulk combinations were also antagonistic except for the equal ratio of bulk CuO (HAI 20) andbulk Al2O3 (HAI 10) that gave additive effect with HAI of 32 Conclusion +e joint actions of nano Al2O3 and CuO with SiOproduced a low toxic effect unlike the high toxicity of their single trials this also indicates that nano Al2O3 and CuO areantagonists Similarly among the bulk metal oxides (SiO Al2O3 and CuO) CuO was the most toxic Bulk SiO and Al2O3 areantagonistic on the effects of CuO on the fish gill +ere is need to properly document the ecological implications of nanoparticlesin the aquatic environment

1 Introduction

+e rapid development of the nanotechnology industry inthe last 20 years is yet to reach its potential [1] +is hasbeen fuelled by the unusual properties that materialspossess at the nanoscale however it is also that theseproperties are in part fuelling concerns regarding theirpotential toxicity and ecotoxicology +e engineeringmaterial applications of NPs can increase their concen-trations in groundwater soil and surface water which

presents the most significant exposure avenues pathwayand fate for assessing environmental risks [2]

Nanoparticles like silicon oxide have several applica-tions in the preparation of nanocomposites to enhancethermal resistance and electrical and mechanical proper-ties Also it is used in various fields of catalysts pigmentstabilization electronics and sensors Nano-aluminiumoxide is used as raw material in solid rocket propellantformulation and explosives Nano-copper II oxide is usedin biomedical applications such as antimicrobial and

HindawiJournal of NanotechnologyVolume 2019 Article ID 7686597 11 pageshttpsdoiorg10115520197686597

plasmonic materials as a component of reformingcatalysts

Studies associated with the use of NPs are limited [3]Miniaturization of materials is of great interest due to thedifference in their physicochemical properties compared tothe bulk materials +ese properties include colour solu-bility conductivity and catalytic activity of engineerednanomaterials [4] In addition to their increased surface areato volume ratio nanoparticles can serve as contaminantabsorbents

Nanotoxicology explains the concepts of the toxico-logical basis of NPs on health and the environment [5]Several studies have reported the harmful effects of NPs andtheir bulk salts on the biota but presence of other con-taminants and constituent mixture in environment has notbeen fully studied yet +e pollution of the aquatic eco-systems by NPs has been of global concern [6] +esepollutants including nanoparticles could increase the level ofmetals in natural water and seriously affect wetland habitats[7] It was reported that the 95 hr LC10 of various nano-particles for fish ranged from 100 μgLminus1 to 1mgmiddotLminus1 whilethe 95 hr LC50 of NPs reach the mgL range [8] Expectedconcentrations of NPs in surface waters range from μgLminus1 tolow mgmiddotLminus1 [8 9]

+e use of fish histopathological alteration index as a toolfor classification and categorization of stages of tissuechanges relative to stress or chemical exposure has beenreported by several ecotoxicologists and scientists [10ndash13]+is has been used to evaluate the effects of contaminants onthe health of fish in the environment and to help establish acausal relation between exposure to toxic substances and thevarious biological responses [10] +e use of incidence andprevalence of fish diseases as associated with contaminantsas indicators of environmental stress provides a definitivebiological endpoint for the history of exposure [10 13 14]

+e fish gill is very sensitive to environmental changesand is easily affected by pollutants at low concentrations[15] +e gills have a large surface area and perform variousvital functions such as respiration osmoregulation andexcretion Due to the fact that they are external structure indirect contact with the external environment they aresensitive to chemical and physical changes of the aquaticenvironment [16] +e gills are the principal sites for gasexchange and other important functions such as ionic andosmotic regulation in addition to acid-base balance histo-pathological changes in the structure of these organs involverespiratory disturbances and electrolyte imbalance [16]

Mansouri et al [15] have reported the most commonhistopathological anomalies in the gill of common carp suchas hyperplasia oedema curvature fusion aneurism andnecrosis after 10 and 20 days exposure to TiO2 NPs(100mgmiddotLminus1) and CuO NPs (25 and 50mgmiddotLminus1) singly andin mixtures +e mixture effect was reported as synergetic+is study was similar to that of [17] that exposed Carassiusauratus (goldfish) to mixtures of suspensions of 20 nm sizeAl2O3 and 50 nm size ZnO Significant morphological al-teration such as hyperplasia (with fusion of lamellae) wasreported in the gills Combined interactions within themixtures could be antagonistic synergetic or additive

depending on the specific properties and type of oxide NPssuch as size and surface area [17] However there are limitedstudies on the joint effects of nanoparticles and their bulkmetallic salts on the fish using histological biomarker

+e aim of this study is to evaluate the toxicity of jointeffect of different heavy metals in mixtures (Al2O3 CuO andSiO2) on fish using histological biomarker

2 Materials and Methods

21 Experimental Chemicals and Preparation of TestSolutions +e bulk and nano sizes of three salts (Al2O3CuO and SiO2) were procured from Sigma Aldrich +eywere stored at room temperatures in the laboratory beforeuse Nano-Al2O3 appeared as grey coloured powder densityof 270 gcm3 and size of 40 nm with 999 purity Nano-CuO has red to yellow powder density of 894 gml at 25degCwith size lt50 nm SiO2 is a brown yellow powder density of233 gml at 25degC with size lt100 nm

Stock suspensions of the uncoated powder were madeusing dechlorinated tap water to obtain various concen-trations of the test solution Respective grams of the metallicoxides were dissolved in 1 L of the dechlorinated tap water toobtain appropriate mixtures to obtain stock solutions(Figure 1) Literature search was used to obtain the testconcentrations +e concentrations of the test compoundswere prepared as single solutions and mixtures of 1 1 1 22 1 and 1 1 1 both for bulk and nanoscale oxides

