Human Health Risk Assessment of Heavy Metals in the Black Sea : Evaluating Mussels

The levels of Fe, Zn, Mn, Cu, Pb, Cd and Hg in the edible tissues of Mytilus galloprovincialis Lamarck, 1819 (Mediterranean mussel) picked up from the Black Sea coasts of Turkey have been determined by Inductively Coupled Plasma – Mass Spectrometer (ICP/MS) with a view to biomonitoring metal contamination in 2015. In this work, a statistically significant difference in the amounts of all studied heavy metals analyzed was noticed amidst sampling areas namely Igneada, Sinop, Samsun and Trabzon so that appraise welfare threat for mussel consumers, utilization M. galloprovincialis as biomonitors. The outcomes of this study were contrasted with the outlines stated by the Ministry of Agriculture, Fisheries and Food (MAFF), the Turkish Food Codex and Commission Regulation (EC) for the harmless consumption restrictions of Bivalves as seafood. Moreover, former works with mussels in the Black Sea countries were reviewed and a summary of heavy metal amounts in mussels from whole the Black Sea waters were presented. In general these available measurements clearly indicated a low level heavy metal in M. galloprovincialis in coastal waters of the Black Sea. In this study the concentrations (mg metal kg-1 wet wt.) of metals ranged from 18-35 for Fe, 8-27 for Zn, 2.84.5 for Mn, 0.5-1.8 for Cu, 0.06-0.31 for Pb, 0.04-0.10 for Cd and 0.03-0.07 for Hg. Considering human health with respect to the investigated heavy metals, the estimated daily intakes (EDIs) did not exceed the permissible intakes. No chronic systemic risk was found since total hazard index (0.521) were quite below critical value 1, and the carcinogenic risk for heavy metals did not exceed the tolerable values. Although there was no health risk to consumed mussels from the Black Sea riparian countries, the amount of mussels consumed is mainly unknown in countries; thus, consumption of about 1 serving of mussels from clean coastal waters per week is enough. current World Environment Journal Website: www.cwejournal.org ISSN: 0973-4929, Vol. 13, No. (1) 2018, Pg. 15-31 cOntAct Levent Bat leventbat@gmail.com University of Sinop, Fisheries Faculty, Department of Hydrobiology, Sinop, Turkey. © 2018 The Author(s). Published by Enviro Research Publishers This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted NonCommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. To link to this article: http://dx.doi.org/10.12944/CWE.13.1.03 Article History Received: 27 November 2017 Accepted: 11 February 2018


