Histopathology based study of Nile tilapia fish (Oreochromis niloticus) as a biomarker for water pollution evaluation in the southern gulf of Lake Tana, Ethiopia

Abstract

In the past decade, the increasing distribution of pollutants in the aquatic environment has been observed, causing integrative effects on fish. Likewise, due to anthropogenic activities, the southern gulf of Lake Tana is an impacted region, and the production of Nile tilapia fish is reduced. For this reason, the aim of this study was to conduct a histopathological-based study of 48 Nile tilapia fishes’ health status at the southern gulf of Lake Tana and aquaculture using a cross-sectional study from February 2023 to May 2023. The study evaluated the histopathology of the gill, liver, gonads, and spleen organs using descriptive statistics accompanied by a 2 × 2 contingency table and t-test analysis. During the study, different histological alterations were detected, and the numbers of fish affected by a specific histological alteration were presented as percentage prevalence; hence, from the total fish examined, hyperplasia (54.15%), followed by pigment deposits (52%), hemorrhage (50%), and immune cell infiltration (50%), respectively, were the most frequently detected alterations. However, Nile tilapias from the southern gulf of Lake Tana were 1.4 (odds ratio) times more likely to show histopathological alterations than those from aquaculture, although statistically, was not significant (p > 0.05). In addition, the study found the mean value of the fish index (95.3) and regressive indices of the gill (13.6), liver (14.8), and gonad (12.3); moreover, the inflammatory indices of the spleen organ (11.3) and mean severity grade value of the gill (2.35) and gonad (1.7) organs, respectively, were obtained from the southern gulf of Lake Tana, and all those values were significantly higher (p < 0.05) from this site as compared to the aquaculture. In general, it has been found that tilapias from the southern gulf of Lake Tana showed higher pathological severity as compared with aquaculture. Among the four target organs evaluated, liver organs were observed to be the most damaged, while gonads were the least impacted organs. Therefore, it has been concluded that tilapia fish are living in abnormal conditions, so to ensure a sustainable fishery, water pollutant sources from Bahirdar city must receive proper attention, and future studies should consider age differences, seasonal variation, and the detection of specific pollutants.

Nile Tilapia Fish

Nile tilapia fish (aquatic chicken) found from Lake Tana (study area)

Introduction

Background

Water bodies are constantly altered by a variety of pollutants from various sources (agricultural, industrial, and domestic wastes). In fact, aquatic bodies can become polluted by microorganisms (bacteria) and parasites, fertilizers, herbicides, pesticides, toxic heavy metals, fuel, and so on [1, 2]. And in the last decade, the increasing distribution of pollutants in the aquatic environment has been observed to cause integrative impacts on the fish’s organs and ultimately affect organ function such as reproduction, endanger their growth and survival, and decrease species diversity in different regions of the world [3, 4]. Furthermore, polluted water has a negative impact on human health, either directly through drinking it or indirectly through eating abnormal fish products that might be impacted by the polluted water [5].

In fact, fish are important for the world’s food supply for humans and as a source of revenue [6]. Moreover, they are used in determining the ecological health of the aquatic environment, and they are considered bio-indicators for water quality deterioration [7]. Among the different types of fish in the universe, Nile tilapia is the most important and widely consumed fish [1, 8]. And it is one of the most often utilized fish in studies of aquatic ecosystem pollution [9]. In Lake Tana, tilapia fish are the most popular and highly productive species, which accounts for 64.96% of the annual catch composition of fish species [10], however, over the past few decades, those fish stocks have dramatically decreased [11]. In fact, both abiotic and biotic markers can be used to assess water pollution in an aquatic ecosystem [12]. These are physicochemical (abiotic) studies and biological (biotic) approaches [13].

A variety of biological monitoring approaches have been used to evaluate the effects of pollutants on the health of fish populations. Among those, necropsy, age and growth analysis, biometric analysis, and moreover, biochemical, immunological, hematological, physiological, and gross pathology, as well as microscopic and histopathological approaches, can be used to assess fish health [14].

However, histopathology as a biomarker is a commonly useful approach for assessing pollution and its impacts on aquatic ecosystems and fish [15]. Histopathology is a microscopic examination of the cells and tissues of an organism and can be investigated both for quantitative and qualitative determination of histological abnormalities [4]. It is an imperative study that can be conducted to assess the overall health of the population in the aquatic ecosystem [16]. Biomarkers for water pollution are early diagnostic tools for biological effect measurement and environmental quality assessment. Although histopathological biomarkers are relatively recent, there are a number of studies on Nile tilapia fish species for aquatic pollution assessments, however, fish histopathology studies in relation to aquatic pollution monitoring are limited in Ethiopia.

Statement of the problem and justification of the study

In fact, Lake Tana is the largest body of water in Ethiopia, and it is a home to several fish species, however, the fast expansion of industries and different anthropogenic activities, it is exposed for different pollutants and putting the survival of fish in jeopardy [13]. Particularly, Southern gulf of Lake Tana is urban impacted region due to different anthropogenic activities and Nile tilapia fishes have been becoming diminished, which may need conservation for it’s future sustainability [17]. In fact, the physicochemical water quality status of Lake Tana including it’s southern gulf is showed an impairment [18, 19]. However, Lomartire et al. [20] reported that physicochemical analysis of water quality gives only a snapshot conditions in time but it does not reflects the presence of the pollutants effect on the fishes. Thus, to carry out the efficient aquatic pollution monitoring program, biological/bio-markers/bio-indicators approaches have found interesting.

As different research results revealed that the quality of water and the well-being of fishes are interconnected and different biological monitoring approaches have been used to evaluate the effects of stress/pollutants on the fish populations. Among those, histopathology is a common and an imperative useful approach for assessing pollution and it’s effects on the fishes, both in a natural and fish farm aquatic ecosystem. as it can be the result of an integrated effects of both the biotic and abiotic contaminats. Moreover, the relevance of each histological lesion relies on how it impacts organ function and the fish’s capacity to live. Therefore, evaluating the pollution level and its effect via histopathological health assessment protocol was interesting and the study may aids to enforce applying measurements to mitigate the stress and diseases in the aquatic environments, safeguared aquatics, safeguared wellbeings of fishes and finally, maintaining a viable fishery or sustainable use and a safe product for human consumption. Furthermore, histopathology of Nile tilapia fish species in respective to water pollution was not investigated in Ethiopia. Therefore, the following objectives are forwarded;

Objectives of the study

General objective: To carry-out histopathology based health assessemnet of Nile tilapia fishes.