22 Experimental Organisms Collection andAcclimatization+e African sharp tooth CatfishAfrican Catfish Clariasgariepinus was used for this study It was selected on thebasis of ease of culture and known responses to pollutantsfrom existing literatures +e fishes were procured from afish farm in Akoka Lagos and transported in open drums tothe Department of Zoology Laboratory Annex at the Bi-ological Garden +e fishes were transported into largeholding tanks (100 L capacity) in the laboratory which werehalf filled with dechlorinated tap water left open for24 hours +e juvenile fishes (mean length 123plusmn 35 cmmean weight 1852plusmn 641 g) were employed for thesublethalchronic toxicity tests Only catfishes of the samebatches were used for the experiment

+e fishes were acclimatized in the lab using the 100 Lcapacity holding tanks at a stocking density of 20 fishes perlitre for the fingerlings and 5 fishes per litter for the juveniles+e acclimatization lasted for 7 days after which the bioassaycommenced +e acclimatization was conducted understandard laboratory conditions (temp 260plusmn 30degC hu-midity 75plusmn 6 photoperiod light dark 12 12 hours) +ebioassay media (dechlorinated tap water) was also of suitablequality (pH 68 DO 85mgL Salinity 00 ppt)

23 Laboratory Bioassay

231 Acute Toxicity Assay +e acute toxicity testrangefinding exercise was conducted over 96 hours with varyingconcentrations in order to achieve mortality +e bioassay

2 Journal of Nanotechnology

containers were made of plastics (6mtimes 4 cmtimes 4 cm) Duringthe acute toxicity test the fingerlings were stocked at 10 fishesper litre of water made up by equivalent concentrations of thestock solution of the bulk and nanoscale heavy metals actingsingly Each setup was in duplicate ie 20 fingerlings perconcentrations

+e fishes were exposed to concentrations of 1mgL10mgL 100mgL 1000mgL and 3000mgL as requiredbased on the response of the test organism +e 96-hour acutetoxicity of the nano and the bulk metals was found to benontoxic up to the concentration of 100mgL and no sig-nificant mortality was found at the highest exposureconcentration

232 Chronic Toxicity Assay +e juvenile catfishes wereemployed in the chronic toxicity tests in which they wereexposed to 7mgL each of the bulk and nano sizes of thethree metallic oxides either singly or in mixtures for 28 days+e chronic toxicity assays were conducted in larger plastics(11 cmtimes 9 cmtimes 75 cm) using 5 L of water because of thelarger sizes of the fishes compared to the acute toxicity testAt the end of the exposure period the fishes were immo-bilized by spinal puncture and dissected to collect the gillsand preserved for histopathological investigation

233 Histopathological Examination +e gills were fixed in10 formalin dehydrated in graded ethanol [18] cleared inxylene embedded in paraffin wax and sectioned at 5 μm ona rotary microtome Slides were stained using the haema-toxylin and eosin technique for light microscopy [18] +ehistopathological changes were evaluated according to [18]and [10] which includes the calculation of the histopath-ological alteration index (HAI) for each fish using the fol-lowing formula

HAI 11113944 I + 1113944 II + 1113944 III (1)

Because I II and III correspond to the number of stagesof change the mean HAI was scored on six-point scale0 = normal tissue 20 =mild damage to the tissue40 =moderate damage to the tissue 60 = partially severedamage to the tissue 80 = severe damage to the tissue10 = irreparable damage to the tissue +ese stages of changeand scoring system or scale are given in Table 1

3 Results and Discussion

31 Histopathological Effects and Joint Action of Single andCombined Exposures of Clarias gariepinus on 28-Day Sub-lethal Concentrations of Nanometallic Salts +e interactionof NPs with other contaminants is dependent on theproperties of the NPs such as size composition mor-phology porosity aggregationdisaggregation and aggregatestructure +e devastating effects of NPs and bulk are mainlydue to the dispersion persistence and bioaccumulation andbiomagnification potentials in addition to their toxicity in thebiological tissues [19]

+e fish gill is the primary target organ affected byNPs Jayaseelan et al and Griffitt et al [20 21] had re-ported physiological alterations such as dysfunction inosmoregulation respiratory gas exchange and body fluidpermeability balance Due to the large superficial area ofthe epithelium per volume ratio the organ is more sus-ceptible to effects of contaminants [22] Histological andbiochemical analysis in previous studies revealed the gillsto be the primary target organ [10 11 21] +e physio-logical alteration could become visible as histologicalalteration as revealed by Griffitt et al [21] who reportedthat NPs produced hypertrophy of epithelial cells in thegills [21]

(a) (b)

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) (HampE stains times400) (b) Histological section of gill of fish exposed to nanosize SiO showing total destructionof primary gill lamella (PGL) and secondary gill lamellae (SGL) eroded epithelium (ERE) and area of severe necrosis (N) and oedema (OD)(HampE stains times100)

Journal of Nanotechnology 3

Table 1 Stages of change and histological alterations of the gill

Alteration score Score description Histological alteration0 Normal tissue No lesion or any alteration (NT)

20 Mild damageMild thickening of gill lamella (GL1)

Epithelial hyperplasia (EH)Hypertrophy (HPT)

40 Moderate damage Moderate thickening of gill lamella (GL2)Oedema (OD)

60 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)

80 Severe damageSevere thickening of gill lamella (GL3)

Complete fusion of secondary lamella (FSGL1)Aneurysm (ANS)

100 Irreparable damage Necrosis (N)Total damage to the lamella (TDL)

Table 2 A 28-day single and joint exposure of Clarias gariepinus to nanosize metals

Conc ratio Nanometals Assumed HAI Toxic level Actual HAI Toxic level Type of joint effect Gill pathology

