Chemical and bacteriological risks of shrimp and clams (Gandofly) from Suez Gulf

Shellfish including shrimp and clams are consumed allover the world for their nutritional value, however, handling in seafood markets may challenge their safety and quality. Shellfish products in the fish markets in Suez Governorate were evaluated throughchemical analysis of total volatile basic nitrogen value, thiobarbituric acid, histamine content, and some heavy metals residues. As well as, microbiological estimation of the total aerobic counts, Enterobacteriaceae counts, Escherichia coli counts, and Staphylococcus aureus counts, in addition of Salmonella detection. Results declared that more than half of the examined samples of shrimp and clams (gandofly) were fit for human consumption based on the permissible limits of the Egyptian standard. Otherwise, some gandofly samples contain high levels of total volatile basic nitrogen (TVB-N), thiobarbituric acid reactive substance (TBA), and may cause scombroid food poisoning with elevated histamine level. The levels of cadmium, lead, and zinc in shellfish samples under study was different with the sequence: Zn>Pb>Cd. Bacteria including E. coli and Staphylococcus aureus were identified. The analysed samples in this study were judged as medium/good quality market samples as more than half of the examined samples were within the Egyptian standard and may indicate that trading of shellfish at Suez markets went under abuse storage temperature.


Introduction
Shellfish is consumed all over the world for its nutritional value. Shrimp is highly important in the human diet due to being rich in proteins, fat-soluble vitamins, omega-3, polyunsaturated fatty acids (Javaheri-Baboli and Velayatzadeh, 2013). However, in developing countries, the importance of shellfish as a human food source may be under-estimated and its safety and quality are questioned. Most of the infectious outbreaks from seafood appear to be due to shellfish rather than finfish (Butt et al., 2004). Shellfish infection takes place due to the fact they are filter feeders that selectively filter out and concentrate contaminant materials, and microorganism of faecal origin and pathogens adapted to the marine surroundings, and chemical substances (Li et al., 2017). There are a variety of indicators to express the freshness and safety of fish can be estimated by chemicals indicators such as trimethylamine (TMA), total volatile basic nitrogen (TVB-N), Thiobarbituric acid, and histamine production (Li et al., 2017).
The microorganisms' development causes the deterioration of the seafood that leads to losses of quality of the final product. Some microbial groups, such as specific spoilage organisms, are responsible for the negative organoleptic characteristics such as unpleasant taste and smell in fish. In this respect, heavy metals pollution is a public health hazard since these contaminants are widely spread within the environment, including marine environments, from either natural or anthropogenic origins (Lozano et al., 2010). Also, the relevant importance of biogenic amines is their potential toxicity associated with high accumulation and consumption of histamine in fish and fisheries products (Hungerford, 2010).
The objective of this study was to determine the quality and safety of shellfish products (shrimp and bivalve molluscs "clams") in the fish markets in Suez Governorate. Assessments were conducted through: i) chemical evaluation of some indicators (total volatile basic nitrogen value, thiobarbituric acid, histamine content, and some heavy metals residues), ii) bacteriological evaluation of total aerobic count,
Samples were wrapped in sterile polyethylene bags, identified, and placed in a clean ice-box. Samples were then rapidly transferred to the food control laboratory, Faculty of Veterinary medicine, Suez Canal University under complete aseptic conditions for further evaluation.

Determination of total volatile basic nitrogen
According to Malle and Poumeryrol (1989), 100 g of the pooled shellfish (shrimp -clams) sample extract was dissolved in 200 mL of 7.5% aqueous trichloroacetic acid and filtrate the mixture to obtain a clear extract, then blend 25 mL of this extract with 6 mL 10% NaOH in a distillation flask. Steam distillation was carried out by using the Kjeldahl-type distillation (Struer TVN)

Determination of thiobarbituric acid
Approximately, 1 g of each sample was taken in a 25 mL test tube and 5 mL of 50% glacial acetic acid in water (AW) as solvent was added. The 0.01% Butylated hydroxyl toluene (BHT) was used to prevent further oxidation of the medium. The samples were shaken for 1h and filtered. The filtrate was centrifuged, when required, and was used for analyses as recommended by Zeb and Ullah (2016).

Determination of histamine
According to Patange et al. (2005), 5 g of shellfish sample transferred to 75 mL centrifuge, homogenized with 20 mL of 0.85% NaCl solution, and centrifuged at 12000×g for 10 mins at 4°C. The extract (1 mL) was diluted to 2 mL with saline and 0.5g of a salt mixture containing 6.25 g of anhydrous sodium sulfate to 1 g trisodium phosphate monohydrate and was shaken briefly to break the protein gel. The tubes were shacked, then centrifuged at 3100×g for 10 mins. The upper butanol layer evaporated until dryness in the stream of nitrogen. The residue was dissolved in 1 mL of distilled water and then mix with p-phenyldiazoniumsulfonate reagent. The colour absorbance was measured after 5 min at 496 nm using distilled water as a reference and the concentration of histamine was obtained from the standard curve.

