Assessment of Cd and Pb levels in commercial fish in Babil, Iraq
Department of Chemistry, Faculty of Science for Women,
University of Babylon, Babylon – Iraq, P. O. Box 309
Abstract
Heavy metal pollution of aquatic environment has become a great concern in
recent years. In this study, cadmium (Cd), and lead (Pb) levels were determined
in muscle of sixteen important consumed fishes in Babylon Province in the
middle of Iraq. Heavy metal concentrations were analyzed after dry digestion by
using anodic stripping voltammetry (ASV). The mean contents of metal,
expressed in mg/kg wet weight, varied from 0.087 to 0.226 for Cd, and 0.62 to
10.91 for Pb. It was concluded that these metallic concentrations are below the
maximum permissible for a safety utilization of these fishes in human nutrition.
Further study is highly recommended since toxic heavy metals have high
tendency to bioaccumulate in various organs of marine organisms especially fish
which reflects the pollution level of marine environment.
Introduction
Fish has long known for its reputation as the established health food for most of
the world’s population particularly developing countries in contrast to meat,
poultry and eggs. The protein content in fish mostly averages from 15 to 20
percent; hence fish provides comparatively cheap and readily
available protein sources in complement with long chains of n-3 fatty acids,
amino acids, vitamins and minerals that further contributes to healthier
nutritional options for a balance dietary intake [1, 2]. Fish is very important
components of protein sources being incorporated into Iraqi diet which
constitutes about 20 to 30 percent of protein consumed in Iraq.
Fish which occupy top level in the aquatic food chain are notorious for its ability
to bio-concentrate heavy metals in its flesh muscles and organs. Thus
it is essential to study the capacity of various fish organs in bio-accumulating
heavy metals since fish plays vital role in human nutrition and to ensure that
unnecessary high level of several toxic trace metals are not being transfer to man
through fish consumption which may directly affect human health [3,4]
Marine pollution indeed is a critical environmental issue of concern across the
globe when growing human population increase the intensities of anthropogenic
threats exert on the environment as a result of industrialisation, municipalities
and agriculture activities [5]. The negative manifestation of anthropogenic
impacts from heavy metal discharge into the aquatic environment have induced
disturbances to the hydrosphere equilibrium which further affects the natural
structure and functions of marine biotic communities. Seafood
especially marine fish are vulnerable to the effects of chemical contaminants
including heavy metals which bio-accumulate and bio-magnifies along the
aquatic food chain [6].
Heavy metal contaminations are one of the pervasive forms of marine pollution
because these metallic elements do not disintegrate rapidly in marine
environment which further impairs the aquatic ecosystems due to the relatively
high densities and toxicity even at low concentrations. These toxic
elemental contaminants cause unhealthy effects to the fish and are transferred
into human metabolism through consumption of contaminated fish that leads to
serious deterioration of human health status [5, 7].
The levels of toxic contaminants in fish are of considerable interest due to its
potential effects on the fish themselves and the organisms that consume
them which including humans. Health advisories such as Food and Drug
Administration (FDA) have recently raised concern on the safety of fish obtained
from commercial sources [8]
Therefore, this study was undertaken to compare the levels of hazardous heavy
metals in edible marine fish purchased from wet markets and supermarkets [8].
Differential pulse anodic stripping voltammetric (DPASV) procedures for the
direct determination of low levels of Cd and Pb in Fish are described. Stripping
electrochemical techniques combine high detection sensitivity, good accuracy
and precision with sufficiently high determination rates, convenience in
application and moderate cost demands for the instrumentation [10–12].
Materials and Methods
Sampling
In October 2010 - April 2011 sixteen commercially important and commonly
consumed marine fish species were randomly acquired in local markets, large
supermarkets, and grocery stores from Babil – Iraq. Purchased samples were
packed in clean zipped polythene bags and transported to the laboratory in an
ice-filled polystyrene insulation box. Fish samples were transferred and stored in
the laboratory freezer at -2ºC to reduce biological deterioration prior to analysis.
Sample preparation
Fish samples were de-scaled and rinsed with ultrapure water before dissection
for the isolation of flesh muscles. Cares were taken during dissection of the
internal organs to prevent any injuries and metal contaminations of the organ
samples by using stainless steel dissecting kits.
