MATERIALS AND METHODS

Study design and location: A cross-sectional survey and experimental design were adopted in this study. The study area included the five states of Abia, Anambra, Ebonyi, Enugu and Imo in south-eastern Nigeria, as shown in Fig. 1. Locally consumed freshwater fish were randomly purchased from fish farmers in two major towns randomly selected in each of the five states. Marine-imported fish were also purchased at the local market in two major towns that were randomly selected in the study area.

Sampling and sample collection: A total of 26 samples that consisted of eight fish species consumed in the five states of Southeast Nigeria were collected and labelled FWFa, FWFb, FWFc, FWFd, FWFe, MWFa, MWFb, MWFc, MWFd, MWFe, MWFf, MWFg and MWFh. Two samples that represented the local breed from each of the five states and 2 samples that represented the eight imported marine species consumed in the study area were collected and are shown in Table 1.

Sample preparation: The fish samples were each washed, de-scaled (where applicable) and oven-dried at 105° C for 10 h.

Fig. 1:	Map of the study area (insert, an enlargement).
	Source: Administrative map of Nigeria

Table 1:	Type and source location of sampled fishes

A portion (2.0 g) of each of the dried homogenised samples was digested in a digestion flask with 20 mL of a mixture of 650 mL of concentrated HNO₃, 80 mL of perchloric acid (HClO₄) and 20 mL of concentrated H₂SO₄ as described in the literature¹³. The digestion flask and the mixture were heated until a clear digest was obtained. The digest was then allowed to cool and was increased to 100 mL by adding distilled water. Aliquots of the diluted solutions were analysed for heavy metals using an Atomic Absorption Spectrophotometer (AAS).

Preparation of reference solution: A series of standard metal solutions in the optimum concentration ranges were prepared. The reference solutions were prepared daily by diluting the single stock elemental solution with water that contained 1.5 mL of concentrated nitric acid/litre. A calibration blank was prepared using the entire reagent but excluding the metal stock solutions.

Sample analysis: The heavy metal analysis was conducted at Springboard Laboratory Awka, Anambra State. The heavy metal analysis was conducted using a Varian AA240 atomic absorption spectrophotometer according to the method described by the APHA¹⁴. Each sample was aspirated into the flame chamber where it was vaporised. The vaporised sample became atomised after the absorption of the AAS’s light beam. The light beam was directed through the flame into the monochromator and onto the detector. Thus, the amount of light absorbed by the atomised element in the flame was measured. Since each metal has a distinct characteristic absorption wavelength, a source lamp composed of that element was used; thus, this approach ensured the method was relatively free from spectral or radiation interference. The amount of energy of the characteristic wavelength absorbed in the flame was proportional to the concentration of the element in the sample.

Health risk assessment: In this study, the health risk estimates of the ingestion of metals from fish consumption were based on the data from heavy metal analysis and other data based on the EPA¹⁵ guidelines. The following assumptions were made:

Estimated daily dose (EDD): The exposure dose caused by the ingestion of fish was calculated using the method proposed by the Agency for Toxic Substances and Diseases Registry (ATSDR)¹⁶

where, EDD is the exposure dose (mg kg^–1 day^–1), C is the contaminant concentration (mg kg^–1), IR is the fish intake rate for Nigeria, i.e., 36600 mg kg^–1 = 36.6E-3 kg day^–1 = 36.6 g day^–1 =13.359 kg y^–1, AF is the bioavailability factor (in percentage) and represents the total amount of an ingested, inhaled, or contacted substance that actually enters the bloodstream and may possibly cause harm to a person. Typically, the bioavailability factor is assumed to be 1 (100%) for screening purposes (i.e., all of a substance to which a person is exposed is assumed to be absorbed) and EF is The exposure factor and is equal to 1. The EF is calculated by multiplying the exposure frequency by the Exposure Duration (ED) and dividing this value by the time period during which the dose is to be averaged, as shown below:

Target Hazard Quotient (THQ): The target hazard quotient (THQ) is used to quantify the amount of metal taken in through ingestion. The target hazard quotient was calculated based on the formula by Wang et al¹⁷. If the THQ or the total hazard index (THI) is below 1, no health risk is likely to occur as a result of the ingestion of fish. However, as the value of the THQ or THI increases, the level of risk associated with the ingestion of the fish also increases.

RESULTS

The analysis of the concentration of metals in the fresh and marine water fish samples consumed in Southeast Nigeria showed that chromium and mercury had the highest mean concentrations, with values of 114.337 and 3.718 mg kg^–1, respectively (Table 2). The Estimated Daily Intake (EDI) for adults was computed and is presented in Table 3. Table 4 presents the target hazard quotient of fish samples consumed in Southeast Nigeria and mercury was noted to have a THQ>1.0.

Table 2:	Elemental concentrations in fresh and marine water fish samples

Table 3:	Exposure dose caused by the daily ingestion of fresh and marine water fish samples

Table 4:	Target hazard quotient and total hazard index of fish samples

DISCUSSION

The heavy metal concentrations for all fish samples did not show a consistent pattern.

Arsenic (As): Arsenic is found as a trace element in fish and other seafood products². Some environmental protection agencies have reported that an intake of 1.0 mg day^–1 of inorganic arsenic is sufficient to induce skin lesions after a few years¹⁸. The highest mean concentration of As in the fish samples was 9.983 mg kg^–1 (dry weight) and was found in MWFg imported from Colombia. Arsenic was not detected in the freshwater fish samples. This value is higher than the 2 mg kg^–1 (dry weight) limit recommended by the European Commission as the maximum permissible limit for marinefish¹⁹. This value is also higher than the value of 4.4 mg kg^–1 which was reported in fish from the Black Sea in Turkey²⁰. However, the finding of the present study does not translate into potential health risks since the target hazard quotient is less than 1.