Specific objectives:

• ⋆ To detect and characterize histological alterations in the selected target organs (gill, liver, gonad and spleen) both in the southern gulf of Lake Tana and Nile tilapia fish aquaculture.

• ⋆ To evaluate the percentage prevalence of histological alterations of the selected target organs associated with study sites.

• ⋆ To quantify and grading severity of histological alterations in all target organs associated with study sites.

Materials and methods

Description of the study area

Lake Tana is the largest lake in Ethiopia, accounting for 50% of the country’s water resource. It is located in the Northwestern part of the Amhara region and situated on the basaltic plateau of the north-western highland of Ethiopia. The south-north maximum length is about 90 km, and the east–west width is about 65 km. The maximum depth of Lake Tana is 14 m, with a mean depth of 9 m, and it is fed by many streams and a few large rivers. The lake covers area of 3150 km² and located at an altitude of 1786 masl [21]. Tana is a crater lake formed two million years ago, due to the volcanic blocking of the Blue Nile River. It gradually becomes harmful to serve as a habitat for aquatic organisms. The capital of the Amhara region, Bahir Dar, is situated on the southern border of the Lake tana [21].

Description of sampling sites

Southern gulf of Lake Tana

This site is located at the corresponding border of Bahirdar city, and this study site was purposefully selected based on its proximity to expected anthropogenic emission sources near and from urban effluents (Bahirdar city) such as residential wastes, wastes from vehicle washing, hotels like the Tana Hotel, Blue Nile Hotel, and Grand Mole Hotel, fish market and processing sites, hospital wastes, different domestic wastes, café and restaurant wastes, and other domestic wastes. The Blue Nile River crossing Bahirdar City receives untreated municipal and industrial wastewater and then drains into the mosaic of mixed wetland habitats of the Abay River. Domestic trash and various urban effluents are discharged from Bahir Dar city into the southern bay or inlet of Lake Tana; moreover, the numbers of Nile tilapia fish’s catches at this site are reduced as compared to the previous catchment amount [13].

Nile tilapia fish aquaculture farm

It is located at the Bahirdar City Fisheries and Aquatic Research Center where southern gulf of Lake Tana is boarded. The aquaculture farm is a solitary settlement; it has periodic and routine water replacement and refreshment; it has a good fish feeding system; undergoes a complete cleaning system annually; there is no direct effluent drainage. Additionally, the farm is enclosed/fenced, and the technique is based on batch growth and species isolation raising (only Nile tilapia). However, no routine chemical or medicinal water treatment is performed in the ponds, and no additional fish health precautions are taken (source: [22]) (Fig. 1).

Fig. 1

A map of lake tana showing it’s gulf of bahir-dar obtained by geographical information software sketching system

Study population and study design

In addition to many unique criteria’s, as Sample size and sampling method section indicated and accessibility from the southern gulf of Lake Tana and Nile tilapia fish in aquaculture site, Nile tilapia was used to conduct a cross sectional study between February 2023 to May 2023.

Sample size and sampling method

Based on referencing previously done fish histopathological study reporties, This study has used a total numbers of 48 tilapia fishes which were taken with simple random sampling method, from each catched numbers of fishes using a legal woven mesh net with the assistance of local fishermen.

Fish sampling protocol and sample preparation

Among the total 48 sampled fishes, 24 were gathered from the southern gulf of Lake Tana, while the remaining 24 were gathered at the Bahirdar Fisheries and tilapia fish in aquaculture site. Six fish from each location were sampled; in total, 12 fish were sampled per sampling period.

During sampling time, to reduce stress and autolysis, sampled fishes were kept alive by a continuous exchange of water from a mobile tap in the field and brought to the Bahirdar Fisheries and Aquatic Research Center laboratory and then both gross lesion observation and organ samples have taken place. The water used in the mobile tap was collected from the study site accordingly. Then after the fishes have taken to dissecting room the weight and length were measured, and euthanized using the cervical dislocation or severing the spinal cord anterior to the dorsal fin [14]. Next, the fish was dissected, and any gross lesions on the target organs were detected and recorded. From each fish sample four organs (liver, gill, spleen, and gonad) were taken for tissue processing, dissected organs (tissue) have sampled and then preserved in a 10% buffered formalin solution containing a sampling plastic bottle, labeled, and pended for laboratory analysis. During the study period, totally, 192 organs were processed for histopathological study.

Sample processing and laboratory activity

Histopathology assessments

Tissue processing was carried out by Leica’s (Leica TP 1020, Germany) automatic tissue processor following standard tissue processing procedures. Histological alterations were qualitatively assessed in all selected organs in which histopathology slides with codes, observed under Light microscope and interpreted/described. Moreover, the coded slides which were not surely identified have checked independently by veterinary pathology experts for comparison and precision, finally results were recorded, then averaged results were provided for the score-value of the tissue alterations.

Quantitative study of the tissue changes was evaluated according to a protocol proposed by Bernet et al. [23] and described in detail as follows. The impact level or severity of histological alteration was expressed by histopathological index analysis. Briefly, this index is based on the lesion score value (a) and importance factors (w) for each considered lesion in the tissue. For each organ, the pathological changes were classified into five reaction patterns, such as circulatory disturbances, regressive changes, progressive changes, inflammatory, and neoplastic responses, and the lesions under each reaction pattern of each target organs were identified and scored. Circulatory disturbances were the result of pathological conditions of blood and tissue fluid flow such as congestion, hyperemia, thrombosis, embolism, and edema. Regressive changes were processes that could terminate in a functional reduction or loss of an tissue/organ, such as atrophy, degenerative changes, and necrosis. Progressive changes are processes that lead to increased activity of cells or tissues, such as hypertrophy and hyperplasia. Inflammatory changes are often associated with processes such as immune cells infiltration and exudates. Lastly, a tumor or neoplasm depicts uncontrolled cell proliferation.