The isolated organs were manually cut into small pieces with stainless-steel
scissor and weighed accurately to 5.00 ± 0.05 g (wet weight basis) into individual
sanitised porcelain crucibles and subsequently subjected to oven drying at 180 C
for 8 hours. The dried samples were later ashed at 550 C for 12 hours inside a
muffle furnace (Carbolite, CWF 1200, UK). The cooled ashes were digested with
1.0 mL of concentrated analytical grade 65% HNO3 (Merck Chemicals,
Germany) and subsequently diluted with ultrapure water to 20 mL. Diluted final
test solution samples were filtered through Whatman® No. 595 filter paper prior
to anodic stripping analysis.
Chemical preparation
All glass wares and porcelain crucibles were soaked and sanitized in aqua regia
of 1:1 analytical grade 37% HCl and 65% HNO3 (Merck Chemicals, Germany)
solution, subsequently rinsed with ultrapure water, and were air-dried for 12
hours prior to usage. Sample blanks were prepared in the similar
way to the test samples for background correction. Standard solutions for Cd and
Pb were prepared from stock solutions (100 ppm). The test solution samples
were then analysed thrice for Cd and Pb using differential pulse anodic stripping
voltammetric (DPASV) [13].
Apparatus
Stripping voltammetric experiments were carried out with a Metrohm
(Herisau, Switzerland) 797 VA Trace Analyzer connected to a Metrohm 797 VA
multimode electrode used in the hanging mercury drop electrode (HMDE) mode.
A platinum rod and a saturated Ag /AgCl electrode were used as auxiliary and
reference electrodes, respectively [13]. pH was measured with a digital pH-meter
WTW, Model 720. Dissolved oxygen was removed from the samples by purging
with purified nitrogen (99.999%) through the measuring vessel for 5 min. During
the experiments, nitrogen was passed over the solution to prevent oxygen
interference.
The optimum experimental conditions were established as follows: the
potential was swept using differential-pulse modulation (DPASV) with a pulse
rate of 3.33 s-1, a scan rate of 10 mV s-1and a pulse amplitude of 50 mV. The
standard additions technique was used to give the concentrations of cadmium
and lead simultaneously when a sweep potential was applied between –1.150 V
and 200 mV (for cadmium –800 mV to –450 mV, and for lead –500 mV to –200
mV). All quoted potentials are referred to the Ag/AgCl electrode.
Statistical analysis
Quantification of metal concentrations in the samples was carried out by
use of the standard addition method. This is the preferred method as the
sensitivity of the stripping voltammetric analysis may vary between samples of
different ionic strength. The best fitting line through the data pairs was calculated
by linear least-squares regression analysis. The concentration of each element in
the sample is equal to the quotient of the intercept and the regression coefficient.
The scatter of the results was examined visually to assess its closeness to a
normal distribution. All data relating to Cd and Pb were approximately normally
distributed.
Obtained data was analysed using two-way ANOVA to determine significant
differences (p<0.05) of statistical means of each heavy metals present
within the organs of twenty selected fish species.. All data were presented in wet
weight in which more useful for health risk considerations since animals as well
as human consume organisms in their natural state. Moreover wet weight was
chosen as it is more convenient to evaluate the safety of fisheries products in
accordance with the objective of this study as to assess and evaluate the safety
of marine fisheries with respect to the level of Cd and Pb detected in twenty
species.
Dietary Exposure Estimates.
Dietary intake of Cd, and Pb through fish consumption was calculated by
multiplying the respective concentration in each marine species by the weight of
that species consumed by an average individual from Babil. For calculations,
when the level of an element was under the LOD, the concentration was
assumed to be half of the respective detection limit (ND ) 1/2-LOD). For health
risk assessment, the provisional tolerable weekly intakes (PTWI) of Cd and Pb
were compared with the intake of these elements through the consumption of fish
by the population of Babil.