Determination of heavy metal residues
Shellfish samples washed by deionized water and dried to determine the concentrations of Cd, Pb and Zn. Each sample (10 g) was digested with a mixture of nitric acid (HNO 3 , Merck, 65%) and perchloric acid (HClO 4 , Merck, 60%) with a 2:1 v/v ratio (Canli and Atli, 2003).Tubes were heated in a water bath adjusted to 70°C , then were cooled at room temperature. Then tubes were diluted with de-ionized water till reach 25 mL and filtered by Whatman filter paper No. 42. The filtrate kept at room temperature until the analysis of heavy metals. All the digest, blank, and standard solutions were aspirated by the Atomic Absorption Spectrophotometer (AAS). Heavy metals (Pb, Cd and Zn) concentrations were analysed by Perkin-Elmer-5100 Atomic Absorption Spectrophotometer.

Bacteriological evaluation 2.3.1 Sample preparation
Shrimp frozen samples were held at 4°C in the fridge for 6-8 hrs to be thawed. Aseptic technique was undertaken throughout the steps of sample's handling. The shell of clams was scrubbed with a sterile stiff bristle brush under cold potable water to remove the mud with special attention to crevices at the junction of the shells and then placed on a clean paper towel to drain. Closed clam was sterilized by alcohol and then flamed. The adductor muscles were cut with sterile scissors and the covers were removed and transfer the soft tissue to a sterile sample container (ISO, 2013). Shellfish samples represented by 25 g of shrimp and clam, separately were added to 225 mL of 0.1% (w/v) sterile buffered peptone water (Oxoid-CM509). The content was homogenized using a Lab Blender (Seward stomacher 400R/UK) for 2 mins. Further tenfold decimal serial dilution up to 10 -7 was carried out.

Determination of total aerobic plate count
The total aerobic plate count was carried out by pour plate method in duplicates, using sterile melted plate count agar medium (HI Media, M091) (ISO, 2013). Plates were promptly incubated (Incucell incubator/ Germany) at 30±1°C for 72±3 hrs. Results were reported and expressed as colony forming unit per gram (CFU/g).

Determination of Enterobacteriaceae
Enterobacteriaceae count was determined on violet red bile glucose agar medium (VRBG) (HI Media, M091), incubated at 37°C for 24±2 hrs (ISO, 2004). Suspected colonies, which showed purplish-red colonies surrounded by a red zone of precipitated bile acids were enumerated to obtain Enterobacteriaceae count per eISSN: 2550-2166 © 2021 The Authors. Published by Rynnye Lyan Resources FULL PAPER gram.

Determination of Escherichia coli
A loopful of sample suspension was aseptically transferred to sterile duplicate plates of MacConkey agar medium (Oxoid, CM015), and incubated at 35±2°C for 18±2 hrs. The colonies grew as pink to brick-red colonies with or without a zone of precipitated bile are lactose fermenting and were picked up and streaked onto sterile duplicate plates of Eosin Methylene Blue agar medium (EMB) (Lap, 61), and incubated at 35±2°C for 18-24 hrs. The typical colony of Escherichia coli is selected were confirmed by Gram's stain, motility test, indole, methyl red, Voges Proskauer, and citrate test (IMVC).

Determination of Salmonella
The technique used in this study to detect Salmonella is carried out according to the method described by USA/FSIS (2004)

Statistical analysis
Statistical analysis of data was carried out using Statistical Package for the Social Sciences (SPSS) version 16.0 (SPSS, Inc., Chicago, IL, USA).

Total Volatile Basic Nitrogenous (TVB-N)
The concentration of TVB-N in the examined samples of shrimp ranged from 52.36 to 68.10 with a mean value of 54.32 ±12.03 (mg N/100 g), while in clams (gandofly) samples ranged from 50.10 to 70.12 with a mean value of 66.47±15.84(mg N/100 g) ( Table  1). Similar results of TVB-N in fish samples were obtained by Ocaño-Higuera et al. (2011).Moreover, 35 (87.5%) of shrimp samples and 29 (72.5%) of clams (gandofly) samples are within the permissible limits for TVB-N which is not more than 65 (mg N/100 g). On the other hand, the number of unaccepted samples is 5 (12.5%) and 11 (27.5%) of the examined samples of shrimps, and gandofly, respectively. which unaccepted according to the maximal permissible limit for TVB-N as recommended by EOS (2005). TVB-N may be a quality index for unprocessed fishery products, whose increase was related to the activity of spoilage bacteria and endogenous enzymes (Bahmani et al., 2011). The formation of TVB-N is associated with the activity of micro-organisms and tends to be high at high microbial populations as observed by Chytiri et al. (2004). Thus, TVB-N levels are affected by the method of catch, postmortem treatment, and storage temperature as well as it differs according to fish species (Nazemroaya et al., 2011).