Each alteration was assigned a factor of 1 to 3, depending on its pathological importance in affecting the organ function and ability of the fish to survive, with 1 assigned to a lesion that was considered easily reversible, such as hemorrhage and congestion, 2 was for moderate lesions, example (atrophy, hyperplasia, and immune cell infiltration) and 3 to a lesion that may lead to partial or total loss of the tissue, for example, necrosis. Every lesion was also assessed using a score ranging from 0 to 6, depending on the degree/extent of distributions of the specific alteration, whereby (0) was unchanged; (2) or mild occurrence; (4) or moderate occurrence; and (6) or more were severe occurrence (diffused lesion), with intermediate values also considered [23].

For each lesion a lesion index was calculated by multiplying its value of importance factor (w) with the score value (a), and the reaction pattern for each organ was calculated as the sum of the lesion indices under that reaction pattern. For example, a circulatory index, regressive index, progressive index, inflammatory index, and neoplastic index were calculated. The sum of the reaction indices per organ yielded the total organ index. Moreover, Total index (fish index) was the sum of all organ indices of an individual fish, that is, the sum of gill, liver, spleen and gonads of a given single fish. These indices are indicative of the extent and intensity of histological alterations in the respective tissue as they convert the observed qualitative histological alterations into a quantitative value [23, 24].

To classify the aquatic study sites according to the severity of the histological response, the organ index results were graded into four grades. The grades were a modification of a scoring scheme developed in studies of Clarias gariepinus and Oreochromis mossambicus in polluted waters by Van Dyk et al. [14] which was developed a system for trout in Swiss rivers by Zimmerli et al. [24]. The modification applied were based on lesion nomenclature, score value provision, lesion type and gonad and spleen organ inclusion. Organ histological indices were classified into four classes, where class 1 (slight histological alteration), class 2 (moderate histological alteration), class 3 (pronounced histological alterations) and 4 were considered more severe alterations. Below on the Table 1 was a detailed description of each class.

Table 1 Histological alteration indices grading protocols

Data management and statistical analysis

The data were entered into an Excel spreadsheet and checked for errors. And data were analyzed by using STATA statistics version 16. The number of fishes affected by a specific histological alteration at each study sites were presented as percentage prevalence. And 2 × 2 contingency table was used to evaluate the proportion of positive individuals in which histological alteration was detected per total number of samples in the study sites. Descriptive research methods were used to derive the means, standard deviations (SD), and ranges of Histopathological alterations indices in each study sites. The Independent Samples T-test was used to compare mean values of histopathological changes severity between the study sites and fish sex. Moreover, one way ANOVA was used to evaluate the presence of mean severity grade difference and significance level between four target organs. The level of significance of all statistical tests was set at 95%. The four grades of organ index results, i.e., index ≤ 15, 16–30, 31–40, and ≥ 41, for each organ were derived and graphically presented to show the percentage of each grade among the studied fish population for both sites.

Ethical considerations

The study had obtained the ethical clearance from college of veterinary medicine and animal sciences ethical research board of university of Gondar.

Results

Nile tilapia fish gross pathological assessment

In the current investigation, target organs of all Nile tilapia fish samples were examined macroscopically. And the study were found very few gross lesions such as mucus deposition, organ enlargement, congestion, gill friability, abnormal distention, paleness, degeneration, yellowish discoloration, roundworm infestation, and infarction. As shown in Fig. 2, some of the gross assessments were forwarded in photo macrographs.

Fig. 2

Macrographs observed from Nile tilapia fish target organs (A-H): (A) infarcted liver ( black arrow) and brown to black diffused fin discolorations (red arrow) (Southern Gulf of Tana); (B) Round worm infestation in the root of gill (blue arrow) and Atrophied & lighten liver (black arrow) (Southern Gulf of Tana); (C) fatty liver (black arrow), necrosis (yellow arrow) (Aquaculture); (D) hyperemic and swollen ovary (blue arrow) (aquaculture); (E) excess mucous on gills (red arrow) (Southern Gulf of Tana); (F) lighten and degenerated gill (bluish arrow) (Aquaculture); (G) yellowish discoloration (rapture of ova) (red arrow) (Aquaculture) and (H) hyperemia (yellow arrow) and mucoid gill (red arrow) (southern gulf of tana)

Histological alterations and occurrence

In this study different tissue alterations were detected in all target organs and the proportion of the positive individuals in which histological alteration is detected per total number of samples in both study sites was assessed, but it did not mean it revealed the severity or all-over impacts, which were analyzed by using the score value and importance factor of tissue alterations. And the results were statistically compared by a 2 × 2 contingency table. And based on the confidence interval, the odds ratio was in the range, and the proportion of occurrence was (x² = 0.34), p-value was not statistically significant (p > 0.05). Therefore, numerically, fish from the southern Gulf of Tana were 1.4 times more likely to have pathological alteration than those in aquarium. The details are reflected below in Table 2.

Table 2 Odds ratio, x², confidence interval, and p-value of histological alteration occurrence per total samples of fishes associated with study sites

Gill histopathological findings and prevalence

In the current study, different gill tissue alterations were detected and the numbers of fish affected by a specific histological alteration in each target organs were presented as percentage prevalence. The study was found that structural alteration (58.3%) which is a regressive change and hemorrhage (58.3%) which is a circulatory change gill tissue alterations were the most prevalent lesions occurred in the southern Gulf of Tana as compared to the aquarium site. And telangiectasia (50%) which is a circulatory response and lamellar hyperplasia (54.2%) which is a tissue progressive change were the most prevalent gill tissue alterations observed at the aquarium site as compared from southern gulf of tana. The % Prevalence of histological alterations in the gill organ associated with study sites are provided in Table 3, and some of the histological alterations have shown in Fig. 3 with their descriptions.

