© 2015 IJSRSET | Volume 1 | Issue 1 | Print ISSN : 2395-1990 | Online ISSN : 2394-4099

Themed Section: Science

Histopathological Studies of Gills and Liver of Clarias Gariepinus Cultured in

Wastewater from Superphosphate Fertilizer Company (SFC)
Barak Zebedee*1, Auta Jehu1, Bingari , Mathias Simon2, Babale Musa Yoane1
Department of Biological Sciences, Ahmadu Bello University Zaria, Nigeria
Department of Biological Sciences, Taraba State University Jalingo, Taraba State, Nigeria

ABSTRACT

The histological effects of Wastewater from Superphosphate Fertilizer Company (SFC) on the fingerlings of Clarias
gariepinus, an important African fish were investigated. Exposure of fingerlings of Clarias gariepinus to the
wastewater on their gills revealed different histopathological alterations such as attenuated primary lamellae,
marked loss of secondary lamellae, loss of epithelial cells, erosion of gill villi while in the liver there was fatty
degeneration, oedema, mild fatty change in treatments with less concentration of the wastewater, moderate steatosis,
necrosis and hyperemia was observed in treatments with the highest concentration of while the gills and liver from
the control remained intact.

Keywords: Histopathology, Wastewater and Technology

I. INTRODUCTION are derived from phosphate rock (Barker and Pilbeam,

Contamination of water results when by-product and 2006). However, world reserves of currently exploitable
chemicals are not properly disposed into the phosphate rock are rather limited and increasingly
environment or conserved in reservoirs. Contamination dissipated (Abelson, 1999).Phosphate fertilizer is of
of soil and water also results from the use of pesticides environmental concern because excess nutrients can lead
and fertilizers which enters water systems from several to ‘algal blooms’ or eutrophication as it is known in
sources including industrial waste, sewage effluent, lakes, ponds, estuaries and very slow moving rivers. The
agricultural input and animal feedlots. It is also a algal bloom reduces light penetration and restricts
metabolic by-product of fish (Abbas, 2006). atmospheric re-oxygenation of the water. When the
dense algal growth dies the subsequent biodegradation
Histopathological study of gills showed structural results in anaerobic conditions and the death of many
abnormalities such as elongation, fusion and hyperplasia aquatic organisms. High phosphate concentrations and
of the lamellae. A number of organ pathologies have algal blooms are generally not a problem in moving
been reported to be indicative of fish health (Heath, streams because such streams are continually flushed out
1995; Hinton et al. 2001 and WGBEC, 2002) but gill and algae do not accumulate (Hodgson, 2004).
histopathology has been reported to have an edge over
them (van den Heuvel et al. 2000, Au, 2004) and has II. METHODS AND MATERIAL
been recognized to be a reliable biomarker of water-
quality-related stress in fish (Teh et al. 1997; van der Study Area
Oost et al. 2003, Costa et al. 2009). This is because of
the notion that histopathological biomarkers reflect fish The collection of the wastewater was from
health more realistically than biochemical biomarkers Superphoshate Fertilizer Company located in Kaduna
and can be better extrapolated to community and State (Lat. 10˚52′N, Long.7˚44′E) and the research work
ecosystem level effects of toxicity (Au, 2004). was carried out in the Department of Biological Sciences,
Phosphorus is one of the primary nutrients essential for Ahmadu Bello University Zaria, Nigeria (Lat.11˚13′N,
plant growth and crop production (Mengel and Kirkby, Long.7˚12′E).
2004). At present, most commercial phosphorus (P)
fertilizers on the market such as single superphosphate Selection of Test Organism