Thiobarbituric acid reactive substances (TBA).
TBA is a widely used as indicator for the assessment of degree of secondary lipid oxidation (Gulsun et al., 2009). Malondialdehyde (MDA) is one of the most abundant aldehydes generated during secondary lipid oxidation and probably the most used as an oxidation marker (Barriuso et al., 2012 (Khodanazary, 2019). Furthermore, 38 (95%) of shrimp samples and 34 (85%) of clams (gandofly) samples are within the permissible limits for TBA, which is not more than 4.5 (mg MA/Kg). Number of unaccepted samples were 2 (5%) and 6 (15%) of the examined samples of shrimp and clams (gandofly) respectively, which unaccepted according to the maximal permissible limit for TBA as recommended by EOS (2005).

Histamine content
Results detected in Table1that the concentration of histamine in the examined samples of shrimp ranged from 9.88 to 22.58 with a mean value of 14.21±3.46 (mg/100 g), while in clams (gandofly) samples ranged from 11.41 to 25.20 with a mean value of16.44±4.10 (mg/100 g). High levels of histamine concentration in shellfish samples compared to this study were obtained by Prester et al. (2010), and lower results were reported by Tam et al. (2017). Also, 36 (90%) of shrimp samples and 32 (80%) of gandofly samples are within the permissible limits for histamine which is not more than 20 (mg/100 g). Otherwise, the number of unaccepted samples is 4 (10%) and 8 (20%) of the examined samples of shrimp and gandofly respectively, which unaccepted according to the maximal permissible limit for histamine recommended by EOS (2005). The quantity of biogenic amines produced in seafood relies on the availability of free amino acids, bacterial growth, presence of decarboxylase enzymes and the desired environment for the decarboxylation action (Brink et al. 1990).

Heavy metal
It is evident from the results shown in Table 1 that the concentration of lead in the examined samples of shrimp ranged from 0.02 to 1.05 with a mean value of 0.51±0.101 (mg/Kg wet wt.), while in clams (gandofly) samples ranged from 0.03 to 1.25 with a mean value of 0.60±0.122 (mg/Kg wet wt.). Nearly similar results for shrimp and fish samples and higher results for mollusc samples were obtained by Ahmed et al. (2015). Most of the shrimp samples 37 (92.5%) and 31 (77.5%) of gandofly samples are within the permissible limits for the lead as recommended by the Egyptian Standard for lead in fish which is 0.3 mg/kg wet wt. (EOS 2010). Lead pollution is introduced from many sources such as wastewater treatment sludges to water and soil, transportation, rain, ice, hail and others. Nearly 98% of lead in the atmosphere initiates from human activates (Amirah et al., 2013).
The concentration of cadmium in the examined samples of shrimp ranged from 0.01 to 0.51 with a mean value of 0.20±0.031 mg/Kg wet wt., while clams (gandofly) samples ranged from 0.01 to 0.71 with a mean value of 0.30±0.042 mg/Kg wet wt. (Table 1). Further, 39 (97.5%) of shrimp samples and 38 (95%) of gandofly samples are within the permissible limits for cadmium as recommended by the Egyptian Standard for cadmium in fish which is 0.05 mg/kg wet wt. (EOS, 2010). Cadmium happens normally in low amounts in the environment and was also utilized in batteries, pigments, and metal coatings. Industrial processes such as smelting or electroplating and the addition of fertilizers can increase the concentration of Cd in the environment (Ahmed et al., 2015).
The concentration of zinc in the examined samples of shrimp ranged from 28.22 to 32.11 with a mean value of 30.14±5.10 mg/Kg wet wt., while in gandofly samples ranged from 29.74 to 38.21 with a mean value of 32.64±6.73 mg/Kg wet wt. (Table 1). Higher results of zinc concentration in shellfish samples were obtained by Ahmed et al. (2015). All shrimp and gandofly samples were within the permissible limits for zinc as recommended by the Egyptian Standard 40 mg/kg wet wt. (EOS 2005). Generally, the effluent discharges from metallurgic, electroplating, and petrochemical industry, runoffs from agricultural areas and dredging of the harbour and municipal wastes are the sources of zinc in coastal waters. Zinc in the marine environment principally related to suspended substance prior to settling in sediments (Sasikumar et al., 2011). Shellfish species that inhabiting bottom levels have higher capacities for the uptake of heavy metals than others inhabiting the top level due to direct contact with the sediment (Gu et al., 2015). Table 2 showed that the mean values of the total aerobic counts were 4.62×10 4 ± 1.7×10 3 (CFU/g) for shrimp samples and 6.3×10 5 ± 2.1×10 4 (CFU/g) for gandofly samples respectively. Higher results of total aerobic count in shrimp samples were reported by Abd-El-Aziz and Moharram (2016). Lower results were mentioned by Asai et al. (2008) and Mohamed-Hatha et al. (2003). Also, lower results in fish samples were obtained by Sanjee and Karim (2016). Examined samples revealed that 33 (82.5%) of shrimp samples and 28 (70%) of gandofly samples are within the permissible limits for the total aerobic count. On the other hand, 7 (17.5%) and 12 (30%) of the examined samples of shrimp and gandofly, respectively, exceeded the upper permissible limit (1×10 6 CFU/g) (EOS, 2005

