Histopathological Markers for Copper Toxicity in Rainbow Trout Fry (Oncorhynchus mykiss

Basrah J. Agric. Sci. 25 (special issued 2), 2012

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Histopathological Markers for Copper Toxicity in Rainbow Trout

Fry (Oncorhynchus mykiss)

Nader A. Salman

1, Jeffery L. Ullman

2, Kevin Snekvik

3, Xiao Q. Lu

2

1Department of Fisheries, College of Agriculture, University of Basrah, Iraq; 2Department of biological Systems Engineering, Washington State university, USA; 3College of Veterinary

Medicine, Washington State University, Pullman, USA.

Abstract. One month old fry of rainbow trout (Oncorhynchus mykiss) of 0.5 g weight were exposed to three levels

of CuSO4.5H20 at 100, 200 and 400 ppb Cu for 24, 48 , 72 and 96 h periods. Samples for histological sections were

taken and prepared for microscopic examination using the standard methods. Due to the small size of the fry, the

whole fish was sectioned longitudinally. Four selected organs were examined including gills, liver, kidney and

alimentary canal. Gills showed mark changes of lamellar hypertrophy appeared as fusion of secondary lamella and

formation of bud-like structures filled with epithelial cells. Hyperplasia and lifting of epithelial cells were also

noticed in the high dose media. Signs of necrosis appeared in fish kept at 200 and 400 ppb Cu exposures.

Hepatocytes showed less alteration represented by morphological deformities especially in the nuclei which has no

crisp shape. Granular cytoplasm was commonly seen. Its relationship with toxicity needs to be confirmed. Up-

normal red blood cells and signs of autolysis was also noticed. Mild changes in both tubules and glomeruli of the

head and rear kidneys were noticed. Evidence of pykcnotic and degeneration of the tubule epithelial cells was seen

in fish exposed to the highest copper concentration. Macrophages along the capillary were also noticed. Some

alteration in tubule structure is mostly related to hypoxia. The less affected organ was the alimentary canal which

showed minor changes mostly in the upper and middle part. Degeneration of epithelial cells which appeared as

vacuoles in the stomach proximal region was noticed. Pyknosis due to starvation was apparent as well. It is

concluded that histopathological signs should be considered among other biomarkers as far as Cu-toxicity is

concerned.

Key Words: Histopathology. Copper toxicity, rainbow trout fry

Corresponding author: [email protected]

Introduction

The contamination of fresh waters with a wide range of pollutants has become a matter of

concern over the last few decades (39). Increased human activities especially with rapid

development of agriculture and industry has resulted in a considerable increase in levels of

pollutant such as heavy metals ( 25). Heavy metals occur naturally in the environment and are

found in varying levels in the ground and surface water. Industrial pollution and anthropogenic

activities do, however, cause an increased discharge of these metals into natural aquatic

ecosystems. Sometimes, aquatic organisms are exposed to unnaturally high levels of these

metals. Fish are relatively sensitive to changes in their surroundings environment. Therefore,

heavy metals, have been recognized as strong biological poisons because of their persistent

nature, toxicity ,tendency to accumulate in organisms and undergo food chain amplification (15,

10). Fish health may therefore reflect and give a good indication of the health status of a specific

aquatic ecosystem.

Copper can exert adverse toxicological effects, when present in high concentrations in

water (32). In fact, it is potentially toxic when the internal available concentration exceeds the

mailto:[email protected]


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capacity of physiological detoxification processes. High concentrations of this heavy metal were

detected in some aquatic ecosystems collecting vineyard runoff water and it is also highly

concentrated in ground water (12) (36). There are also anthropogenic sources of environmental

contamination by copper including mining, smelting, foundries, municipal waste incinerators,

burning of coal for power generation and a variety of copper-based products used in building and

construction (26)(43).

