Population Genetic Study on the European Flounder (Platichthys flesus) from the Southern Baltic Sea Using SNPs and Microsatellite Markers.

The European flounder (Platichthys flesus), which is closely related to the recently discovered Baltic flounder (Platichthys solemdali), is currently the third most commercially fished species in the Baltic Sea. According to the available data from the Polish Fisheries Monitoring Center and fishermen's observations, the body condition indices of the species in the Baltic Sea have declined in recent years. The aim of the present study was to obtain information on the current patterns of genetic variability and the population structure of the European flounder and to verify whether the Baltic flounder is present in the southern Baltic Sea. Moreover, we aimed to verify whether the observed decline in the body condition indices of the species in the Baltic Sea might be associated with adaptive alterations in its gene pool due to increased fishing pressure. For this purpose, 190 fish were collected from four locations along the central coastline of Poland, i.e., Mechelinki, Wladyslawowo, the Vistula Lagoon in 2018, and the Slupsk Bank in 2020. The fish were morphologically analyzed and then genetically screened by the application of nineteen microsatellite DNA and two diagnostic SNP markers. The examined European flounder specimens displayed a high level of genetic diversity (PIC = 0.832-0.903, I = 2.579-2.768). A lack of significant genetic differentiation (Fst = 0.004, p > 0.05) was observed in all the examined fish, indicating that the European flounder in the sampled area constitutes a single genetic cluster. A significant deficiency in heterozygotes (Fis = 0.093, p < 0.05) and overall deviations from Hardy-Weinberg expectations (H-WE) were only detected in fish sampled from the Slupsk Bank. The estimated effective population size (Ne) among the sampled fish groups varied from 712 (Slupsk Bank) to 10,115 (Wladyslawowo and Mechelinki). However, the recorded values of the Garza-Williamson indicator (M = 0.574-0.600) and the lack of significant (p > 0.05) differences in Heq > He under the SMM model did not support the species' population size changes in the past. The applied SNP markers did not detect the presence of the Baltic flounder among the fish sampled from the studied area. The analysis of an association between biological traits and patterns of genetic diversity did not detect any signs of directional selection or density-dependent adaptive changes in the gene pool of the examined fish that might be caused by increased fishing pressure.

Levels, time trends, and distribution of dioxins and polychlorinated biphenyls in fishes from the Baltic Sea.

Fish absorb dioxins from the environment through water and contact with sediments but the main source is food. These contaminants also enter the human body with food, including through the consumption of fish. This study presents the dioxin and polychlorinated biphenyl (PCB) concentrations in 145 samples of four fish species (herring, sprat, sea trout, salmon) caught in the southern Baltic Sea. The study results permit assessing whether the efforts made in recent years to reduce dioxin and PCB emissions into the environment have translated into decreases in concentrations of these pollutants in Baltic Sea fishes and whether these raw fish materials meet food law requirements. The safest fish species with regard to dioxins were designated, as were those of which consumption should be limited. The influence of fish age (length) and weight on dioxin and dioxin-like polychlorinated biphenyl (dl-PCB) concentrations was investigated. The correlation between fat content and dioxin concentration was determined. The Baltic Sea region and fishing ports from which the least contaminated fish come were also determined. The studies indicated that dioxin and PCB concentrationsin fishes from Polish fishing areas are currently lower than they were twenty years ago.

Occurrence and tissue distribution of alkylphenols (APs) in selected waterbirds from the Southern Baltic.

The aim of the present study was to determine the concentrations of nonylphenols (NPs) and 4-t-octylphenol (4tOP) in the muscles, liver, and kidneys of selected waterbird species. Three species with different feeding habits were selected, i.e., greater scaup (Aythya marila), great crested grebe (Podiceps cristatus), and great cormorant (Phalacrocorax carbo) to investigate the potential effects of diet on the level of contaminants tested. The determination and quantification of analytes were performed using gas chromatography with mass spectrometric detection (GC-MS). The highest NP concentrations were noted in the kidneys of greater scaups and great crested grebes (208.3 and 160.8 µg kg-1 ww, resp.), which were six to fourteen-fold higher than those in the muscles (15.0 and 25.6 µg kg-1 ww, resp.) and livers (22.9 and 13.8 µg kg-1 ww, resp.) of these species. In greater scaups, the mean concentration of NPs in the livers was lower than in the muscles, while in great crested grebes, it was the opposite and higher concentrations were noted in the muscles. The mean concentrations of NPs in the muscles and livers of great cormorants were at similarly low levels (12.5 and 9.7 µg kg-1 ww, resp.). The concentrations of 4tOP in all samples were low, ranging from

Arsenic speciation in fish from Baltic Sea close to chemical munitions dumpsites.

