Competency of lemon and vinegar in chelation of some heavy metal residues

in muscles of salted sardine and herring fish

Enas , A. Abbas., Ommaima, A. Saleh* and El-Kewaiey I. A. **
Biochemistry Unit, Animal Health Research Institute and Food Hygiene Unit.
Damanhour Provincial Lab.* Biochemistry Unit Damanhour Provincial Lab.**
Abstract
In this study 30 Samples of both salted sardine and herring fish were purcashed from
Damanhour City markets. Two main factors were studied; the effect of immersion in lemon
or vinegar (house-hold concentration, 5% acetic acid) and the difference in species on heavy
metal levels in fish muscles. Samples were digested and analyzed by Atomic Absorption.
Spectrophotometer to determine heavy metals levels [cadmium (Cd), lead (Pb) iron (Fe) and
zinc (Zn)] before and after treatment. Fe exceeded the permissible limits in all non-treated
sardine and herring tissues and in lemon-treated herring tissues and Pb in non-treated herring
tissues while Cd and Zn were within the permissible limits set by Egyptian Organization for
Standardization (E.O.S.Q.C., 1993) in sardine and herring tissues before and after treatment.
A significant increase (p<0.05) in Pb and Zn levels in herring tissues in all tested samples
compared to sardine tissues and for Cd in lemon-treated tissue, while non significant
differences were observed in Fe level between the two fish species. These results indicating
that herring is more contaminated with heavy metals than sardine. This study revealed a
significant decrease (P<0.05) in Cd level in lemon-treated sardine compared to the non treated
edible muscles tissue, significant decreases in Pb level (P < 0.05) in lemon or vinegar-treated
sardine as well as lemon-treated herring, while non significant decrease in vinegar-treated
herring and significant decreases in Fe and Zn levels in both lemon and vinegar-treated
sardine and herring muscle tissue were observed. These findings confirm the effective role of
. vinegar and lemon in heavy metal chelation
Introduction
Some heavy metals act as nutrients for the human body such as Fe and Zn but others,
such as Pb, Cd, cause severe health problems. These problems range from impairing the liver
detoxification pathways to interfering with energy production at the cellular level. Certain
foods have the ability to help remove these heavy metals. Chelation is a chemical reaction
that involves the binding of dissolved metals (e.g., Cd, Pb, Fe and Zn) with an organic
ring-forming compound. These organic ring compounds include many organic acids
such as acetic acid, citric acid, and ascorbic acid. All of these natural acids, which are
classified as weak acids, have the ability to seize metal atoms such as Cd and Pb.
Chelating agent chemicals used as food additives, are capable of combining with metal ions to
render them ineffective (Hajeb, et al., 2014).

1
 Citric acid is an excellent chelating agent, binding metals. It is used to remove limescale
from boilers and evaporators. It can be used to soften water, which makes it useful in soaps
and laundry detergents. By chelating the metals in hard water, it lets these cleaners produce
foam and work better without need for water softening. Citric acid is the active ingredient in
some bathroom and kitchen cleaning solutions. A solution with a 6% concentration of citric
acid removes hard water stains from glass without scrubbing. In industry, it is used to
dissolve rust from steel. It can be used in shampoo to wash out wax and coloring from the
hair. Illustrative of its chelating abilities, citric acid was the first successful eluant used for
total ion-exchange separation of the lanthanides, during the Manhattan Project in the 1940s.
In the 1950s, it was replaced by the far more efficient ethylene diamine tetra acetic acid
EDTA. It can be used to slow setting of Portland cement. It can delay setting time
substantially (Frank, 2005).
Salted and smoked fish are widely consumed all over the world because of its high
protein, low saturated fat, fat soluble vitamins, essential minerals, omega 3 and 6 and fatty
acids known to support good health (Ikem and Egiebor 2005). Salted (Sardines) and smoked
(Herring) fish are traditionally eaten in Egypt, especially in Spring feast (Sham El-Neseem).
The aim of this work was to study the effect of two main factors; the effect of
immersion of two species of salted fish in lemon or vinegar (house-hold concentration, 5%
acetic acid) and the difference in species on heavy metal levels in edible musles tissue.