Fishes exposed to toxicants undergo stress, which is a state of re-established

homeostasis. Fish are responding to various stressors by a series of biochemical and

physiological stress reactions, so called secondary stress responses comparable to those of higher

vertebrates (22). Biochemical and physiological biomarkers are frequently used for detecting or

diagnosing sublethal effects in fish exposed to different toxic substances (37). The toxicity of

Cu2 is a stress factor for fish, but the physiological responses to stress can be alleviated by a

variety of acclimation mechanisms (23). Usually, the process of Cu2 accumulation is

characterized as a damage-repair model (19), and it includes three phases: an initial shock phase,

a recovery phase, and then acclimation itself. The last phase includes increased tolerance. The

initial shock phase corresponds to physiological regulation. The damage phase is usually short

lived (a few days) and consists of assorted disturbances to internal physiological homeostasis.

The toxicity tests are, therefore, necessary in water pollution evaluation because chemical and

physical measurements alone are not sufficient to assess potential effects on aquatic biota (35).

It has been noted that heavy metals had a negative impact on all relevant parameters and

caused histo-pathological changes in fish. After conducting a survey on Poecilia reticulata

(guppy) and Oryzias latipes (medaka) to examine the usefulness of histopathology for the

identification of toxic effects of environmental contaminants in fish (42), they concluded that

histopathology provides useful data in characterizing toxic effects in fish. Histological study

appears to be a very sensitive parameter and is crucial in determining cellular changes that may

occur in target organs, such as the gills, liver, kidney and gut (1). Sub-lethal copper exposure

may cause damage to numerous tissues and organs, including liver, gills, kidneys. If any of these

changes can be related to exposure concentration or time and show enough specificity it can be

used as a biomarker (33). The aim of this research is to evaluate the usefulness of histological

biomarkers in gills, kidney and gut for toxicity of acute exposure of rainbow trout fry to copper.

Materials and Methods

Copper exposure trials

Copper stock solution used in the toxicity tests was prepared by dissolving Cu2SO4.5H2O into a

desirable concentration using Milli Q water. Exposure of rainbow trout fry was conducted in

glass aquaria (28 x 72 x 42 cm) under controlled temperature (13 ± 1 oC) and artificial aeration.

Forty randomly chosen rainbow trout fry raised in our fish laboratory were acclimatized to each

test aquarium for 24h with aeration. Afterwards, the fish were exposed to copper by spiking the

metal with different concentrations (0, 100, 200 and 400µg/L) into the aquarium.

Food was not provided during the acclimation and in the course of exposures. Copper

concentration in water was analysed after copper stock solution was spiked into the aquarium

and was then analysed after the exposure experiment was completed. At the end of the exposure

(24, 48, 72 and 96 h), the whole fish were anaesthetised using a solution containing 0.1% of

Tricaine anesthetics and then fixed in 10% phosphate buffered formalin for histological

examination.


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Preparation of histological sections

The whole larva were fixed in Bouin's fluid for 24 h, dehydrated in graded ethanol

concentrations, cleared in xylene and infiltrated with paraffin using Tissue Tek II automatic

tissue processor. They were embedded in paraffin using Shandon Histocentre embedding

machine. Sections (5m) were cut using a Microm microtome.

The sections were attached to slides and stained with haematoxylin and eosin (H&E)

using Shandon Linistain GLX automatic staining machine. The slides were examined using

Leica DMLS microscope and photographs were taken using Wild Leitz MPS46/52 photoautomat

using magnification x 100 for scanning all the slides and x 400 for examination of any

pathological changes found. Presence of histological changes and their severity were determined

for each fish. All histological processing and examination of slides were performed at the

College of Veterinary Medicine, Washington State University.

Results

1. Gills

The gills of rainbow trout fry of the control group revealed normal structures of developing gill

filaments and well organized secondary lamella with normal epithelial cells as seen in Fig.1

under low and high magnification. Fry exposed to 100 ppb Cu showed mild fusion in the gills

and hyperplasia of some secondary lamella (Fig. 2). Under exposure to 200 ppb Cu, gills of

rainbow trout fry began to show signs of stress by revealing fused and curved secondary Lamella

filled with epithelial cells as a sign of hyperplasia (Fig.3).