SinglySiOAl2O3CuO

20020100

HighLow

Moderate

NANANA

NANANA

NANANA

EGL hypertrophyOedemaNecrosis

1 1 SiO+Al2O3SiO+CuO

220300

HighHigh

2060

LowLow

AntagonisticAntagonistic Hypertrophy SSGL


220300

HighHigh

8060

LowLow

AntagonisticAntagonistic EH SSGL


220300

HighHigh

16060

ModerateLow

AntagonisticAntagonistic PEC EPL necrosis

1 1 1 SiO+Al2O3+CuO 30 or 22 High 60 Low Antagonistic EGL SSGLToxic level HAI Xlt 10 low 10geXlt 20 moderate Xge 20 high

(a) (b)

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 (HampE stains times400) (b) Histological section of gillexposed 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 (HampE stains times100)


In this study the most histopathological lesions in fishgills such as hyperplasia oedema curvature shorteningand fusion of gill lamellae aneurism and necrosis aredescribed and quantified using an index as used in severalstudies [10ndash13] Tables 2 and 4ndash7 and Figures 1ndash12 showthe histopathological effects and joint action of single andcombined exposures of Clarias gariepinus to 28-daysublethal concentrations of nanometallic salts Single ex-posure to nano-SiO nano-Al2O3 and nano-CuO producedvarying effects after 28 days Nano-SiO was the most toxicwith histopathological alteration index (HAI) of 200followed by nano-CuO (HAI 100) and nano-Al2O3 (HAI

20) +e gill alterations include high frequencies of ero-sion of gill lamella (EGL) hypertrophy (HPT) oedema(OD) and necrosis (N)

+e histopathological alteration index (HAI) showedthat the gills are affected by nanoparticles of these metaloxides and similar damages have been reported in other fishspecies exposed to Cu NPs [22 23] TiO2 NPs [24] and otherNPs such as cobalt (III) oxide (Co2O3) nanoparticle [24] andcolloidal silver nanoparticle [25]

If the effect of the joint exposures of nano-SiO withanother nanometallic salt of equal ratio (1 1) were to be theaddition of individual effect then the HAI of SiO+Al2O3

(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) (HampE stains times100) (b) Histological section of gill exposed to bulk Al2O3 showing primarygill lamella (PGL) shortened secondary gill lamella (SSGL) hypertrophy (HPT) and some areas of mild necrosis (N) (HampE stains times400)

(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) (HampE stains times400) (b) Histological section of gill exposed tobulk SiOCuO (1 1) showing secondary gill lamella (SGL) hypertrophy (HPT) and epithelial lifting (EPL) (HampE stains times400)


and SiO+CuO should be 220 and 300 respectively +eactual combined effects of SiO+Al2O3 and SiO+CuO wereHAI 20 and 60 respectively +e gill alterations includelow frequencies of hypertrophy (HPT) and shortening ofsecondary gill lamella (SSGL) +is implies that joint actionsof Al2O3 and CuO with SiO produced a low toxic effectunlike the high toxicity of their single trials this also in-dicates 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 nodifference in the toxic level +e actual combined effects of

SiO+Al2O3 and SiO+CuO were HAI 80 and 60 re-spectively +e gill alterations include low frequencies of gillepithelial hyperplasia (EH) and shortening of secondary gilllamella (SSGL)

Increasing the concentrations of Al2O3 and CuO in themixtures (SiO+Al2O3 and SiO+CuO) in ratio 1 to 2 stillhad no change in the toxic level for CuOwith HAI of 60 butthere was moderate toxicity for Al2O3 with HAI of 160 +egill alterations include moderate frequencies of uncontrolledproliferation of epithelial cells (PEC) gill epithelial lifting(EPL) and necrosis (N)

(a) (b)

Figure 5 (a) Histological section of gill exposed to nano-SiOCuO (1 1) showing primary gill lamella (PGL) shortened secondary gilllamella (SSGL) blood vessel oedema of primary lamella and eroded epithelium (HampE stains times100) (b) Histological section of gill exposedto nano-SiOAl2O3 (1 1) showing primary gill lamella (PGL) eroded secondary gill lamella (ESGL) and hypertrophy (HPT) (HampE stainstimes100)

(a) (b)

Figure 6 (a) Histological section of gill exposed to bulk SiOAl2O3 (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 (HampE stains times100) (b)Histological section of gill exposed to bulk Al2O3CuO (1 1) showing primary gill lamella (PGL) total destruction of secondary gill lamellaoedema (OD) aneurysm (ANS) and necrosis (N) (HampE stains times100)


Mixtures of the three nanometallic salts (SiO Al2O3 andCuO) in ratio 1 1 1 produced low toxic effect (HAI 60)compared to the addition of the individual effect assumed tobe HAI 30 or 22 +e gills have low frequencies of epitheliallifting (EPL) and shortening of secondary gill lamella(SSGL)

32 Histopathological Effects and Joint Action of Single andCombined Exposures of Clarias gariepinus on 28-Day Sub-lethal Concentrations of BulkMetallic Salts Tables 3 and 4ndash7and Figures 1ndash12 show the histopathological effects and jointaction of single and combined exposures of Clarias gariepinus

to 28-day sublethal concentrations of bulk metallic saltsSingle exposure to bulk SiO bulk Al2O3 and bulk CuOproduced varying effects after 28 days Bulk CuOwas themosttoxic with histopathological alteration index (HAI) of 200followed by bulk Al2O3 (HAI 100) and bulk SiO (HAI 60)+e gill alterations include high frequencies of erosion of gilllamella (EGL) hypertrophy (HPT) oedema (OD) stunted gilllamella (SGL) aneurysm (ANS) and necrosis (N)

Major alterations in fish under acute and chronic ex-posure to NPs and their bulk are changes in the morphologyof the lamellar epithelium+e epithelium layer of secondarylamella becomes oedemawith reduced surface area resultingin asphyxiation +is change might also indicate acute

