Heavy metal concentration and public health risk in consuming Sardinella maderensis (Sardine), Sarotherodon melanotheron (Tilapia), and Liza falicipinis (Mullet) harvested from Bonny River, Nigeria

Contamination of the river by oil spill had put the health of the people who consume fish at risk. Five fish samples each of Liza falicipinis (Mullet), Sarotherodon melanotheron (Tilapia) and Sardinella maderensis (Sardine) (n=15) were collected monthly from the Bonny Estuary from February to July 2018. The samples were oven dried at 100°C and analyzed using Atomic Absorption Spectrophotometer (Model pm ver 2.02 Avanta) to determine the concentration of Zinc (Zn), Lead (Pb), Copper (Cu), Cadmium (Cd), Nickel (Ni), Chromium (Cr), Vanadium (V) and Mercury (Hg). The result show that there is significant difference (F7, 130 = 65.6, P < 0.0001) in heavy metal concentration in the three fish species. Zinc had the highest concentration followed by Lead, Copper, Nickel and Cadmium. There was no seasonal difference (F1, 136 = 1.58, P > 0.05), although dry season had higher concentration. Sardine had the highest concentration of heavy metal followed by tilapia and mullet. Result of EDI indicates higher intake during the dry season (that is February). Highest EDI was recorded in tilapia (5673.1 ± 5632.5 mg/kg). Similarly, highest Cd, Cu, Cr, Ni and Zn were recorded in sardine whereas Pb was highest in tilapia. Mullet had highest daily intake for February and March. Sardine had the highest THQ for Cd, Cu, Cr, Ni and Zn, whereas tilapia had high values in Cd and Ni while mullet had high values in Pb. The implication of this result is that dry season is not the best time to consume these three fish species.


INTRODUCTION
Studies have shown that despite the health benefit of deriving protein from consuming fish, human health can be compromised if heavy metals such as Lead, Cadmium, Arsenic, and Mercury are present (Bosch et al., 2016). Furthermore, concentration of heavy metals varied significantly between species and organs, and mercury was found to accumulate more in the muscle (Farkas et al., 2000;Cai et al., 2019). Lead and Nickel were shown to cause the greatest risk to health, while tilapia species had no risk of consumption (Taweel et al., 2013). Heavy metal pollution in the aquatic ecosystem has long been recognized as a serious environmental *Corresponding author. E-mail: aroloyen@yahoo.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License concern. Anthropogenic activities such as oil exploration and exploitation, discharge of municipal and industrial wastes into the aquatic environment are major sources of pollution. These heavy metals may accumulate in the tissues of marine organisms and are transferred to humans through consumption (Ali and Khan, 2019) Some metals (copper, iron and zinc) at low concentrations are of nutritional importance and are essential for healthy living; in contrast, metals such as Mercury, Cadmuim and Lead have not been known to play beneficial role in human metabolism and are considered as chemical carcinogens even at very low levels of exposure (Luch, 2005). Generally, heavy metals which are present in the environment in minute concentrations become part of various food chains through biomagnifications; leading to concentration increase to a level that may prove toxic to both humans and other living organisms. Dietary is the main route of human exposure to heavy metals except for occupational exposures (Moslen and Miebaka, 2016).
Fish are excellent source of protein and its lowcalorific value makes it a healthier alternative to red meats or poultry; it is also rich in omega-3 polyunsaturated fat which has been known to reduce cholesterol levels in man (Kaushik and Seiliez, 2010 ).
The Bonny Estuary in the Niger Delta region empties directly into the Atlantic Ocean at Bonny Island. Bonny is the host to the Liquified Natural Gas (LNG) Company, which utilizes the gas flared during crude oil exploration to produce methane, a cooking gas. In this operation crude oil is spilled into the river and contaminates the drinking water source leading to proliferation of water borne diseases and food poisoning. Gas and exhausts flared into the atmosphere cause soot, which lead to respiratory diseases such asthma, bronchitis, cough, and catarrh. Large scale oil and gas production, transportation, industrialization are the order of the day in this location leading to cases of oil spills, gas flares, industrial discharges, agricultural runoff and domestic effluents (Babatunde et al., 2019;Numbere, 2018). Despite these activities, artisanal fisheries in the estuary largely support the livelihood of its inhabitants who have little or no knowledge of the extent of pollution in the estuary.
For riverine communities such as the inhabitants of the study area, seafood is being served at almost every meal. Fish is consumed either freshly prepared or smoked. Shellfish, especially Tympanotonus fuscatus are used largely as a condiment in most meals eaten in the Niger Delta (Gomna and Rana, 2007). Therefore, it is expedient to determine the concentrations of heavy metals in this seafood in order to ascertain the potential health risk consumers may face.
Heavy metals are non-biodegradable, inorganic chemicals that tend to accumulate overtime in the bodies of organisms due to inability to metabolize them. Thus, it is important to determine the heavy metal concentrations in seafood in order to evaluate the impact of these metals on the health of the consumers. In Nigeria, most studies on metal accumulation in edible biota have failed to consider the risk associated with exposure through the food chain. However, few works have been reported on the health risk associated with the consumption of certain seafood and vegetables in Nigeria. Fernandez et al. (1999) reported that there is a cancer risk associated with eating contaminated fish. Similarly, Moslen and Miebaka (2017) reported on negligible health risk associated with consumption of tilapia (Sarotherodon melanotheron) harvested from Azuabie creek, upper Bonny estuary. There are limited data on health risk assessment with regards to heavy metal level and consumption of fish species Liza falicipinis, Sarotherodon melanotheron and Sardinella maderensis harvested from the Bonny Estuary (River). The objectives of the study therefore, are: (1) To determine the heavy metal concentrations in three fish species; (2) To compare the heavy metal concentration in dry (February-March) and wet (April-July) seasons; (3) To determine the month with the highest heavy metal concentration, and (4) To determine the health risk assessment in consuming the fishes.