Table 3 Prevalence of gill histological alterations associated with study sites

Fig. 3

Photomicrographs of gill histological sections (a–h): (a) nearly normal gill histology (aquaculture); (b) Epithelial lifting/separation/detachments of secondary lamellae (black arrows) and hemorrhage (yellow arrow) (southern gulf of tana, 10x); (c) fat degenerations at gill arch (star), benign tumor (red arrow) (southern gulf of tana, 10x); (d) lamellar necrosis (star) (southern gulf of tana);(e) curling/curving of secondary lamellae (black arrow) (aquaculture, 10x); (f) epitheliocyctis (blue arrow), telangiectasia (Dilatation) of the venous sinus observed at the apex of secondary lamellae (red arrow) (aquaculture, 10x); (g) cyst surrounded by fibrous connective tissue (black arrow) (southern gulf of tana) and (h) lamellar hyperplasia/ fusion of secondary lamellae (double black arrow (aquaculture, 10x). All sections were stained with H & E

Liver histopathological findings and prevalence

This study was detected different liver tissue alterations in both study sites. However, the current study was found that nuclear alteration (58.3%) which is a regressive change and immune cell infiltration (50%) which is an inflammatory change were the most frequently occurred liver tissue alterations in the southern Gulf of Tana as compared to the aquarium site. Moreover, congestion (50%) and telangiectasia (50%) which are a circulatory response were the most prevalent liver tissue alterations at the aquarium site. Moreover, from the total samples examined immune cell infiltration (50%) followed by sinusoid dilation and congestion were the most frequent liver tissue alteration. And the percentage prevalence of histological alterations in the liver organ associated with study sites are provided in Table 4. And some of the histological alterations have shown in Fig. 4 with their descriptions.

Table 4 Percentage prevalence of liver histological alterations associated with study sites

Fig. 4

Photomicrographs of liver histopathological sections of Nile tilapia (a-h): (a) normal histology of the liver (aquaculture, 10x); (b) congestion (star) (aquaculture, 40x); (c) yellowish brown pigment deposition of hepatopancrease (black arrow) and necrosis (yellow arrow) (southern gulf of tana, 10x); (d) vacuolar degeneration (black arrow) (southern gulf of tana, 40x); (e) sinusoidal congestion (black arrows) (aquaculture, 40x); (f) mild kupffer cells aggregation (black arrow) (aquaculture, 40x); (g) hepatopancrease necrosis or losing of hepatopancrease (black arrow) (southern gulf of tana, 10x) and (h) nuclear alterations (black arrows) (aquaculture, 40x). All sections were stained with H & E

Gonads histopathological findings and prevalence

In the present study, different gonadal alterations were detected. And the numbers of fish affected by a specific histological alteration were presented as percentage prevalence in related with study sites. The current study was found that degeneration/necrosis (58.3%), followed by crumbled oocytes and gonadal membrane alterations which are a regressive changes were frequently observed tissue alteration in the southern Gulf of Tana. Moreover, from the total samples evaluated hemorrhage (45.8%) was the most frequent gonads alteration. The percentage prevalence of histological alterations in the gonads organ associated with study sites are provided in Table 5. And some of the histological alterations have shown in Fig. 5 with their descriptions.

Table 5 Percentage prevalence of gonad histological alterations with study sites

Fig. 5

Photomicrographs of gonads in histopathological sections of Nile tilapia (a-h): (a) normal ovary histology (aquaculture, 10x); (b) Atretic, liquefied/dissociated oocyte (black arrow) (aquaculture, 10x); (c) oocyte hyaline degeneration at the periphery (Atresia of vitellogenic oocyte (disintegrated vitelline envelope) (blue arrow), hemorrhage (black arrow), immune cell infiltration (green arrow) (Southern Gulf of Tana, 10x); (d) crumpled oocytes (black arrows) (aquaculture, 10x); (e) Oocyte vacuollation/ liquefaction (black arrows) (southern Gulf of tana, 10x); (f) tests normal histology (aquaculture, 10x); (g) Accumulation of yellowish-brown pigment with (mmc) aggregation (black arrow) (southern gulf of tana, 10x); (h) Reduced number of spermatocytes (red arrow), edematous fluid (black arrow) (southern gulf of tana, 10x). All sections were stained with H & E

Spleen histopathological findings and prevalence

In the current study, among the observed tissue alterations in the spleen, the progressive reaction pattern, particularly MMC hyperplasia (54.15%) and pigment deposits (58.3%) which are a regressive change were the most frequently encountered spleen lesions detected at the southern gulf tana. Moreover, necrosis (50%) which is a regressive response and immune cell infiltration (50%) which was an inflammatory change were the most observed alterations in the aquarium site. Moreover, from the total samples examined mmc hyperplasia (54.15%) followed by pigment deposits (52%) were the most frequent detected spleen tissue alterations. The percentage prevalence of histological alterations in the spleen associated with study sites are provided in Table 6. And some of the histological changes have shown in Fig. 6 with their detail descriptions.

Table 6 Percentage prevalence of spleen histological alterations and association with study sites

Fig. 6

Photomicrographs of spleen histological sections (a-f): (a) normal spleen histology with red pulp (black arrow) and white pulp (blue arrow) (aquaculture, 10x); (b) aggregated MMC with hypertrophied Macrophage (red arrows) and hemosiderin deposition (yellow arrow) (southern gulf of tana, 40x); (c) aggregate of exhausted/atrophied MMC (star) (aquaculture, 40x); (d) Vacuollation (red arrow) and immune cell infiltration (yellow arrow) (southern gulf of tana, 10x); (e) Mono nuclear cell infiltration (black arrow) and necrosis (immune cell depletion) (yellow arrow) (southern gulf of tana, 40x); (f) Mild MMC infiltration with yellowish Brown-pigmented (yellow arrow) and extended Necrosis (blue arrow) (aquaculture, 10x). All sections were stained with H & E

Quantitative histopathological assessment

Organs reaction pattern indices

Based on Bernet et al. [23] protocol and formula, gill, liver, spleen, and gonad organs reaction patterns severity were analyzed, and each target organs histopathological reaction pattern index results were presented as mean and standard deviation. Independent sample t-test was used to compare the mean values of these reaction indices. This study was observed regressive reaction patterns of the gill, liver, and gonad organs, and in the spleen, inflammatory responses were higher in the southern Gulf of Tana and statistically significant (p < 0.05) however the remained values were statistically not significant. The details are reported in Table 7 below.