Total Enterobacteriaceae counts
The mean values of the Enterobacteriaceae count in this study were 2×10 2 ± 1×10 2 (CFU/g) for shrimp samples and 92×10 1 ± 2×10 2 CFU/g for clams (gandofly) samples respectively (Table 2). Nearly similar results of Enterobacteriaceae count in shrimp samples were reported by Abd-El-Aziz and Moharram (2016). Lower results were stated by Onyango (2009) in fish samples. Higher results of Enterobacteriaceae count in mollusc samples were showed by Goulas et al. (2005). Shrimp samples 38 (95%), and 35 (87.5%) of gandofly samples are within the permissible limits for Enterobacteriaceae count. Otherwise, 2 (5%) and 5 (12.5%) of the examined samples of shrimp and gandofly, respectively, exceeded the upper permissible limit which is 10 2 (CFU/g) as recommended by the Egyptian Standard for Enterobacteriaceae count in shellfish EOS (2005). Enterobacteriaceae count is well-known as an alternative index of seafood quality as its relation to ice storage, rinsing and evisceration (Zambuchini et al., 2008).

Escherichia coli counts
The mean values of the E coli count in this study were 3×10 2 ± 1×10 2 (CFU/g) for shrimp samples and 50×10 2 ± 2×10 2 CFU/g for clams (gandofly) samples respectively (Table 2). Furthermore, 40 (100%) of shrimp samples and 38 (95%) of clams (gandofly) samples are within the permissible limits which should be free from pathogenic E. coli. On the other hand, 2 (5%) of gandofly samples were not in accordance with the Egyptian standards for pathogenic E. coli count in shellfish EOS (2005). E. coli may transfer to the foods due to poor hygienic conditions, cross-contamination, or contaminated water (Huss et al., 2003). Ineffectively cleaned and sterilized boat decks and fish vessels are also known to contaminate the catch with E. coli (Iyer, 2005). Often cited as a potential cause for E. coli contamination is, the quality of the ice used for conservation and the food processing plants (Bagge-Ravn et al., 2003).

Salmonella
In this study, Salmonella spp. was not detected in all examined shellfish samples of shrimp and clams (Table  2), which agrees with the Egyptian standards that 25 g of shellfish samples must be free from Salmonella spp. (EOS, 2005). The same result was obtained by Marceddu et al. (2017), where all examined seafood samples were free from Salmonella. The possible source of Salmonella contamination in fish farms is may due to bad water quality, farm run off and faecal contamination from wild animals. Generally, sewage pollution of shellfish harvest beds resulted in more shellfish-related outbreaks of Salmonella infections (Iwamoto et al., 2010).

Staphylococcus aureus counts
Results of S. aureus counts were 8×10 2 ± 2×10 2 CFU/g for shrimp samples and 2.4×10 3 ± 3×10 2 CFU/g for gandofly samples respectively (Table 2). Higher results of S. aureus count in mollusc samples were stated by Obire et al. (2017). Moreover, 36 (90%) of shrimp samples and 34 (85%) of gandofly samples are within the permissible limits for S. aureus count. Otherwise, 4 (10%) and 6 (15%) of the examined samples of shrimp and gandofly, respectively, were exceeded the upper permissible limit which is 10 3 CFU/g as recommended by the Egyptian Standard for S. aureus count in shellfish (EOS, 2005). Food processing with poor hygienic practice is highly associated with infection of S. aureus enterotoxin (Hassanien and Abdel-Aziz, 2017). Food handlers with a hand infection or with cold or with a sore throat may transfer enterotoxigenic strains of Staphylococcus to food (Hammad et al., 2012).