Overviews of heavy metals residues in some frozen fish and its biocontrol by probiotic

Overviews of heavy metals residues in some frozen fish and its biocontrol by probiotic

Asmaa, E. Hassan.¹, Amany, M.Salama.² and Amira, A. Elokle ¹

¹Food Hygiene department and ²Chemistry department Animal Health Research Institute, Agriculture Research center, Dokki, Egypt.

Abstract:

 Heavy metals are the most significant pollutants in aquatic networks due to their toxicity, accumulation, and bio-magnification by marine organisms. Environmental pollution is the universal problem.

So, A total of 100 random samples of frozen fishes which include Mackrel (Scomber), Horse mackerel (Trachurus trachurus) (30 of each) and Hiring (Clupea harengus)(40 samples) were collected from different fish markets in Cairo governorate, Egypt, for determination of (Lead, Cadmium, Copper, Zinc, Iron, Arsenic, Nickel and tin) levels by atomic spectrophotometer. The obtained results revealed that all elements were within the permissible limit.

High level exploratory work was led expected to survey the corruption impact of one of Lactobacillus rhamnosus (10⁷ CFU/ml) on lead and cadmium levels in experimentally inoculated fish for zero, 8, 16 and 24 hours. Within 24 hours of interacting with L. rhamnosus, the levels of lead and cadmium were reduced by 83.6 and 71.1 percent, respectively. Appropriately, normal examination of heavy metals levels in aquatic environment and creatures is recommended public and worldwide, with stringently proposal to safe removal of plant squanders.

Key words: heavy metals, frozen fish, probiotics.

1-Introduction:

Fish is viewed as a fundamental wellspring of protein due to its high natural worth, polyunsaturated unsaturated fats, nutrients, and minerals including calcium and phosphorus. Because red meat is so scarce and fish is cheaper than chicken and meat, Egypt is becoming a nation that eats more fish  (Morshdy et al., 2019).

Fish is significant piece of a solid eating routine because of numerous nourishing advantages as they contain great protein, low cholesterol level, omega-3 unsaturated fats, minerals as well as nutrients. Additionally, their lower costs potentiate their nutritive qualities. An even eating routine incorporates an assortment of fish and shellfish can add to heart wellbeing and youngsters' development and improvement. Weighty metals are normally happening components despite the fact that their various modern, horticultural, drug and mechanical applications have prompted their wide circulation in the climate, raising worries over their likely impacts on environments and human wellbeing (Tchounwon et al., 2012). The contamination of sea-going climate with heavy metals is a general wellbeing peril during ongoing years. Food residues have been linked to immune suppression, chemical hypersensitivity, and breast cancer, as demonstrated by scientific researchers; decrease sperm count and fruitlessness (Sharp, 1999).

 Heavy metals are dissolved and easily accumulate in the various parts of aquatic living organisms, including fish, once they enter the aquatic system. Consumers of contaminated fish then take in these heavy metals. (Authman, 2015).

Copper, cadmium, and lead, among other heavy metals, may be highly toxic to aquatic life when introduced into the water supply (Ambreen et al., 2015).

Moreover, fish are situated toward the finish of the sea-going order of things and may aggregate metals and pass them to people through food causing constant or intense infections (Al- Yousul et al., 2000).

Lead has hepatotoxic effects and raises liver function test parameters significantly (Adeyemi et al., 2009).

Cadmium is toxic to the kidneys, initially causing damage to the kidney tubules. It can also damage bones, either directly by affecting bone tissue or indirectly through renal dysfunction  (Wang et al., 2008).

Copper is a fundamental trace element that, when ingested in large quantities, is extremely harmful to organisms and organs. Toxic levels of copper sulfate in the liver cause oxidative damage in the form of granular degeneration, hepatocyte necrosis, and impairment of the cell lining of the remark cords. These biochemical changes in malondialdehyde and glutathione levels are evidence of this (Emin et al., 2010).

Zinc is remarkable component that is minimal fundamental for human wellbeing. An excess of Zn can cause conspicuous medical conditions, for example, skin disturbances, stomach cramps, sickliness, retching and queasiness. Arteriosclerosis is brought on by high levels of zinc, which damage the pancreas and disrupt protein metabolism (Afshan et al., 2014).