IJSRSET151140 | Received: 30 Jan 2015 | Accepted: 27 Feb 2015 | January-February 2015 [(1)1: 184-189] 184
The organism selected for this study was internationally The physico-chemical parameters of the dilution water
recommended for ecotoxicological assessments, and (de-chlorinated tap water from Ahmadu Bello University
have significant importance in aquatic food chains, Zaria) were monitored daily. These parameters include,
facility of handling in the laboratory, and sensitivity to pH, Temperature, Total Dissolved Oxygen and
potentially toxic compounds (OECD, 1992; OECD, Electrical Conductivity. The pH and Electrical
2002). It has also been suggested by Doudoroff et al Conductivity were determined using a pH/Hanna multi
(1959) that economically important local fish species be parameter instrument (model H13952). Temperature was
used in toxicity assay. Clarias gariepinus, family determined by the use of mercury in glass thermometer
Claridae, is a fish of high commercial value and are very and Dissolved Oxygen was determined by the modified
common in Nigerian freshwaters. It is of high nutritive Winkler Azide Method (Lind, 1979; APHA, 1985;
value. On the basis of availability, commercial and 1990).
ecological importance, Clarias gariepinus was chosen
for this study. Acute toxicity bioassay

Collection of Wastewater The renewal method of bioassay 96h were conducted in

the laboratory following methods by Spraque (1973) and
Collection of samples involved collection of wastewater APHA (1985) to determine the toxicity of wastewater
samples at the point of discharge into the reservoir against Clarias gariepinus. The bioassay test was
before releasing it into the environment from Federal carried out in 12 glass tanks each of size 30.5 X 30.5 by
Superphosphate Company. About 100L of the sample 92.5cm into which approximate quantity of wastewater
were taken in plastic containers and transported to the from Superphosphate Fertilizer Company was taken to
Department of Biological Sciences, Fisheries & give a final volume of 25.0L. The nominal
Hydrobiology Laboratory, Ahmadu Bello University concentrations of wastewater used were 0.0, 5, 25, 50,
Zaria, and it was preserved using Trioxonitrate (V) acid. 75 and 100.

Collection and maintenance of fish The mixture of the toxicant and dilution water was
allowed to stand for 30minutes before the introduction
Fingerlings of Clarias gariepinus were obtained from of test fishes.
Miracle Farms ABU Zaria, in cold boxes with water to
the Department of Biological Sciences, Fisheries & The fishes were randomly distributed to each glass tank
Hydrobiology Laboratory, Ahmadu Bello University as suggested by Spraque (1973). Each tank contained 10
Zaria. They were acclimatized for two weeks in 4 fish. Each assay was replicated simultaneously and
oval/rectangular shaped bath tubs, separately containing repeated once to determine reproducibility. Aeration of
water of about 150L. The fish were fed on Coppen’s the test water was suspended a few minutes prior to
pelleted commercial feed of 2mm in size at 5% biomass addition of the toxicant and during the test. The toxicant
of 35% crude protein diet three times daily. and the test water were renewed after 48hours during
each series to test. The fish were starved 24hours prior
De-chlorination and changing of Test water to the start and during each assay test

Water was renewed partially thrice a week using de- Histopathology

chlorinated tap water. Water was aerated during holding
and acclimation. No aeration was carried out during the Gills and liver obtained from fish that have just died (in
assay. Each time of renewal (during acclimation), three case of those concentrations and from fish that survived
quarters of the water was siphoned out using a rubber the acute exposure were removed and fixed in formo-
hose of 5mm bore and topped up with de-chlorinated saline (Alan et al, 1983). The tissues were washed in
water. running tap water for at least 2 hours to remove traces of
formalin. This was followed by dehydration using
Physico-chemical parameters of dilution water successive percentages of alcohol (30, 50, 70, 90 and

International Journal of Scientific Research in Science, Engineering and Technology (ijsrset.com)

