Sample selection

Bosnia and Herzegovina (BiH) is a continental country with a narrow exit to the Adriatic Sea (Fig. 3). Despite this fact, most fish and seafood eaten in BiH are bought in grocery stores, usually frozen or canned, probably because it is more easily accessible throughout the year. Thus, we purchased our samples (n = 37) from retail in June 2019. We chose different fish and seafood species widely available and consumed by the local population. The toxic metals content was determined in samples of European hake (Merluccius merluccius, Linnaeus 1758)—frozen, Atlantic bluefin tuna (Thunnus thynnus, Linnaeus, 1758) – frozen and canned, Atlantic mackerel (Scomber scombrus, Linnaeus, 1758)—canned, Patagonian squid (Loligo gahi, Orbigny, 1835)—frozen, blue mussel (Mytilus galloprovincialis, Lamark 1819)—frozen, black tiger shrimp (Penaeus monodon, Fabricius 1798)—frozen and Indian white prawn (Penaeus indicus, H. Milne Edwards, 1837)—frozen.

Figure 3

Geographical location of Bosnia and Herzegovina.


All chemicals used during the analytical procedure were of ultrapure grade. Nitric acid (68%) and hydrochloric acid (35%) were purchased from Fisher Scientific (UK). Sodium borohydride granules were from Merck Millipore (USA). Matrix modifiers for THGA-AAS (palladium nitrate 2 g/L, magnesium nitrate 10 g/L, and di-ammonium hydrogen phosphate 25 mg/L) were from CARLO ERBA Reagents (Italy). Metals standard solutions (Pb and Cd 1,000 mg/l in HNO3 0.5 mol/l Certipur and Hg 1,000 mg/l in HNO3 2 mol/l Certipur) were from Merck Millipore (USA), and Mussel tissue (elements) ERM certified Reference Material was from Sigma-Aldrich (UK). Samples digestion was performed in the Berghof microwave oven with PTFE vessels.

Ultrapure MilliPore water (Mili-Q Direct 8) was used in all operations. Sample analysis was performed on PinAAcle 900T PerkinElmer AAS with THGA graphite furnace and flow injection for atomic spectroscopy (FIAS) system.

Sample preparation

The analysis of metals content was performed in an accredited laboratory for food analysis at the Veterinary faculty in Sarajevo, BiH. Samples were prepared according to standards EN 13804:201326 and EN 13805:201427. In brief, an accurately weighted aliquot (0.50 ± 0.1 g) of a homogenized sample (edible part) was transferred in the PTFE vessels for microwave digestion and 6 mL of nitric acid was added. Frozen samples were thawed to room temperature before homogenization. Digestion was performed in the microwave oven by temperature-controlled program: heating to 160 °C for 5 min, holding time 5 min, ramp time 1 min to 190 °C, holding time 15 min, cooling to 100 °C for 10 min. After cooling to room temperature the content of the vessel was transferred to a volumetric flask (20 mL) and diluted with ultrapure water to the mark. This solution was used for the analysis of Pb and Cd, while it was further diluted with 3 mol/L hydrochloric acid (1:10) for determination of Hg content.

Toxic metals analysis

The content of Pb and Cd was measured by GFAAS (Graphite Furnace Atomic Absorption Spectrometry) with a mixture of matrix modifiers [NH4H2PO4 and Pd(NO3)2 for Pb; NH4H2PO4 and Mg(NO3)2 for Cd], according to the EN 14084:200328. The content of Hg was determined by FIAS technique with NaBH4 and 3 mol/L HCl according to the in-house validated method (wavelength 253,65 nm; slit 0,7 nm; cell temperature 100 °C; pump speed 120; carrier gas flow 100 ml/min). The content of metals was quantified from the calibration curve.

Quality control/quality assurance

Quality control was performed by analysis of one aliquot of reference material, as well as one laboratory reagent blank with each batch of samples. All the samples were analyzed in duplicate and metals content was presented as an average. The differences between duplicates were ≤ 6.14%. Analytical method parameters are shown in Table 3. Blank did not contain detectable concentrations of measured metals. The recovery was calculated as the percentage of the true (certified) concentration of a metal in the certified reference material recovered during the analytical procedure. The recovery values were in the range of 80–110% (Table 3), which is acceptable for the levels of the target analytes, indicating the absence of a significant analytical bias.