Table 7 Mean, SD and p-value of each target organs reactive pattern histopathological indices in both study sites

Target organs and fish histopathological indices

During the study period, histological alterations severity was evaluated quantitatively and each target organs and fish index values were analyzed, and presented as mean, SD, and range. Again, independent sample t-test was used to analyze the index values among the study sites and fish sex. Comparing the mean values of these indices, the southern Gulf of Tana had statistically higher values (p < 0.05) for gill (27.3), gonad (18.5), liver organ (30), and fish index or total index (95.3), respectively as compared to aquarium sites however the other values were not statistically significant although there were numerically difference in between. However, the spleen organ mean index value was found to be comparably similar between both study sites. And also, index values were statistically derived between sexes of tilapia fishes sampled; however, significant differences were not observed. Furthermore, among all target organs examined liver (26.1) was the most impacted while gonads (14.69) were least impacted. index results have forwarded in detail on the Table 8.

Table 8 Mean, SD, Range, and P-values of target organ and fish indices associated with study sites and sex variables

Target organs histopathological grades and prevalence

In order to evaluate the severity grade of tissue alterations in each study site, the quantified histopathological findings were further graded according to modification of the protocol developed by van Dyk et al. [14], which is given in Table 1. And the current study was involved on evaluation of percentage prevalence of each organs index grades. Severity grade 1 in gill, liver and gonad was the most frequent histopathological index grade observed at the aquarium site as compared from southern gulf of Lake Tana. And this study has found that less frequent but severe alterations of organ tissue (grade 4), were observed only in gills and livers from the southern Gulf of Tana. In the aquarium, severe alterations (grade 4) were not detected in any of the examined organs. one way ANOVA was used to compare the presence of difference in the mean values of severity grade between four target organs, the result indicated that the presence of significant different between target organs (p < 0.05). The details have forwarded below, both in Table 9.

Table 9 Percentage prevalence of organs histopathological grades associated with study sites and the presence of mean severity grade difference between four target organs and significance status

Mean value of target organs histopathological index severity grades

The present study was assessed the mean values of already graded each target organs histological lesion indices). And the mean value of each organ severity or index grade associated with study sites and sex were analyzed by using an independent sample T-test. And the mean value of the severity grade results were significant for the gill and gonad organs (p-value < 0.05). However, mean severity values of the spleen and liver organs were not significantly different with study sites, moreover, organ severity associated with sex differences was not statistically significant (p > 0.05). Furthermore, among four organs liver (2.23) organs were the most impacted while gonads (1.59) were least impacted organs. The details of the mean and SD of organ severity grade and its significance associated with sites and sex have given in Table 10.

Table 10 Mean, SD of organ severity level and its significance state associated with site and fish sex

Discussion

In fact, Lake Tana is the largest lake in Ethiopia, and several fish species are found there. However, through time, the expansion of industries and other anthropogenic activities has been causing alteration, pollution, or being made poorer in this aquatic ecosystem, putting the survival of fish at risk [13]. In fact, aquatic pollution has significantly increased over the past decade [25]. Off course, it is obvious that Nile tilapias are the most popular and highly productive fish species of Lake Tana [10], however, over the past few decades, those fish stocks have dramatically decreased in number [11]. The current study was focused on assuring the presence of water pollution and its impacts on the health of Nile tilapia fish species using a histopathological investigation.

Although it was not the objective of the current study, the presence of gross pathological conditions in each sampled fish’s target organs was examined correspondingly; however, only a few gross lesions were observed. However, for an overall assessment of chronic, acute, apparent/unapparent, mild, moderate, and severe pathological conditions, histopathology of the gill, liver, gonad, and spleen organs of each sampled individual was carried out. In the current study, the histopathological assessment conducted involved both qualitative and quantitative scenarios. The qualitative assessments were applied to the detection, identification, and description of histopathological alterations that were observed in the target organs. And then qualitative histological alterations were given quantitative values as previously recommended by the Bernet et al. [23] protocol.

The application of the lesion index of the Bernet protocol associated with histological alterations and image analyses allowed for the evaluation of the presence of pollutants and their effects on tilapia fish. After the establishment of the index values of the tissue alterations in the organs and fishes, it is easier to compare the level of injuries between the target organs on the same individual, between two or more different species, and also among groups of the same species from different locations studied [24].

The present study has found that circulatory, regressive, progressive, and inflammatory disturbances were observed in different percentages of prevalence in the two study sites. However, tissue change, which was classified under a regressive reaction pattern, was the most prevalent change that was observed at the southern Gulf of Lake Tana as compared to aquarium sites. This might be due to the fact that the fact that the southern Gulf of Lake Tana is loaded with toxic chemicals like pesticides and heavy metals or pathogenic bacteria more than the aquarium site or the absence of some management practices that are applied to aquaculture fish, like providing concentrate feeds that boost the adaptability or immunity of the fish to different pollutants or stress. The accumulation of heavy metals in fish leads to damage to the organ structure due to their potential toxicity, and continuous exposure may affect fish health [26].

During this study neoplastic/tumor lesions were very rare. In fact, tumors are the result of old age or long-term or chronic exposure to a combination of potentially carcinogenic pollutants. Such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, pesticides, and heavy metals [1]. Unfortunately, the current study sampled and analyzed fish that were up to 23 cm in length and had a maximum weight of 218.8 g. Off course, by local fishery men, literally, it was explained that there was frequent catchment of fish in the southern gulf of Lake Tana and no fish were found to be larger. Normally, Nile tilapias reach up to 60 cm in length and can exceed 5 kg in weight [27]. This may be the reason for the limited occurrence of neoplastic changes during this study. The current study did not include the length, weight, and age of fish to compare tissue alteration occurrence and severity. It was due to a loss of reference, which used and included standardized length, weight, and age of Nile tilapia for histopathological study for aquatic environment health assessment.

The current study evaluated the occurrence of overall histological alterations found in each study site per whole sample; however, this does not imply that it provides information on the severity of changes or Bernet-based tissue change analysis. So, as indicated in Table 2, fish from the southern Gulf of Tana were 1.4 times more likely to show pathological alterations than those in aquariums. This might be due to the presence of high levels of pollutants and a poor management system in the southern gulf of Lake Tana rather than aquaculture.

Gill is vulnerable to the presence of various sources of pollutants as it is in direct contact with the external environment. Gill is also considered the primary target of contaminants, and gills are also indirectly exposed through circulation. And changes in fish gills are among the most commonly recognized responses to environmental pollutants [25, 28]. Oxygen declines in water lead to digestive, respiratory, reproductive, and physiological abnormalities in the functions of aquatic life [1]. Respiratory distress caused by aquatic environmental changes or aquatic pollution may induce vasodilatation and the appearance of edema and detachment of the gill epithelium [29].