185
100%). They were then infiltrated in chloroform and 5.5 -9.5 as recommended by WHO(1984)/FEPA(1991)
blocked in paraffin wax 58-600C melting point. Samples and this acidic nature of the toxicant most have led to
were embedded in fresh molten wax using L-shaped the mortality of the fish during the bioassay; Wynne et
embedding moulds. Sections of 8µm thickness were cut al. (2001) reported that, pH has profound effects on
and stained in haematoxylin and eosin (H&E). water quality affecting the ability of bacteria which
Permanent slides were prepared with these sections and require slightly acidic pH to degrade toxic substances to
microphotographs taken with a magnification of X400. less harmful forms. The temperature remained fairly
This were examined and compared with those for constant in all the test tanks. The temperature remained
control. Data that was obtained from physico-chemical fairly constant in all the test tanks. Exposure period also
analysis in wastewater was subjected to Analysis of had an effect on the temperature during the period of
Variance (ANOVA) using Microsoft Excel for Windows study. Parameters such as pH, temperature and DO in
2007 were used to test differences between levels of the control were significantly higher than those in the
treatment and to separate means respectively. test tanks (p< 0.05) and this may lead to negative
environmental problems, low 2.60-3.50 DO mean values
III. RESULT AND DISCUSSION of led to the death of some fishes in the treatment tanks.
Alberto et al., (2005), observed that the first responses
Physico-chemical Analysis of Wastewater Used: of fish to environmental hypoxia (low DO levels) were
Acute Exposure changes in ventilation and cardiovascular functions
stimulated by cathecholamines released from andregenic
The values for the physico-chemical parameters for nerves and chromaffin tissues where they are both
the wastewater used for the research in the test synthesized and stored also Gabriel et al. (2007)
tanks are shown in Table 1. These parameters of the reported that the DO level in the control were
test water vary slightly (p<0.05) during the bioassay. significantly higher than those in the treatment. Electric
Water quality parameters analysed were temperatur conductivity (EC) showed that at higher concentration of
e, electrical conductivity, pH and dissolved oxygen. the toxicant the value of EC was high; this might be due
to high concentration of the ions that were found in the
Table 1: Mean water quality parameters for control and
treatment tanks in Acute Bioassay wastewater.

Water 100 75.00 50.00 25.00 5.00 0.0 Histopathology for Gills
quality (ml/L) (ml/L) (ml/L) (ml/L) (ml/L) (ml/L)
parameters
pH 3.10 3.61 3.80 3.95 4.10 7.15 From the histological sections of the gill are presented in
Temperature 33.52 32.67 31.18 31.00 30.42 33.10 plate II and III, gill of control fish has a normal structure
(0C)
Dissolved
with the filament and lamellae consisting of filaments
2.20 2.50 2.65 2.70 3.00 5.60
Oxygen attached to cartilaginous gill bar with fingerlike
(ml/L) projections (secondary lamellae) on each side of the gills
Electrical 1013 724 492 390 250 170
Conductivity (plate II). Sections of gill from different concentration of
(µS/cm) the wastewater from SFC were the most damaged as
observed in plate III. At 100ml/L wastewater there was
pH increased with increase in concentration and the oedema of gills epithelium and mutilation of the gill
exposure time, the control was neutral as against that of rakers. At 5ml/L concentration of wastewater there was
the tanks with the toxicant which was acidic. less damage to gills. Damage done to the gills was dose-
Concentrations of the toxicant had effect on the pH dependent.
value and affected the quality of water and the
environment, from this study pH had a range between
4.16-4.82 during the acute study which revealed that the
toxicant was acidic as observed from the pH scale which
was against the lamella. General observations showed
evidence of proliferative lesions in the gill pH scale of

International Journal of Scientific Research in Science, Engineering and Technology (ijsrset.com)

186
 changes observed. Some of the observed changes
include marked loss of secondary lamellae, attenuated
PL primary lamellae and loss of epithelial cells as observed
in the tank with 100ml/L of toxicant and this was due to
high concentration of Pb (0.91 mg/L), effect on the gills
of fish might affect the respiratory system of the fish and
SL the liver too was affected which led to the death of fish
as was revealed by the histopathological studies when
compared with tissues gotten from the control fish,
Findings too by Dhanapakiam, et al, (1998) on
histopathological alterations in the fish gills were been
used in biomonitoring the effects of various pollutants in
Figure 1 : Plate I: Photomicrograph of Gill cells of control Fish, PL
the aquatic environment. Fafioye et al.,(2004) reported
and SL intact (PL= primary lamellae, SL=secondary lamellae). X40
similar findings on histopathological response of Clarias
gariepinus to plant extracts under brief exposure.
PL

SL Other pathological alterations observed in this study (i.e.