The most severe cases appeared in fish fry exposed to 400 ppb Cu as they showed fused

and curved secondary lamella filled with epithelial cells, desquamation of the epithelial lining,

telangiectasia, haemoragic and hyperplasia at the secondary lamellae. Necrosis and presence of

necrotic blood cells in the vessels were another reasons of inflammatory signs. Furthermore, the

gills were losing the normal ordination due to epithelial lifting (Figs. 4 and 5).

To understand the effect of exposure time to mild levels of copper (100 and 200 ppb Cu)

on rainbow trout fry, slides of fish exposed for a prolonged period of time (96h) were also

examined. Gills of those fish suffered from cell swelling leading to hypertrophy and mild

necrosis, in addition to the presence of few necrotic cells (Fig. 6).

Those finding reflect that prolonged exposure to mild concentrations of copper can also

resulted in stressful effects on the fry’s gills.


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Fig.1:a Fig.1:b

Fig. (1): a Normal larvae gill in the Control treatment after 24 h at low magnification H &

E (100x) to show the normal structure of the gills. B: Normal structure of the larval

gills of the control group under higher magnification H & E (400x) to show the

secondary lamella looking like buds.

Fig. (2): Larval gills at 100 ppb Cu for 24h under H&T (400x)to show mild changes with

Individual (small numbers) cell swelling causing hypertrophy but no increase in

numbers and mild necrosis.


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Fig. (3): Gills of trout fry exposed to 200 ppb for 24 h under H&E (400x) showing more

changes of mild Necrosis, Hypertrophy and 5% hyperplasia.

Fig. 4:a Fig. 4:b

Fig. (4): Gills of larvae exposed to 400 ppb Cu after 4 h under H & E (400x) showing in a:

fusion of secondary lamella SL, filled with epithelial cells leading to hyperplasia. Dead

epithelial cells are noticed with mild necrosis. In b: Gills showed Inflammatory

Neutrophils (WBC), secondary lamellar SL buds filled with epithelial cells leading to a

case of epithelial hyperplasia. Signs of Necrosis in some cells (Necrotic Cells) with mild

inflammation.


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Fig. (5): Gills of Larvae exposed to 400 ppb CU after 28h under H&E (400x) showing

severe malfunctioning signs such as: Stuffing of epithelial cells, lifting, hyperplasia,

necrosis is spreading, dilating causing epithelium take off, deterioration leading to loss

of the normal ordination, blood cells RBC coming out of vessels, and finally dead cells

covering SL.

Fig. (6): (left) Gills of larvae exposed to 100 ppb Cu for 96h under H&E (400x) showing

hypertrophy, little hyperplasia and few necrotic cells. (right) Gills of larvae exposed to

200 ppb for 96h H&E (400x) showing hyperplasia , slight centric necrosis, hypertrophy

(puncture) and fusion of secondary lamella.

2. Gut

Normal stomach and intestine histological sections were seen in rainbow trout fry in the control

group which serve for comparison with those of fry exposed to copper. All stomach and intestine

structures appeared normal (Fig.7). Fry exposed to the higher concentration of copper (400 ppb)

for the initial 4 h showed a signs of pyknosis, i.e presence of pyknotic nuclei of cells which

might be due to starvation. The surface epithelium looked fine at this early stage of exposure as


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seen in Fig. (8). On contrary severe structural changes occurred especially in the proximal upper

stomach in fry exposed to the same concentration, but for a longer period of time (28h). As seen

in Fig. (9) those fry suffered from accumulation of vacuoles and degeneration of epithelial cells in

comparison with fry of the control group. Gastric gland showed necrosis of cell (base structure) with

pyknotic nuclei. Absorptive area and muscle layers were mostly affected with vacuolations in the

mucosal membrane.

Broken villi were present with small discharged mucosal part in the lumen. It seems that

the histopathological impacts of copper exposure was limited and was most severe in the upper stomach

region and extends to the middle part only. Nothing occurred in the intestine region as the epithelium

looks fine.

