=Paper=
{{Paper
|id=Vol-1498/HAICTA_2015_paper94
|storemode=property
|title=Influence of Sublethal Lead Concentrations on Glucose, Serum Enzymes and Ion Levels in Tilapia (Oreochromis mossambicus)
|pdfUrl=https://ceur-ws.org/Vol-1498/HAICTA_2015_paper94.pdf
|volume=Vol-1498
|dblpUrl=https://dblp.org/rec/conf/haicta/KayaAY15
}}
==Influence of Sublethal Lead Concentrations on Glucose, Serum Enzymes and Ion Levels in Tilapia (Oreochromis mossambicus)==
https://ceur-ws.org/Vol-1498/HAICTA_2015_paper94.pdf
Influence of Sublethal Lead Concentrations on Glucose,
Serum Enzymes and Ion Levels in Tilapia (Oreochromis
mossambicus)
Hasan Kaya1, Mehmet Akbulut2, Sevdan Yılmaz3
1
Çanakkale Onsekiz Mart University, Marine Sciences and Technology Faculty, Department
of Basic Sciences, Çanakkale-Turkey, e-mail: hasankaya@comu.edu.tr
2
Çanakkale Onsekiz Mart University, Marine Sciences and Technology Faculty, Department
of Basic Sciences, Çanakkale-Turkey, e-mail: mehakbulut@comu.edu.tr
3
Çanakkale Onsekiz Mart University, Marine Sciences and Technology Faculty, Department
of Aquaculture, Çanakkale-Turkey, e-mail: sevdanyilmaz@comu.edu.tr
Abstract. In this study, alterations in glucose, blood enzymes (alkaline
phosphatase (ALP), lactate dehydrogenase (LDH), alanine transaminase
(ALT), aspartate aminotransferase (AST)) and serum ion (P+++, Mg+, Cl-, Ca++,
Fe++) levels were investigated in Tilapia (Oreochromis mossambicus), which
were semi-statically exposed to different lead concentrations in vivo. The fish
were exposed to low (0.5 mg/L), medium (2.5 mg/L) and high (5 mg/L)
concentrations of lead during 14 days. At the end of the experiment,
biochemical blood parameters such as glucose, ALP, LDH, AST, chloride and
magnesium increased (p<0.05). While, LDL and calcium levels decreased
(p<0.05); ALT, cholesterol, albumin, iron and phosphor were fluctuated
(p<0.05). Consequently, it was found that exposure of O. mossambicus to lead
concentrations affected serum biochemical parameters negatively.
Keywords: lead, toxicity, glucose, serum enzymes, ion levels, Oreochromis
mossambicus
1 Introduction
Lead is a persistent contaminant in the natural environment that can enter the water
column through geologic weathering and volcanic action, or by various
anthropogenic activities including mining and smelting of lead-ores, burning of coal,
effluents from storage battery industries, automobile exhausts, metal coating and
finishing operations, fertilizers, pesticides and from additives in pigments and
gasoline (WHO, 1995).
Contamination of water through anthropogenic practices is the primary cause of
lead poisoning in fish (Sorensen, 1991). Due to its nondegredable nature, it’s get into
the environment and eventually enters to fish and human body system. When it can
enter to the body lead can accumulate to soft tissues such as liver, kidney, nervous
system and brain of fish (Berman, 1980). It is well documented that lead can impair
858
the health of humans and other organisms by neurotoxicity, renal toxicity, and
deleterious effects on the hematological and cardiovascular systems (ATSDR, 2006).
In studies examining the toxicity of lead on fish, it was determined that lead inhibited
to Na+, K+-ATPase enzyme activity and caused to oxidative stress in tilapia (Kaya
and Akbulut, 2015), it damage Ca2+ and Na+ homeostasis in trout fish at
concentrations found in ecosystems (Rogers et al. 2003; Patel et al. 2006), and
caused hematological (Kaya et al. 2013) and neurological (Davies et al. 1976) effects
in fish under chronic conditions. However, no studies evaluating the effects of
sublethal lead concentrations on the biochemical parameters of fish could be found.
The present study aimed to investigate the effects of water-borne lead on fish with
special reference to the blood glucose, serum enzymes and ions.