L erosion of gill villi, oedema, matting of gill filaments,
necrosis and hyperemia (vascular congestion) agrees
with reports by Dhanapalkiam et al.(2004), Harper and
Wolf, (2009) Pathan et al.(2010), their report was that
specific reactions to acute localized oxygen deprivation
of gill tissues). Au, (2004), Tang and Au, (2004)
Nordberg et al. (2005), reported that gill lesions such as
oedema formation and shortened gill filaments did not
Figure 2 : Plate II: T.S of gill of Clarias gariepinus exposed to acute
concentration (50ml/L). (PL=primary lamellae), complete filament
only indicate possibilities of impaired respiratory
detachment. X40 functions but impaired osmo-regulatory functions too.

GF The reduced availability of oxygen to respiratory

surfaces like gills was a direct consequence of hypoxic
conditions traceable to anthropogenic impacts on water
quality of aquatic systems (Adeogun et al. 2011,
Adeogun and Chukwuka, 2012).

Sub- lethal studies on the fish showed loss of epithelial

cells along with marked attenuation of primary and
secondary lamellae at the highest concentration of
19ml/L and slight attenuation of primary and secondary
lamellae at the lowest concentration of 5ml/L.

Similarly, Dhanapalkiam et al. (2004) reported that the

Figure 3 : Plate III Gill filaments of C. gariepinus from (100ml/L) gill lesions in the carp, Labeo rohita exposed to sub-
after 8weeks of exposure showing Complete erosion of filament(GL= lethal concentrations of tannery effluent and reported
gill filament)X40
severe damages to gill architecture including swelling of
primary and secondary epithelial cells. Peebua et al.
The gills of fish exposed to the acute concentration
(2008) also reported histopathological alteration in the
of the toxicant and those of the control fish exposed
gill of Oreochromis niloticus exposed to alachlor and
for 96hrs were compared and there were slite
observed gill alterations ranging from oedema of

International Journal of Scientific Research in Science, Engineering and Technology (ijsrset.com)

187
theepithelial system to hypertrophy and hyperplasia of cell. Necrosis and mild statuses was observed in fish
epithelial cells. Pathan et al. (2010) also reported exposed to 9ml/L concentration of the toxicant, other
pathological changes and lesions ranging from epithelial observations made revealed mild fatty change and focal
hypertrophy, swelling in pillar and mucous cells to peri portal inflammation. Cengis et al. (2001) reported
hemorrhage in gill lamella of a freshwater fish Rasbora some histopathological alteration in the liver of
daniconius exposed to paper mill effluent which was mosquito fish (Gambusia affins) such as sinusoid
high in organic content. enlargement, haemorrhage, pikonosis, vacuolization of
cell cytoplasm, infiltration of mononuclear lymphocytes,
Histopathology for Liver hyperthrophy and congestion after exposure to sub-lethal
concentrations of endosulfan.
Plate IV shows the features of liver sections of control
fish. Observations showed hepatocytes and other cells IV. CONCLUSION
systematically arranged. The epithelium of the veins is
lined with various epithelial cells. Plate V that is fatty The acute and sub-lethal histopathological degeneration
degeneration of the liver. In fish exposed to 75ml/L of observed include necrosis, attenuated primary lamellae,
wastewater, necrotic hepatocytes were observed. loss of epithelial and mucus cells, marked loss of
Oedema of the liver was dose dependent. secondary lamellae, oedema, haemorrhage, vascular
congestion, hyperplasia and hypertrophy in a variety of
anatomical sites including gills. The liver showed mild
CV fatty change, moderate steotosis and mild inflammatory
infiltrate.