Results
An anodic stripping voltammogram of a digested fish sample is shown in
Figure 1; well defined peaks for cadmium, and lead were observed, indicating
that the digestion of the sample was relatively complete. The peak height of the
voltammetric signal increases linearly with the deposition time in the range of 30
– 180 s for all four metals studied allowing thus the adaptation of the deposition
time to the level of the metal
|
C Cu,
Cu,Z
CC
ZnCu,Cu,
Zn700
Cu, Zn 1200
Zn
_
Zn
25
|
20
|
15
|
10
|
5
|
|
Pb
|
|
|
Cd
Cd
Cd Cd
Cd
|
|
|
|
|
-1200
|
Potential (mV)
|
|
-800
|
|
-400
|
000
Potential (mV)
Fig. 1 Typical voltammogram of dry digested fish (5.00 g) after
dilution to 500 ml. The peaks represent 1 µg g
–1
Cd, and 4 µg g –1
Pb, Deposition potential –1200 mV; deposition time 60 s for Cd,
Current (nA)
Current
(nA) (nA)
Current
|
Cu
|
Current (nA)
|
30
|
|
|
600 200
500
600500
800
500
400
500400
600
400
300
400300
400
300
200
300
200
200
200
100
200
100
000
100
000
100
Cu
|
Pb
Pb
|
700
600
600
700
1000
Zn
The calibration graph obtained for the two elements was determined in the
concentration range of 0 – 300 ppb.The calibration curves were linear over the
entire range with a correlation coefficient lies between 0.9950 and 0.9957 for the
two elements. Based on the calibration curve, the limits of detection were also
determined. The limit of detection is the analyte concentration giving a signal
equal to the blank signal, plus three standard deviations [14]. The limits of
detection were 0.15 µg kg -1 Cd, and 0.30 µg kg –1 Pb.
The precision and accuracy of the proposed method were checked with Orchard
leaves (NBS Standard Reference Material 1571) after dilution to 1000ml. Table 1
lists the analytical data obtained by DPASV, indicating that this method was
reliable for analysis.
-----------------------------------------------------------------------------------------------------------Table1: Determination of cadmium,and lead in reference material by the
recommended procedure.
Cd/mg kg -1
Pb/mg kg -1
Measured value
116 ± 13
48 ± 5
Certified value
110 ± 10
45 ± 3
Each value is the mean ± s of five runs.
Recovery tests were performed as follows: half of a batch of ten samples from
fish mixture was spiked with aliquots of each analyte prior to analysis. The whole
batch was then subjected to the digestion / analysis procedure. The results are
compiled in Table 2. from this table it can be seen that the recovery is
satisfactory for all the elements.
Table 2: Recovery for ten 1 g samples of a fish mixture after dry digestion and
dilution to 30 ml. (Concentrations are given as g kg-1 fresh weight).
Mean fish
Spike added
Total
Found
Recovery (%)
Cd
11.2
50.0
61.2
66.1
108
Pb
224.6
600
824.6
857.6
104
The mean concentrations of Cd and Pb in the 16 species of fish analyzed in this
study are depicted in Figures 1-2. For each species, three composite samples
were analyzed. The ranges of the respective concentrations are summarized in
Table 3.
Figure 1. Cadmium concentrations (mg/kg of fresh weight) in fish. Data are given
as arithmetic mean values corresponding to three composite samples for each
species.
------------------------------------------------------------------------------------------------------------
Figure 2. Lead concentrations (mg/kg of fresh weight) in fish. Data are given as
arithmetic mean values corresponding to three composite samples for each
species.
-----------------------------------------------------------------------------------------------------------
Table 3. Cadmium, and Lead Concentrations in Various Fish Species Purchased
in Babil, Iraq*.
