INTRODUCTIONFish is a highly nutritive part of human diet. Firstof all, it is the primary source of polyunsaturated fattyacids (PUFA), especially eicosapentaenoic acid (EPA)and docosahexaenoic acid (DHA). PUFAs are knownto 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 fisheriesdue to its many rivers, streams, lakes, and ponds [3].Fish and rice are the basis of the traditional menuin the state of Tripura. Unfortunately, fish can becontaminated with various harmful substances, e.g.salt, dust, organic toxins, heavy metals, microbes,pesticides, preservatives, etc. [4, 5]. As a result, fishmay pose a serious threat to human health. For instance,formaldehyde, which is often used as an antimicrobialpreservative, is considered to be carcinogenic tohumans, which was also confirmed by the InternationalAgency for Research on Cancer (IARC) [6, 7]. Still,this substance is often used to process foods [8]. Insmall quantities, formaldehyde is involved in humanmetabolism. However, high doses can cause pain,vomiting, coma, and possible death [9]. According tothe United States Environmental Protection Agency,the acceptable daily intake of formaldehyde is about0.2 mg/kg b.w. [10]. In addition to being used as apreservative, low concentrations of formaldehydecan also serve as an antiseptic solution due to itsantibacterial and antifungal properties. If inhaled, itdamages respiratory organs and may cause dizzinessand suffocation, not to mention eye, nose, and throatirritation [11]. Exposure to formaldehyde increases theincidence of lung and nasopharyngeal cancer. Ingestionof 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 andmarine fish [8].Heavy metals, such as lead, chromium, mercury,arsenic, etc., are well-known contaminating chemicalsthat cause water and soil pollution [12]. These metalshave no beneficial effects on human health. On thecontrary, they are generally considered as one of themost toxic elements for humans and animals.Similarly, pesticides in fish products are a sign ofcontamination and pose a serious threat to human healthas they accumulate in human body fats [13]. Acutesymptoms of pesticide poisoning include numbness,incoordination, headache, dizziness, tremor, nausea,abdominal cramps, respiratory depression, etc. [14].Exposure to organochlorine pesticides mainlyoccurs through contaminated food, especially fish orshellfish obtained from contaminated rivers and streams.A long term exposure to moderate levels of aldrin anddieldrin can cause headache, irritability, vomiting, anduncontrollable muscle movements. Excessive dieldrinhas been established as a 2–7-fold higher risk factor forbreast cancer. The lethal dose (LD50) of dieldrin per dayis approximately 10 mg per 1 kg of body weight [15].Carbofuran and its major metabolites (3-hydroxycarbofuranand 3-ketocarbofuran) can cross theplacental barrier and affect the maternal-placentalfetalunit [16]. Alachlor is another toxic chemical thatdissipates from soil mainly through volatilization,photodegradation, and biodegradation. It is readilyabsorbed through gastrointestinal tract and distributed toblood, spleen, liver, kidney, brain, stomach, and ovaries.The LD50 of alachlor is between 1.910 and 2.310 mg/kgin mice [17]. Therefore, timely detection of these toxicelements in fish is essential for human health.Tenualosa ilisha, Clupeidae family, is the mostpopular fish in the Bay of Bengal. Hilsa, as they call itin India, occupies the top position among edible fishesdue to its unique taste and delicious properties. T. ilishais rich in amino acids, minerals, and fats. In addition,the fish has a high content of high density lipoproteinand a low level of low density lipoprotein, which makesit beneficial for human health [18]. Because of its highlipid content, it cannot be sun-dried. As a result, it ispreserved by salting, which is simple and cheap [19].Both fresh and salted Hilsa are very popular among thecommon people of Tripura. Cooking methods for freshand salted Hilsa include boiling, frying, roasting, etc.Suitable cooking methods minimize the nutrient loss andalso improve