(a) (b)

Figure 7 (a) Histological section of gill exposed to nano-SiOAl2O3 (2 1) showing secondary gill lamella (SGL) blood vessel and epithelialhyperplasia (HPS) (HampE stains times400) (b) Histological section of gill exposed to bulk SiOAl2O3 (2 1) showing primary gill lamellashortened secondary gill lamella (SSGL) and blood vessel (BV) (HampE stains times400)

(a) (b)

Figure 8 (a) Histological section of gill exposed to nano-SiOCuO (2 1) showing fused secondary gill lamella (FSGL) secondary gill lamella(SGL) and epithelial hyperplasia (HPS) (HampE stains times400) (b) Histological section of gill exposed to bulk SiOCuO (2 1) showinghypertrophy (HPT) blood vessel (BV) and epithelial hyperplasia (HPS) (HampE stains times400)


inflammation due to the failure of the epithelial sodiumpump [26] +e epithelial oedema and fusion affect tox-icokinetics and distribution of pollutants in the gill epi-thelium [27]

Furthermore changes in the structure of the lamellarepithelium cause changes in the volume of gas and ionexchange [28 29] Similar studies by Ostaszewska et al andJohari et al [30ndash32] reported these histological changessuch as swelling of goblet cell villus deformation hyper-plasia inflammation necrosis and vacuoles in the in-testinal tissues

If the effect of the joint exposures of bulk CuO withanother bulk metallic salt of equal ratio (1 1) were to be theaddition of individual effect then the HAI of CuO+ SiO

and CuO+Al2O3 should be 260 and 300 respectively +eactual combined effects of CuO+ SiO and CuO+Al2O3were HAI 140 and 320 respectively +e gill alterationsfor CuO+ SiO include moderate frequencies of hypertro-phy (HPT) erosion of gill lamella (EGL) and gill epitheliallifting (EPL) +e gill alterations for CuO+Al2O3 includemoderate frequencies of oedema (OD) aneurysm (ANS)total damage to gill lamella (TDL) and necrosis (N) +isimplies that joint actions of SiO and Al2O3 with CuOproduced a low toxic effect unlike the high toxicity of theirsingle trials this also indicates that bulk SiO and bulk Al2O3are antagonists

Increasing the concentration of CuO in the mixtures(CuO + SiO and CuO +Al2O3) in ratio 2 to 1 makes no

(a) (b)

Figure 9 (a) Histological section of gill exposed to bulk Al2O3CuO (2 1) showing oedema (OD) and epithelial hyperplasia (HPS) (HampEstains times400) (b) Histological section of gill exposed to bulk Al2O3CuO (1 2) showing primary gill lamella (PGL) epithelial lifting (EPL)and hypertrophy (HPT) (HampE stains times400)

(a) (b)

Figure 10 (a) Histological section of gill exposed to nano-Al2O3CuO (1 2) showing primary gill lamella (PGL) curved secondary gilllamella (CSGL) and shortened secondary gill lamella (SSGL) (HampE stains times400) (b) Histological section of gill exposed to nano-SiOCuO(1 2) showing secondary gill lamella (SGL) inflammation of lamella (INF) and blood vessel (HampE stains times400)


difference in the toxic level +e actual combined effects ofCuO + SiO and CuO +Al2O3 were HAI 60 and 60 re-spectively +e gill alterations include low frequenciesof gill epithelial hyperplasia (EH) hypertrophy and ne-crosis (N)

Increasing the concentrations of SiO and Al2O3 in themixtures (CuO+ SiO and CuO+Al2O3) in ratio 1 to 2makesno difference in the toxic level for Al2O3 +e actual com-bined effects of CuO+Al2O3 were HAI 80 +ere wasmoderate toxicity with SiO with HAI of 140 +e gill

alterations include moderate frequencies of epithelial hy-perplasia (EH) and oedema (OD)

Mixtures of the three bulk metallic salts (SiO Al2O3 andCuO) in ratio 1 1 1 produced low toxic effect (HAI 120)compared to the addition of the individual effect assumed tobe HAI 26 or 30 +e gills have low frequencies of epitheliallifting (EPL) and shortening of secondary gill lamella(SSGL)

Aneurysm is one common alteration in this study+is isthe swelling of the blood vessel in the gill tissues which could

(a) (b)

Figure 11 (a) Histological section of gill exposed to bulk SiOCuO (1 2) showing epithelial hyperplasia (HPS) necrosis (N) and hy-pertrophy (HPT) (HampE stains times400) (b) Histological section of gill exposed to bulk SiOAl2O3 (1 2) showing mild necrosis (N) andsecondary gill lamella (SGL) No serious damage is observed (HampE stains times400)

(a) (b)

Figure 12 (a) Histological section of gill exposed to nano-SiOAl2O3 (1 2) showing curved secondary gill lamella (CSGL) epithelial lifting(EPL) and hypertrophy (HPT) (HampE stains times400) (b) Histological section of gill exposed to nano-SiOAl2O3CuO showing total de-struction of gill architecture Blood vessel is seen (HampE stains times400)


disturb blood flow [32 33] +e histological responses in thegills of fish are mostly caused by circulatory disturbances asaneurism regressive and progressive changes and hyper-plasia [34 35]

4 Conclusion

+e joint actions of Al2O3 and CuOwith SiO produced a lowtoxic effect unlike the high toxicity of their single trials thisalso indicates that Al2O3 and CuO are antagonists Similarlyamong the bulk metal oxides (SiO Al2O3 and CuO) CuOwas the most toxic +e joint actions of SiO and Al2O3 withCuO produced a low toxic effect unlike the high toxicity oftheir single exposures Bulk SiO and bulk Al2O3 are an-tagonistic on the effects of CuO on the fish gill +ere is aneed to properly document the ecological implications ofnanoparticles in the aquatic environment

