Content of Trace Elements and Human Health Risk Assessment via Consumption of Commercially Important Fishes from Montenegrin Coast.

Muscle tissues of Mullus barbatus and Merluccius merluccius were analyzed for the presence of selected trace elements (As, Hg, Cd, and Pb) to determine the value of the daily intake of trace elements from fish consumption and to assess the risk to human health. The mean concentrations of As in the muscle tissue of M. barbatus and M. merluccius for the entire period were 19.689 mg/kg wet weight (ww) and 8.356 mg/kg ww, Hg 0.497 mg/kg ww and 0.153 mg/kg ww, and Pb 0.031 mg/kg ww and 0.025 mg/kg ww, respectively. The concentrations of Cd in all fish sampled were below the detection limit (<0.02 mg/kg ww). The evaluation of the potential health risk assessments based on the target hazard quotient (THQ) and estimated daily intake (EDI) indicated that the intake of As in both fish species and Hg for M. barbatus could pose an appreciable risk to human health. The calculated hazard index (HI) was higher than 1 for both fish species. The continuous monitoring of trace elements' concentrations in fish is strongly recommended, as the results demonstrate potential health risks due to the presence of As and Hg.

Microplastics in mussels from the Boka Kotorska Bay (Adriatic Sea) and impact on human health.

This study evaluated the microplastic abundance, shape, color, size and chemical composition of microplastic in mussels and estimated human exposure to microplastic through consumption of mussels collected from Boka Kotorska Bay (Adriatic coast of Montenegro). Microplastic was found in 53.3% of the studied mussels, with an average microplastic abundance of 2.53 ± 1.1 items/individual. Most of the ingested microplastic were fibers (63.7%), which were blue in color. FT-IR revealed that 98% of the examined particles were plastic, with seven polymers identified, of which polyethylene, polypropylene, and polyethylene terephthalate were the most abumdant polymers in mussels. Three of the polymers detected in mussels (polyamide, polyvinyl chloride and polystyrene) are classified as hazardous by the European Chemical Agency with warning or danger signals. With one serving of mussels, consumers would ingest 22.7 microplastic particles, while the annual dietary intake of microplastic via consumption of mussels was estimated at 99 MP/year.

Single and combined potential of polystyrene microparticles and fluoranthene in the induction of DNA damage in haemocytes of Mediterranean mussel (Mytilus galloprovincialis).

In this study, the possible 'vector effect' within the exposure of Mediterranean mussels (Mytilus galloprovincialis) to polystyrene microplastics with adsorbed fluoranthene was investigated by applying the multibiomarker approach. The major focus was placed on genotoxicological endpoints as to our knowledge there are no literature data on the genotoxicity of polystyrene microparticles alone or with adsorbed fluoranthene in the selected experimental organisms. DNA damage was assessed in haemocytes by comet assay and micronucleus test. For the assessment of neurotoxicity, acetylcholinesterase activity was measured in gills. Glutathione S-transferase was assessed in gills and hepatopancreas since these enzymes are induced for biotransformation and excretion of lipophilic compounds such as hydrocarbons. Finally, differences in physiological response within the exposure to polystyrene particles, fluoranthene, or particles with adsorbed fluoranthene were assessed by the variation of heart rate patterns studied by the noninvasive laser fibre-optic method. The uniform response of individual biomarkers within the exposure groups was not recorded. There was no clear pattern in variation of acetylcholinesterase or glutathione S-transferase activity which could be attributed to the treatment. Exposure to polystyrene increased DNA damage which was detected by the comet assay but was not confirmed by micronucleus formation. Data of genotoxicity assays indicated differential responses among the groups exposed to fluoranthene alone and fluoranthene adsorbed to polystyrene. Change in the heart rate patterns within the studied groups supports the concept of the Trojan horse effect within the exposure to polystyrene particles with adsorbed fluoranthene.

Evaluation of Physiological State of Pen Shell Pinna nobilis (Linnaeus, 1758) by a Non-Invasive Heart Rate Recording under Short-Term Hyposalinity Test.