Table 3 Analytical method parameters.

Precautionary measures were taken to prevent possible contamination of the samples. All glassware was cleaned by soaking in 1% nitric acid overnight and rinsing with ultrapure water before use.

Risk assessment evaluation

The risk assessment was estimated based on the Target Hazard Quotient (THQ), hazard index (HI) and contribution to the Tolerable Weekly Intake or Benchmark Dose.

THQ represents the ratio of exposure level to a substance over a specified period to reference dose (RfD) of that particular substance. Thus, THQ ≥ 1 indicates potential health hazards associated with the consumption of certain food. THQ values were calculated by the formula given by U.S. EPA29:

$$ {text{THQ}} = frac{{{text{EF}} cdot {text{ED}} cdot {text{FIR}} cdot {text{C}}}}{{{text{RfD}} cdot {text{BW}} cdot {text{TA}}}} cdot mathop {10}nolimits^{ – 3} $$


where EF is the exposure frequency (365 days year−1), ED is the exposure duration equivalent to the average human lifetime (70 years)30, FIR is the fish and seafood ingestion rate (g day−1), C is the metal concentration in fish tissue (mg kg−1), RfD is the oral reference dose for contaminant (mg kg−1 day−1), BW is the average body weight (70 kg for adults), and TA is the exposure time for non-carcinogens (365 days year−1 ED). The oral reference dose for Cd, Hg, and Pb is 1 × 10–3, 1 × 10–4 and 3.5 × 10–3 mg kg−1 day−1, respectively31,32. The RfD value for methylmercury was used since in fish and seafood this metal is almost exclusively present in methylated form (90% of total Hg content)33. Since official data on dietary habits of the BiH population is not available, we used the fish and seafood ingestion rates (FIR) reported by Gicevic et al.14. They found that mean ingestion rate was 18.4, 9.8, and 36.0 g day−1 for age groups 18–40, 41–60 and ≥ 61 years, respectively and 25.4, 15.4, and 19.8 g day−1 for socio-economic status groups “low”, “medium” and “high”, respectively. The geometric mean concentrations of analysed metals in all samples (0.108 mg kg−1 for Cd, 0.077 mg kg−1 for Hg and 0.030 mg kg−1 for Pb) and maximum concentrations (0.918 mg kg−1 for Cd, 0.624 mg kg−1 for Hg and 0.278 mg kg−1 for Pb) were combined with different fish and seafood consumption rates reported for different age and socio-economic status groups in order to assess the estimated daily intake of metals.

To evaluate the potential risk of adverse health effects from a mixture of toxic metals the hazard index (HI) was calculated as the sum of THQ for each metal:

$$ {text{HI}} = mathop {{text{THQ}}}nolimits_{{{text{Cd}}}} + mathop {{text{THQ}}}nolimits_{{{text{Hg}}}} + mathop {{text{THQ}}}nolimits_{{{text{Pb}}}} $$


When HI < 1.0, it is unlikely that there will be obvious adverse effects, while HI > 10 indicates high risk and chronic or even acute effect34.

Estimated weekly intake of metals via fish and seafood was also compared to corresponding Tolerable Weekly Intake (TWI) for Cd (2.5 μg kg−1 b.w.)21 and Hg (1.3 μg kg−1 b.w.)18. Since EFSA and other food safety authorities no longer recommend the use of previously established TWI for Pb, we used two BMDL (Benchmark Dose Lower Confidence Limit) values for Pb: BMDL10 (0.63 μg/kg b.w.) and BMDL01 (1.5 μg/kg b.w.) for chronic kidney effects and cardiovascular effects35. The contribution to the TWI (%TWI) or BMDL (%BMDL) was calculated for mixed seafood consumption using formula:

$$ {text{% TWI or % BMDL}} = frac{{{text{EWI}}}}{{{text{TWI}} cdot {text{BW}}}} cdot {100} $$


where EWI is estimated weekly intake of a metal (µg week−1), calculated as a product of the geometric mean concentration of each metal (µg g−1) and weekly fish and seafood consumption (g).

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