Data Availability

All data have been provided in the manuscript For anyfurther data that may be needed contact the correspondingauthor

Conflicts of Interest

+e authors declare that they have no conflicts of interest

Supplementary Materials

Tables 4ndash7 list data used in calculating and estimating theresults summarised in Tables 2 and 3 (SupplementaryMaterials)

References

[1] K Savolainen H Alenius H Norppa L PylkkanenT Tuomi and G Kasper ldquoRisk assessment of engineerednanomaterials and nanotechnologiesmdasha reviewrdquo Toxicologyvol 269 no 2-3 pp 92ndash104 2010

[2] M Farre K Gajda-Schrantz L Kantiani and D BarceloldquoEcotoxicity and analysis of nanomaterials in the aquaticenvironmentrdquo Analytical and Bioanalytical Chemistryvol 393 no 1 pp 81ndash95 2009

[3] P Christian F Von der Kammer M Baalousha andT Hofmann ldquoNanoparticles structure properties prepara-tion and behaviour in environmental mediardquo Ecotoxicologyvol 17 no 5 pp 326ndash343 2008

[4] S W Wijnhoven S Dekkers M Kooi W P Jongeneel andW H Jong Nanomaterials in Consumer Products Update ofProducts on the European Market in 2010 National Institutefor Public Health and the Environment Netherlands 2010

[5] R D Handy N van den Brink M Chappell et al ldquoPracticalconsiderations for conducting ecotoxicity test methods withmanufactured nanomaterials what have we learnt so farrdquoEcotoxicology vol 21 no 4 pp 933ndash972 2012

[6] D G McNeil and J Fredberg ldquoEnvironmental water re-quirements of native fishes in the middle river catchmentkangaroo island south Australiamdasha report to the SA de-partment for waterrdquoAquatic Sciences vol 528 pp 50ndash57 2011

[7] G B Yu Y Liu S Yu et al ldquoInconsistency and compre-hensiveness of risk assessments for heavy metals in urbansurface sedimentsrdquo Chemosphere vol 85 no 6 pp 1080ndash1087 2011

[8] R D Handy T B Henry T M Scown B D Johnston andC R Tyler ldquoManufactured nanoparticles their uptake andeffects on fish-a mechanistic analysisrdquo Ecotoxicology vol 17no 5 pp 396ndash409 2008

[9] F Gottschalk T Sonderer R W Scholz and B NowackldquoPossibilities and limitations of modeling environmentalexposure to engineered nanomaterials by probabilistic ma-terial flow analysisrdquo Environmental Toxicology and Chemistryvol 29 pp 1036ndash1048 2010

[10] V Poleksic M Lenhardt I Jaric et al ldquoLiver gills and skinhistopathology and heavy metal content of the Danube sterlet(Acipenser ruthenus Linnaeus 1758)rdquo Environmental Toxi-cology and Chemistry vol 29 no 3 pp 515ndash521 2010

[11] J K Saliu S A Oluberu I I Akpoke and U D UkwaldquoCortisol stress response and Histopathological AlterationIndex in Clarias gariepinus exposed to sub-lethal concen-trations of Qua Iboe crude oil and rig washrdquo African Journalof Aquatic Science vol 42 no 1 pp 55ndash64 2016

[12] B Akinsanya O U Utoh and U D Ukwa ldquoToxicologicalphytochemical and anthelminthic properties of rich plantextracts on Clarias gariepinusrdquo Journal of Basic amp AppliedZoology vol 74 pp 75ndash86 2016

[13] U D Ukwa J K Saliu and A O Osibona ldquoCombined effectsof intestinal infestation and extrinsic stress on host energy inMalapterurus electricushost-parasite system in Lekki LagoonNigeriardquo Iranian Journal of Ichthyology vol 5 no 1pp 43ndash54 2018

Table 3 A 28-day single and joint exposure of Clarias gariepinus to bulk size metals

Concratio Bulk metals Assumed

HAIToxiclevel

ActualHAI

Toxiclevel

Type of jointeffect Pathology

SinglyCuOSiOAl2O3

20060100

HighLow

Moderate

NANANA

NANANA

NANANA

Hypertrophy EGL OD SGL ANSnecrosis

1 1 CuO+ SiOCuO+Al2O3

260300

HighHigh

140320

ModerateHigh

AntagonisticAdditive

Hypertrophy EGL OD EPL ANS NTDL


260300

HighHigh

6060

LowLow

AntagonisticAntagonistic

EH necrosisHypertrophy


260300

HighHigh

14080

ModerateLow

AntagonisticAntagonistic EH oedema

1 1 1 CuO+ SiO+Al2O3 30 or 26 High 120 Low Antagonistic EGL SSGLToxic level HAI Xlt 10 low 10geXlt 20 moderate Xge 20 high


[14] J K Saliu B Akinsanya U D Ukwa J Odozie and Y GaniuldquoHost condition parasite interaction and metal accumulationin Tilapia guineensis from iddo area of Lagos lagoon NigeriardquoIranian Journal of Ichthyology vol 1 no 4 pp 286ndash295 2014

[15] B Mansouri A Maleki S A Johari B ShahmoradiE Mohammadi and B Davari ldquoHistopathological effects ofcopper oxide nanoparticles on the gill and intestine ofcommon carp (Cyprinus carpio) in the presence of titaniumdioxide nanoparticlesrdquo Chemistry and Ecology vol 33 no 4pp 295ndash308 2017

[16] C C C Cerqueira and M N Fernandes ldquoGill tissue recoveryafter copper exposure and blood parameter responses in thetropical fish Prochilodus scrofardquo Ecotoxicology and Environ-mental Safety vol 52 no 2 pp 83ndash91 2002