Overall, the present study found different histological alterations in the gill organs such as hemorrhage, telangiectasia, edema, immune cell infiltration, goblet cell proliferation, pigment deposition, structural alteration (curling), necrosis, fatty degeneration, fibrosis, lamellar hyperplasia, epitheliocyctis, neoplasia, and cysts, as Table 3 has shown. The current study could certainly agree with Abalaka [30], Roselin et al. [31], Abiona et al. [15], and Steckert et al. [29], who reported histological changes such as hyperplasia, edema, degeneration, aneurysm (telangiectasia) or dilation of blood vessels, epithelial lifting and clubbing (structural alteration), hemorrhage, immune cell infiltration, and edema.

Additionally, Tayel et al. [28] reported that gill histological lesions in the Nile tilapia fish, including hemolysis in primary lamellae, necrosis, degeneration, epithelial separation, curling, hemosiderin deposition, and aneurysm (telangiectasia), could be seen in a polluted aquatic ecosystem. In addition, lamellar fusion and hyperplasia of the mucous cells of the tips of the primary lamellae, hypertrophy and elongation of the secondary lamellae, edema of the cartilage of the primary lamellae, congestion in the gill arch, mononuclear cell infiltration in the gill arch, and clumping of gill filaments were observed in gill tissue due to aquatic pollution effects [1, 32].

In the present study of gill organs, results from aquaculture, the occurrence of aneurysms and hemorrhage were 50% and 41.7%, respectively. Similarly, these results agreed with those of Roselin et al. [31] study. In aquaculture study, Steckert et al. [29] reported that lamellar hyperplasia, immune cell infiltration, lamellar structural alterations, hemorrhage, and telangiectasia were the most frequently occurring gill tissue lesions,similarly, the present study certainly agreed with this author’s finding. In fact various gill histological alteration could be caused by due to sewage water effluent and low oxygen level [25]. Epithelial hyperplasia is a non-specific response induced by many gill irritants in pollution exposed fish, including ammonia and heavy metals [30]. Ammonia concentration exceeding 2.0 mg/L are reported to cause gill tissue damage, extreme lethargy, and death in exposed fish [15]. The elevated ammonia level might be due to the presence of possible urban sewage effluent [30]. Additionally, Tilapia fish exposed to higher Ammonia concentrations show pathological conditions such as Congestion of the central vein and telangiectasia of secondary lamellae, vacuolar degeneration of the secondary lamellae, mononuclear cell infiltration, Degeneration, sloughing, and necrosis of the lamellar epithelium [33]. The physico-chemical water quality status of Lake Tana, such as ammonia, is highly fluctuating and increasing from time to time [34]. Hence, the current study observed gill histological alterations may be due to the above-mentioned condition.

Histological Changes in Nile Tilapia Exposed to Pesticide (pollutant) can be epithelial lifting, Disruption of the secondary lamellae, and blood congestion [31, 33]. Excessive aneurysms can impair respiratory efficiency, especially at higher temperatures when dissolved oxygen levels are low and metabolic oxygen demand is high [30]. Epitheliocyctis (epithelial cell swelling) is an infectious-contagious abnormality commonly associated with bacteria and Chlamydia-like organisms that affect the gills of the fish. Recent studies have shown that these agents can cause widespread morbidity in cultured fish [35]. And the observed tissue alterations from the aquaculture site may be also associated with bacteria and Chlamydia-like organisms.

Parasitic infestation could causes different degrees of lesions in the gills such as secondary lamellar epithelial hyperplasia and lamellar fusion. Moreover, it is known that parasite attachment structures cause severe tissue lesions [33]. Chronic infections, especially parasitic ones, can provoke the inflammatory response with the migration of neutrophils, thrombocytes, macrophages, lymphocytes, and eosinophilic granulocytes along the gill filaments and in the central filament vessel [29]. Likewise, in the current study during necropsy round worm and tape worm parasites were detected at the gill root and fish viscera and the observed gill alterations may be due to parasitic. Proliferation of mucous cells, associated with excess mucus secretion, seems to occur more frequently as a result of exposure to metals [36]. The presence of edema and edematous separation of the respiratory epithelium and lamellar clubbing may be due to water-borne toxins, and this departure from normal structure affects the functional efficiency of the gills for gas transport and ionic regulation [35]. In fact, Lake Tana is observed as it is polluted by heavy toxic metals.

General, Gill histological alterations are considered as non-specific biomarker, which means that many different organic and inorganic contaminants or any pathogens can cause tissue changes. However, they are recognized as a valid and fast method to determine the damage caused in fish by the pollutant exposure, and are one of the first major target organs of pollutants [16].

Liver is the most associated organ in detoxification and biotransformation processes, and due to its function, position, and blood supply, it is one of the organ mostly affected by contaminants in the aquatic environment [35]. Many contaminants tend to accumulate in the liver much higher levels than in other organs [15].

Several histopathological findings are observed in the liver of Nile tilapia due to domestic, industrial, and agricultural pollutants [29]. Moreover, the histological alterations in the liver could be a direct result of the parasitic infection from sewage effluents [28]. Hence, during this study period, a roundworm and tapeworm infestation was observed in most of the sampled fish, which was observed at the root of the gills and visceral as Fig. 4B has shown.

The present study has observed different histological alterations in the liver organ such as necrosis, nuclear alteration, brown pigment deposits, congestion, telangiectasia, edema, immune cell infiltration, mmc hyperplasia, mmc hypertrophy, fibrosis, and fatty degeneration. The current study was able to observe congestion and immune cell infiltration were the most frequently observed tissue alterations in the liver from aquaculture. This result agreed with that of Steckert et al. [29] results.

The histological alterations of liver from the natural habitant of Nile tilapia fish can include degeneration in hepatic cells, atrophied hepatocytes, Sinusoidal congestion, dilation in blood vessels, fibrosis, hemorrhage, hemosiderin deposition, inflammatory cell infiltration, Melanomacrophage centers aggregation, hyaline degeneration, and Granulomatous lesion [28]. Tilapia fish exposed to higher Ammonia concentration show pathological conditions such as Vacuollation of hepatic cells in the liver, Congestion of blood vessels, activation of the Melanomacrophage centers, and pyknotic nuclei in hepatocytes [28].