Study area
The Niger Delta of Nigeria is situated in the Gulf of Guinea between latitudes 3° and 5° N and longitudes 5° and 8° E. It is the largest delta in Africa with an area of about 70,000 km 2 ; it is rich in biodiversity and maintains the largest drainage system into the Atlantic Ocean in West Africa (Akpomuvie, 2011). The sampling site is Bonny River located in the Niger Delta area of Nigeria. It is a 127 km long tidal estuary and lies on the eastern flank of the Niger Delta between latitudes 4.25° and 4.50° N and longitudes 7.00° and 7.15° E ( Figure 1). It is a place of residence and fishing activities by local inhabitants.

Research design
Five fish samples each of Liza falicipinis (Mullet), Sarotherodon melanotheron (Tilapia) and Sardinella maderensis (Sardine) (n=15) were collected monthly for six months, that is February 2018 to July, 2018 from artisanal fishermen along the Bonny Estuary Samples were preserved in an ice box and transported immediately to the laboratory for analysis ( Figure 2).

Sample collection and preparation
Each fish sample was placed on a petri dish and properly washed with distilled water and labeled. The samples were oven dried at 100°C to ensure that all the water content were removed to get a constant weight. Each sample was homogenized using clean mortar and pestle. Thereafter, 3.0 g of each sample were weighed. 20 ml of aqua regia prepared by mixing 30 ml of 10% hydrochloric acid and 70 ml of 10% Trioxonitrate (V) acid (HNO 3 ) were added and digested in a water bath. 10 ml of de-ionized water was added to the content of each flask and was allowed to cool. The content of each flask was later filtered with Whatman 0.05 μm filter paper.
The salinity, temperature, conductivity, TDS and pH of the four sample sites along the Bonny Estuary was taken in-situ whereas for the DO, the water sample was collected using amber colored bottle   and was determined by the Modified Azide or Winkler's method (APHA, 1985). To a 70 ml BOD bottle filled with 0.5 ml manganese sulphate (Winkler I) and 0.5 ml Alkali-iodide-azide reagent (Winkler II) were added. After about 10 min, 0.5 ml concentration H 2 SO 4 was added, re-stoppered and mixed for complete dissolution of precipitate. A 50 ml portion was placed in an Erlenmeyer flask, five drops of freshly prepared starch solution added and titrated with 0.025N Na 2 S 2 O 3 (Sodium thiosulphate) solution. The titration was continued to the first disappearance of the blue color.
DO mg/L was calculated using: Where V is the volume in ml and N is the normality of sodium thiosulphate solution used in titration.

Laboratory analysis
Heavy metals determination in sample was then performed using Atomic Absorption Spectrophotometer (Model pm ver 2.02 Avanta). The analysis of the standard, reagent blanks and replicates were run in the same way and concentrations determined using standard solutions prepared in the same acid matrix. For each of the metals under study, atomic absorption spectrophotometer (model pm ver 2.02 Avanta) was calibrated using standard of the metals. Metals of interest are: Zinc (Zn), Lead (Pb), Copper (Cu), Cadmium (Cd), Nickel (Ni), Chromium (Cr), Vanadium (V) and Mercury (Hg).