Iron is the primary driver of malignant growth because of iron inebriation is through the age of free revolutionaries which assault DNA bringing about cell harm, change and threatening change which thusly cause a variety of sicknesses  (Grazuleviciene et al., 2009).

Arsenic acts to coagulate protein, structures edifices with coenzymes and represses the creation of adenosine tri phosphate (ATP) during breath (INECAR, 2000). It is conceivably cancer-causing in mixtures of all its oxidation states and significant level openness can cause passing (USDOL, 2004).

Nickel can cause respiratory problems and is a carcinogen (Anonymous 2003; Agency for Toxic Substance and Disease Registry, 2004).

Tin could cause unusual digestion of the fundamental components like zinc, copper and iron. Furthermore it diminishes calcium content in bone and prompts organ harm especially in the kidney (Rader,  1991 ) and was carcinogenic at doses up to 2000 mg in mice with increased incidence of hepatocellular adenomas (ATSDR, 2003).

Such countless preliminaries were completed to control these components and their difficult issue, one of these preliminaries is probiotics. Probiotics—literally, "for life"—are bacteria or yeasts that are thought to benefit the host organism's health. Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, and Bacillus are the six different genera of probiotic organisms used in interventions. The goal of the review was to compile all of the information that was known about the safety of such interventions. Alone or in mix, used to lessen the gamble of, forestall, or treat sickness in research studies (Hempel et al., 2011). The decrease of the harmful perils might be credited to that the metal particles tie to the cell wall and extracellular polysaccharides by suitable or latent cells including adsorption, particle trade, complexation, chelation and microprecipitation (Veglio et al., 1997).

Probiotic strains of lactobacillus can bind to heavy metals, making them useful for heavy metal detoxification (Milatovic et al., 2017). These days, probiotics are viewed as a promising age to mitigate weighty metal harmfulness (Giri et al., 2018).

Consequently, the point of this study is to determine a heavy metals residue (Lead, Cadmium, Copper, Zinc, Iron, Arsenic, Nickel and tin) in frozen fish as Mackerel, Horse mackerel and hiring moreover its biocontrol by the probiotic. The general wellbeing significance and the perilous harmful impacts of these heavy metals were talked about as well as the intriguing suggestions to limit fish contamination with these heavy metals were referenced.

2. Material and Methods: -

2.1. Collection of samples:

A sum of 100 random samples of frozen fish nearly similar in the weight and size which include Mackrel (Scomber), Horse mackerel (Trachurus trachurus) (30 of each) and Hiring (Clupea harengus) (40 samples) were collected from different fish markets in Cairo governorate, Egypt, for determination of (Lead, Cadmium, Copper, Zinc, Iron, Arsenic, Nickel and tin) residue levels.

2.2. Preparation of fish samples for residual detection of heavy metals:

All the fish samples were separately kept at -20 °C then allowed to thaw, washed with running water, then placed on an aluminum foil to avoid contact with the working bench. The samples dissected with sterile scissors to get muscles. The muscles were transferred into sterile sample beakers, labeled for digestion and analysis of heavy metals.

2.3. Procedures for detection of heavy metals residue:

1. Every fish homogenized then one gram of the muscle tissue was digested with 10 ml of nitric / sulfuric / perchloric acids (8: 1: 1).
2. Kept for 4 hours at room temperature then heated in water bath at 40-45°C for one hour then the temperature was increased to 75°C until complete digestion of the tissue.
3. Allow samples to cool at room temperature, and then dilute the digest to 20 ml with deionized water and filtered through 0.45 µl Whatman filter paper.
4. The clear filtrate of each sample can be kept in refrigerator till analysis using atomic absorption spectrometry following the guidelines of AOAC (2002) Official Method 999.10.
5. The digest, blanks and standard solutions were aspirated by Atomic Absorption Spectrophotometer (AAS) and analyzed for their concentrations of such elements. The apparatus has an auto sampler, digital absorbance and concentration readout capable of operating under the conditions recommended by the instrument instructions.

2.4. Quantitative determination of heavy metals residue:

Absorbency of elements was directly recorded from the digital scale and their concentrations were calculated according to the following equation:   C=R x (D/W).

Where, C= Concentration of the element (wet weight)

R= Reading of digital scale of AAS.

D= Dilution of the prepared sample.

W= Weight of the sample.

1. B. The concentration of each element in the blank solution was also calculated and subtracted from each analyzed sample.