A non-invasive laser fiber-optic method based on infrared sensors for heart rate (Hr) recording was applied to assess the physiological condition of Pinna nobilis. During 2017, the specimens of P. nobilis were sampled at three sites within the Boka Kotorska Bay, Montenegro and used for ex situ experiments with short-term reduction/restoration of ambient salinity to evaluate their physiological adaptive capacity based on heart rate recovery time (Trec). Mean Trec for specimens from Sv. Nedjelja (reference site), Dobrota and Sv. Stasije were 72 ± 3, 91 ± 7 and 117 ± 15 min, while the coefficients of variation (CV) were 0.12, 0.13 and 0.17, respectively. Resting heart rate (Hrrest) and Trec showed statistically significant differences between the groups of mussels from Dobrota and Sv. Stasije in comparison to the reference site. Statistically significant correlations were observed between Trec and shell length/width, which was not the case in comparison between Hrrest and shell length/width. The lower adaptive capacity within the P. nobilis specimens from Dobrota and Sv. Stasije in comparison to the reference site could occur due to stress induced by deterioration of environmental conditions, which could have led to impairment of the physiological state of the mussels evaluated by Hr. All the specimens of P. nobilis survived the experimental treatments; afterwards, they were successfully transplanted at the Dobrota site. The experimental unit with sensor technology applied in this study can provide Hr recording in real time and could have an application in monitoring the physiological/health state of P. nobilis individuals maintained in aquaria.

Microplastics in fish and sediments from the Montenegrin coast (Adriatic Sea): Similarities in accumulation.

The accumulation of microplastics (MPs) in the biotic and abiotic components of the marine environment poses a major threat to marine ecosystems worldwide. The objective of this study was to document, for the first time, differences in MP accumulation in the gastrointestinal tract of two commercially important fish species and to evaluate the possible correlation between MP accumulation in the biotic (fish) and abiotic (sediment) components of the marine environment of the Montenegrin coast (Adriatic Sea). Samples were collected from two areas of the Montenegrin coast, Boka Kotorska Bay and the coastal part of the open sea. The frequency of MP ingestion was 58.6 % for Mullus barbatus and 54 % for Merluccius merluccius, while the average number of ingested MPs was 2.9 ± 0.5 and 3.2 ± 1.0 items/individual, respectively. Average MP abundance in surface sediments from Boka Kotorska Bay and the coastal part of the open sea was 315 ± 45 and 435 ± 258 MPs/kg of dry sediment, respectively. Most MPs identified were filaments, followed by fragments and films, while the most abundant polymers found in fish and sediments samples were polypropylene and polyethylene. The present results indicate that MP pollution in the study area is reflected in the accumulation of MPs in the biotic (fish) and abiotic (sediment) components of the marine environment. Measures need to be taken to reduce the input of plastics/MPs into the marine environment.

Distribution and characterization of microplastics in marine sediments from the Montenegrin coast.

Purpose: Plastic pollution in the world has led to an abundance of microplastics (MPs) and has been identified as a potential factor that can lead to serious environmental problems, especially in oceans and seas. Information on the current status of MPs pollution along the Montenegrin coast is insufficiently investigated. This study monitors the abundance, distribution, and sources of MPs, and identifies present polymers in the surface sediment of the Montenegrin coast, as well as comparison with previous research. Materials and methods: Ten sampling sites along the Montenegrin coast were selected to collect surface sediment samples. The upper layer of sediment (0-5 cm) was collected by a Petite ponar grab. The samples were dried, and density separation was performed using a NaCl solution. The abundance and morphological characteristics of MPs were determined using an optical microscope (DP-Soft software), while FT-IR analysis was done to identify the polymer type. Results and discussion: Microplastics were identified in all sediment samples with an average abundance of 307 ± 133 (SD) MPs/kg in dry sediment. The highest abundance of MPs was found in locations in the vicinity of highly populated areas, near wastewater discharges, and areas with high fishing and tourist activities. The most dominant shape types of MPs in all samples were filaments and fragments. The most common colors of MPs were blue and red, while the dominant MPs sizes were 0.1-0.5 mm and 0.5-1.0 mm. Of the eight identified polymers, PP, PE, and PET were the most common. Conclusion: This study reveals MPs characteristics (abundance, distribution, shape type, colors, size, polymers type) in surface sediment along the Montenegrin coast, as well as the most significant sources of MPs pollution, and provides important data for further research on MPs to identify the effects of MPs pollution on the quality, health, and functionality of the marine environment.