introduction
The Marine Strategy Framework Directive (MSFD) of the European Parliament and of the Council constituted a structure for community movement in the area of marine ecological strategy.It established that European Union (EU) Member States have to define Good Environmental Status (GES), to set environmental target, to develop operative monitoring programmes and to assess every six years the environmental situation of their sea water, by 2020, using eleven qualitative descriptors 1 .Turkey has signed this agreement as well.The main goals of the MSFD are to keep safe and to take care of the marine ecosystem and to stop and to reduce inputs from pollutants.The quantity of pollutants with the inclusion of heavy metals in the marine ecosystem and their impacts necessity to be evaluated considering the impacts and threatening to the ecosystem in Article 8(1)(b)(ii) of Directive 2008/56/ EC.Pollutants in biota for people consumption do not pass over amounts based by Community acts or other germane rules (Descriptor 9; Directive 2000/60/EC) 2 .The Black Sea is a European sea now because of Bulgaria and Romania joint the European Union.
Due to its geographical position and limited water change with the rest of other oceans and seas in the World, the Black Sea has been one of the unique basins most polluted with the heavy metals.The total volume of the sea is 547.000km 3 , 87% of which is covered with oxygen-poor water 3 .Due to the excessive input of freshwater, which causes low salinity in the top waters of the Black Sea, and to the inflow of saline Mediterranean waters into the deep basin, stable salinity stratification has been occurred.In the past three decades, pollution of the Black Sea by pollutants has turn out an important issue 4 .These pollutants transport tremendous amount of chemicals particularly the heavy metals have the propensity to cumulate inside the primary food chain and ascend by courtesy of the high-rise trophic grade and consequence in unfavorable impact on the sea resources thence allow of commercial loss.Heavy metals are perceived as lasting and damaging chemicals of aquatic ecosystem in consequence of their toxicity.It is known that nonessential metals are toxic to alive biota insomuch as at low levels, whilst biologically necessary metals may happen toxic at somewhat high levels 5,6 .For example, mercury is one of the heavy metals of actual concern in pollution works onwards the emanation in Japan of Minamata disease in the 1950s.This disease was a result of eating fish and shrimps polluted by methyl mercury from the wastewaters discharged by chloralkali factories.Regional folks of Minamata, who eaten up extremely on seafood, were at hazard of exposure to methyl mercury led to intense neurological harm and died more than 900 people.Furthermore almost two million people off the region suffered welfare causes 7 .Other instance is the itai itai disease in Fugawa, Japan in 1955.It was the reaction of eating fish and bivalves that were Cd polluted from waste waters unloaded by close mining 8 .Heavy metals are of environmental concern in the Black Sea due to the presence of populations of edible mussels that are known to accumulate these elements.M. galloprovincialisis much effective at changing nominal worth supplies into great standard animal protein 9 .Mussels are used as a food source in the Black Sea countries.It was reported that mussels production in entire Black Sea coast as 4321 t (Turkey: 243 t; Bulgaria: 3391 t; Romania: 81 t; Ukraine: 605 t; Russia: 1 t) in 2015.80.6% of the total production was occurred aquaculture and 19.4% of capture 10 .Monitoring of heavy metals in mussels was achieved in the Black Sea for many decades.
The common mussel (Mytilus galloprovincialis L.) is one of the most important Mollusca on the rocky and gravel bottoms of the Black Sea.They have an important role in the recycling of nutrients and particles, using suspended materials from the water column.At the same time mussels accumulate metals in their soft tissue and they are valuable indicators of heavy metal pollution of marine ecosystems 9 .Clearly M. galloprovincialis has potential as bio-monitor species for heavy metal pollution in marine waters.Because this organism is a benthic and sessile and it is incessantly exposed to pollutants.Moreover mussels ingest particles contaminated with heavy metals while feeding, are existing year throughout, oft take place in major densities, one important in food chains, and have a broad geographical range.The purpose of the present work is to investigate the amounts of Fe, Zn, Mn, Cu, Pb, Cd and Hg in M. galloprovincialis and to check the outcomes of the current work with the utmost allowable levels of these elements recommended by European Union 11 and BAT et al., Curr.World Environ., Vol.13(1) 15-31 (2018)   Turkish Food Codex 12,13 and to compare the results with those obtained in preceding studies and to assess the potential health risk for consumers in the Black Sea coasts based on their intake.The present study also provides an overview of the studies in the Black Sea riparian countries and assesses recent levels of heavy metal pollution.

Materials and Methods
The material was collected in 2015 from mussel (M.galloprovincialis) (Figure 1) habitats by the small rocky habitats amid four sampling regions namely Igneada, Sinop, Samsun and Trabzon coast of the Black Sea (Figure 2).There has been increased population density in the coastal cities giving rise to more pollution.The rapid urbanization that has been taking place in coastal area is one of the very significant causes of the Black Sea pollution.Among the developments affecting coastal pollution in the cities, many other factors play major roles.Some of these include domestic wastes, incorrect urbanization, fishing activities, an increase in the number of tourists and inadequate disposal of wastes.In terms of industrialization and urbanization Igneada and Sinop coasts are relatively considered uncontaminated.Areas significantly affected by industrial pollution in the Black Sea coast of Turkey are Samsun and Trabzon.Especially the fertilizer industry is located in Samsun and the cement industry in Trabzon 14 .
Samples were carried right away from the stations to the Hydrobiology Laboratory of Fisheries Faculty, Sinop and next they were put apart in clean seawater in aquariums (20x20x25 cm) for 24 h to defecate the contents in alimentary canals.After removal of the bowel contents, the samples were separated with respect to their sizes and were allocated into edible tissues.The specimens were put in nylon bags in a deep freeze at -21°C up to their analysis.To acquire three homogeneous samples of each biological sample for analysis, a portion of edible muscle tissue was taken from mussel, and was washed with HNO 3 and rinsed with deionized water.The samples digested with Suprapur® HNO 3 (nitric acid) using a microwave digestion system (Milestone Systems, Start D 260) for analysis.In parallel, blanks and certified reference material samples were processed similarly to verify the accuracy and precision of the method.Heavy metal analysis in mussels was performed by accredited Çevre Food Analysis Laboratory Environmental Industrial Analysis Industry and Trade Inc.Samples were analyzed by an ICP-MS, Agilent Technologies, 7700X and application of m-AOAC 999.10 (AOAC: Association of Official Analytical Chemists; reference number TÜRKAK Test TS EN ISO IEC 17025 AB-0364-T) and CSN EN 15763 European Standard methods.It was used for qualitative and quantitative elemental analysis.Advantages of ICP-MS are extremely low detection limits, wide linear range and uncomplicated spectra.The sensitivity of the method was determined according to the detection limits established for the spectrometer, which were < 0.001 μg/L for Mn, Fe, Pb and Cd, < 0.01 Zn and Hg and < 0.0001 μg/L for Cu.
The operational conditions of ICP-MS set for the analysis of the metals are shown in Table 1.
The accuracy of the analytical method in the current work was assessed using the Standard Reference Material (SRM) 2976 (mussel tissue) from National Institute of Standards and Technology (NIST).Results of the measurements were in good agreement between the certified and the analytical values, together with the Relative Standard Deviation (RSD) percentage which always range of 10%.The certified reference values were 33 ± 2, 171.0 ± 4.9, 1.19 ± 0.18, 0.82 ± 0.16, 137 ± 13, 61.0 ± 3.6 and 4.02 ± 0.33 mg/kg for Mn, Fe, Pb, Cd, Zn, Hg and fig.1: Mytilus galloprovincialis L.
All samples were analyzed in triplicate and the results were expressed as mg kg -1 wet weight.
Bw is the body weight of an adult (kg).The estimated weekly intakes (EWI) were calculated from EDI.

