Alterations in the Leucocytes and Serum Biochemistry in grey mullet (Mugil
cephalus L,) fingerlings Exposed to Sub lethal doses of Lead for
Hussien. M. EL-Shafei
General Authority for Fish Resource Development,
Grey Mullet Mugil cephalus L, fingerlings were chronically exposed to sub lethal concentrations of lead(Pb) (0.1 and 0.4 mg/L for
twenty eight. The changes in the leucocytes and serum glucose, protein, and total cholesterol of the fish were determined every seven
days in a renewable static bioassay system. At the end of the study, these parameters were significantly (p < 0.05) elevated in the Pb
- exposed groups when compared with the control group. Showing a pronounced leuocytosis in the Pb-exposed fish. The magnitude
of increase was influenced by increasing of exposure period and Pb concentration. The Pb exposed fish were significantly (p<0.05)
hyperglycemic and hyperbcholesteremic. The serum glucose levels on the 7th day were 26.50 ± 2.12 g/dl and 30.50 ± 0.70 g/dl in the
fish exposed to 0.1 and 0.4 mg Pb /L respectively. On the 28th day , the serum glucose concentrations were 52.50 ± 2.12 and 70.00 ± 2.83
g Pb /dl in the groups exposed to 0.1 and 0.4 mg Pb/L , respectively. The cholesterol concentration increased from 113.5±3.53 mg Pb /dl
on day 7 to 208.0 ± 1.80 mg/dl on day 28 in the group exposed to 0.1mg Pb /L. When the fish was exposed to 0.4mg Pb /L lead acetate,
the cholesterol concentration increased from 131.5 ±3.54 in the first week to 288± 5.19mg Pb /dl on 28th day of the study. The serum
protein concentration was also significantly (p< 0.05) increased in the Pb – exposed groups when compared with the control group, it
increased from 4.04±0.06mg Pb /dl on day 7 to 5.30±0.05 mg/dl on day 28 in the fish exposed to 0.1mgPb /L. When the fish treated with
0.4mg Pb /L , the serum protein increased from 4.45±0.37 mg/dl on day 7 to 6.18±0.19 mg Pb /dl on day 28, respectively. These changes
are indicative of stress imposed on the fish by lead and could be used as indices of lead poisoning.
lead, hyperglycemia, cholesterol, protein, leukocyte
Lead(Pb) is one of the most widely used heavy metals that has
wide applications in such products as storage batteries (lead
accumulator), electric cable sheaths, alloys, pesticides, paints,
petrol and rubber products among other uses  . In water bodies,
Pb forms complexes with sediments or organic materials  .
and in the process enters the food chain. The problem posed by
Pb in the aquatic system is complex due to its non-degradability
and interactions with other materials to form complexes that
may potentiate their toxic effects Lead deposits in various
fish organs: liver, kidneys and spleen, but also digestive tract and
gills . Accumulation of lead in different fish species has been
determined in several works, leading to disorders in fish body.
When C. batrachus exposed to 5 ppm of lead nitrate for 150 days,
it exhibited marked inhibition of gonadal growth and showed
decrease in cholesterol and lipid levels in brain, testis and ovary
whereas the liver showed an elevation of both .  observed a
very high number of red let cells (RCs) in the epidermis of common
carp and rainbow trout kept in lead polluted water. Hepatocyte
vacuolization, hepatic cirrhosis, necrosis, shrinkage, parenchyma
degeneration, nuclear psychosis and increase of sinusoidal spaces
were the distinct changes observed in the liver of lead-exposed fish
. The characteristic symptoms of chronic lead toxicity include
changes in the blood parameters with severe damage to erythrocytes
and leucocytes and damage in the nervous system . Low levels
of Pb pollution could cause some adverse effects on fish health and
reproduction . Also, lead was found to impair the embryonic
and larval development of fish species .  monitored the
effect of lead on the Chinese sturgeon, Acipenser sinensis.
They observed deformities as body (spinal) curvatures. The authors also
reported reduced ability of locomotion and foraging by deformed
juveniles. The purpose of the study was therefore to investigate the
effect of sub lethal lead on the leucocyte and some biochemical
parameters in grey mullet Mugil cephalus,L- with a view to using
them as biomarkers of lead toxicity.
MATERIALS AND METHODS :
Collection and handling of experimental of fish. :
The fish fingerlings were bought from Fish Farms at Manzalla
lake Damietta , and transported to a wet laboratory in two 50 L
plastic fish transport containers during march 2015 . Once in the
wet laboratory, fish was introduced into 200 L plastic container for
acclimatization for two week . The water was continually aerated
to ensure that the dissolved oxygen level remained above 6 mg/L
and fish were fed on a 32% crude protein diet at 3% body weight
daily at 10.0 h. The laboratory water was analyzed for different
physico-chemical parameters (APHA, 2010) and lead. No fish
mortality was recorded during acclimatization. The LC50 value
for lead was statistically determined 
Experimental design and in vivo studies:
One hundred and twenty fingerlings of Mugil Cephalus L, (mean
length of 12.00±2.25 cm and average weight of 35±3.20g) were
used for this study. They were divided into three groups of forty
fingerlings per each. Each group was further divided into two
replicates, twenty fish per replicate. Fish were exposed to 0.1
mg Pb /L and the second group was exposed to 0.4 mg Pb /L.