2 Material and Methods
2.1 Experimental design
Tilapia fish used in this study (n=144) were obtained from Çanakkale Onsekiz
Mart University (Marine Sciences and Technology Faculty, Aquaculture
Department), Çanakkale, Turkey, and were adapted to ambient conditions in 12 stock
aquariums, each with dimensions of 45x28x80 cm and containing 80 L of rested¸
Çanakkale city tap water, for 4 weeks. Fish weighting 45.2±5 g (mean±SD) were
divided into 12 experimental aquariums, each containing 12 fish, and an
experimental design with three replicates was established. Feeding was interrupted
24 h before the start of the experiments to help maintain water quality. During the
experiment, the fish were fed twice a day with feed at about 2% of their body weight
and their behavior was observed during each feeding. Care was also taken to ensure
that all of the feed added to the tanks was eaten and that fecal waste was quickly
removed from the tanks at every water change. In the experiment, fish were exposed
to the following sublethal concentrations of lead: low, 0.5 mg/L; medium, 2.5 mg/L;
and high, 5 mg/L. The control group was maintained in freshwater only.
Concentrations were determined by considering in Ay et al. (1999). The experiment
had a semi-static regime, and water was changed every day: a 75% change in the
morning and a 25% change in the evening (modified from Smith et al. 2007). After
each water parameters were as follows: temperature, 25.4±0.3◦C (mean±SE);
dissolved oxygen, 6.31±0.11 mg/L; pH, 7.15±0.04; hardness, 125.0±6.2 mg/L
CaCO3; total ammonia, 0.151±0.02 mg/L. The electrolyte composition of the
dechlorinated Çanakkale tap water was measured as 0.310±0.005, 0.049±0.001,
0.534±0.001, and 0.828±0.006 mmol/L for Na+, K+, Mg+, and Ca2+, respectively.
Fish were randomly sampled on days 0, 7, and 14 for blood biochemistry analysis.
The experiments were performed in accordance with the guidelines for fish research
established by the Animal Ethics Committee at Çanakkale Onsekiz Mart University.
859
2.2 Preparation of the Pb(NO3)2 Solution and Application
The heavy metal salt, Pb(NO3)2 (99.5% purity; Sigma-Aldrich, Steinheim,
Germany), was used in the experiment. To obtain the needed concentrations, the
main stock solution was prepared in ultra-distilled water and appropriate dilutions
made from it.
2.3 Blood Sampling
In the experiment, total 12 fish on the first day (from the stock aquarium), 6 fish
from each aquarium on the 7th and 14th day were used for blood analysis. For blood
sampling, fish were anaesthetized with MS222 (Smith et al. 2007). They were well
wiped and cleaned in order to avoid mucus mixing into the blood, and then, blood
was taken from the fish through the caudal vein by a 5 ml plastic syringe, without
harming the fish (Val et al. 1998). Then, a sample of blood was transferred to EDTA
tubes, BD Microtainer®, UK for hematological analysis. Plastic biochemistry tubes
(Kima-vacutest®, Italy) were used for biochemical analysis. Blood serum was
isolated by centrifugation (4000xg, 10 min) and it was stored below -20°C.
2.4 Biochemical Analysis
For biochemical analysis, the blood collected was centrifuged at 4000 rpm for 10
minutes and blood serum was separated (Bricknell et al. 1999). Then, the serum
extracted was analyzed on the spectrophotometer (T80+UV/VIS) using an analyzer
(Bioanalytic Diagnostic Industry, Co). The biochemical parameters that were
detected during the test included glucose (GLU), alkaline phosphatase (ALP),
aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate
dehydrogenase (LDH), calcium (Ca2+), magnesium (Mg+), iron (Fe++), phosphorus
(P+++) and chlorine (Cl-).
2.5 Statistical analysis
ANOVA with Dunnett post-test (one-way ANOVA for comparison between
exposure groups and control group) was used. The statistical analysis was made by
using SPSS 17.0, and the significance level was considered to be 0.05 (Logan, 2010).