----------------------------------------------------------------------------------------------------concetrationn (mg/kg of fresh wt)
Species
Cd
Pb
-----------------------------------------------------------------------------------------------------Acanthopagrus
‫الشانك‬
0.140
5.72
Barbus xanthopterus
Cyprinus carpio
Barbus barbulus
Liza abu
‫الكطان‬
0.087
5.9
‫الكارب‬
0.169
4.27
‫نباش دكاك الصخر‬
)‫(ابو براط‬
‫الزوري‬
0.133
3.48
0.139
6.19
‫الشلق‬
0.226
6.76
‫الشبوط‬
0.207
10.91
‫الزبيد ي‬
0.107
0.62
Hypothalmichthys molitirix
‫السلفر‬
0.107
2.07
Silurus Triostegus
‫الجري‬
0.172
2.72
Aspinsvorux
Barbus grypus
Pampus argenteus
Frozen Hake
‫سمك المجمد‬
0.106
1.47
Lizabu
‫خشني‬
0.143
0.53
Cyprinus carpio
‫السمتي‬
0.213
3.88
Frozen fish fillet
‫سمك فيليه‬
0.097
8.20
Barbus xanthopterus
‫الكطان‬
0.087
5.9
Grassiase
‫الفلسي‬
0.152
3.65
-----------------------------------------------------------------------------------------------------------Average
0.210
4.39
-----------------------------------------------------------------------------------------------------------* For each species and metal, three composite samples were analyzed.
The highest Cd concentrations were found in aspinsvorux 0.226 and barbus
grypus 0.207 mg/kg of fresh weight, respectively. The mean Cd concentration of
our measurements was quite similar to the mean found in other studies.
The highest Pb concentrations were found in Barbus grypus and Liza abu 10.91
and 6.19 mg/kg of fresh weight, respectively.
DISCUSSION
Fish is among the dominant bioindicator species used for acute toxicity assay of
pollutants such as heavy metals since much attention has been drawn
due to the wide occurrence of metal pollution in aquatic system. The rapid
development of industries and agricultures have promote the increase of
environmental pollution although heavy metals in aquatic system can be naturally
produced by slow leaching from rocks and soil into water which occurs
at low levels. Cd and Pb are among the aquatic metal pollutants which usually
present at significant levels in water system which may pose high toxicities on
the aquatic organisms [3].
In recent years, a notable number of surveys carried out in different countries
have determined the concentrations of metals in various edible marine species
and estimated human exposure by their consumption. However, comparison
among studies is not always easy, as the dietary habits depend on each specific
region or country. Moreover, fish and seafood species in the different surveys are
not generally the same. Bearing this in mind, we have compared the current
results with those of recent studies in which some species analyzed in the
present study were also included.
Cd and Pb have higher tendencies to bioaccumulate in the fish kidney and liver
tissues due to the similar functions of kidney and liver as the organs that
involve in the detoxification process. The presence of free protein-thiol group
content and metallothioneins binding proteins in kidneys and livers forms strong
fixation with the heavy metals [15]. Fish kidney located along the dorsal wall of
the fish body mainly contains excretory tissues meanwhile fish liver acts as major
site for homeostasis
Marine fish are exposed to waterborne heavy metal fractions when marine fish
drinks considerable amount of sea water. Therefore, gills serve as the
important route of heavy metal cationic exposure from surrounding sea water.
The large surface area of gills further facilitates the adsorption of Cd and
Pb onto the surface of gills during respiration and osmoregulation processes.
Metallothioneins binding proteins were also found in fish gills which trapped
heavy metals compounds [17].
Fish intestine compared to the other organs acts as a transient site for heavy
metal bioaccumulations in fish body. Fish intestine involved
in the uptake of particulate heavy metal fractions via alimentary tract in which the
rate of heavy metals uptake being controlled by specific transport system
through simple diffusion mechanism across the intestinal epithelium. Cd and Pb
form complexation with the intestinal amino acids and small peptides
with respect to high affinity for protein thiol-group which present within the fish
intestine [18].
Cadmium concentrations in our samples were, in general terms, of the same
order of magnitude as those found in the recent literature. However, Juresa and
Blanusa [19], and Storelli et al. (20) have reported Cd concentrations of (0.002
mg/kg ) and (0.005 and 0.02 mg/kg) respectively in muscle tissues of swordfish
and tuna from the Mediterranean Sea much lower than that of the present study.
With respect to Pb, in comparison with the average concentrations found in the
present study (4.39mg/kg), Juresa and Blanusa [17], Licata et al.[(21] and Storelli
et al.[20] have also reported much lower values (0.007 – 0.15 mg/kg).