Histological changes such as degeneration/ necrosis, Lymphocytic infiltrations, congestion, changes in the hepatopancrease, and hyperemia under the influence of various pesticides and other toxic heavy metal bioaccumulation as a results of water pollution [35]. Metals released into the aquatic ecosystem may be accumulated in aquatic organisms through the cumulative effects of bio-concentration and bioaccumulation via the food chain pathway and become toxic when concentration reaches a considerably high level. And unfortunately, the presence of heavy metals such as Zn, Cu, Pb, Mn, Cr, and Cd was reported higher in Lake Tana [18].

The presence of pollutants causes fatty degeneration and vacuollation of hepatocytes [15, 30]. Moreover, leukocyte infiltration indicate the presence of inflammation in hepatic tissues, could be observed in fish exposed to cadmium [9]. The accumulation of hemosiderin in liver cells may be attributed to the rapid and continuous destruction of erythrocytes [33]. A Pyknosis/nuclear alteration is considered an early sign of necrosis exposed to environmental toxicants such as pesticides. Necrosis of liver tissue probably can result from the excessive work required by the fish to get rid of the water-soluble fraction from its body during the process of detoxification [35]. MMCs, are a subtype of pigmented macrophages found in different organs, mainly in the liver, spleen, and occasionally in the gills and gonads [29]. Several authors have suggested that the involvement of MMCs in various disease processes and the changes brought about in them by factors such as chemical exposure [1].

Histological changes of the fish gonads as a response to aquatic environmental stress have been shown as a biomarker-indicative tool to assist in the bio-monitoring process of aquatic ecosystems [35]. Water pollution has a serious inhibitory effect on fish reproduction. Different pollutants, such as heavy metals, pesticides, and different types of bacteria, have histopathological effects on the reproductive tissue of fish gonads [37]. The effects of pollutants on fish reproduction have been investigated by numerous researchers, who confirmed adverse impacts on fish organs reproductive capacities.

In the present study, necrosis, crumpled oocytes, deposits, focal reduction of sperm cells, edema, congestion, gonad membrane alteration, mmc hyperplasia, immune cell infiltration, and fibrosis gonadal alterations were detected. Different microscopic alterations in tilapia fish gonads, such as atresia or degeneration of the oocyte (shrinkage of the oocyte), vacuollation and necrosis, inter-follicular edema, partial lysis of the oocyte, crumbled oocytes, and oocytes with a thicker vitelline envelope (thickening of the ovarian wall) were reported [33].

Pollution results in a decline in gonad activity, which is clearly reflected in decreasing sperm in ripe testes and ripe oocyte degeneration. These effects may upset the development of germ cells and decrease the fish’s ability to reproduce [37, 38]. A reduction in oocytes are characterized by the presence of a small number of oogonia and immature oocytes and a few early degenerating vitellogenic oocytes with complete dissolution of ooplasmic and nuclear materials in the ovaries. And the nuclei of the oogonia underwent complete karyolysis, vacuollation, and shrinkage of the nucleus in the ovary. Moreover, prominent inter-follicular spaces are observed in the ovaries, which are probably formed due to shrinkage of the oocytes [35]. Gonads (testis and ovary) deformation from their ideal shapes is found to indicate the effect of pollution such as pesticides and endocrine-disrupting metals (cadmium, arsenic, lead, mercury, and uranium) [25, 39]. Additionally, atresia, characterized by the disintegration of the nucleus, vitelline envelope breakdown, increase in number and size of follicular cells, and liquefaction of yolk globules, is associated with exposure to environmental contaminants.

A scientific investigation verified the link between pollution and the rise in follicular atresia, which is brought on by the gonadotropin hormone and other estrogen hormone decreased activity. Pollution exposure, particularly exposure to heavy metals, causes the reproductive hormone to malfunction, which subsequently affects the vitellogenin receptors, causing oocyte disruption [39]. The ovaries of fish subjected to sub-lethal concentrations of nickel and textile mill effluent have shown atretic oocytes and an increase in the inter-follicular space [35]. Aggregates of pigmented cells can be found in both the ovaries and testes. These structures, also referred to as chromatophores or macrophage aggregates, are more numerous in fish collected at contaminated sites.

Heavy metals especially, Pb and Cd can disrupt and impair reproduction due to their remarkable degree of bioaccumulation and consequently exert serious problems. Testes of the fish collected from the polluted water revealed an impairment of spermatogenesis and lobular structures, suppressing sperm production [39].

Over the years, spleen organs in many species, including fish, have been studied due to their importance in immunity-related roles [40]. Recent studies show that histological findings of spleen organs are biomarker parameters for determining the presence of stress in fish caused by poor water quality habitants.

The present study has observed different histological alterations in the spleen, such as pigment deposition (hemosiderin), necrosis, degeneration, lymphocyte depletion, mononuclear immune cell infiltration and/or melanomacrophages center infiltration, hypertrophy, congestion, hemorrhage, and blood vessel inflammation.

In the spleen of Nile tilapia, lymphocyte depletion, as well as an increase in the size of the spleen, Necrosis, MMCs abnormalities, Vacuollation, and hemosiderin, has often been associated with environmental contamination [31, 40]. The present study could detect that MMC infiltration was highest in fish spleens from the aquaculture study site. Similarly, the current study is agreed with [29]. The number, size, and pigment contents of macrophages change in poor health conditions in fish, under stress, and in response to environmental contaminants. The size, number, and histopathological appearance of MMCs and the levels of macrophage activities such as phagocytosis is considered important parameters among immunological biomarkers, especially in determining the effect of environmental pollutants and in bacteriological infections. The current study was detected depletion of the lymphocytes and exhaustion of the MMCs, it results in a reduction in number and size with scant pigmentation of the MMCs and this result agreed with [32]. Bacterial infections are the main cause of death in aquaculture. Aeromonad, Pseudomonad, and Edwardsiella are naturally present in aquaculture and act to cause diseases in fish [41].

