Introduction. Fish can sometimes be contaminated with several highly toxic substances at once, e.g. heavy metals, pesticides, and preservatives. In this regard, it is essential to determine the presence of these harmful chemicals in fish products. The research objective was to analyze the level of organochlorine pesticide residues and other toxic substances in Tenualosa ilisha L. Study objects and methods. The study featured organochlorine pesticide residues and other toxic substances in raw and cooked samples of fresh and salted T. ilisha, which is a popular dish in Northeast India, especially in the state of Tripura. The analysis involved tests for formaldehyde, pesticides, and heavy metals. Formaldehyde content was estimated using high-performance liquid chromatography, pesticides content – by low-pressure gas chromatography/tandem mass spectrometry, and heavy metals – by inductively coupled plasma/mass spectrometry. Results and discussion. The salted samples had a high content of formaldehyde, though it remained within the normal range. Both fresh and salted samples demonstrated high concentrations of heavy metals such as zinc, copper, and selenium. The salted sample appeared to have a high content of toxic organochlorine pesticide residues. Frying and boiling of fresh and salted fish decreased formaldehyde and organochlorine pesticide residue contents but did not reduce heavy metal content. Conclusion. T. ilisha was found to be quite safe for human health.
Tenualosa ilisha, formaldehyde, pesticides, heavy metals, cooking, chromatography, spectrometry
INTRODUCTION
Fish is a highly nutritive part of human diet. First
of all, it is the primary source of polyunsaturated fatty
acids (PUFA), especially eicosapentaenoic acid (EPA)
and docosahexaenoic acid (DHA). PUFAs are known
to decrease the risk of cardiovascular diseases [1].
Additionally, fish provides proteins, fats, amino acids,
essential minerals (mainly iron), as well as vitamins A,
B group, and D [2].
Northeast India has huge potential for fisheries
due to its many rivers, streams, lakes, and ponds [3].
Fish and rice are the basis of the traditional menu
in the state of Tripura. Unfortunately, fish can be
contaminated with various harmful substances, e.g.
salt, dust, organic toxins, heavy metals, microbes,
pesticides, preservatives, etc. [4, 5]. As a result, fish
may pose a serious threat to human health. For instance,
formaldehyde, which is often used as an antimicrobial
preservative, is considered to be carcinogenic to
humans, which was also confirmed by the International
Agency for Research on Cancer (IARC) [6, 7]. Still,
this substance is often used to process foods [8]. In
small quantities, formaldehyde is involved in human
metabolism. However, high doses can cause pain,
vomiting, coma, and possible death [9]. According to
the United States Environmental Protection Agency,
the acceptable daily intake of formaldehyde is about
0.2 mg/kg b.w. [10]. In addition to being used as a
preservative, low concentrations of formaldehyde
can also serve as an antiseptic solution due to its
antibacterial and antifungal properties. If inhaled, it
damages respiratory organs and may cause dizziness
and suffocation, not to mention eye, nose, and throat
irritation [11]. Exposure to formaldehyde increases the
incidence of lung and nasopharyngeal cancer. Ingestion
of formaldehyde damages the gastrointestinal tract [11].
The normal range of formaldehyde is 2–50 mg/kg,
and the maximum can reach 60 mg/kg in fruits and
marine fish [8].
Heavy metals, such as lead, chromium, mercury,
arsenic, etc., are well-known contaminating chemicals
that cause water and soil pollution [12]. These metals
have no beneficial effects on human health. On the
contrary, they are generally considered as one of the
most toxic elements for humans and animals.
Similarly, pesticides in fish products are a sign of
contamination and pose a serious threat to human health
as they accumulate in human body fats [13]. Acute
symptoms of pesticide poisoning include numbness,
incoordination, headache, dizziness, tremor, nausea,
abdominal cramps, respiratory depression, etc. [14].
Exposure to organochlorine pesticides mainly
occurs through contaminated food, especially fish or
shellfish obtained from contaminated rivers and streams.