2.5. Experimental part:

 The effect of Lactobacillus rhamnosus on the concentrations of lead and cadmium in fish samples experimentally inoculated with.

2.5.1. Preparation of bacterial suspension:

Lactobacillus rhamnosus strain (from National Research center) was enriched in Brain Heart Infusion (BHI) broth for 24hr at 37ºC to prepare an overnight culture. One mL of the cultivated bacterial suspension was decimally diluted in sterile peptone water (0.1%, w/v) and counted by spread cultivation on BHI agar. A volume of the culture broth corresponding to approximately 1×10⁷ CFU/ml bacteria was centrifuged (500 rpm / 15min) and the bacterial pellets were washed twice with deionized water (Halttunen et al., 2007).

2.5.2. Binding assay:

The bacterial pellet (10⁷CFU/ml), 30 mg/Kg ionic lead, and 10 mg/Kg ionic cadmium solutions were mixed with fish samples free from heavy metal residues as indicated by Halttunen et al., (2008).

2.5.3. Experimental design:

Metal-contaminated fish served as the control positive group (G1). Fish inoculated with L. rhamnosus (10⁷ CFU/g) and lead (30 mg/kg) (G2), and fish inoculated with L. rhamnosus (10⁷ CFU/g) and cadmium (10 mg/kg) (G3). (G2) and (G3) were used as the treated groups. The samples were acidified with ultrapure HNO3 (Lars, 2003), and examined at zero, 8-, 16-, and 24-hour time points for measuring the free metal by atomic absorption spectrophotometer.

2.5. Statistical analysis:

Statistical analysis was assessed using the SPSS (v.13, SPSS Inc., Chicago, IL). Results were recorded as mean ± standard errors (SE). The value of P < 0.05 was used to indicate statistical significance. One-Way ANOVA test was applied to compare differences among means. Paired samples t-test was used to compare samples before and after adding probiotic strain.

3. Results:-

Table 1: Heavy Metals concentrations (ppm "mg/kg") in the examined frozen fish samples:

There were significance P ≤0.05 between the same letters in the same row.

Table (2): The residual levels of heavy metal (ppm "mg/kg") in analyzed fish samples as compared with the permissible limit

Table (3): Impact of L. rhamnosus culture (10⁷ CFU/g) on the degrees of lead experimentally inoculated to fish (30 mg/Kg).

Table (4): Impact of L. rhamnosus culture (10⁷ CFU/g) on the degrees of Cadmium experimentally inoculated to fish (10 mg/Kg).

4. Discussion: -

Heavy metals are environmental and food-safe contaminants. These have long biological half-lives and are not biodegradable  (Heidarieh et al., 2013). As a result, the purpose of the current study was to survey and investigate the concentrations (ppm or mg/kg) of various heavy metals in some frozen fish that was sold commercially in Egyptian markets represented by mackerel fish (Scomber), Horse Mackerel (Trachurus trachurus) and Hiring(Clupea harengus).

Regarding, the results in table (1) revealed that the mean value of lead levels were 0.074± 0.004 , 0.082±0.004 and 0.12±0.01 ppm with minimum values of 0.04, 0.07 and 0.05ppm while the maximum values were 0.12, 0.13 and 0.26 ppm  in mackerel fish(Scomber), Horse Mackerel (Trachurus trachurus) and Hiring(Clupea harengus), respectively. There were significant differences between mackerel (Scomber) and Hiring (Clupea harengus) also between Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus).

In table (2) there were no samples exceed the permissible limit as indicated by FAO, (1983) is 0.5 mg kg, and Joint Expert Committee on Food Additives (JECFA, 2000) allowed up to 3.0 mg kg. Moreover, the maximum Residual Limit of lead (0.30 mg/Kg) stipulated by Egyptian Organization of Standardization "EOS" (2010).

Heavy metals are a class of elements with a high density and are poisonous at low concentrations (Koller and Saleh 2018). Metals, once consumed, can gather in the body of a living creature to levels adequate to become poisonous (Ojuederie and Babalola 2017).

Low results were recorded by  Sireli et al., (2006) and  Ekpo et al., (2008). Nearly similar results obtained by  Shokr et al., (2019), while higher results by  Abubakar et al., (2015),  Ali et al., (2016) and  Ahmed et al., (2019) estimated  Pb ranged between 2.76 and 4.63 ppm for commercial marine fish species .