Oceanographic characteristics of the Adriatic Sea - Support to secondary HAOP spread through natural dispersal.

Vessels, specifically ballast water and hull fouling, are a major vector for the introduction of non-indigenous species (NIS) in European seas. The Mediterranean is one of the world's marine regions where their invasion is heaviest. The shallow Adriatic basin is a highly sensitive area that is already experiencing its consequences. The secondary spread of NIS over a wider area through natural dispersion is a complex process that depends on a wide range of oceanographic factors. This work analysed the dataset of the BALMAS project, in whose framework twelve ports in the Adriatic Sea were subjected to a Port Baseline Survey (PBS), to estimate the natural spread of NIS organisms from their port of arrival to the wider Adriatic basin. Its findings indicate that the prevailing water circulation patterns facilitate the natural dispersal of harmful aquatic organisms and pathogens (HAOP).

The impacts of seawater physicochemical parameters and sediment metal contents on trace metal concentrations in mussels-a chemometric approach.

The concentrations of Al, Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Ni, Pb, Sr, Zn, and Hg were studied in Mytilus galloprovincialis collected from the coastal area of Montenegro. The impact of seawater temperature, salinity, dissolved oxygen, total organic carbon (TOC), and metal content in sediment samples on the metal contents in mussels collected from three locations in four different seasons was analyzed by a Pearson correlation coefficient (r), principal component analysis (PCA), and cluster analysis (CA). These analyses were used to discriminate groups of samples, elements, and seawater parameters, according to similarity of samples chemical composition in different seasons, as well as the impact of seawater parameters and surface sediment composition on the mussels' element concentrations. Synergistic interactions occurred between seawater TOC, Fe, and Al concentrations in mussels. Compared with other studies, which are usually performed under constant laboratory conditions where mussels undergo only one stress at a time, this study was performed in nature. The analyses showed the importance of considering simultaneously acting environmental parameters that make determining of separate impacts of each factor selected very difficult and complex.

Determination of trace metals in Mytilus galloprovincialis along the Boka Kotorska Bay, Montenegrin coast.

The concentrations of six trace metals, cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb), zinc (Zn) and mercury (Hg) were determined in the soft tissues of the mussel Mytilus galloprovincialis and bottom sediments collected from the coastal area of Boka Kotorska Bay (Montenegrin coast). The mussels and sediment samples were collected at three sites along the Boka Kotorska Bay from the autumn of 2014 to the winter of 2016. The concentrations (mg/kg dw) of these metals in the mussel samples ranged from 1.01 to 2.87 for Cd, 6.08 to 13.2 for Cu, 1.37 to 3.55 for Pb, 85.6 to 241 for Zn, 0.72 to 4.05 for Cr and 0.098 to 0.269 for Hg. The concentrations (mg/kg dw) of these metals in the sediment samples ranged from 0.16 to 0.39 for Cd, 4.32 to 48.2 for Cu, 7.02 to 49.8 for Pb, 22.0 to 158 for Zn, 93.1 to 317 for Cr and 0.035 to 0.497 for Hg. The metal content in the mussel samples and the metal content in the sediment collected from three locations over four different seasons were analyzed using the Pearson correlation coefficient (r) and cluster analysis (CA). A correlation between the metal levels found in the mussel soft tissues with those found in the sediments, for both 2014 and 2015, were established. The metal pollution indexes (MPI) for the sediment and mussels were compared, indicating that the most polluted sediment was at the location Institute of Marine Biology Kotor (IBMK) and the most polluted mussels were sampled from the Zanjice location.