A long term exposure to moderate levels of aldrin and
dieldrin can cause headache, irritability, vomiting, and
uncontrollable muscle movements. Excessive dieldrin
has been established as a 2–7-fold higher risk factor for
breast cancer. The lethal dose (LD50) of dieldrin per day
is approximately 10 mg per 1 kg of body weight [15].
Carbofuran and its major metabolites (3-hydroxycarbofuran
and 3-ketocarbofuran) can cross the
placental barrier and affect the maternal-placentalfetal
unit [16]. Alachlor is another toxic chemical that
dissipates from soil mainly through volatilization,
photodegradation, and biodegradation. It is readily
absorbed through gastrointestinal tract and distributed to
blood, spleen, liver, kidney, brain, stomach, and ovaries.
The LD50 of alachlor is between 1.910 and 2.310 mg/kg
in mice [17]. Therefore, timely detection of these toxic
elements in fish is essential for human health.
Tenualosa ilisha, Clupeidae family, is the most
popular fish in the Bay of Bengal. Hilsa, as they call it
in India, occupies the top position among edible fishes
due to its unique taste and delicious properties. T. ilisha
is rich in amino acids, minerals, and fats. In addition,
the fish has a high content of high density lipoprotein
and a low level of low density lipoprotein, which makes
it beneficial for human health [18]. Because of its high
lipid content, it cannot be sun-dried. As a result, it is
preserved by salting, which is simple and cheap [19].
Both fresh and salted Hilsa are very popular among the
common people of Tripura. Cooking methods for fresh
and salted Hilsa include boiling, frying, roasting, etc.
Suitable cooking methods minimize the nutrient loss and
also improve the digestibility of food [20, 21].
Although some aspects of nutritional composition
of T. ilisha have already been reported, the estimation
of toxic elements in both raw and cooked samples of
fresh and salted T. ilisha has not yet been explored
scientifically [22]. The objective of the present
experiment was to analyze the level of organochlorine
pesticide residues and other toxic materials in raw and
cooked samples of fresh and salted T. ilisha.
STUDY OBJECTS AND METHODS
Sample preparation and cooking. Fresh and salted
samples of Tenualosa ilisha L. were obtained from the
local market of Battala, Tripura. The samples were
cleaned to remove dust particles. The fresh samples were
washed and gutted. In case of fresh samples, the main
purpose was to obtain the maximal amount of flesh
portion, so the samples were cut approximately parallel
to the backbone. The flesh portion was cut into small
pieces and prepared for boiling and frying. The salted
samples were washed with water and cut into small
pieces for further cooking. Fresh and salted samples
were boiled for 20 min or deep-fried in vegetable oil for
15 min at 240°C.
Sample preparation to determine formaldehyde.
The formaldehyde content was determined according
to Claeysa et al. [23]. Blank and spiked formalin was
added into five-gram samples. After adding 5 mL of
acetonitrile, the samples were sonicated for 30 min
at 25–30°C and shaken for 30 min in a shaking water
bath at 150 rpm at room temperature. Then, they were
centrifuged at 6000 rpm at 22°C for 5 min and filtered
through a Whatman filter paper (90 mm). After 5 mL
of the upper layer of the extract was carefully removed,
2.5 mL DNPH solution (dinitrophenylhydrazine)
and vortex were added. Recrystallization of DNPH
was carried out by dissolving 10 mL of anhydrous
acetonitrile acetate to obtain a saturated solution. The
samples were derivatized by shaking at 150 rpm at
40°C for 1 h in a shaking water bath. After incubation,
the supernatant was filtered with a syringe micro filter
(0.45 μm).
Analytical condition of HPLC. A 10-μL sample
solution was analyzed by using a C-18 column (250×
4.6 mm, 5 μm) with a 60% methanol solution as mobile
phase and analyzed at 355 nm. The flow rate was 1 mL/
min and the operating time was 13 min.