The average quantity of fish consumed by ordinary Iraqis is ranging from 10 to
30 gram daily. This implies that the consumption of fish contributes 2.1 to 6.3 µg
of cadmium and 43.9 to 131.7µg of lead daily. The FAO/WHO Joint Expert
Committee on Food Additives recommended a provisional maximum tolerable
daily intake of Cd and Pb from all sources (food, air and water) of 1 – 1.2 and 3.5
– 4 g kg-1 body mass respectively. These values correspond to a provisional
daily intake of 60 – 72 and 210 – 240 µg (assuming an average Iraqi weight of 60
kg). According to these directives the daily intake of Cd and Pb by Iraqi
consumers from fish alone is below the FAO/WHO Provisional Tolerable Daily
Intakes. However, if other Pb and Cd sources are included the daily intake may
exceed the recommended levels..
Continuous exposure to Cd and Pb results in their gradual accumulation in
human vital organs, which may cause profound biochemical and neurological
changes in the body. Lead poisoning is recently considered the most important
environmental health problem for young children [23]. Consequently, it
would be recommendable that monitoring studies are periodically performed to
assess the temporal trends in human exposure to these toxic elements through
fish and seafood consumption.
The presence of mucous layer coating the fish skin surface served as a barrier
which protects the integrity of fish flesh muscle tissues from surrounding
contaminants. The mucous layer serves as the first line of defense against the
entrance of heavy metals into fish flesh muscle tissues by forming complexes
with the heavy metals. Therefore fish flesh muscle tends to bioaccumulate lesser
metals compared to the other fish organs [24, 25, 26].
Recently, Nor Hasyimah, A.K.,et. al.[27], found less Cd in flesh muscle compared
to other organs [Table 4], meanwhile there were no significant differences
(p>0.05) of Pb level in brain and flesh muscles samples examined in fish
collected from both wet markets and supermarkets. This variation was believed
due to differences in levels of fish exposure to heavy metal contaminants.
Table 4. Concentrations of cadmium in different fish species collected from
wet markets and supermarkets in Klang Valley, Malaysia
The presence of blood brain barrier in fish brain serves to protect the vulnerable
brain tissues from toxic metal perturbations which further prevents fish against
neurotoxicity effects. Moreover, the metallothioneins found within the fish brain
tissues are not as inducible as compared to the metal binding proteins found in
fish kidney and liver. This further supported the lesser amount of Cd and Pb
being detected in the fish brain compared to kidney and liver [18].
Fish flesh muscle is the edible part of fish and frequently employed in assessing
human health risks in relation to marine fish consumptions. The concentrations of
Cd and Pb detected in fish flesh muscle tissues were the lowest compared to
other organs.
The effects of freezing on cells, storage temperatures, fish stress and rigor mortis
with relation to cellular metabolism during perimortem and postmortem are
speculated for higher Cd and Pb contents in supermarket samples based on the
findings of this study. Freezing method is important in determining
the size of ice crystals that can rupture cell walls which allow movement of
cytoplasmic fluid that contain metals when thawed. Quick freezing method
reduces the formation of large ice crystals which aims to yield good quality
products in terms of textures, colouration, freshness and tenderness of meat.
Contrastingly, slow freezing method causes the fluid in fish tissues to form large
ice crystals that damages the delicate tissue cells.
Freezing also results in concentration of dissolved organic and inorganic salts
increases while liquid water is converted into ice [28].
Conclusion
The edible fish flesh muscle in all sixteen fish species purchased from wet
markets and supermarkets were not heavily contaminated with Cd and Pb as the
concentrations were well situated within the permissible safety limits.
Factors from post-harvest handling such as freezing on pre-mortem and postmortem fish that can affect cellular metabolic activities might be
attributable to the significant difference of Cd and Pb contents in fish sampled
from wet markets and supermarkets. Furthermore, the scenario of the actual
freezing and storage practices by fish trawlers were unknown and this can also
influence both metal concentrations, Cd and Pb in fish captured.
However, the potential of dietary hazards due to Cd and Pb exposures upon
consumption of these selected fish species sold in wet markets and
supermarkets within Babil are possible to occur in the future as further study is
highly recommended since toxic heavy metals have high tendency to
bioaccumulate in various organs of marine organisms especially fish which
reflects the pollution level of marine environment.