Materials and Methods

Collection of samples :

Thirty samples of both salted sardine and salted smoked herring were purcashed from
Damanhour City markets. Two main factors were studied; the effect of immersion in lemon
and vinegar on heavy metal levels in two species of salted fish (herring and sardine) and the
effect of fish species on heavy metal residues, in their muscle tissues. After removal of
skin and subcutaneous fat, muscle samples were taken from the middle dorsal layer. One
gram of edible muscle tissue samples were subjected to analysis for the detection of heavy
metals before and after good immersion in diluted solutions of freshly prepared lemon juice
or vinegar (house-hold concentration, 5% acetic acid) for 15 min.; then getting rid of the
liquid part (lemon or vinegar solutions containing most of the metals); the method comprises
of the following steps: 1) Addition of deionized water and acid to a heavy metal-containing
substance to form a liquid having a pH equal to or lower than 4 and stirring the liquid;
2) extraction of heavy metals substance in the liquid, as heavy metal ions according to the

2
 method reported by (Elnimr, 2011) of removing heavy metals from a heavy metal containing
substance
: Digestion of samples and estimation of metals
Each tissue sample was ground and homogenized according to Al-Baggou (2002). One
gram from each homogenized sample was digested in a Pyrex beaker with 7 ml of a mixture
of ultra pure concentrated nitric acid : perchloric acid (HNO3: HCLO4) at a ratio of (6:1) and
was placed on a hot plate at 135 C◦ for 2h. The liquor formed was evaporated slowly to near
dryness, cooled and dissolved in 1 ml of 20% nitric acid and diluted with 5 ml of the
deionized water. The solution was analyzed for lead (Pb), cadmium (Cd), iron (Fe), and zinc
(Zn) were determined by using Unicam 969 Atomic Absorption spectrometer England
according to Iwegbue (2008).
Statistical analyses:
Data were statistically analyzed using analyses of variance (F-test) followed by
Duncan’s multiple range test. A probability at level of 0.05 or less was considered significant.
Standard errors were also estimated using IBM SPSS statistics program version 20.The
differences in the mean concentrations for each heavy metal between treated and non treated
samples of the same fish species (F1 ) and between fish species( sardine and herring muscle
tissues) with the same treatment (F2 ).
Results and Discussion
Table 1: Cadmium concentraions (ppm) in muscles of salted Sardines and Herring before
.and after treatment with lemon and vinegar
Treatment Before After treatment After treatment F1- EOSQC(1993)
Fish treatment with Lemon with Vinegar Prob. Permissible Limit
Species

Sardine 0.03a ± 0.22 0.002b ± 0.004 03a. ±0 0.16 *000.

Herring a 0.04 ± 0.23 0.06a ± 0.15 ±0.03a 0.20 441. ppm 0.5

.F2-Prob 834. * 015. 303. *000.

Data are represented as means of 15 samples ± S.E. * Significantly difference using ANOVA test at P
<0.05. Mean in the same row with different letters are significantly different (Duncan multiple range
test P < 0.05). F1: between different treatment F2: between different species.
Data presented in, Table (1) revealed a significant decrease (P<0.05) in Cd level in
lemon -treated sardine compared to the non-treated edible muscles tissue, Table (2) exhibited
a significant decreases in Pb level (P < 0.05) in lemon or vinegar treated sardine as well as
lemon treated herring, while revealed non significant decrease in vinegar treated herring. A
significant decreases in Fe and Zn levels in both lemon and vinegar treated sardine and
herring muscle tissue was revealed in Tables (3 and 4).

3
 Moisture content may be play an important role in the increasing of metal contents in salted
fish, as it decreased by 10-15% after ripening which may be attributed to the loss in the water
holding capacity of fish protein as a result of proteolysis (Foda et al., 1986), consequently ash
content increased by 10-15% as a result of loss of moisture content and the diffusion of salt in the

fish during salting and ripening process (Awad, 1999). .Table 2: Lead

concentraions (ppm) in muscles of salted Sardines and Herring before and after
treatment with lemon and vinegar.
Treatment Before After treatment After treatment F1- EOSQC (1993)
treatment with Lemon with Vinegar prob Permissible Limit
Fish
Species
Sardine ±0.03a 1.93 b 0.03 ± 0.59 c 0.03 ± 1.24 *000.
Herring 0.04a ± 2.10 b0 0.03 ± 76. a 0.09 ± 1.92 *000. ppm 2
.F2 -prob *005. *001. *000.
Data are represented as means of 15 samples ± S.E. * Significantly difference using ANOVA test
at P <0.05. Mean in the same row with different letters are significantly different (Duncan
multiple range test P < 0.05). F1: between different treatment F2: between different species