3 Results
Glucose and serum enzymes obtained from the study were given in Table 1.While
the glucose levels registered an increase in medium and high concentrations on 7th
and 14th days compared to the control group, they did not show any differences with
the control group in low concentrations. While the ALP increased across all groups
on day 7 compared to the control group, on day 14, the low and medium
860
concentrations registered an increase. The enzyme AST was revealed to be high
across all groups on day 7 and on day 14, it was found out to be higher in low and
medium concentrations compared to control group. While the ALT activity was
revealed to be lower on day 7 compared to control group, on day 14, a decrease in
low and medium dose was determined compared to the control group as an increase
in high group was experienced. While the enzyme LDH showed similarities with
control group in every group, (p>0.05), it increased across all groups on day 14.
Serum electrolytes during the study are given in Table 1. While the Ca2+, one of
the serum ions of the study decreased on days 7 and 14 across all groups compared to
control group, the Mg+ increased in medium and high concentrations on day 7. On
the other hand, while Fe++ showed a decrease in low and high doses on day 7
compared to control group, the medium dose increased. On day 14; a decrease
compared to the control group in low and medium doses was determined while an
increase was registered in high doses. The Cl- electrolyte showed an increase on days
7 and 14 across all groups compared to the control group. P+++ on the other hand,
showed an increase in low and high concentrations on day 7 and showed a decrease
on day 14 in high group compared to the control group.
861
Table 1. Effects of different concentrations of lead on glucose, serum enzymes and some serum minerals. Exposure groups are represented as follows
control: 0 (Control), low (0.5 mg/L), medium (2.5 mg/L), high (5 mg/L); ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine
aminotransferase; LDH, lactate dehydrogenase, Ca2+, calcium; Mg+, magnesium; Cl-, chloride; Fe++, iron; P+++, phosphor. The differences among the
times shown with the small letters for each ion are significant (p<0.05).
GLU ALP AST ALT LDH Ca2+ Mg+ Fe++ Cl- P+++
(mg/dL) (U/L) (U/L) (U/L) (U/L) (mmol/L) (mmol/L) (µg/dL) (mmol/L) (mmol/L)
Init 172.044±0 16.71±2.0 58.22±2.9 10.621±0. 3.026±0.0 79.681±0. 162.035±4.6 5.281±0.4
5.13±0.26 6.33±0.87
ial .78 1 3 50 5 8 7 3
Cont 167.199±4 5.73±0.77 6.52±0.44 16.58±0.6 61.17±4.1 10.599±0. 3.006±0.1 77.134±1. 163.107±6.6 5.371±0.2
rol .10c d c
6a 9 39a 8b 58b 2c 0b
182.484±4 9.52±0.47 6.56±0.12 13.05±0.7 73.17±2.7 9.149±0.3 3.130±0.0 61.087±1. 345.095±4.6 7.607±0.5
Low
7th .98c c c
7ab 0 7b 3b 36c 8b 2a
Medi day 215.884±7 18.76±1.7 18.21±0.9 13.26±1.3 74.38±3.0 7.391±0.1 3.583±0.1 129.717±2 379.362±2.5 6.044±0.1
um .68b a
9b 9ab 2 6c 1a .8a 8a 9b
271.954±2 13.41±0.8 38.15±1.6 9.34±0.75 85.29±2.7 8.186±0.2 3.707±0.0 59.148±1. 390.79±6.63 8.366±0.4
High
.80a 7b a b
7 7bc 6a 31c a
3a
Cont 172.900±2 5.89±0.72 6.80±0.60 17.88±1.4 54.27±3.0 10.373±0. 3.258±0.0 78.374±2. 170.763±8.0 5.642±0.5
rol .60c b d
6b 3b 28a 7ab 66b 4c 8a
182.424±5 10.19±1.1 21.81±2.8 2.12±0.15 145.03±4. 9.616±0.2 3.473±0.0 58.289±0. 161.338±4.6 6.459±0.3
Low
14th .91c 2a 6b c
21a 0a 5a 86c 8c 7a
Medi day 206.338±7 8.90±0.77 14.86±1.0 7.50±0.61 163.30±5. 6.080±0.0 2.962±0.1 61.463±0. 392.107±6.6 5.874±0.1
um .67b a
5c c
60a 07c 2b 94c 8a 3a
229.235±5 3.13±0.19 85.48±2.7 27.77±1.6 142.41±3. 7.638±0.0 2.924±0.0 97.392±3. 346.97±1.26 3.961±0.1
High
.94a b
8a 1a 24a 07b 2b 31a b
0b
862
4 Discussion
The glucose is the primal source of the energy that is required for the vital actions
and its level in serum is regulated through the endocrine system (Dange, 1986). In
fish, in addition to the stress induce such as hunger, dense stocking etc; the pollutants
such as metals also increase the secretion of the cortisol, epinephrine and
glucocorticoid thus leading to the changes in carbohydrate metabolism (Sastry and
Subhadra, 1985). Under the influence of lead metal and the environmental
concentrations, it was revealed that the glucose levels in serum increased compared
to the control group. It is thought that such an increase forms hyperglycemia and in
addition, leads to damages in liver and hormonal irregularities (insulin deficiency).