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List of Freshwater Fishes for Iraq
Order
Family
Cypriniformes Cyprinidae
Cypriniformes Cyprinidae
Species
Status
FB name Name
Acanthalburnus introduced Blackbro
microlepis
w bleak
Acanthobrama misidentificat
lissneri
ion
Cypriniformes Cyprinidae
Acanthobrama
telavivensis
Acanthopagrus
berda
native
Alburnus
caeruleus
Cyprinodontifor Cyprinodontid Aphanius
mes
ae
dispar dispar
Cyprinodontifor Cyprinodontid Aphanius
mes
ae
mento
Cyprinodontifor Cyprinodontid Aphanius
mes
ae
sophiae
Cypriniformes Cyprinidae
Aristichthys
nobilis
Cypriniformes Cyprinidae
Aspius vorax
Cypriniformes Balitoridae
Barbatula
frenata
Cypriniformes Balitoridae
Barbatula
panthera
Cypriniformes Balitoridae
Barbatula tigris
Cypriniformes Cyprinidae
Barbus
esocinus
Cypriniformes Cyprinidae
Barbus grypus
native
Perciformes
Sparidae
Cypriniformes Cyprinidae
native
Picnic
seabream
native
native
native
introduced Bighead
carp
native
native
Shilik
native
native
native
Mangar
native
Shabbo
ut
Cypriniformes Cyprinidae
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Carcharhinifor
Barbus
misidentificat
longiceps
ion
Cyprinidae
Barbus
native
xanthopterus
Cyprinidae
Barilius
native
mesopotamicus
Cyprinidae
Caecocypris
endemic
basimi
Cyprinidae
Capoeta
native
damascina
Cyprinidae
Capoeta trutta
native
Cyprinidae
Carasobarbus
native
luteus
Cyprinidae
Carassius
introduced Goldfish
auratus auratus
Carcharhinid Carcharhinus
native
Bull
Biss
Gattan
Himri
mes
Siluriformes
ae
Clariidae
leucas
Clarias
gariepinus
shark
introduced North
African
catfish
Cypriniformes Cyprinidae
Ctenopharyngo introduced Grass
don idella
carp
Cypriniformes Cyprinidae
Cyprinion
native
tenuiradius
Cypriniformes Cyprinidae
Cyprinus carpio introduced Common
carpio
carp
Cyprinodontifor Poeciliidae
Gambusia
introduced Mosquitofi
mes
affinis
sh
Cyprinodontifor Poeciliidae
Gambusia
introduced Eastern
mes
holbrooki
mosquitofi
sh
Cypriniformes Cyprinidae
Garra rufa
native
Siluriformes
Heteropneust Heteropneuste introduced Stinging
idae
s fossilis
catfish
Cypriniformes Cyprinidae
Hypophthalmic introduced Silver
hthys molitrix
carp
Mugiliformes Mugilidae
Liza abu
native
Abu
Hishni
mullet
Cypriniformes Cyprinidae
Mesopotamicht
native
Bunni
hys sharpeyi
Siluriformes
Bagridae
Mystus
native
pelusius
Cypriniformes Balitoridae
Nemacheilus
misidentificat
insignis
ion
Salmoniformes Salmonidae Oncorhynchus
not
Rainbow
mykiss
established trout
Perciformes
Cichlidae
Oreochromis
not
Nile
niloticus
established tilapia
niloticus
Perciformes
Cichlidae
Sarotherodon
introduced Mango
galilaeus
tilapia
galilaeus
Perciformes
Sillaginidae Sillago sihama
native
Silver
sillago
Siluriformes
Siluridae
Silurus
native
triostegus
Cypriniformes Cyprinidae
Squalius
native
lepidus
Beloniformes
Belonidae
Strongylura
strongylura
native
Spottail
needlefish
Clupeiformes
Clupeidae
Tenualosa
ilisha
Typhlogarra
widdowsoni
native
Hilsa
Shour
shad
Iraq blind
barb
Cypriniformes Cyprinidae
Anoding stripping voltammetry
native
(V A Computrace 797 Metrohm)