Table 3 : Iron concenntraions (ppm) in muscles of salted Sardines and Herring before
.and after treatment with lemon and vinegar
Treatment Before After treatment After treatment F1- EOSQC(1993)
Fish treatment with Lemon with Vinegar prob Permissible Limit
Species
Sardine 40.56±0.18a b 29.54±2.99 26.62±1.51b *009.
Herring 43.11±1.94a b 34.28±1.82 c ±1.15 27.82 *000. ppm 30
F2-prob 539. 201. 544.
Data are represented as means of 15 samples ± S.E. * Significantly difference using ANOVA test at P
<0.05. Mean in the same row with different letters are significantly different (Duncan multiple range
test P < 0.05). F1: between different treatment F2: between different species.
Table 4 : Zinc concenntraions (ppm) in muscles of salted Sardines and Herring before and
.after treatment with lemon and vinegar

Treatment After treatment After treatment F1-prob EOSQC (1993)

Fish Before treatment with Lemon with Vinegar PermissibleLimit
Species
Sardine a 0.74 ± 11.07 b 0.42 4.05± b 0.19 ± 3.68 *000.
Herring a 0.83 ± 16.02 b 5.82±0.34 b 0.39 ± 6.76 *000. ppm 40
F2 –prob *000. *004. *000.
Data are represented as means of 15 samples ± S.E. * Significantly difference using ANOVA test at P
<0.05. Mean in the same row with different letters are significantly different (Duncan multiple range
test P < 0.05). F1: between different treatment F2: between different species.
Tables (1-4) revealed that Fe exceeded the permissible limits in all non-treated sardine
and herring and in lemon treated herring tissues and Pb in non-treated herring tissues while

4
Cd and Zn were with in the Egyptian Organization for Standardization and Quality Control
(E .O.S.Q.C., 1993) permissible limits in all examine tissue. Results from Table (3) indicate
that samples of salted non treated sardine and herring were found to contain iron element in a
concentration of40.56 and 43.11 ppm respectively is much above the recommended limits set
by (E .O.S.Q.C., 1993), which is 30 ppm. Nfon et al. ( 2009) found that the maximum
accepted concentrations for Cd, Hg and Pb were not exceeded in any of the muscles tested.
The concentration of cadmium in the muscles of all fish was very low. The high mean
concentration of lead in the fish caught in the Baltic was found in the muscles (0.053 mg kg-
1), but the maximum observed values still did not exceed acceptable levels and is lower than
the amounts in the present study, while Cd and Pb content in the samples examined by (EL-
Tahan et al.; 1999) were higher than the permissible level. The maximum accepted
concentrations for Cd and Pb were not exceeded in any of the muscles tested by Pokoroska
et al. (2012). Jorhem and Sundström, (1993) reported 0,014 mg/kg lead content and 0,008
mg/kg cadmium content in Baltic herring which is lower than the amount recorded in the
present study this may be due to salting and smoking process. Smoked fish is produced by
burning of some wood. Smoke were carried by some heavy metals which where adsorbed on
smoked fish. Moreover salted and smoked fish may be exposed to different heavy metals
during its life and before catching and processing (Polak-Juszcza, 2008). This may clarify
the increase in heavy metal concentration recorded in this study. Heavy metal contents in
analytical and industrial salt used in fish salting were 0.009, 0.174;0.000, 0.224, 2.510, and 7,
3.240, 34.221 , 11.227,60.123 for Cd, Pb, Cu, Zn and Fe respectively (Tuzen, 2003). Sardine
is produced by salting fish for a relatively long period. As this process concumes a large
amount of salt , there is a great probability that a polluted, unfit salt that contains some
heavy metals may used to reduce the cost this also explain the increase in its concentration
observed in the present study (Salah El-Dien et al., 2005). Table (3) showed that Fe levels
were 29.54 and 26.62 in lemon and vinegar treated sardine and 34.28 and 27.82 ppm in lemon
and vinegar treated herring, respectively. Yosef and Ghada (2011) reported that Pb level was
almost double the concentration of the limit set by FAO/WHO in muscle of salted fish using
industrial salt. Generally, the contents of Pb were found higher than the limit set by
E.O.S.Q.C in both species of examined fishes. Concerning Pb element, its concentrations
ranged from 0.322 to 0.974 μg/g,in fresh fish and from 0.448 to 1.226 μg/g, in salted fish by
analytical salt while from 1.230 to 1.864 μg/g,in case of salted fish by industrial one. Cd
contents varied from 0.093 to 0.246 μg/g, in fresh fish and from 0.164 to 0.273 μg/g, in salted
fish by analytical salt while from 0.281 to 0.712 μg/g, in case of salted fish by industrial one
which is similar to our results (table 1&2). The level of Fe in muscles of sardines (Canli and
Atli, 2003) in μg/g was 39.607. The results were comparable with Fe levels obtained in this
work. Fe content in herring muscles reported by Nfon et al. (2009) ranged from 5.85 to 14.3