The enzymes of dehydrogenize and phosphatase are important and critical
enzymes in terms of biological processes and thus they are responsible for the
detoxification and biosynthesis of macro molecules (Yousef et al. 2007). In tilapia;
increases in LDH and ALP activities (both are blood serum enzymes) indicate that
liver damage due to the presence of lead metal. The increases are thought to be
occurring due to the fact that as the result of the liver damage, the liver cytoplasm
leaks into the blood stream (Wang and Zhai, 1988). Rahman et al. (2000) reported
that in fish that were exposed to the pollutants, the increase results from the LDH
enzyme, mixing into blood due to the necrosis in liver. The transaminase enzymes
such as ALT and AST play an important role in the metabolism of protein and amino
acids. In this study, while the AST activity was found out to be increasing in all
concentrations, time and concentration varied increases and decreases were
registered in ALT enzyme activity. It is thought that the increases in such enzyme
activities are resulted after the enzymes are introduced to the circulatory system due
to the damages in liver tissue. ALT, AST, ALP and LDH serum enzymes that were
assessed within the scope of this study are prone to be used as sensitive biomarkers in
ecotoxicological studies because of their characteristic of being an early warning
mechanism against the heavy metal based pollution in aquatic ecosystems (Vaglio
and Landriscina, 1999).
The aquatic organisms have to preserve the osmotic pressure of the plasma in
order to survive the ever changing environmental conditions and to maintain the
water and ion homeostasis. In bony fish, there are advanced structures to provide the
aforementioned regulations and such structures keep the inorganic ion concentrations
of the fish in close levels. The changes that may occur in electrolyte levels may
induce stress and thus may lead to the damages (Sjöbeck and Larsson, 1979).
Calcium is an ion that has various roles in ion regulation, membrane permeability,
muscle and neuron cell functions and skeletal bone metabolism and in the blood
clotting. The most important serum electrolyte for the lead toxicity is calcium. In this
study, the calcium levels of tilapia fish that were subjected to lead concentrations
were observed to be lower compared to the control group. Such decreases are found
out to be consistent with the literature and the decreases in blood calcium levels
(hypocalcaemia) was observed. In this study, unlike the calcium ions, increases were
revealed in magnesium electrolyte under the impact of lead. The previous study
863
indicates an inverse relationship between calcium and magnesium ions (Marshall,
2002). Chloride plays an important role in osmotic pressure and ion balance as well
as in acid–base equilibrium. In this study, under the effect of water-borne lead
concentrations, CI- ion levels of experimental groups registered increases compared
to the control group. Na+ and Cl- levels in fish are responsible for the osmolarity.
Changes in such ions may cause increase in gill permeability and damages to
osmoregulation. It is known that in some studies, under the effect of the pollutants,
the inhibitions that occur in the gill Na+, K+-ATPase enzyme activity lead to the
disarray in ion regulation (Haux and Larsson, 1979).
The changes in parameters that were examined within the scope of this study such as
glucose, LDH, ALP, ALT and AST showed that sublethal lead concentrations inflict
damage to the liver as the changes in magnesium; calcium and chloride indicate gill
damages.
Acknowledgments. This study was supported by the Çanakkale Onsekiz Mart
University Scientific Foundation (BAP) (project no: 2010/26).
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