Moreover table (1) revealed that the mean cadmium levels were 0.041±0.001 ppm , 0.015 ± 0.003 ppm   and 0.031± 0.003 ppm with minimum values of 0.032 , 0, 0.01 ppm while the maximum values were 0.048 , 0.041, 0.049 ppm in mackerel fish(Scomber), Horse Mackerel (Trachurus trachurus) and Hiring(Clupea harengus), respectively. There were significant differences between the three fish species.

These results are almost identical to those obtained by Ayeloja et al., (2014). However, Ekpo et al., (2008) and  Badr et al., (2014) recorded higher results.

An acceptance limits for Cd 0.1 mg kg was set up by FAO, (1983), EU, (2001) and WHO, (2004).

 Moreover the maximum residual limit of Cd (0.05 mg/Kg) stipulated by Egyptian Organization of Standardization "EOS" (2010). (Table 2) according to that all samples not exceed this permissible limit.

In addition to table (1) mentioned that the mean copper residual levels were 0.06± 0.02, 0.14± 0.06 and 0.13±0.02 ppm with minimum value of  0, 0 and 0.04 ppm. However the maximum values were 0.18, 0.92 and 0.31ppm in mackerel (Scomber), Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus), respectively. There were no significant differences between any fish species.

According to table (2) none of the examined muscle samples exceeded the limit of FAO, (1983) 70 mg kg, FAO/WHO, (1989) and (MAFF (Ministry of Agriculture, Fisheries and Food), 1995) which expressed that the permissible limit of copper should not be in excess of 30 mg/kg. Moreover EU, (2001) mentioned that the permissible limit of copper 10 mg/kg. 

Higher results obtained by Kaoud and EL-Dahshan (2010) and Ali et al., (2016).

The level of Tin residues were 4.92± 0.7 , 4.66± 0.4 and 0.92± 0.2 ppm as mean value with minimum values of 1.6 , 2.38 ppm and  0 (not detectable ) while the maximum values were 10.6, 7.81 and 2.57 ppm in mackerel (Scomber), Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus), respectively. These differences in the accumulation levels may contribute to the sample origin, feeding habit and the age of fish. There were significant differences between mackerel (Scomber) and Hiring (Clupea harengus) also between Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus).

 In table (2) the permissible limit of tin 250 mg/kg and 200 mg/kg by WHO (JECFA, 2006) and (EU, 2006), respectively. So, all samples were accepted.

Lower results were obtained by Morshdy et al. (2013) and Morshdy et al. (2021).In table (1) the mean values of Arsenic residues (Ar) were 0.003± 0.002, 0.12± 0.05 and 0.16 ± 0.04 ppm with minimum values of 0 (not detectable) while the maximum values were 0.04, 0.5 and 0.44 ppm in mackerel (Scomber), Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus), respectively. There were significant differences between mackerel (Scomber), Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus), also between Horse Mackerel (Trachurus trachurus) and mackerel (Scomber), moreover between Hiring (Clupea harengus) and mackerel (Scomber).

In table (2) the Permissible limit for Arsenic was set up at 1.0 mg kg by  FAO, (1983) and California Environmental Protection Agency (CEPA) (1995-97), so there weren’t samples exceed these Permissible limit

In a study which was conducted by Javaheri Babooli and Velayatzadeh (2013) the mean concentrations of Arsenic in fish samples 0.117±0.07 ppm which was similar to our results.

In another study which was conducted by  Heidarieh et al., (2013) arsenic concentrations in fish samples was 21.38±3.31 ppm which was higher than our results.

Moreover in table (1) the Nickel (Ni) mean values were 0.034± 0.006 , 0.05 ±0.008 and 0.09±0.02 ppm with no detectable minimum values and 0.06, 0.13 and 0.28 ppm as maximum values ppm in mackerel (Scomber), Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus), respectively. There were significant differences between mackerel (Scomber) and Hiring (Clupea harengus) also between Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus).

However in table (2) there weren’t any sample exceed the permissible limit which 0.5–0.6 mg/kg set by the  WHO, (1985) and 186 mg/kg by FAO/WHO, (1989).

 Ni is an accretive body toxin and its concentration in the environment should remain as low as possible.