the digestibility of food [20, 21].Although some aspects of nutritional compositionof T. ilisha have already been reported, the estimationof toxic elements in both raw and cooked samples offresh and salted T. ilisha has not yet been exploredscientifically [22]. The objective of the presentexperiment was to analyze the level of organochlorinepesticide residues and other toxic materials in raw andcooked samples of fresh and salted T. ilisha.STUDY OBJECTS AND METHODSSample preparation and cooking. Fresh and saltedsamples of Tenualosa ilisha L. were obtained from thelocal market of Battala, Tripura. The samples werecleaned to remove dust particles. The fresh samples werewashed and gutted. In case of fresh samples, the mainpurpose was to obtain the maximal amount of fleshportion, so the samples were cut approximately parallelto the backbone. The flesh portion was cut into smallpieces and prepared for boiling and frying. The saltedsamples were washed with water and cut into smallpieces for further cooking. Fresh and salted sampleswere boiled for 20 min or deep-fried in vegetable oil for15 min at 240°C.Sample preparation to determine formaldehyde.The formaldehyde content was determined accordingto Claeysa et al. [23]. Blank and spiked formalin wasadded into five-gram samples. After adding 5 mL ofacetonitrile, the samples were sonicated for 30 minat 25–30°C and shaken for 30 min in a shaking waterbath at 150 rpm at room temperature. Then, they werecentrifuged at 6000 rpm at 22°C for 5 min and filteredthrough a Whatman filter paper (90 mm). After 5 mLof the upper layer of the extract was carefully removed,2.5 mL DNPH solution (dinitrophenylhydrazine)and vortex were added. Recrystallization of DNPHwas carried out by dissolving 10 mL of anhydrousacetonitrile acetate to obtain a saturated solution. Thesamples were derivatized by shaking at 150 rpm at40°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 samplesolution was analyzed by using a C-18 column (250×4.6 mm, 5 μm) with a 60% methanol solution as mobilephase and analyzed at 355 nm. The flow rate was 1 mL/min and the operating time was 13 min.Standard curve preparation. A stock formaldehydesolution (6.2%) was used to prepare standard solutionswith concentrations of 0.838, 1.68, 2.51, 3.35, and5.03 ppm by diluting with distilled water. Theabsorbance was then measured using a spectrophotometerat 415 nm. The molar concentration offormaldehyde 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 thecell.In case of matrix-free calibration, the limit ofdetection (LOD) was 0.117 ppm and the limit ofquantification (LOQ) was 0.384 ppm.Recovery test. The known concentration offormaldehyde (5, 10, and 25 mg/L) was spiked in fishmatrix. Recovery was calculated as follows:% of recovery = concentration of formaldehydequantified in the sample × 100/spiked concentration (2)Pesticide determination. The fish samples werehomogenized with an Ultra-Turrax T25 homogenizerand stored in a freezer at –20°C. A standard pesticide Table 1 Formaldehyde content in raw and cooked samples of fresh and salted Tenualosa ilishaFormaldehyde Fresh T. ilisha Salted T. ilishaRaw ± SEM Boiled ± SEM Fried ± SEM Raw ± SEM Boiled ± SEM Fried ± SEMContent, mg/kg 10.32 ± 2.11 9.24 ± 2.40a 9.02 ± 1.80a 12.14 ± 1.51 11.23 ± 1.10b 10.58 ± 3.70bFive samples were taken to calculate the standard error meana P < 0.05 when compared with raw fresh T. ilishab P < 0.05 when compared with raw (uncooked) salted T. ilishaTable 2 Pesticide residues in raw and cooked samples of fresh and salted Tenualosa ilishaPesticides T. ilisha (fresh) T. ilisha (salted)Raw ± SEM Boiled ± SEM Fried ± SEM Raw ± SEM Boiled ± SEM Fried ± SEMAldrin 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. ilishailisha L. The salted sample contained a higher amountof formaldehyde (12.14 mg/kg) than the fresh sample(10.32 mg/kg), which could be because formaldehydeserved as a preservative. However, its content reducedafter boiling and frying. Again, cooking had someeffect on the formaldehyde content. This toxic elementis known to degrade after thermal