[17] M Benavides P Coelho C Lodeiro and M S Diniz ldquoEffectsof exposure of oxides of nanoparticles (Al2O3 and ZnO) singlyand mixtures on Carassius auratus gillsrdquo Microscopy andMicroanalysis vol 21 no 6 pp 18-19 2015

[18] J Schwaiger R Wanke S AdamM Pawert W Honnen andR Triebskorn ldquo+e use of histopathological indicators toevaluate contaminant-related stress in fishrdquo Journal ofAquatic Ecosystem Stress and Recovery vol 6 no 1 pp 75ndash861997

[19] M Golobic A Jemec D Drobne T Romih K Kasemets andA Kahru ldquoUpon exposure to Cu nanoparticles accumulationof copper in the isopod Porcellio scaber is due to the dissolvedCu ions inside the digestive tractrdquo Environmental Science andTechnology vol 46 no 21 pp 12112ndash12119 2012

[20] C Jayaseelan A Abdul Rahuman R Ramkumar et al ldquoEffectof sub-acute exposure to nickel nanoparticles on oxidativestress and histopathological changes in Mozambique tilapiaOreochromis mossambicusrdquo Ecotoxicology and EnvironmentalSafety vol 107 pp 220ndash228 2014

[21] R J Griffitt R Weil K A Hyndman et al ldquoExposure tocopper nanoparticles causes gill injury and acute lethality inzebrafish (Danio rerio)rdquo Environmental Science amp Technologyvol 41 no 23 pp 8178ndash8186 2007

[22] A Maleki N A Azadi B Mansouri F Majnoni Z Rezaeiand F Gharibi ldquoHealth risk assessment of trace elements intwo fish species of Sanandaj Gheshlagh Reservoir IranrdquoToxicology and Environmental Health Sciences vol 7 no 1pp 43ndash49 2015

[23] G A Al-Bairuty B J Shaw and R D Handy Histopatho-logical effects of metal and metallic nanoparticles on the bodysystems of rainbow trout (Oncorhynchus mykiss) PhD thesisSchool of Biomedical and Biological Sciences University ofPlymouth Plymouth UK 2013

[24] L Song M G Vijver W J G M Peijnenburg T S Gallowayand C R Tyler ldquoA comparative analysis on the in vivo toxicityof copper nanoparticles in three species of freshwater fishrdquoChemosphere vol 139 pp 181ndash189 2015

[25] G Federici B Shaw and R Handy ldquoToxicity of titaniumdioxide nanoparticles to rainbow trout (Oncorhynchusmykiss) gill injury oxidative stress and other physiologicaleffectsrdquo Aquatic Toxicology vol 84 no 4 pp 415ndash430 2007

[26] B Mansouri and S A Johari ldquoEffects of short-term exposureto sublethal concentrations of silver nanoparticles on histo-pathology and electron microscope ultrastructure of zebrafish(Danio Rerio) Gillsrdquo Iranian Journal of Toxicology vol 10no 1 pp 15ndash20 2016

[27] R N Mitchell and R S Cotran ldquoCell injury adaptation anddeathrdquo in Robbins Basic Pathology V Kumar R S Cotranand S L Robbins Eds pp 3ndash32 Saunders New Delhi India2000

[28] R Bhuvaneshwari K Padmanaban and R Babu RajendranldquoHistopathological alterations in muscle liver and gill tissuesof zebrafish Danio rerio due to environmentally relevantconcentrations of organchlorine pesticides (OCPs) and heavymetalsrdquo International Journal of Environmental Researchvol 9 pp 1365ndash1372 2015

[29] W Jiraungkoorskul E S Upathama and M KruatrachuealdquoHistopathological effects of roundup a glyphosate herbicideon Nile tilapia (Oreochromis niloticus)rdquo Science Asia vol 28pp 121ndash127 2002

[30] T Ostaszewska M Chojnacki M Kamaszewski andE Sawosz-Chwalibog ldquoHistopathological effects of silver andcopper nanoparticles on the epidermis gills and liver ofSiberian sturgeonrdquo Environmental Science and PollutionResearch vol 23 no 2 pp 1621ndash1633 2016

[31] H Kaya F Aydın M Gurkan et al ldquoA comparative toxicitystudy between small and large size zinc oxide nanoparticles intilapia (Oreochromis niloticus) organ pathologies osmoreg-ulatory responses and immunological parametersrdquo Chemo-sphere vol 144 pp 571ndash582 2016

[32] S A Johari M R Kalbassi I J Yu and J H Lee ldquoChroniceffect of waterborne silver nanoparticles on rainbow trout(Oncorhynchus mykiss) histopathology and bioaccumulationrdquoComparative Clinical Pathology vol 24 no 5 pp 995ndash10072015

[33] F Flores-Lopes and A +omaz ldquoHistopathologic alterationsobserved in fish gills as a tool in environmental monitoringrdquoBrazilian Journal of Biology vol 71 no 1 pp 179ndash188 2011

[34] K S Rajkumar N Kanipandian and R +irumuruganldquoToxicity assessment on haemotology biochemical and his-topathological alterations of silver nanoparticles-exposedfreshwater fish Labeo rohitardquo Applied Nanoscience vol 6no 1 pp 19ndash29 2012

[35] J C Van Dyk M J Marchand G M PieterseI E Barnhoorn and M S Bornman ldquoHistological changes inthe gills ofClarias gariepinus (Teleostei clariidae) from apolluted South African urban aquatic systemrdquoAfrican Journalof Aquatic Science vol 34 no 3 pp 283ndash291 2009