Fig. (7): Normal stomach (left) and normal intestine (right) of rainbow trout fry of the

control group after 24h.

Fig. (8): Stomach (left) and intestine (right) of rainbow trout fry exposed to 400 ppb Cu 400

for 4 h (100x). It shows signs of pyknosis (Pyknotic nuclei) due to starvation, while the

surface epithelium looks fine.


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Fig. (9): Proximal upper region of the stomach (400x) of rainbow trout fry of the control

group (left) which looks normal compared with that of fry exposed to 400 ppb for 28 h

with accumulation of vacuoles and degeneration of epithelial cells.

3. Kidney

Normal kidney structure of was noticed in rainbow trout fry of the control group after 24 h. The

tubules and glomeruli are still immature due to the early age of the fry. Few haemopoietic cells

and white blood cells can also be seen (Fig. 10). Kidney of larva exposed to 400 ppb Cu for 4-8 h

showed mild histopathological changes such as dilation, collapse and degeneration of tubule

epithelial cells leading to Inflammation (Fig. 11 A). karyolitic of nucleic material and vacuole

formation of the tubular epithelial cells was also noted. In another slide the same larva revealed

the presence of cell debris and microphages along the capillary in addition to some changes in

the tubule structure. Few necrosis leading to hypoxia changes because of alteration in the gills

imposed by the severe exposure to high concentration of copper (Fig. 11 B).

Fig.(10): Normal kidney structure (H&E 100 x) of rainbow trout fry in the control group

after 24 h. Normal immature tubules and glomeruli are noticed with haemopoietic cells and white blood cells.


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A B

Fig. 11: Kidney of larvae (H&E 400x) exposed to 400 ppb Cu for 4 h.

A : Dilated and collapse of tubules, degenerating of epithelial cells and Inflammation.

B: Cell debris – microphages along the capillary, few necrosis from the gills, changes in

tubule structure, hypoxia changes because of alteration in gills.

Discussion

Findings of the present study indicated clearly that histopathological markers can be used as

clear evident of copper toxicity in rainbow trout fry. As seen in the results, the three studied

organs showed marked alterations in fish exposed to the highest copper concentration of 400

ppb for 34,48 and 96h. Mild changes were also noticed among fish fry exposed to the medium

(200 ppb) and low (100 ppb) copper concentrations. There was an evident of severe changes with

increasing the copper concentration and with prolonging the exposure time intervals. Findings of

the present study agreed with other studies investigating the histopathological changes in various

tissues in response to copper toxicity in many other fish species (42, 33, 1).

In aquatic environments with elevated levels of copper the primary method of uptake of

the metal is via the gills (13). Gills are the first target of waterborne pollutants due to the

constant contact with the external environment, as well as the main place for copper uptake (7)

(31). Copper diffuses passively across the gill epithelia into the bloodstream, although active

transport may also occur (44). Once copper has entered the bloodstream it is transported to the

liver which is the primary site of accumulation of the metal within fish (33). It is well known that

changes in fish gill are among the most commonly recognized responses to environmental

pollutants (21, 20, 5). Histological study of the gills shows a typical structural organization of the lamella in the

untreated fish. However, fish exposed to copper shows several histological alterations, namely

lamellar epithelium lifting, epithelium proliferation, lamellar axis vasodilation, fusion of

lamellae and lamellar aneurisms. These gill histological alterations has been observed by several

authors in fish submitted to copper (16, 8, 9). Changes in the gills of the juvenile common carp,

Cyprinus carpio (L.) exposed to copper have been reported by (14). The exposure has resulted in

separation of epithelial secondary gill lamellae, hyperplasia, fusion of secondary lamellae and

necrosis. The results of (24) on gold fish Carassius auratus which was subjected to sub-lethal


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concentration of copper sulphate for 96 hr, also reported hyperplasia in the top of the filament.