The third group was exposed to tap water only and it served as
control group. The water in the experiment was changed every day
to maintain the same Pb concentrations. Three fish from each
replicate experiment was killed every seven days for twenty eight
days for the analysis.
Blood Collection :
The blood of the fish was collected every seven days through both
cardiac puncture  . The blood was collected into three different
vials . One of the vials containing fluoride oxalate was use to
collect blood used for glucose determination. The other two vials
were without anticoagulant and were used to collect the blood for
the biochemical test, and thin blood smear.
White Blood Cell Count (WBCC):
The leucocyte count was done using the Neubauer microscopic
counter after diluting the blood with Turk’s dilution fluid. The
differential white blood cell count was done by preparing a thin
blood smear and staining same with Geimsa Romanosky stain
. The stained blood was left for 25 minutes for the Geimsa
stain to act on it. Thereafter the stained blood was flooded with
distilled water and rocked gently to evenly mix the distilled water
and stain. The stained blood was washed with water and allowed to
dry. The slide was viewed with a binocular microscope to identify
the leukocyte species which was calculated as a percentage.
Biochemical analyses :
The blood serum was obtained by centrifuging the blood sample
at 5000 rpm for five minutes and the protein in the samples was
spectrophotometrically determined using Biuret method  at
540 nm. The blood glucose was determined by  method and
the cholesterol concentration was determined by the method of
Statistical Analysis :
The data was statistically analyzed using two way analysis
of variance (ANOVA) followed by LSD post hoc test at 95%
confidence interval .
Water quality parameters, cations , anions, and background metals
in acclimation. were presented in table(1)
The changes in the total leucocytes and differential white blood
cell count Pb- exposed to Fish are presented in (Table 2) . The
leucocytes count in the control group did not vary ( p > 0.05)
significantly during the study. The leucocytes counts increased
significantly with increasing Pb concentrations and exposure
period. Five different subspecies of leucocytes (lymphocytes,
monocytes, neutrophils, eosinophils and basophils) were identified
in the fish during the study. The lymphocyte and the monocytes
constituted the a granulocytes identified while neutrophil,
eosinophils and basophils were the granulocytes recorded due to
the presence of granules in their cytoplasm. The lymphocytes were
the most abundant leucocytes group identified in the blood of the
fish exposed to lead acetate . Both small and large lymphocytes
were found during the study and they accounted for more than 80
% of the white blood cells. The lymphocytes increased significantly
( p < 0.05) in the lead-exposed fish when compared with the
control group .
The lymphocytes were significantly different (p>
0.05) in the treatment groups and the lymphocytosis was both
concentration and duration dependent. The monocytes decreased
in the lead-exposed fish on the 7th day and thereafter, it increased
significantly in the treatment groups ( p <0.05) when compared
with the control group . The basophiles were the most abundant
granular leucocytes in the peripheral blood of Mugil cephalus,L-
exposed to lead acetate . The proportion was highest in the first
week and on day 21 of the study. The neutrophils are the second
largest granular white blood cell in the fish while the eosinophils
are the least abundant subpopulation.
As shown in the Table 2, in the intervention group, the total average score of MoCA-test taken before the intervention was 24.39 (26 points), and it did not reach significance. The total score value of, 24.05, pre-intervention failed to reach significance, but increased to (<26.11) after intervention, which was a statistically significant value (p <0.01).
The results taken at the same time points showed little differences in the control group scores, both of which were below the cutoff value (<26 points).
Effect on serum glucose :
The glucose concentration in the control group did not vary
throughout the study (Table 3). The serum glucose level on day
7 were 26.50 ± 2.12 g/dl and 30.50 ± 0.70 g/dl in the fish exposed
to 0.1 and 0.4 mg Pb /L, respectively. On day 28, the serum
glucose concentrations were 52.50 ± 2.12 and 70.00 ± 2.83 g/dl
in the groups exposed to 0.1 and 0.4 mg Pb /l lead, respectively.
There was concentration and duration significant increase (p <
0.05) in the treatment groups when compared with the control and
the values differed also in the treatment groups (p < 0.05) at each
Effect on serum protein :
The results showed that the protein level in the control did not
vary (p > 0.05) throughout the study while the serum protein in the
Pb – exposed fish did not differ ( p > 0.05) from the control value
during the first 14 days ( Table 3). Generally, there was progressive
concentration and duration dependent increases in the serum
protein in the Pb–exposed fish as it increased from 4.04±0.06mg/
dl on day 7 to 5.30±0.05 mg/dl on day 28 in the fish exposed to
0.1mg Pb /L. When the fish treated with 0.4mg Pb /L, the serum
protein increased from 4.45±0.37 mg/dl on day 7 to 6.18±0.19 mg/
dl on day 28, respectively. Statistical analysis showed that serum
protein levels in the treatment groups differed significantly ( p
<0.05) at the end of the study.