5
mg kg-1 which was lower than the amounts in the present study. Fe which ranged between
52.524-133.842 μg/g in fresh fish and between 58.527-173.333 μg/g in salted fish by
analytical salt while between 177.773- 189.380 μg/g in salted fish by industrial salt.
Pertaining to Zn element, its content was much below the recommended limits set by
FAO/WHO which is 150 ppm. Those findings were similar to Zn levels recorded in this
study which are 16.02 and 11.07 ppm in non treated sardine and herring and 4.05 and 3.68
ppm in lemon and vinegar treated sardine and 5.82 6.76 ppm in lemon and vinegar treated
herring (Table 4) (Yosef and Ghada, 2011). The zinc content levels in fish tissue ranged from
3.2 to 44.6 mg kg-1 (Nfon et al. 2009) which coincide with this study in sardine and herring
muscles tissue (Table 4). Protasowicki (1991) observed higher amounts of zinc in the
muscles (9.0 mg kg-1) of Baltic herring. On the other hand, imported sardine contained
0.002-3.868 ppm zinc (Abou-Arab et al., 1995).
On a basis of species, different levels of metals were reported in different species
(Tuzen, 2003). This explain the result presented in our study (Table 1- 4) which showed a
significant increase (p<0.05) in Pb and Zn levels in herring tissues in all tested samples
compared to sardine tissues and for Cd in lemon treated tissue while non significant
difference was observed in Fe level between the two fish species. These results indicating that
herring is more contaminated with heavy metals than sardine. The bioaccumulation of the
monitored metals varied between fish species. These differences in bioaccumulation between
fish species stem from their affinity for metal uptake from the aquatic environment and their
place in the food chain.
Many organic acids can act as chelating agents, including acetic acid (vinegar),
ascorbic acid (vitamin C) and citric acid (present in lemon). Citric acid complexes with many
multivalent metal ions to form chelates. as it more than one so, it control metal contamination and
prevent metal to undergo any reactions. It does not affect color, stability, and texture of food product.
Chelating agents are organic compounds that sequester iron from further reactions by binding
sufficiently tightly to the iron, removing it either as a free agent in solution or as scale. 0.1%
citric acid solution to remove heavy metals from fish. They reported increased amounts of
heavy metal removed from fish in a shorter time due to the reductive property of citric acid.
The carboxyl and hydroxyl groups of citric acid can each lose a proton and coordinate to a
metal ion to form a metal chelate. Metals form stable complexes with citric acid, and the
typical structure of citric acid with a metal. Ascorbic acid, Vitamin C, naturally occurring in
lemon and found in trace amounts in fish muscle tissue. It is an antioxidant, and used as
permitted food additive for this purpose. Chelation involves the formation or presence of two
or more separate coordinate bonds between a poly dentate (multiple bonded) ligand and a
single central atom. Usually these ligands are organic compounds, and are called chelants ,
chelators , chelating agents, or sequestering agents. The chelate effect describes the enhanced