Cadmium (Cd): The highest concentration of Cd in marine water fish was seen in the sample MWFf (0.172 mg kg^–1) which was imported from Canada. For the freshwater fish, the highest concentration was observed in the sample FWFe (0.085 mg kg^–1) from the Imo River in, Nigeria. The concentrations of Cd in all the fish samples, however, were below the NCBP concentration of 2.1 μ g g^–1 (dry weight) which is the threshold considered to be harmful to fish and predators^21,22. Akan et al ²³ reported Cd concentrations of 0.11-1.03 μ g g^–1 in the flesh of Clarias anguillaris and Synodontis budgetti from the River Benue in Nigeria. Severe toxic symptoms can result from the ingestion of Cd and symptoms have been reported for Cd values ranging from 10-326 mg g^{–1 9}. Fatal ingestions of Cd result in shock and acute renal failure and can occur from ingestion levels exceeding 350 mg g^{–1 24}.

Chromium (Cr): Chromium is an essential trace element but has been reported as a known carcinogen if ingested at a daily dose greater than 0.5 mg kg^–1 of body weight²⁵. Chromium was observed in varying concentrations in some fish samples but not in FWFc, FWFd, FWFe, MWFa or MWFb (where Cr was below the detectable limit). The highest concentration was observed in MWFc (114.337 mg kg^–1) which was imported from Uruguay. This value was more than the values reported in earlier studies²⁰ which ranged from <0.1-0.73 mg kg^–1 dry weight. An intake of 0.03 mg kg^–1 body weight has been suggested for adults who are 70 years old or older²⁶. The results of the present study indicate a potential health hazard since the target hazard quotient was greater than 1.

Cobalt (Co): Cobalt is an essential trace element but the excessive intake of Co has been noted to lead to health complications²⁷. The highest concentration from our study was in FWFd (0.113 mg kg^–1). This concentration is similar to the values reported by Gorur et al.²⁰ and Hamidalddin and AlZahrani², with range of <0.05-0.30 mg kg^–1. The result of the current study showed that the level of cobalt in fish did not pose any health risks.

Copper (Cu): Copper is an essential trace element. It facilitates iron uptake and serves as a constituent of respiratory enzyme complexes in the human body. The highest mean concentration was observed in FWFd (0.188 mg kg^–1) from the Imo River in Nigeria. The highest mean value in the present study was below the permissible limit set by the FAO/WHO (i.e., 3 mg kg^–1 body weight)²⁰. However, mean value observed in the present study was less than the value reported in the study by Usero et al.²⁸, with range of 0.4-1.5 mg kg^–1.

Lead (Pb) : Lead is a potential carcinogen and can cause adverse health effects²⁹. In the present study, Pb was detected in all the samples. The highest mean concentration was observed in FWFd (0.783 mg kg^–1). Current study showed lead values in all samples. These values were found to be below the permissible limit of 2.0 mg kg^–1 in dry fish weight³⁰. Turkmen and Ciminli³¹ reported higher values of Pb in fish products from Turkey which ranged from 0.09-6.95 mg kg^–1 but Gorur et al.²⁰ reported lower Pb values in fish products from the Black Sea in Turkey (range: <0.001-0.06 mg kg^–1).

Manganese (Mn): Manganese is an essential trace element but its excessive intake has a negative effect on fertility, the central nervous system and embryo and foetal development³². Manganese was detected in all samples at levels below the permissible limit of 2.5 mg kg^–1. The highest mean concentration was observed in FWFc (0.595 mg kg^–1) which was from the Ugwuaji River in Nigeria. This observed value was within the range (mg kg^–1, dry weight) reported in previous studies^20,33,34.

Mercury (Hg): Acute or chronic exposure to Hg can produce adverse health effects during human developmental stages. In the present study, the highest concentration of Hg was observed in FWFb (3.718 mg kg^–1) which came from the Afikpo River in Nigeria and the minimum value from all samples was 0.79 mg kg^–1 (MWFd) which was imported from Norway. The Canadian Food Inspection Agency (CFIA) has set the Hg limit at 0.2 mg kg^–1 (dry weight) of fish²⁶. In the present study, all fish samples had mercury concentrations higher than the permissible limit of 0.2 mg kg^–1 set by the CFIA and had THQ values> 1. The consumption of these fish products has potential health risks from the perspective of possible bioaccumulation. The mercury concentration in fish products was reported to range from 0.004-0.056 mg kg^–1 dry weight in Port Klang³³.

Nickel (Ni): Nickel is an essential trace element in humans and is believed to contribute to physiological processes as a co-factor in the absorption of iron from the intestines. However, at higher levels in the human body, nickel can become toxic or act as a carcinogen³⁵. In the present study, Ni was below the detectable level.

Zinc (Zn): Zinc is an essential trace element whose deficiency results in retarded growth, loss of taste, dermatitis, alopecia, hypogonadism and decreased fertility⁹. An excessive intake of Zn can cause acute poisoning. The highest mean concentration of Zn in our study was 21.213 mg kg^–1 and was observed in MWFg which was imported from Colombia. The concentration of Zn across all samples was lower than the maximum permissible limit of 30 μ g g^{–1 36}.

CONCLUSION

The present study identified the presence of a wide range of heavy metals in fish samples consumed in Southeast Nigeria. All fish products had heavy metal concentrations below the permissible limits, except for the heavy metals of chromium and mercury. However, only mercury had a target hazard quotient>1.0; therefore, the consumption of fish products has the potential to cause adverse health impacts in humans. Further studies are required to ascertain the source of mercury contamination in the freshwater fish samples. Different regulatory agencies are urged to conduct periodic heavy metal assessments to avert possible adverse public health hazards.