Effect on serum cholesterol :
The serum cholesterol level in the control group did not vary (
p < 0.05) throughout the study (Table 3). When compared with
the control group , the cholesterol concentration was significantly
higher (P < 0.05) in the Pb -exposed fish. Also, the cholesterol level
differed significantly (p < 0.05) in the treatment groups throughout
the study. The cholesterol concentration increased significantly
from 113.5±3.53 mg/dl on day 7 to 208.0 ± 1.80 mg/dl on day 28
in the group exposed to 0.1mgPb/L. When the fish was exposed to
0.4mgPb/L, the cholesterol concentration increased significantly
from 131.5 ±3.54 mg/dl in the first week to 288± 5.19 mg/dl on
28th day of the study.
Water quality parameters, cations , anions, and background metals in acclimation.
Effect of different lead concentration on the differential white blood cell count of grey mullet Mugil cephalus L.
change in some Biochemical parameters in grey mulle Mugil cephalus L
exposed to different Pb doses for
different exposure periods.days.
Value in the same column with the same superscript (lower case) are not significantly different (p = 0.05)between
different concentrations within the same exposure duration. Values with different numeric superscripts differ
significantly (P=0.05) between different exposure periods within the same concentration.
The results of the study show that lead has significant effects
on the leucocyte count of Mugil cephalus L- and the small
lymphocytes decreased in number while the neutrophils increased.
On the contrary,  found decreased leucocyte counts in Clarias
gariepinus exposed to 0.45mg Pb /L for 4 to 5 days. The general
leucocytosis reported in this study is consistent with the observation
of  in the dogfish exposed to 50μg/L Cd for 4 days.
Changes of blood glucose are a good indicator of metal stress in
fish  and alterations in the glucose level might be related to
renal injury, liver damage, and lack of nutrition . This study
showed a dose- dependent increase of glucose level after first 15-
days. The observed increase in the serum glucose level in Mugil
Cephalus L, exposed to lead in this study is in accord with report
of some earlier workers. Similar increase in plasma glucose was
also reported in Prochidolus lineatus exposed to lead  and in
Oreochromis niloticus exposed to copper . It has been widely
reported that hyperglycaemia in fish arises due to the stimulation
of catecholamines and corticosteroids 
The increased serum glucose level in this study is an evidence
of stress due to lead exposure as  argued that coping with
such stress is an energy demanding process that requires the
fish to mobilize metabolically energy substrates through intense
gluconeogenesis. Glucose being one of such known substrates is
mobilized through gluconeogenesis to meet this challenge 
. Heavy metals have been reported to act antagonistically with
glucocorticoids by inhibiting the receptors thereby disrupting
the osmotic and mineral regulatory mechanisms . Serum
increase in glucose due to toxicants has have been associated with
hypothalamus-sympathetic-chromaffin cells  instead of the
hypothalamus-pituitary-internal axis  that is known to have
tremendous influence on carbohydrate metabolism.
The results of this study showed that the serum total protein
increased significantly in Mugil cephalus, L ,exposed to lead.
Similar increases in the serum protein level were reported in
Oreochromis niloticus exposed to metals . Also increased
plasma protein was reported in Mugil exposed to 0.5ppm copper
and cadmium  . On the contrary, decreased tissue protein was
reported in Oreochromis niloticus treated with cadmium  and
in Cyprinus carpio when exposed to heavy metals  .
The enhanced serum cholesterol in this study is an indication of
hypercholesteremia in the fish due to the stimulatory effect on
the cholesterol biosynthetic pathway. Reduced serum cholesterol
level has been reported in Oreochromis niloticus exposed to some
heavy metals ,  and in Lepomis macrochirus exposed to
methyl mercuric chloride .Cholesterol concentration in the
serum of cadmium exposed fish also showed a different pattern.
Another study showed a reduction in cholesterol within 15-days,
possibly due to tissue damage in the kidney. On the contrary,
in Oreochromis niloticus, an increase in cholesterol was seen
during a 21 day period due to cadmium .
This alteration in cholesterol concentration could be due to
hazardous effects of metals on cell membrane. The observed
elevated cholesterol in this study could have resulted in part to the
adverse effect of lead on the liver leading to altered cholesterol
metabolism resulting in increased serum cholesterol. This
according to  could be due to liver and kidney failure that
resulted in the release of cholesterol into the blood stream. Thus,
increase in cholesterol levels are good indicators of environmental
stress in fishes.
Generally, the leucocytosis observed in this study gives indication
that exposing the fish to lead predisposes it to secondary infections.
Also, the reported hyperglycemia, increased serum protein and
cholesterol levels are indications of altered carbohydrate, lipid
and protein metabolism in the fish due to lead exposure. Serum
parameters which provide information as to state of the internal
environment of the fish are known to respond quickly to changes in
the water quality. The changes in these biomarkers are a reflection
of organ dysfunction in the fish due to metal exposure and these
biomarkers could be used in ecotoxicological assessment and as
early warning indicators of pollution.
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