6
affinity of chelating ligands for a metal ion compared to the affinity of a collection of similar
non chelating (mono dentate) ligands for the same metal (Hajeb, et al., 2014). Fe is held by
the chelating agent. The chelated iron from the citric acid treatment remains in the ferric state.
This important chemical property makes citric acid and citrates useful in controlling metal
contamination that can affect the color, stability, or appearance of a product or the efficiency
of a process. Citric acid, with its one hydroxyl and three carboxyl groups, is a multidentate
ligand. Two or more of these sites are utilized to form a ring structure. The normal molar ratio
of metal-to-ligand is 1:1. With some metal ions, under certain conditions, more than one ring
can be formed allowing a higher metal-to-ligand ratio. Citric acid is readily available from
many sources as lemon. When a metal ion is chelated by a ligand such as citric acid, it is no
longer free to undergo many of its chemical reactions. Under pH conditions that may
precipitate a metal hydroxide, the citrate complex may be soluble. The pH affects the
speciation of metal ions in solution and the metalbinding sites of the chelating agent. The pH
has always been one of the factors investigated in studies performed on heavy metal
reduction in foods. The absorption of heavy metal ions by chelating agents depends on
the pH of the solution. It is a natural, indeed, a vital process that goes on continually in our
bodies. Organic molecules that are catalytically decomposed in the presence of metal ions
can be made stable by chelating the metal ions with citric acid (Staal, 1989). Salts and esters
of carboxylic acids are called carboxylates. When a carboxyl group is deprotonated, its
conjugate base, a carboxylate anion is formed. Carboxylate ions are resonance stabilized and
this increased stability makes carboxylic acids more acidic than alcohols. Carboxylic acids
can be seen as reduced or alkylated forms of carbon dioxide; under some circumstances they
can be decarboxylated to yield carbon dioxide (Voest et al., 1994).
The second best chemical chelating agent is acetic acid. Acetic acid formal chemical
name, ethanoic acid. An organic acid of formula CH3.COOH. It is the main component, 3-
6%, other than water, of vinegar. Used in fish technology in preparation of marinades. The
preparation of marinades, typically involves the preservation of fish by treatment with
solutions of dilute acid (usually acetic acid or vinegar) and salt, a process that gives the fish a
characteristic flavour and texture. Carboxylic acids are organic acids characterized by the
presence of at least one carboxyl group. The general formula of a carboxylic acid is R-COOH,
where R is some monovalent functional group. A carboxyl group (or carboxy) is a functional
group consisting of a carbonyl (RR'C=O) and a hydroxyl (R-O-H), which has the formula
-C(=O) OH, usually written as -COOH or -CO2H. Chelation therapy can be used to remove
heavy metals, such as Pb, Cd, Fe and Zn. This treatment uses chelating agents to bind and
precipitate metals and remove them from the body. Carboxylic acids are are proton (H+)
donors. They are the most common type of organic acid. Among the simplest examples are,

7
acetic acid CH3-COOH, which gives vinegar its sour taste (Nic, et al., 2006). This may
explain the observed decrease in examined heavy metal levels in most treated groups and may
confirm the important role of weak organic acid (lemon and vinegar) in getting rid of some of
heavy metals presented in our foods especially in salted fishes examined in this study.
Addition of acetic acid to fish tissue helped to reduce level of heavy metals. Interestingly,
there was a marked reduction of heavy metal levels in fleshy parts of the fish pre-treated with
5% acetic acid (similar to house-hold concentration). When Basa fish was immersed in 5%
acetic acid , the cadmium level was reduced from 0.12 μg/g in flesh to 0.07 μg/g in
homogenate (41.6%) and the lead level was reduced from 0.83 to 0.41 μg/g (51.9%). In Bolti
fish, pretreatment of fleshy parts by acetic acid reduced the Cd level from 0.12 to 0.07 μg/g
(58.3%) and the lead level was reduced from 0.83 to 0.02 μg/g (24%). The same finding was
observed in Karmout fish, where pretreatment with acetic acid lowered the cadmium level
from 0.17 to 0.05 μg/g (29.4%) and the lead level was lowered from 1.51 to 0.82 μg/g
(54.3%). Reduction of heavy metals in fish pre-treated with acetic acid could be due to
formation of insoluble acetate salts of these metals. Fish liquid contained a considerable
amount of metals, leaked with liquid by filtration, and thus should be discarded to minimize
metal exposure (Elnimr, 2011). The heavy metal removal was not inhibited by elevated NaCl
concentrations in salted fish. NaCl in the presence of other chelating agents improved
removal process (Li et al., 2013 and Hajeb et al., 2014).
Conclusion and Recommendation
Iron was found to exceeded the permissible limits in all non-treated sardine and herring
tissues and lead in non-treated and in lemon treated herring tissues while cadmium and zinc
were with in the permissible limits set by Egyptian Organization for Standardization and
Quality Control (E .O.S.Q.C., 1993) .Therefore both fish species may pose health hazards for
consumers before treatment. These results were probably due to the usage of impure salts
during salting process. The bioaccumulation of the monitored metals also varied between fish
species in most detected samples. It was found that herring is more contaminated with heavy
metal than sardine. Salted fish by industrial salt had greater concentrations of most studied
metals than those of treated one. Both lemon and vinegar were found to be efficient in
removal of large amount of some heavy metal residues in salted fish. Catching of fish from
uncontaminated water, controlling the industrial and agriculture effluents into lakes and
surface water; using salts of food grade quality and using organic acid immersion technique
are our recommendation to produce a hygienic salted fish for human consumption.