Standard curve preparation. A stock formaldehyde
solution (6.2%) was used to prepare standard solutions
with concentrations of 0.838, 1.68, 2.51, 3.35, and
5.03 ppm by diluting with distilled water. The
absorbance was then measured using a spectrophotometer
at 415 nm. The molar concentration of
formaldehyde was determined as follows:
( A = 1€) Rh HG Rh n = n ( − ⋅ 1zC) Rh HG Rh n = n − ⋅ zL (1)
where A is absorbance, € is molar absorption coefficient,
C is molar concentration, and L is length of the
cell.In case of matrix-free calibration, the limit of
detection (LOD) was 0.117 ppm and the limit of
quantification (LOQ) was 0.384 ppm.
Recovery test. The known concentration of
formaldehyde (5, 10, and 25 mg/L) was spiked in fish
matrix. Recovery was calculated as follows:
% of recovery = concentration of formaldehyde
quantified in the sample × 100/spiked concentration (2)
Pesticide determination. The fish samples were
homogenized with an Ultra-Turrax T25 homogenizer
and stored in a freezer at –20°C. A standard pesticide Table 1 Formaldehyde content in raw and cooked samples of fresh and salted Tenualosa ilisha
Formaldehyde Fresh T. ilisha Salted T. ilisha
Raw ± SEM Boiled ± SEM Fried ± SEM Raw ± SEM Boiled ± SEM Fried ± SEM
Content, mg/kg 10.32 ± 2.11 9.24 ± 2.40a 9.02 ± 1.80a 12.14 ± 1.51 11.23 ± 1.10b 10.58 ± 3.70b
Five samples were taken to calculate the standard error mean
a P < 0.05 when compared with raw fresh T. ilisha
b P < 0.05 when compared with raw (uncooked) salted T. ilisha
Table 2 Pesticide residues in raw and cooked samples of fresh and salted Tenualosa ilisha
Pesticides T. ilisha (fresh) T. ilisha (salted)
Raw ± SEM Boiled ± SEM Fried ± SEM Raw ± SEM Boiled ± SEM Fried ± SEM
Aldrin 11 ± 2 10 ± 2* 10 ± 1* 78 ± 9 76 ± 7** 68 ± 8**
Alachlor n.d. n.d. n.d. 88 ± 5 82 ± 6** 78 ± 4**
Carbofuran 17 ± 3 12 ± 2* 10 ± 4* 64 ± 3 62 ± 2** 58 ± 3**
Dieldrin 26 ± 5 22 ± 2* 18 ± 3* 66 ± 4 64 ± 2** 62 ± 4**
Endosulfan sulfate 17 ± 4 15 ± 2* 14 ± 3* 34 ± 9 32 ± 7** 31 ± 8**
o,p′-DDT+ p,p′-DDD 47 ± 8 46 ± 6* 45 ± 8* 118 ± 13 113 ± 17** 112 ± 12**
p,p′-DDT 19 ± 6 17 ± 2* 16 ± 1* 38 ± 4 36 ± 7** 32 ± 8**
Hexachlorobenzene n.d. n.d. n.d. 67 ± 5 65 ± 2** 64 ± 6**
The pesticide values are expressed as spiking level (5 ng/g)
n.d. – not detected
* P < 0.05 when compared with raw fresh T. ilisha
** P < 0.05 when compared with raw (uncooked) salted T. ilisha
ilisha L. The salted sample contained a higher amount
of formaldehyde (12.14 mg/kg) than the fresh sample
(10.32 mg/kg), which could be because formaldehyde
served as a preservative. However, its content reduced
after boiling and frying. Again, cooking had some
effect on the formaldehyde content. This toxic element
is known to degrade after thermal treatment [26]. The
concentration of formaldehyde mainly depends on
different levels of trimethylamine n-oxide (TMAO) [27].
Tri-methylamine, di-methylamine, and formaldehyde are
formed after the breakdown of TMAO [28].