Human exposure to trace elements via consumption of mussels Mytilus galloprovincialis from Boka Kotorska Bay, Montenegro.

In order to assess human health risks via consumption of potentially toxic mussels, the concentrations of Al, Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Ni, Pb, Sr, Zn and Hg were studied in Mytilus galloprovincialis collected from the coastal area of Montenegro. By two approaches for the human health risk assessment (HHRA), considering oral reference doses by the United States Environmental Protection Agency (US EPA) and provisional tolerable intakes by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), it has been revealed that despite the fact that trace element levels do not exceed the defined limits for mussels, they can be limiting factors for the mussel consumption. Specifically, it was noticed that the levels of Co, Pb, Cd and Li could be the limiting factors for the consumption of mussels from this coastal area. Al and Li data obtained in this study are especially important since these two elements have not been previously studied in M. galloprovincialis. Furthermore, taking into account the significant differences in concentrations of elements in different seasons, the study confirmed the starting assumption that in the calculations for the HHRA the average concentrations of elements in samples taken in different seasons during a longer period should be used.

Comparative assessment of cardiac activity and DNA damage in haemocytes of the Mediterranean mussel Mytilus galloprovincialis in exposure to tributyltin chloride.

This study gives an insight in sensitivity of heart rate (Hr) of Mytilus galloprovincialis as a physiological biomarker. Impact of tributyltin chloride (TBT-Cl) on Hr was studied in parallel with evaluation of mutagenic, genotoxic and cytotoxic potential of TBT-Cl (10, 100 and 1000µg/L) within 96h treatment in static conditions. Mutagenic potential was assessed by SOS/umuC assay while genotoxicity was assessed in haemocytes of M. galloprovincialis by using the comet assay and the micronucleus test. Benzo(a)pyrene (B(a)P) was used as a positive control. Hr variations detected in TBT-Cl treatments can be linked to data obtained in the genotoxicological assays indicating that Hr can be considered and used as a reliable physiological biomarker for detecting the presence of organotin compounds. However despite the observed genotoxic potential of B(a)P, a noteworthy Hr response was not observed which further questions the potential of Hr in the detection of different types of pollutants.

Can the origin of some metals in the seagrass Posidonia oceanica be determined by the indexes of metals pollutions?

To assess metal pollution, Fe, Mn, Cu, Zn, Pb, Ni, Co, As, Cd, and Hg contents in samples of the seagrass Posidonia oceanica and surface sediment, collected at eight locations along the Montenegrin coast, were determined. The metal pollution index (MPI) and metal enrichment factor (EF) were then calculated. MPI and EF were lower in sediment than in P. oceanica at the same locations. This was very evident for EF values of Hg and Cd. Based on the Pearson's correlations and EF values, it was possible to conclude that the last two metals' content in the seagrass did not originate from the crustal sources or natural weathering processes.

Trace metal concentrations in Mediterranean blue mussel and surface sediments and evaluation of the mussels quality and possible risks of high human consumption.

The concentrations of trace metals (Fe, Zn, Mn, Ni, Cu, Co and Cr) were determined for the first time in Mediterranean blue mussels (Mytilus galloprovincialis) and surface sediments. The mussel and sediment samples were collected from five sites on the Montenegrin coastal area (southeastern Adriatic Sea) in the fall of 2005 and 2006. The collected samples were digested with a microwave digestion system. The maximum Fe, Zn, Mn, Ni, Cu, Co and Cr mean concentrations in the mussel samples were 603.0, 345.0, 85.0, 18.9, 17.2, 9.1 and 4.2mg/kg of sample dry weight, respectively. The maximum Fe, Mn, Cr, Ni, Zn, Cu and Co concentrations in the sediment samples were 40867, 943.0, 382.0, 336.0, 67.2, 24.8 and 16.9mg/kg of sample dry weight, respectively. A correlation between the metal levels found in the mussel soft tissues with those found in sediments, for both 2005 and 2006, could be established, except for the levels of Zn observed.