Results and Discussion
The concentrations (mg metal kg -1 wet wt.) of the elements ranged from 18-35 for Fe, 8-27 for Zn, 2.8-4.5 for Mn, 0.5-1.8 for Cu, 0.06-0.31for Pb, 0.04-0.10for Cd and 0.03-0.07for Hg (Figure 3).Sinop is small city and contaminants load less than those in both Samsun and Trabzon.Relying on metal levels, Samsun demonstrated the greater levels of Fe, Cu, Cd and Hg whereas Zn and Pb levels were higher in Trabzon and Mn was maximum value in Igneada.
Present study provides the information on the accumulation of elements in the edible parts of mussels from the Turkish Black Sea coasts.In general, the measured heavy metals showed a low tendency to accumulate in the tissues.However, in this study, concentrations of the heavy metals in edible parts of mussels were compared Turkish Food Codex 12,13 , the Commission Regulation 11,15 and MAFF 16 standard values (see Table 1).The heavy metal levels in edible part of the mollusks are below the proposed limit values for human consumption.
Considering tolerable limits, outcomes in all studied areas indicate that those who consume mussels in the Black Seaseem to have no health problems.Besides, the Provisional Tolerable Weekly Intake (PTWI) amount is evaluate of the level of a metal that may be taken by people duringtime of life out perceptible risk.PTWI is set up by the Joint Food and Agricultural Organization (FAO) for the United Nations / World Health Organization (WHO) Expert Committee on Food Additives (JECFA).
The mean daily mussels' consumption in Turkey is 1 g per person 17 .However, this amount is higher in the Black Sea coastal cities of Turkey.Therefore both minimum and maximum values of Estimated Weekly Intakes (EWI) and Estimated Daily Intakes (EDI) for a 70 kg adult person on basis of the current workoutcomes are calculated and presented in Table 2.