Human health risk assessment
The values of fish metal accumulation and constants were used to calculate the estimateddaily intake of metals (EDI), chronic daily intake for the metals (CDI), target hazard quotients (THQ) and Hazard quotient (HI). The calculations were based on the standard assumption for integrate USEPA risk analysis, FDA, and USDOE standard.

Estimated daily intake (EDI) of heavy metals
EDI is the estimated daily intake of metal concentration in the fish expressed in mg kg -1 . Calculations were made based on the standard assumption from USEPA-IRIS (2016), considering the Ingestion rate for fish to be 0.10 kg/fish person/day and an adult average body weight of 70 kg as reported by Moslen and Miebaka (2017).

Target hazard quotient (THQ)
The non-carcinogenic fish ingestion equation is as follows: RfD is an estimate of a daily oral exposure for human population, which is a non-carcinogenic effect during a lifetime; generally used in EPA's non-cancer health assessment (Uche et al., 2017; Table  1).

Target hazard quotient
The human health risks from consumption of fish by the populace were assessed using the target hazard quotient (THQ). It is defined as the ratio between estimated daily intake (EDI) and reference oral dose (RfD). THQ is used to express the risk of non-carcinogenic effects (Yu-Jun et al., 2011). If the ratio is equal to or greater than 1, an exposed population experiences health risks.

THQ =
Where: EDI= Estimated daily intake for the toxicant (mg/kg-day); and RfD =chronic reference dose for the toxicant (mg/kg-day).
Although the THQ-based assessment method does not provide a quantitative estimate for the probability of an exposed population experiencing a reverse health effect, it does, however, provide an indication of risk level due to exposure to pollutants (Guerra et al., 2012;Mahmoud and Abdel-Mohsein, 2015). The THQ has been recognized as a useful parameter for evaluating risk associated with consumption of metal-contaminated fish (USEPA, 2011a).

Hazard index (HI)
Hazard Index (HI) is used to evaluate the potential risk to humans

Statistical analysis
An analysis of variance (ANOVA) was conducted since there were multiple samples (n=15) and monthly replications to determine whether there was a significant difference in heavy metal concentration for the three fishes (tilapia, mullet and sardine). The data was first log transformed to ensure that they were normal and the variances were equal. Bar graphs were then used to illustrate the significance and difference in concentration in the fish species. All analyses were done in R (R Development Core Team, 2014). Tukey HSD test was then done to determine where the significance lies (Logan, 2010).

Physico-chemistry of study area
The temperature of the study area range between 27 and 30˚C. The dissolved oxygen ranges between 2 and 6 mg/L. The total dissolved solid (TDS) ranges between 11.56 and 15.86mg/L and the conductivity ranges between 20.98 and 27.32 µohm/cm (Table 2).

Heavy metal concentration in three fish species
The result (Figure 3) (Figure 3).

Heavy metal concentration in three fish species in dry and wet seasons
There was no significant difference in heavy metal concentration in the three fish species in wet and dry season (F 1 , 136 = 1.58, P >0.05; Figure 4). However, sardine had the highest concentration of heavy metal followed by tilapia and mullet in both dry and wet seasons. The order of heavy metal concentration in fish sample is sardine>tilapia>mullet.

Monthly heavy metal concentration in three fish species
Heavy metal concentration in sardine was the highest for each month apart from March, where tilapia was slightly higher than other species. However, the ANOVA result ( Figure 5) indicates that there is no significant difference (F 5 , 132 = 0.4, P > 0.05) in heavy metal concentration across months. Table 3 indicates the calculation of the cumulative intake of heavy metal by fish species. The EDI result indicates that there was higher intake of heavy metal by fish  species during the dry season than during the wet season (Table 3). Highest EDI was recorded in tilapia (5673.1 ± 5632.5 mg/kg). Similarly, Table 4 shows the estimated daily intake of individual heavy metals (that is, Cd, Cu, Cr, Pb, Hg, Ni, V and Zn) by the three fish species sampled at the Bonny River. Furthermore, highest Cd, Cu, Cr, Ni and Zn were recorded in sardine (Table 3) whereas Pb was highest in tilapia. The order of  daily intake of heavy metal in fish samples is sardine>tilapia>mullet.