V. REFERENCES

[1] Abbas, H.H. (2006). Acute toxicity of ammonia to common

carp fingerlings (Cyprinus carpio) at different pH levels.
Pakistan Journal of Biological Sciences 9 (12): 2215-2221
[2] Abelson, P.H. (1999). A potential phosphate crisis. Science
283:2015.
[3] Adeogun, A. O. and A. V. Chukwuka (2012). Altered
reproduction in Clarias gariepinusexposedto Industrial
Figure 4 : Plate IV: T.S of the cells of control fish showing with Effluents. American Journal of Agricultural and Biological
homogenous cytoplasm. (CV= central vein, PC parenchyma cells) Sciences 7(1): 61-70.
X100 [4] Adeogun, A. O. and A. V. Chukwuka (2012). Altered
reproduction in Clarias gariepinusexposedto Industrial
Effluents. American Journal of Agricultural and Biological
ES Sciences 7(1): 61-70.
[5] Adeogun, A.O., A.V. Chukwuka and Ibor, O.R(2011). Impact
of Abattoir and Saw-Mill Effluents on Water Quality of Upper
Ogun River (Abeokuta).American Journal of Environmental
Sciences 7 (6): 525-530.
[6] Alan, J.A.S., Genaci, J.R., and Holdson, P.V. (1983).
Histopathological and Physiological Response of Rainbow
Trout, Salmo gaidneri (R) to sub-lethal levels of lead. Water
Research, 17:1115-1118.
[7] Alberto, A., A. F. M. Camargo, J. R. Verani, F. T. Costa and M.
N. Fernandes (2005). Healthvariables and gill morphology in
the tropical fish Astyanax fasciatus from a sewagecontaminated
river. Ecotoxicology and Environmental Safety 61: 247-255.
Figure 4 : Plate V: T.S of the liver cells of Clarias gariepinus, [8] A.P.H.A. (1985).Standard methods for the examination of water
extensive areas of necrosis exposed to 50ml/L (ES=sinusoids) X100
and wastewater. 16thedition. American Public Health
The acute study of liver exposed to the toxicant revealed Association, New York. 1268pp
necrosis and marked fatty changes at 50ml/L. Liver [9] A.P.H.A. (1990).Standard methods for the examination of water
statuses was also observed which was as a result of and wastewater. 16thedition. American Public Health
glycogen accumulation as a result of breakdown of liver Association, New York. 1268pp

International Journal of Scientific Research in Science, Engineering and Technology (ijsrset.com)