Assessments total Hazard index (thi) of Heavy Metals in Mussels
Hazard from metals entering owing to ingestion may be defined using a THI as the rate of the estimated daily intake (EDI) mg/kg of body wt. and the reference dose (RfD mg/ kg.).The THI was computed by using the equation below: If THI > 1.0, so the EDI of a certain element overruns the RfD, pointing out that there is a possible hazard associated with that element.The EDI depends on both the metal amount and the quantity of consumption of seafood.The EDI of elements was calculated using the equation below: Where: C metal is the metal levels in mussels; W mussel represents the daily mean consumption of mussels; remarked no opposed influence to the people..0 1.5 ----0.5 Turkish Food Codex 12 1.0 1.5 20 50 0.5 The Commission Regulation 11 1.0 1.5 ----0.5 Turkish Food Codex 13 1.0 1.5 ----0.5 Heavy metals are one of the most significant contaminant of the aquatic environmental. Hey metals are higher in areas close to the shore, especially in industrial areas and where large cities are located.Monitoring of the marine coasts has resulting due to the need to keep safe living organisms and human health through food web.Pollution monitoring studies are utility road for judging the actual situation of coastal environments, for determining tendency in contaminants especially heavy metals over space and time, and for assigning potential sources of contamination to protect coming problems.More than 40 years, Mytilus spp. ha been successfully studiedas biomonitors of heavy metal contamination in marine coasts 4,18 , since these bivalves meet most of the required criteria for biomonitor species 19,20,21,22 .
In relation to other coasts of countries in the Black Sea, heavy metal levels in M. galloprovincialis in this current study were compared with other studies (Table 4).Big disparities in heavy metal levels in mussels were seen among different regions.In Russian coasts of the Black Sea Fe, Mn, Cu, Pb, Cr, As and Ni values were highest in Blue Bay.Zn and Cd values were highest in Inal Bay and Hg values were observed in Crimean coasts.When the maximum values of the metals are taken into consideration, the order of the metal values obtained in all studies conducted on the Russian coast is; Fe> Zn> Cr> Cu> Pb> Mn> As> Cd> Ni> Hg.The metal values conducted on the border between Ukraine and Romania were found as Cu> Ni> Cd> Cr> Pb.
Unfortunately there was not found available literature in Georgian coasts of the Black Sea.
In Romanian the highest values of Fe, Mn, Cu, Cd, Pb and Ni were observed on VamaVeche coasts and Hg value on the highest North Efoire coasts.When the maximum values of all metals are taken into consideration, the order of the metal values in the Romanian coast is as follows: Fe> Mn> Zn> Cd> Cu> Cr> Pb> Ni> Hg.
In the highest values of Fe, Mn, Cr and As were found in the coasts of the southwestern Bulgaria of the Black Sea with the highest values of Zn and Cd, and Gulf of Varna, Cu and Pb were highest in Cape Galata coasts.The order of the metal values obtained in the studies carried out on the Bulgarian coast is found as Zn> Fe> Cu> Mn> As> Cd> Cr> Ni> Pb.Stream, which takes up the used waters of Adapazarı and also carries the runoff of Lake Sapanca to the Sakarya and the industrial enterprises in the Adapazarı area.These creeks and streams take up the wastewater of many cities and join the Sakarya River 14 .
The highest Zn value was 1742.56 μg/g dry wt. 64, followed by 630 μg /g dry wt. 56, in the coast of Trabzon in the Turkish Black Sea.However the highest Cu was 654.13 μg /g in Artvin μg /g dry wt. 64followed by 260 μg /g in Trabzon μg/g dry wt. 56.The highest Pb value was 179.15 μg/g dry wt.(approximately 24.88 μg/g wet wt.) 64 , followed by 3.16±0.0824.88 μg/g wet wt. 59in the coast of Trabzon.
Comparisons of heavy metal studies on the coasts of the Black Sea are given in in the risk assessment (see Table 4).For this aim if heavy metal levels in M. galloprovincialis are given as dry wt., they were transformed to wet wt.dividing by 7.2 as factor and all outcomes are given on a wet weight basis as μg/g wet wt. 67.
Health risk from metals intake via diet may be estimated using a Risk Quotient (RQ) as the ratio of the calculated metal dose and the reference dose (see Table 5).
Rf. D values were developed by US EPA and Agency for Toxic Substances and Disease Registry (ATSDR) for seafood consumption as estimates of daily exposures to a contaminant that are probably without a noticeable risk of injurious effects to the general population during a lifespan of exposure.
It is concluded that estimated RQ of Cd, Pb, Hg, Fe, Zn, Mn and Cu in the M. galloprovincialis do not hazard any apparent threat to human, where the total hazard index (THI) = 0.521 were below the value of 1.

conclusions
The mean concentrations of total heavy metals in mussels from Sinop coasts were lower than those in other cities in the Turkish Black Sea coasts.Samsun shows the higher concentrations of Fe, Cu, Cd and Hg whereas Zn and Pb levels were higher in Trabzon and Mn was maximum value in Igneada.However these amounts were quite down the limit founded by EU legislation for non-essential heavy metals Hg, Cd and Pb.Thus, the mussels' consumption in the Black Sea countries diet doesn't pose a risk for population in terms of these studied heavy metals.Considering public health in adult persons with respect to the investigated heavy metals, the estimated daily intakes (EDIs) did not exceed the tolerable intakes.There was no health risk since the target hazard quotients (THQs) were far below critical value 1.Therefore, consuming M. galloprovincialis does not involve any danger to the public health in terms of studied heavy metals.
O ve r a l l c o n c l u s i o n i s t h a t t h e m u s s e l s M. galloprovincialis are appropriate bio-monitors to state change in metal contamination in whole the Black Sea coasts.