Target hazard quotient (THQ)
Higher THQ was obtained during the dry season (Table  5). Mullet had highest daily intake for February and March. Sardine has the highest THQ for Cd, Cu, Cr, Ni and Zn, whereas tilapia had high values in Cd and Ni while mullet had high values in Pb (Table 6). The order of hazard for fish samples is sardine>tilapia>mullet

DISCUSSION
Aquatic organisms such as fishes are susceptible to heavy metal contamination through body contact or filter feeding. These heavy metals bioaccumulate in their tissues and organs (Farkas et al., 2000;Cai et al., 2019) and are later transferred to humans who consume them thereby posing as danger to their health (Taweel et al., 2013). The disposal of waste into water bodies, and spillages from oil and gas explorations are some of the causes of increased pollution of rivers. The liquefied natural gas (LNG) situated in Bonny is being supplied by  Refinery. Part of this pipelines run offshore through the swamps, and serve as entry points of pollutants into the aquatic environment. High zinc concentration has been reported in studies of fish (Eremasi et al., 2018). Zinc concentration is often high in the waters and coastal zones of the Niger Delta ( Figure 3) because of its multiple entry points such as through the earth crust along mangrove forest and as corrosion products from metallic substances used as alloys for pipelines. However, Zinc at low concentration is of nutritional importance. Cadmium on the other hand, is toxic and carcinogenic at low concentration (Duruibe et al., 2007). Cadmium when released to the environment can lead to the outright death of fishes or can bioaccumulate in their bodies, and is later transferred to humans who consume them. Sardine species were found to have higher concentration of heavy metal probably because of their scale less body which leads to more diffusion of metallic substances from the aquatic environment into their bodies. Nevertheless, the three most harmful metals of Vanadium, Mercury and Chromium were not found in the bodies of fishes sampled. These metals are very toxic, that is, they have carcinogenic and teratogenic effect on humans (Saxena et al., 2020). Mercury, which is one of the most harmful metals, can cause genetic abnormalities in new born babies if consumed by pregnant mothers. It can also cause brain damage and mental disorders if consumed via sea food (e.g. fishes, periwinkles, crabs etc). This study shows that seasons have effect on heavy metal concentration in aquatic media and aquatic organisms. This is in line with studies of Farkas et al. (2000) that revealed higher Cd concentration in eel fish in autumn season. Autumn weather in temperate region is similar to dry season in the Niger Delta area of Nigeria.
There is a concentrating effect of dryness ( Figure  4), which tends to increase heavy metal concentrations in waters and within organisms that reside in them. The result shows that the consumption of fishes during the dry season is riskier than during the wet season. Although statistically, the result is not significant, but physiological effect is not controlled by statistical significance, because minute concentration can have magnified effects in the body. In the same vein, although there is no significant difference in terms of season, dry season was shown to have slightly higher concentration (Figure 4). Dryness of the environment causes high evaporation from the river surface leading to increase in heavy metal concentration. February, which is a dry season month, had the highest concentration of heavy metal in sardine ( Figure 5). All the results for heavy metal and health risk analysis had revealed that sardine should be thoroughly monitored because of its ability to accumulate high heavy metal concentration before being sold to the public for consumption. Previous studies had indicated that dry season has a higher intake (EDI) of heavy metals by fishes (Miebaka and Moslen, 2016). This thus predispose the fishes to bioaccumulating more heavy metals. This makes the fishes to be risky to human health if consumed.
The result indicate that consumption of the three fishes especially Mullet during the dry season is also hazardous to human health, However, sardine is more hazardous because of its ability to bioaccumulate high amount of heavy metals. Furthermore, sardine has the highest health risk of the three fishes sampled whereas February and March are the riskiest months to consume sardine, which are dry season and the ending of the dry season months, respectively.

CONCLUSION AND RECOMMENDATION
The implication of this result is that the total hazard quotient even though not too high may keep increasing to a harmful level that will be detrimental to the health of the public. This is because stochastic environmental change can push the concentration limit of the heavy metals in the fishes from a non-harmful to a harmful level. Therefore, residents living around the Bonny River should be careful in the way they consume sea food so as to prevent the accumulation of heavy metals in their bodies which may be too risky for their health. Furthermore, there should be constant monitoring of all sea food harvested from the Bonny Estuary. This will help to reduce heavy metal poisoning.