Higher concentrations of Ni have been detected to be 1.2-3.4 mg/kg by  Mendil et al., (2005) and 3.40 mg/kg by  Nisbet et al., (2010).

Like other living things, fish need zinc to survive; and numerous enzyme structures contain it. Also, it is recognized that it has critical capabilities in safeguarding film structure. Since it is a fundamental component, fish have a capacity of enduring zinc (Eisler, 1981).

The mean values of Zinc (zn) in table (1) were 0.22± 0.02, 0.38± 0.02 and 0.3± 0.02 ppm with minimum values of 0.06, 0.27 and 0.14 ppm while the maximum values were 0.35, 0.51 and 0.5 ppm  in mackerel (Scomber), Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus), respectively. There were significant differences between the three fish species.

The maximum permissible limit of Zn in fish and fish product was 100 mg/ kg proposed by the FAO/WHO, (1989) while FAO, (1983) and EU, (2001) 30 mg/ kg. None of the examined muscle samples exceeded the limit in table (2).

The higher zinc concentrations obtained were (19.55 mg/ kg) by Nisbet et al., (2010) but Tüzen, (2003) found that concentrations of zinc were 9.50 mg/ kg.

Eventually in table (1) the mean values of Iron (Fe) residues were 0.44± 0.06, 0.6± 0.03 and 0.7± 0.05 ppm with minimum values of 0, 0.37 and 0.36 ppm while the maximum values were 0.9, 0.83 and 1.04 ppm in mackerel (Scomber), Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus), respectively. There were significant differences between mackerel (Scomber), Horse Mackerel (Trachurus trachurus) and Hiring (Clupea harengus), also between Horse Mackerel (Trachurus trachurus) and mackerel (Scomber), moreover between Hiring (Clupea harengus) and mackerel (Scomber).

 So the whole samples accepted according to  FAO/WHO, (1989), (Table 2)

Higher concentrations of Fe ranged from 21.17 to 33.78 mg/kg obtained by Nisbet et al., (2010).

The recorded varieties between the got results and different records might be credited to the distinction in the territories of test's assortment, period of fishes as a huge consider bioaccumulation cycle, and kinds of the analyzed fishes.

• Biocontrol of heavy metals by probiotic:
• Impact of rhamnosus culture (10⁷ CFU/g) on the degrees of lead and cadmium experimentally inoculated to fish:

The toxic nature of heavy metals (bioaccumulation and bio magnification) in food chain, so the tainting of the climate with these serious poisons, related straightforwardly to general wellbeing (Hussain et al., 2012).The cadmium and lead are reported amongst the top ten toxic metals in the Priority List of Hazardous Substances (ATSDR, 2007) for causing a grossly biological impact by bioconcentration, bioaccumulation phenomena. Certain species of lactic acid bacteria (LAB), as well as other microorganisms, can bind metal ions to their cells surface or transport and store them inside the cell. As a result, interactions between metal ions and LAB have been extensively studied over the past few years to develop their use in new biotechnology processes in addition to their health benefits and probiotic properties.   LAB has a high potential for absorption of toxic and essential metal ions in model aqueous solutions, which can be used to improve food quality and safety  (Mrvčić et al., 2012).

An exploratory review was led to examine the lead and cadmium levels debasement under the impact of L. rhamnosus in fish model. Results as classified in Tables (3 and 4) showed an extraordinary reducing impact in lead and cadmium levels, separately. Within 24 hours of the incubation, they were reduced by 83.6 and 71%; besides, higher corruption level on lead was recorded than cadmium levels in experimentally inoculated fish.

Nearly similar results obtained by Samir et al., (2021) who recorded reduction in lead and cadmium levels experimentally inoculated in fish fillet 84.3 and 72% within 24h of the incubation.

5. Conclusion: -

When the results of this study were compared to fish standards set by the United States and other countries, it was found that none of the frozen fish samples examined went above what was allowed by those standards. In addition, L. rhamnosus demonstrated a promising method for decreasing the accumulation of heavy metals (lead and cadmium) in fish tissues.

Because of the medical advantages and extraordinary significance of fish in human eating routine, consideration ought to be given to their pollution with harmful or possibly poisonous ecological synthetics like weighty metals. Utilization of fish ought to be directed particularly in weak populaces, for example, kids and pregnant ladies as well as in exorbitant fish-eating populaces.