treatment [26]. Theconcentration of formaldehyde mainly depends ondifferent levels of trimethylamine n-oxide (TMAO) [27].Tri-methylamine, di-methylamine, and formaldehyde areformed after the breakdown of TMAO [28].The normal range of formaldehyde is 2–50 mg/kg, sothe formaldehyde content in the present experiment waswithin the normal range for both fresh and salted fishsamples.Table 2 demonstrates the pesticide content (spikinglevel 5 ng/g) in the raw and cooked samples of freshand salted T. ilisha. Alachlor and hexachlorobenzenewere detected neither in the raw nor in the cookedsamples. On the other hand, the levels of aldrin, alachlor,carbofuran, dieldrin, endosulfan sulfate, o,p′-DDT+p,p′-DDD, p,p′-DDT, and hexachlorobenzene werehigher in the salted sample. After cooking, the pesticidecontent went down. The toxic organochlorine pesticidesresidue was higher in the salted sample, which may bedue to the fact that these substances were added to thefish as preservatives. After cooking, the organochlorinepesticides residue decreased because cooking processincreases volatilization, hydrolysis, or other chemicaldegradation and leads to the decomposition by applyingheat [29, 30].Table 3 shows the heavy metal content in the raw andcooked samples of fresh and salted Hilsa. The mercurycontent was found to be 0.101 mg/kg in the fresh sampleand 0.102 mg/kg in the salted sample. However, it wentdown after cooking. The above result is acceptablefor fish, considering that the proposed upper limitfor mercury is 0.5 mg per 1 kg of fresh weight. Thecadmium content was high in both fresh and processedsamples. For general fish muscle, cadmium level is0.05 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, thecopper concentration was 54.02 and 53.88 mg/kg infresh and salted T. ilisha, respectively. The chromiumcontent did not exceed 1.0 mg/kg for all raw and cookedsamples of fresh and salted T. ilisha. Zinc is anotheressential nutrient. In moderate quantities, it improvesimmune system and metabolism, whereas a high levelof zinc can be harmful. According to FAO, the limit forzinc is 30 mg/kg [32].The limit of zinc acceptability exceeded the normin both raw and cooked samples of fresh and salted fish.Selenium is an essential trace element required in smallamounts for animals and humans. However, a highercontent of selenium is toxic. Cooking appeared to haveno significant effect on selenium content. The nickelcontent was within acceptable limits. Cooking had nosignificant effect on lead contamination either. Arsenic,another toxic element, can be found as a contaminantin fish, shellfish, and other seafood. In the presentexperiment, the arsenic content was within the normalrange. According to FAO/WHO, the maximal intake ofarsenic is 15 μg/kg b.w. Both raw and cooked samplesof fresh and salted T. ilisha contained a high amount ofarsenic [33].CONCLUSIONThe organochlorine pesticide residues were higherin the salted samples of Tenualosa ilisha L., but boilingand frying reduced their amounts. The salted samples ofT. ilisha contained a high amount of such heavy metalsas zinc, copper, and selenium. Fresh T. ilisha proved tobe safe for human health as thermal treatment had somepositive effects on the potentially harmful substances.CONTRIBUTIONDr. Kuntal Manna designed the research; SanchariGoswami collected the samples, performed the analysis,and wrote the manuscript.CONFLICT OF INTERESTThe authors declare no conflict of interests regardingthe publication of this article.ACKNOWLEDGMENTSThe authors are grateful to New Jersey FeedLab Inc., 1686 Fifth St, Trenton NJ 08638, USA, forproviding instrument facility. The authors would liketo express their gratitude to State Biotech Hub, TripuraCentral University-799022, for providing workingfacility and to Tripura State Pollution Control Board,Kunjaban, Gorkhabasti, Agartala-799006, for providingthe instrumental facility for mineral analysis. Theauthors are also indebted to Mr. W. Somraj Singh andMr. Bikash Debnath, Research Scholar, Department ofPharmacy, Tripura Central University, for editing themanuscript.