conflict of interest
We declare that we have no conflict of interest.

figure 2 :
figure 2: Mussels sampling area from the turkish Black Sea coasts

table 4 :table 4 :
comparison of heavy metal amounts in M. galloprovincialis from the Black Sea coasts analyzed with literature data.Mean concentrations, standard deviations and ranges are indicated.comparison of heavy metal amounts in M. galloprovincialis from the Black Sea coasts analyzed with literature data.Mean concentrations, standard deviations and ranges are indicated.
et al., Curr.World Environ., Vol.13(1) 15-31 (2018) table 4: comparison of heavy metal amounts in M. galloprovincialis from the Black Sea coasts analyzed with literature data.Mean concentrations, standard deviations and ranges are indicated.
BAT et al., Curr.World Environ., Vol.13(1) 15-31 (2018) table 4: comparison of heavy metal amounts in M. galloprovincialis from the Black Sea coasts analyzed with literature data.Mean concentrations, standard deviations and ranges are indicated.

table 1 : icP-MS operating conditions for the analysis Operating conditions value
Table 3 that the calculated EWIs and EDIs of heavy metals in the current work are quite down the advised PTWIs and PTDIs and

table 3 : Estimated Weekly intakes (EWi) and Estimated Daily intakes (EDi) of heavy metals in M. galloprovincialis from the turkish Black Sea coasts Metals PtWi a PtWi b
a PTWI (Provisional Tolerable Weekly Intake) in mg/week/kg body wt.b PTWI for 70 kg adult person (mg/week/70 kg body wt.) c PTDI (Permissible Tolerable Daily Intake) (mg/day/70 kg body wt.) d EWI (Estimated Weekly Intake) (mg/week/ kg body wt.) e EDI (Estimated Daily Intake) (mg/day/ kg body wt.)

table 5 : Hazard analysis for the minimum and maximum amounts of elements in M. galloprovincialis (adopted from Stankovic et al., 9 )
64ggest freshwater supplies of the Black Sea came from the north shore.River Danube, Dnieper and Dniester are the major rivers flowing into the Black Sea, Danube being the most pollutant one.Wastes from the European countries carried by the Danube and heavy metals carried by rivers flowing through Russia and Ukraine to the Black Sea have been cited as playing a very big role the increase of the metals in the Black Sea.Due both to natural causes and to the pollution deposited in it by large rivers carrying wastewaters from the industrialized countries, especially the heavy metals load of the Black Sea is significantly high14.In general, the average heavy metal amounts in the Black Sea mussels are below the acceptable values, but in some studies the maximum values are well above this value.In Turkish coasts of the Black Sea, the highest values of Fe in Rize, Zn and Pb in Trabzon, As and Ni in Giresun and Cu in Artvin were found.The order of the metal values obtained in all studies conducted on the Turkish coast of the Black Sea are listed as Fe> Zn> Cu> Mn> Pb> Ni> As> Cr> Cd> Hg.The highest values of Zn, Cu and Pb in in mussels from the Turkish Black Sea were determined by Baltas et al.,64found on the shores of Giresun, Trabzon, Artvin and Rize.The values are higher than Turkish Food Codex, the Commission Regulation and MAFF.As regards these exceed the permissible limits; yet, these organisms should not be used as food.by the fertilizer, iron and steel, paper and cellulose, and cement industries.The fertilizer industry is located in Samsun, the iron and steel industry at Eregli and Karabük, the paper and cellulose industry at Çaycuma and the cement industry in Bartın, Trabzon and Ünye.The mining operations at Murgul and Zonguldak are also responsible for pollution as is urbanization, which has contributed to pollutants in Trabzon especially.Industrial pollution dominates the household heating in Samsun, particularly due to nitrogen based fertilizers and copper factories, 15 km east of the city.The major sources of pollution of the Sakarya River are Seydi Creek, Ankara Creek, Çark bes = Risk Quotient for the best case scenario ( X min / LOC ) RQ wes = Risk Quotient for the worst case scenario ( X max / LOC ) PTDI = Permissible Tolerable Daily Intake (in μg/person/day) PTDI= (PTWIx70 kg)/(7 days) PTDI ( μg/person/day) -Cd (70), Pb (250), Hg(40), Fe (5600), Zn (7000), Mn (2000-5000),Cu (3500)