The normal range of formaldehyde is 2–50 mg/kg, so
the formaldehyde content in the present experiment was
within the normal range for both fresh and salted fish
samples.
Table 2 demonstrates the pesticide content (spiking
level 5 ng/g) in the raw and cooked samples of fresh
and salted T. ilisha. Alachlor and hexachlorobenzene
were detected neither in the raw nor in the cooked
samples. On the other hand, the levels of aldrin, alachlor,
carbofuran, dieldrin, endosulfan sulfate, o,p′-DDT+
p,p′-DDD, p,p′-DDT, and hexachlorobenzene were
higher in the salted sample. After cooking, the pesticide
content went down. The toxic organochlorine pesticides
residue was higher in the salted sample, which may be
due to the fact that these substances were added to the
fish as preservatives. After cooking, the organochlorine
pesticides residue decreased because cooking process
increases volatilization, hydrolysis, or other chemical
degradation and leads to the decomposition by applying
heat [29, 30].
Table 3 shows the heavy metal content in the raw and
cooked samples of fresh and salted Hilsa. The mercury
content was found to be 0.101 mg/kg in the fresh sample
and 0.102 mg/kg in the salted sample. However, it went
down after cooking. The above result is acceptable
for fish, considering that the proposed upper limit
for mercury is 0.5 mg per 1 kg of fresh weight. The
cadmium content was high in both fresh and processed
samples. For general fish muscle, cadmium level is
0.05 mg per 1 kg of fresh weight [30].
Copper is essential for maintaining good health,
but a long term exposure may cause toxic effects, e.g.
Wilson’s disease [31]. In the present experiment, the
copper concentration was 54.02 and 53.88 mg/kg in
fresh and salted T. ilisha, respectively. The chromium
content did not exceed 1.0 mg/kg for all raw and cooked
samples of fresh and salted T. ilisha. Zinc is another
essential nutrient. In moderate quantities, it improves
immune system and metabolism, whereas a high level
of zinc can be harmful. According to FAO, the limit for
zinc is 30 mg/kg [32].
The limit of zinc acceptability exceeded the norm
in both raw and cooked samples of fresh and salted fish.
Selenium is an essential trace element required in small
amounts for animals and humans. However, a higher
content of selenium is toxic. Cooking appeared to have
no significant effect on selenium content. The nickel
content was within acceptable limits. Cooking had no
significant effect on lead contamination either. Arsenic,
another toxic element, can be found as a contaminant
in fish, shellfish, and other seafood. In the present
experiment, the arsenic content was within the normal
range. According to FAO/WHO, the maximal intake of
arsenic is 15 μg/kg b.w. Both raw and cooked samples
of fresh and salted T. ilisha contained a high amount of
arsenic [33].
CONCLUSION
The organochlorine pesticide residues were higher
in the salted samples of Tenualosa ilisha L., but boiling
and frying reduced their amounts. The salted samples of
T. ilisha contained a high amount of such heavy metals
as zinc, copper, and selenium. Fresh T. ilisha proved to
be safe for human health as thermal treatment had some
positive effects on the potentially harmful substances.
CONTRIBUTION
Dr. Kuntal Manna designed the research; Sanchari
Goswami collected the samples, performed the analysis,
and wrote the manuscript.
CONFLICT OF INTEREST
The authors declare no conflict of interests regarding
the publication of this article.
ACKNOWLEDGMENTS
The authors are grateful to New Jersey Feed
Lab Inc., 1686 Fifth St, Trenton NJ 08638, USA, for
providing instrument facility. The authors would like
to express their gratitude to State Biotech Hub, Tripura
Central University-799022, for providing working
facility and to Tripura State Pollution Control Board,
Kunjaban, Gorkhabasti, Agartala-799006, for providing
the instrumental facility for mineral analysis. The
authors are also indebted to Mr. W. Somraj Singh and
Mr. Bikash Debnath, Research Scholar, Department of
Pharmacy, Tripura Central University, for editing the
manuscript.
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