CorrosionInternational Journal of

Hindawiwwwhindawicom Volume 2018

Advances in

Materials Science and EngineeringHindawiwwwhindawicom Volume 2018


Journal of

Chemistry

Analytical ChemistryInternational Journal of


ScienticaHindawiwwwhindawicom Volume 2018

Polymer ScienceInternational Journal of



Advances in Condensed Matter Physics


International Journal of

BiomaterialsHindawiwwwhindawicom

Journal ofEngineeringVolume 2018

Applied ChemistryJournal of


NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of


High Energy PhysicsAdvances in

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

TribologyAdvances in



ChemistryAdvances in


Advances inPhysical Chemistry


BioMed Research InternationalMaterials

Journal of


Na

nom

ate

ria

ls


Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom

plasmonic materials as a component of reformingcatalysts

Studies associated with the use of NPs are limited [3]Miniaturization of materials is of great interest due to thedifference in their physicochemical properties compared tothe bulk materials +ese properties include colour solu-bility conductivity and catalytic activity of engineerednanomaterials [4] In addition to their increased surface areato volume ratio nanoparticles can serve as contaminantabsorbents

Nanotoxicology explains the concepts of the toxico-logical basis of NPs on health and the environment [5]Several studies have reported the harmful effects of NPs andtheir bulk salts on the biota but presence of other con-taminants and constituent mixture in environment has notbeen fully studied yet +e pollution of the aquatic eco-systems by NPs has been of global concern [6] +esepollutants including nanoparticles could increase the level ofmetals in natural water and seriously affect wetland habitats[7] It was reported that the 95 hr LC10 of various nano-particles for fish ranged from 100 μgLminus1 to 1mgmiddotLminus1 whilethe 95 hr LC50 of NPs reach the mgL range [8] Expectedconcentrations of NPs in surface waters range from μgLminus1 tolow mgmiddotLminus1 [8 9]

+e use of fish histopathological alteration index as a toolfor classification and categorization of stages of tissuechanges relative to stress or chemical exposure has beenreported by several ecotoxicologists and scientists [10ndash13]+is has been used to evaluate the effects of contaminants onthe health of fish in the environment and to help establish acausal relation between exposure to toxic substances and thevarious biological responses [10] +e use of incidence andprevalence of fish diseases as associated with contaminantsas indicators of environmental stress provides a definitivebiological endpoint for the history of exposure [10 13 14]

+e fish gill is very sensitive to environmental changesand is easily affected by pollutants at low concentrations[15] +e gills have a large surface area and perform variousvital functions such as respiration osmoregulation andexcretion Due to the fact that they are external structure indirect contact with the external environment they aresensitive to chemical and physical changes of the aquaticenvironment [16] +e gills are the principal sites for gasexchange and other important functions such as ionic andosmotic regulation in addition to acid-base balance histo-pathological changes in the structure of these organs involverespiratory disturbances and electrolyte imbalance [16]

Mansouri et al [15] have reported the most commonhistopathological anomalies in the gill of common carp suchas hyperplasia oedema curvature fusion aneurism andnecrosis after 10 and 20 days exposure to TiO2 NPs(100mgmiddotLminus1) and CuO NPs (25 and 50mgmiddotLminus1) singly andin mixtures +e mixture effect was reported as synergetic+is study was similar to that of [17] that exposed Carassiusauratus (goldfish) to mixtures of suspensions of 20 nm sizeAl2O3 and 50 nm size ZnO Significant morphological al-teration such as hyperplasia (with fusion of lamellae) wasreported in the gills Combined interactions within themixtures could be antagonistic synergetic or additive

depending on the specific properties and type of oxide NPssuch as size and surface area [17] However there are limitedstudies on the joint effects of nanoparticles and their bulkmetallic salts on the fish using histological biomarker

+e aim of this study is to evaluate the toxicity of jointeffect of different heavy metals in mixtures (Al2O3 CuO andSiO2) on fish using histological biomarker

2 Materials and Methods

21 Experimental Chemicals and Preparation of TestSolutions +e bulk and nano sizes of three salts (Al2O3CuO and SiO2) were procured from Sigma Aldrich +eywere stored at room temperatures in the laboratory beforeuse Nano-Al2O3 appeared as grey coloured powder densityof 270 gcm3 and size of 40 nm with 999 purity Nano-CuO has red to yellow powder density of 894 gml at 25degCwith size lt50 nm SiO2 is a brown yellow powder density of233 gml at 25degC with size lt100 nm

Stock suspensions of the uncoated powder were madeusing dechlorinated tap water to obtain various concen-trations of the test solution Respective grams of the metallicoxides were dissolved in 1 L of the dechlorinated tap water toobtain appropriate mixtures to obtain stock solutions(Figure 1) Literature search was used to obtain the testconcentrations +e concentrations of the test compoundswere prepared as single solutions and mixtures of 1 1 1 22 1 and 1 1 1 both for bulk and nanoscale oxides

22 Experimental Organisms Collection andAcclimatization+e African sharp tooth CatfishAfrican Catfish Clariasgariepinus was used for this study It was selected on thebasis of ease of culture and known responses to pollutantsfrom existing literatures +e fishes were procured from afish farm in Akoka Lagos and transported in open drums tothe Department of Zoology Laboratory Annex at the Bi-ological Garden +e fishes were transported into largeholding tanks (100 L capacity) in the laboratory which werehalf filled with dechlorinated tap water left open for24 hours +e juvenile fishes (mean length 123plusmn 35 cmmean weight 1852plusmn 641 g) were employed for thesublethalchronic toxicity tests Only catfishes of the samebatches were used for the experiment

+e fishes were acclimatized in the lab using the 100 Lcapacity holding tanks at a stocking density of 20 fishes perlitre for the fingerlings and 5 fishes per litter for the juveniles+e acclimatization lasted for 7 days after which the bioassaycommenced +e acclimatization was conducted understandard laboratory conditions (temp 260plusmn 30degC hu-midity 75plusmn 6 photoperiod light dark 12 12 hours) +ebioassay media (dechlorinated tap water) was also of suitablequality (pH 68 DO 85mgL Salinity 00 ppt)