According to (3), the main histopathological changes observed in gills exposed to the highest

copper concentration were edema, lifting of lamellar epithelia and an intense vasodilatation of

the lamellar vascular axis.

These changes in the gill epithelial layer are considered as a mechanism of defense. It can

be manifested as hypertrophy and hyperplasia of the gill epithelium causing increase in the

thickness of the secondary lamellae, mucous cell hypertrophy and proliferation, mucous

hypersecretion, proliferation of eosinophilic granule cells and hyperplasia of interlamellar cells.

They increase the distance across which waterborne pollutants must diffuse to reach the

bloodstream (4) and reduce the branchial superficial area in contact with the external milieu so

that diffusion barriers to the pollutant can be established (18), (38). Hyperplasia results in fusion

of secondary lamellae which could be protective for a larger vulnerable gills surface area (41).

These findings agree with those of (34) who observed hyperplasia of the gill epithelium in

Carassius auratus exposed to 0.1 mg/l Cu for 25 days. Hyperplasia of the gill epithelium is a

measure to protect the gill filaments from any irritation in the surrounding water (11). According

to (40) changes in the architecture of gill morphology like loss, fusion, clubbing of secondary gill

lamellae, and detachment of gill rakers following softening of gill shaft in fish under lethal

exposures indicating reduced respiratory surface area.

Alterations occurred in the upper proximal part of stomach in the present study can be

summarized as: presence of pyknotic nuclei, vacuolations in the mucosal membrane, degeneration of epithelial cells, necrosis in the gastric gland and broken villi with small

discharged mucosal part in the lumen. Other studies have reported similar alterations in addition

to enlargement of epithelial cells and autolysis. They postulated that CuSO4 cause damage not

only to the absorptive area of the stomach but they also disturb the physiological processes and

absorption of digested food. Similar results were reported by (17) in Lepomis microchirus using

DDT as a pesticide. The vacuolations observed in the stomach disturb the texture of the tissue

which may modify the digestive enzyme secretion which induce the digestive process before

absorption. Similar results were earlier reported by (30) in Chana gachua exposed to DDT.

The present histopathological changes induced in the kidney of rainbow trout fry in

response to copper exposure are more or less similar to those observed in different fishes by

different toxicants but the extent of damage varies depending upon the dose of toxicants,

duration damage varies depending upon the dose of chemical, duration of exposure, toxicity of

chemical and susceptibility of the fish (27, 28, 29). Those changes include dilation, collapse and

degeneration of the tubular epithelial cells, karyolitic of nucleic material, formation of vacuoles,

presence of cell debris and microphages in addition to changes in the tubular structure and few

cases of necrosis. Furthermore, (6) reported that kidney of winter flounder subjected to

considerable pathological changes in response to copper exposure.

In another study, histopathological alterations produced in the kidneys of Sarotherodon

galilaeus fish following exposure to gradually ascending sublethal concentrations of copper,

zinc and lead(2). These include slight changes in the proximal convoluted tubular epithelium

which were slightly vacuolated and the nuclei of its cells were karyorhectic. Hypertrophy, cloudy

swelling and hydropic degeneration of some renal tubules as well as congestion of kidney blood

vessels were common among fishes exposed to the medium levels of metals. Atrophied cellular

components of the Bowman's capsules and enlarged Bowman's spaces were also observed in

such treated fishes. Upon studying the effects of linuron herbicide on the rainbow trout

(Oncorhynchus mykiss), the occurrence of small cytoplasmic vacuoles, nuclear deformation in


36

the epithelium of the first and second segments of the proximal tubule was reported (29). The

kidney cells were observed to have been massively destroyed, the renal corpuscles of the kidney

were scattered resulting in their disorganization and consequently obstruction to their

physiological functions. Such alterations were not seen in the present study.

Acknowledgement

The sponsorship of International Institute of Education (IIE) and SRF-Iraq program to the

fellowship of the first author in Washington State University, Pullman is highly appreciated.

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Histopathological Markers for Copper Toxicity in Rainbow Trout Fry (Oncorhynchus mykiss

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