Overall, the formation of MMCs can show the response of aquatic organisms to stressors such as pathogens, toxic agents, variations in water temperature, or bio-indicators of pollution [29]. And Haemosiderosis is a pathological condition that occurs due to the deposition of hemosiderin in the tissue/organs. It is related to an increased rate of erythrocyte destruction in the red pulp of the spleen. Hemosiderin is a golden yellow to brown granular crystalline pigments derived from hemoglobin and stored in cells. Deposition of hemosiderin pigments has been observed in the spleen of fish exposed to sodium cyanide [32]. Pesticides could cause necrotic alterations in the spleen tissues of fish species [42]. Likewise, Lake Tana is polluted by Pesticides.

By Using Bernet et al. [23] histopathological health assessment protocol, the study found the mean value of total fish index 95.3 from southern gulf of lake tana and from the aquarium site it was 65.5 and it was significantly higher (p < 0.05) at the southern gulf of tana as compared to aquarium site. Zimmerli et al. [24] reported that histological manifestations may be explained by the higher burdens of anthropogenic influences. Therefore, the current study may be reflected that the southern gulf of Lake Tana is currently experiencing a lower aquatic environmental health status due to the higher burden of anthropogenic activities, and other factors like feed quality or quantity might also further expose the fish to many stressors. And fish from aquariums have good management interventions like proper feeding, water refreshments, and fencing,those factors may improve the adaptability nature of the Nile tilapia and result in lower severity of histological changes.

This study was found grade four severity levels in the liver and gill organs which indicated that those organs are highly impacted organs exceptionally. In fact, the liver is mostly associated with the detoxification and biotransformation processes, and due to its function, position (visceral), and blood supply, it is also one of the organs most affected by contaminants in water [43, 44]. Moreover, some authors reported findings of higher heavy metal accumulation in the liver of Nile tilapia in Lake Tana even though their impacts were not studied [18]. Moreover, the current study tried to identify pathological changes severity in relation to the sex of the sampled fish. However, statistically, it could not be found to be different. This may be due to that there is no specific pollutant uniquely that could affect sex differences, or it may be due to absence of any risk factor that intended for the occurrence fish health problem in sex differences.

Lake Tana, more specifically, the southern Gulf, is most likely located in an urbanized area that has been impacted by urbanization, indiscriminate industrial activities, hotel, restaurant, and/or café sewage, and hospital sewage, as well as the disposal of domestic waste [17]. The aforementioned human activities lead to a decline in water quality, which can further affect fish health (Nile tilapias).

The current study evaluated the results of organ alteration indices in terms of the mean severity of the histological response or severity of pathological changes in each study site accompanied with a severity grading system that was developed by van Dyk et al. [14] modification. The lowest grade (grade 1) indicates slight histological alterations of organ tissue, so the southern gulf of Lake Tana site has been shown to be associated with higher organ grades, indicating a higher tendency for severe histological changes. In the present findings, the statistical association between poor habitant quality and organ histopathological mean grades was found to be significant, at least for gills (2.35) and gonads (1.7), but liver (2.5) was fairly non-significant in relation to sites. This may be due to gills are the main route of toxicant penetration into the fish organism,thus, they are the first organs that come into contact with environmental pollutants and are sensitive subjects for detecting the effects of water toxicants on the fish organism; thus, they are the primary markers for aquatic pollution and fish health conditions [16, 45]. And gonads and gills organs may be have higher level of bioaccumulation of toxic chemicals or infected with pathogenic microbes and ultimately, this results implied that the southern gulf of tana is highly impacted aquatic environment by different pollutants as compared to aquarium. And relatively Nile tilapia aquarium is certainly at good environmental health condition and this implied that widening practices of fish farming shall be encouraged accompanied with intensive management intervention.

Fish health is typically more degraded in regions where anthropogenic activity has damaged the environment and absence of application of husbandry practices for aquatics and their habitants in comparison to less polluted sites. And aquatic regions influenced by human activities had higher organ indices and, consequently, higher histopathological grades. The current study found higher mean values of the histopathological grades at the southern gulf of Lake Tana which can be attributed to ongoing human effects in the urbanized wetlands of Lake Tana or southern gulf of Tana and indicated the presence of higher level of pollutants in this aquatic environment.

In general, once it is able to integrate the impacts of both biotic (pathogens) and abiotic (chemical) elements, the advantage of using fish histopathology as a biomarker resides in its intermediate location in the hierarchy of biological responses [23, 24]. Although the histological alterations identified may be the result of a range of different environmental stressors, the most likely causative agent identified is the high prevalence of parasitic infections [46]. At the same time, the integrative nature of the histopathological alterations implies that they often cannot be assigned to a specific causative factor. Additionally, even if they are present in a small number of specimens, more severe and visible abnormalities are typically mentioned in histopathological surveys of fish populations [25]. Therefore, using Nile tilapia fishes as a biomarker, based on Bernet et al.’s [23] histopathology protocol, southern Gulf of Tana environmental quality is characterized by reduced overall health, which could be an indicator of lower aquatic environmental quality.

Conclusion and recommendations

The present study was conducted to undergone a histopathological study of Nile tilapia fishes health status which was the first in it’s kind in Ethiopia on this fish. The study has found different pathological changes in both study sites, however, the total histopathological fish index result was significantly higher at the southern gulf of Lake Tana as compared to aquaculture site. Moreover, among the four target organs evaluated, liver organs were the most damaged while gonads were least impacted organs. Therefore, it can be suggested that southern gulf of Lake Tana is a lower aquatic environment health status which may indicate the presence of higher level of pollutants in this site as compared to the aquaculture site. Therefore, the study observed that, the presence of ongoing pollution at both study sites, more particularly at the southern gulf of Lake Tana, and the reduction of the Nile tilapia fish’s catchment number may be due to the presence of reproductive organ effects, which have potential implications for the Nile tilapia fish’s future sustainability. Although histological alterations cannot be stressor-specific, they can effectively assist in assessments as an indicator of fish health in relation to aquatic pollution. So that, before entrance to Lake Tana, effluents from the city of Bahirdar should be properly handled and monitored to control the water quality and fish health, as well as ensure the sustainability of the fishery; Intensive management, such as treating drainage water before its entrance into Nile tilapia farms, should be applied to monitor the water quality and fish health to ensure the sustainability of the Nile tilapias and Future studies should consider age differences, seasonal variation, and the detection of specific pollutants with specific organ alterations via bio position or bioaccumulation.