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8
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‫الملخص العربى‬
‫كفاءة الليمون والخل فى التخلص من بقايا بعض المعادن الثقيلة فى عضلتا أسماك السردين والرنجة المملحة‬
‫ إبراهيم على القويعى‬- ‫ أميمه عبد الفتاح صالح‬- ‫إيناس أنور عباس‬
‫معهد بحوث صحة الحيوان –قسم صحة الغأذية معمل فرعى دمنهور–معمل فرعى دمنهور‬-‫قسم الكيمياء الحيوية‬
‫عينة من الرنجه المدخنة‬15 ‫ عينة من السردين المملح و‬15 ‫ عينةة من السماك المملحة‬30 ‫فى هذه الدراسة تم تجميع‬
‫دقيقة وأيضا‬15 ‫ و لمدة‬%5 ‫والمملحة المعروضة للبيع فى مدينة دمنهور وقد تم دراسة تأثير النقع فى محلول الليمون أو الخل بنسبة‬
‫ وقد تم قياس عناصر الكادميوم والرصاص والحديد‬. ‫تأثير الختلفا فى النوع على مستوى بعض المعادن الثقيلة فى أنسجة العضلتا‬
. ‫والزنك بواسطة جهاز المتصاص الذرى قبل وبعد المعالجة‬
‫وقد دلت النتائج على تخطى مستوى الحديد الحدود المسموح بها فى أنسجة عضلتا السردين والرنجة المملحة قبل المعالجة‬
‫وفى أنسجة الرنجة بعد المعالجة بالليمون والرصاص فى أنسجة الرنجة قبل المعالجة بينماكان مستوى الكادميوم والزنك فى الحدود‬
.‫المسموح بها فى كل أنسجة السردين والرنجة قبل وبعد المعالجة‬

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 ‫وقد اظهر التحليل الحإصائى نقص ملحوظ فى تركيز الكادميوم فى أنسجة السردين بعد المعالجة بالليمون ونقص ملحوظ فى‬
‫تركيز الرصاص فى السردين بعد المعالجة بالليمون والخل والرنجة بعد المعالجة بالليمون ونقص ملحوظ ايضا ة فى الحديد والزنك فى‬
‫السردين والرنجة بعد المعالجة بالخل والليمون ‪.‬‬
‫وكان هناك زيادة فى مستوى الرصاص والزنك فى أنسجة الرنجة عن السردين فى كل العيناتا‪ .‬وبالنسبة لمستوى الكادميوم‬
‫كان أعلى فى أنسجة الرنجة عن السردين فى النسجة المعالجة بالليمون ‪ .‬بينما ل يوجد إختلفا معنوى فى مستوى الحديد بينهما‪.‬‬
‫وتؤكد هذه الدراسة الدور الفعال للليمون والخل فى التخلص من بعض المعادن الثقيلة كما اظهرتا أيضا ة إختلفا تراكم‬
‫المعادن الثقيلة على أساس النوع حإيث أظهرتا النتائج أن أسماك الرنجة كانت اكثر تعرضا للتلوث بالمعادن الثقيلة بالمقارنة بالسردين ‪.‬‬

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