23 Laboratory Bioassay

231 Acute Toxicity Assay +e acute toxicity testrangefinding exercise was conducted over 96 hours with varyingconcentrations in order to achieve mortality +e bioassay






HAI 11113944 I + 1113944 II + 1113944 III (1)





(a) (b)


















SinglySiOAl2O3CuO

20020100

HighLow

Moderate

NANANA

NANANA

NANANA



220300

HighHigh

2060

LowLow



220300

HighHigh

8060

LowLow



220300

HighHigh

16060

ModerateLow



(a) (b)







(a) (b)


(a) (b)







(a) (b)


(a) (b)







(a) (b)


(a) (b)








(a) (b)


(a) (b)








(a) (b)


(a) (b)




4 Conclusion


Data Availability






References
















HAIToxiclevel

ActualHAI

Toxiclevel


SinglyCuOSiOAl2O3

20060100

HighLow

Moderate

NANANA

NANANA

NANANA



260300

HighHigh

140320

ModerateHigh




260300

HighHigh

6060

LowLow




260300

HighHigh

14080

ModerateLow





























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls








HAI 11113944 I + 1113944 II + 1113944 III (1)





(a) (b)


















SinglySiOAl2O3CuO

20020100

HighLow

Moderate

NANANA

NANANA

NANANA



220300

HighHigh

2060

LowLow



220300

HighHigh

8060

LowLow



220300

HighHigh

16060

ModerateLow



(a) (b)







(a) (b)


(a) (b)







(a) (b)


(a) (b)







(a) (b)


(a) (b)








(a) (b)


(a) (b)








(a) (b)


(a) (b)




4 Conclusion


Data Availability






References
















HAIToxiclevel

ActualHAI

Toxiclevel


SinglyCuOSiOAl2O3

20060100

HighLow

Moderate

NANANA

NANANA

NANANA



260300

HighHigh

140320

ModerateHigh




260300

HighHigh

6060

LowLow




260300

HighHigh

14080

ModerateLow





























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls



















SinglySiOAl2O3CuO

20020100

HighLow

Moderate

NANANA

NANANA

NANANA



220300

HighHigh

2060

LowLow



220300

HighHigh

8060

LowLow



220300

HighHigh

16060

ModerateLow



(a) (b)







(a) (b)


(a) (b)







(a) (b)


(a) (b)







(a) (b)


(a) (b)








(a) (b)


(a) (b)








(a) (b)


(a) (b)




4 Conclusion


Data Availability






References
















HAIToxiclevel

ActualHAI

Toxiclevel


SinglyCuOSiOAl2O3

20060100

HighLow

Moderate

NANANA

NANANA

NANANA



260300

HighHigh

140320

ModerateHigh




260300

HighHigh

6060

LowLow




260300

HighHigh

14080

ModerateLow





























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls








(a) (b)


(a) (b)







(a) (b)


(a) (b)







(a) (b)


(a) (b)








(a) (b)


(a) (b)








(a) (b)


(a) (b)




4 Conclusion


Data Availability






References
















HAIToxiclevel

ActualHAI

Toxiclevel


SinglyCuOSiOAl2O3

20060100

HighLow

Moderate

NANANA

NANANA

NANANA



260300

HighHigh

140320

ModerateHigh




260300

HighHigh

6060

LowLow




260300

HighHigh

14080

ModerateLow





























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls








(a) (b)


(a) (b)







(a) (b)


(a) (b)








(a) (b)


(a) (b)








(a) (b)


(a) (b)




4 Conclusion


Data Availability






References
















HAIToxiclevel

ActualHAI

Toxiclevel


SinglyCuOSiOAl2O3

20060100

HighLow

Moderate

NANANA

NANANA

NANANA



260300

HighHigh

140320

ModerateHigh




260300

HighHigh

6060

LowLow




260300

HighHigh

14080

ModerateLow





























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls








(a) (b)


(a) (b)








(a) (b)


(a) (b)








(a) (b)


(a) (b)




4 Conclusion


Data Availability






References
















HAIToxiclevel

ActualHAI

Toxiclevel


SinglyCuOSiOAl2O3

20060100

HighLow

Moderate

NANANA

NANANA

NANANA



260300

HighHigh

140320

ModerateHigh




260300

HighHigh

6060

LowLow




260300

HighHigh

14080

ModerateLow





























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls









(a) (b)


(a) (b)








(a) (b)


(a) (b)




4 Conclusion


Data Availability






References
















HAIToxiclevel

ActualHAI

Toxiclevel


SinglyCuOSiOAl2O3

20060100

HighLow

Moderate

NANANA

NANANA

NANANA



260300

HighHigh

140320

ModerateHigh




260300

HighHigh

6060

LowLow




260300

HighHigh

14080

ModerateLow





























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls









(a) (b)


(a) (b)




4 Conclusion


Data Availability






References
















HAIToxiclevel

ActualHAI

Toxiclevel


SinglyCuOSiOAl2O3

20060100

HighLow

Moderate

NANANA

NANANA

NANANA



260300

HighHigh

140320

ModerateHigh




260300

HighHigh

6060

LowLow




260300

HighHigh

14080

ModerateLow





























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls





4 Conclusion


Data Availability






References
















HAIToxiclevel

ActualHAI

Toxiclevel


SinglyCuOSiOAl2O3

20060100

HighLow

Moderate

NANANA

NANANA

NANANA



260300

HighHigh

140320

ModerateHigh




260300

HighHigh

6060

LowLow




260300

HighHigh

14080

ModerateLow





























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls





























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls




Antagonistic Effects of Sublethal Concentrations of Certain...

Documents

Transcript of Antagonistic Effects of Sublethal Concentrations of Certain...