188
[10] Au, D.W.T. (2004). The application of histo-cytopathological [28] OECD (Organization for Economic Cooperation and
biomarkers in marine Pollution monitoring: a review. Marine Development). (2002). Guideline for the testing of chemicals:
Pollution Bulletin 48: 817–834 fish, acute toxicity test No. 203, http://www.oecd.org.
[11] Barker, A.V. and Pilbeam, D.J., (2006).Handbook of plant [29] Pathan, T.S., P.B. Thete, S.E. Shinde, D.L. Sonawane and Y.K.
nutrition. CRC Press, Boca Raton, FL. Khillare (2010). Histopathological changes in the Gill of
[12] Cengis, E.I., E and Balei, K. (2001). The Histopathological Freshwater Fish, Rasbora daniconius Exposed to Paper Mill
Effects of Thiodan on the Liver and gut of mosquito fish, Effluent. Iranian Journal of Energy & Environment.1 (3):170-
Gambusia affins. Journal of Environmental Sciences and Health 175.
part B; Pesticides food contaminants and agricultural wastes. [30] Peebua P., Kruatrachue, M.,Pokethitiyook, P., and Singhakaew,
Taylor and Francis 36(1) 75-85 S. (2008). Histopathological alterations of Nile tilapia,
[13] Costa, P. M., M. S. Diniz, S. Caeiro. J. Lobo, M. Martins, A. M. Oreochmisroniloticus in acute and subchronic alarchlor. Journal
Ferreira, M.Caetano, C. Vale, T. A. DelValls, M. H. Costa. of Environmental Biology 29(3) 325-33
(2009).Histological biomarkers in liver and gills of juvenile [31] Spraque, J.B. (1973). Measurement of pollutants to fish III. Sub-
Soleasenegalensis exposed to contaminated estuarine sediments: lethal effects and
A weighted indices approach.Aquatic Toxicology 92: 202–212. safe concentrations. Water research.5: 245-266.
[14] Dhanapakiam, P. et al. (1998) A Study on the histopathological [32] Tang, J.Y.M. and Au, D.W.T. (2004). Osmoregulatory failure is
changes in gills of Channa punctatus in Cauvery River water J. a possible cause of fish kill upon sub-bloom exposure to
Environ Biol. 19(3) -265-269. Chattonella marina. Environ. Toxicol.Chem.
[15] Dhanapalkiam, P., V. Sampoorani, M..Kavitha, V. K. [33] Tang, J.Y.M. and Au, D.W.T. (2004). Osmoregulatory failure is
Ramasamy, A.Chandrakala and K. C. Aruna (2004). Gill lesion a possible cause of fish kill upon sub-bloom exposure to
in the major carp, Laboe rohita exposed to lethal and sublethal Chattonella marina. Environ. Toxicol.Chem.
concentrations of tannery effluent. J. Environ. Biol. 25(3): 333- [34] Teh, S. J., S. M. Adams and D. E. Hinton (1997).
336. Histopathologic biomarkers in feral freshwater fish populations
[16] Doudoroff, D. and Fry, F.E.J. (1959).How should we determine exposed to different types of contaminant stress.
dissolved oxygencritical for freshwater fishes? Trans 2nd Aquat.Toxicol.37: 51-70002E
Seminar.Biology on problems f water pollution. W.S.P.H.S.W. [35] van den Heuvel, M.R., M. Power, J. Richards, M., MacKinnon
pp 1-10. and D. G. Dixon (2000). Disease and gill lesions in yellow
[17] Fafioye , O.O., A debisi, A.A. and Fagade, S.O. (2004) Toxicity perch (Perca flavescens) exposed to oil sands mining-associated
of Parkia biglobosa and Raphia vinifera extracts on Clarias waters. Ecotoxicol. Environ. Saf.46: 334–341.
gariepinus juveniles. African Journal of Biotechnology, 3:627- [36] van der Oost, R., J. Beyer and N. P. Vermeulen (2003). Fish
630. bioaccumulation and biomarkers in environmental risk
[18] Federal Environmental Protection Agency (FEPA), assessment: a review. Environmental Toxicology and
(1991).Guidelines and Standards for EnvironmentalPollution Pharmacology 13: 57–149
Control in Nigeria, pp: 238. [37] World Health Organisation (WHO), (1984). Guidelines
[19] Gabriel, U.U., Amakiri, E.U. and Ezeri, G.N.O. (2005). forDrinking Water Quality. 3rd Edn. Health Criteriaand
Haematology and Gill pathology of Clarias gariepinus exposed Supporting Information. WHO, Geneva, pp: 668.Retrieved
to refined petroleum oil, kerosene under laboratory condition. from:
Journal of Animal and Veterinary Advances, 6(3): 461-465. http://www.who.int/water_sanitation_health/dwq/fulltext.pdf.
[20] Harper C. and J. C. Wolf (2009). Morphologic Effects on the
Stress Response in Fish. Institute for Laboratory Animal
Research (ILAR) Journal. 50(4):246-255
[21] Heath, A.G., (1995). Water Pollution and Fish Physiology,
second ed. CRC Lewis Publishers, Boca Raton, FL. pp. 125–
140.
[22] Hinton, D.E., H. Segner and T. Braunbeck.,(2001).Toxic
responses of the liver.In: Schlenk, D., Bensen, W.H. (Eds.),
Organs. In: Toxicity in Marine and Freshwater Teleosts, Vol. 1.
Taylor and Francis, London, pp. 224–268.
[23] Hodgson, E. (2004). A textbook of Modern Toxicology (3rded).
A John Wiley and Sons Inc. Publication.
[24] Lind, O.T. (1979). A handbook of common methods of water
analysis for Limnology. C.V. Morshy Publishers, St. Louis,
U.S.A. 199pp
[25] Mengel, K. and E.A. Kirkby. (2004). Principles of plant
nutrition. Springer-Verlag, New York, NY.
[26] Nordberg, G. F., B. A. Fowler and M. Nordberg (2005).
Handbook on Toxicology of Metals. 3rd edition. 969pp.
[27] OECD (Organization for Economic Cooperation and
Development). (1992). Guideline for the testing of chemicals:
fish, acute toxicity test No. 203, http://www.oecd.org

International Journal of Scientific Research in Science, Engineering and Technology (ijsrset.com)

189