Chemical weapons that were dumped in seas and oceans after World War II, including the Baltic Sea, are sources of pollution of marine areas. Sunken containers can corrode, unseal, and numerous compounds pass into the environment, including toxic forms of arsenic, which are then taken up by marine animals. This study aims to quantify concentration of total arsenic, inorganic arsenic (III + V), and organic compounds arsenobetaine, monomethylarsonic acid, dimethylarsinic acid in the muscle tissues of cod, herring, sprat, and flounder and the associated risk to consumer health. Sprat muscle (0.636 mg kg-1) had the highest content of total arsenic, significantly less was noted in the muscles of herring (0.460 mg kg-1) and flounder (0.588 mg kg-1), and the least was in cod (0.390 mg kg-1). Toxic inorganic arsenic compounds were present in the fish tested at levels below 0.02 mgkg-1 and constituted from 3.45 to 5.75% of total arsenic. Arsenobetaine dominated among organic forms, and concentrations of it, like total arsenic, varied depending on the fish species. Consumer health risk was determined with the estimated daily intake, the target hazard quotient, and the carcinogenic risk. Estimated daily intake values for inorganic arsenic in herring, cod, sprat, and flounder were below the reference dose at 0.51 × 10-5 mg kg-1 b. w. day. The target hazard quotient factor of 0.0017 indicated there was no threat. Carcinogenic risk values were within the permissible range of 10-6 to 10-4. Current data indicate that inorganic arsenic compounds pose no risk to the health of consumers of Baltic fishes.

The occurrence and distribution of nonylphenols and nonylphenol ethoxylates in different species of fish.

The aim of this study was to analyze the accumulation of nonylphenols (NPs) and nonylphenol ethoxylates (NPEOs) in the muscles, liver, and bile of flounder (Platichthys flesus), cod (Gadus morhua), and eels (Anguilla anguilla). The flounder and cod were caught in the Gulf of Gdansk (the Baltic Sea), while the eels were sampled in the Vistula and Szczecin lagoons (the Baltic Sea) and in the inland waters of the Masurian Lake District. NP concentrations in muscles were low in all the samples analyzed and ranged from 14.2 to 28.2 µg-1 kg ww. In contrast, a wide range of NP concentrations were observed in livers, which seemed to depend on both the species and the feeding status of the fishes. NP levels in flounder and eel livers were from three to twenty times higher than those in the muscles, but they were below the limit of quantitation (LOQ) in all the cod liver samples. The mean concentration of NPs in the liver of flounder caught in the Gulf of Gdansk was 222 µg kg-1 ww, while in that of the eel ranged from 57 µg kg-1 ww in fish caught in the Masurian Lake District to 519 µg kg-1 ww in eels caught in the Vistula Lagoon. NPs were detected in bile in only a few eel samples, which indicated that bile analysis has limited applications for estimating NP contamination in marine fish. The NPEOs in all the samples analyzed were below the LOQ.

The occurrence of endocrine disrupting compounds in off-shore sediments from the southern Baltic Sea.

This paper presents the study on the occurrence and spatial distribution of polybrominated diphenyl ethers (PBDEs), butyltin compounds (BTCs), bisphenol A (BPA), and alkylphenols (APs) in sediments. The study focused mainly on off-shore surface sediments collected from the southern Baltic Sea. The pollutant concentrations were as follows:

The occurrence of organic contaminants in European eel (Anguilla anguilla) in Poland: an environmental quality assessment.

The objective of the present study was to provide information on the levels of pollutants in the tissues of eels caught in Polish waters. The contaminants included in the study are those which have not yet been widely studied in eel stocks, but which arouse concern in relation to the environment. An overview of the pollutant levels in eels caught in other European waters was also conducted. The results are evaluated in terms of environmental quality and consumer health. The mean concentrations of ΣPBDEs and ΣHBCDs in muscles of eels sampled in Polish waters were between 1 and 2 ng g(-1) ww. The mean TBT concentrations were between 2 and 4 ng g(-1)ww with the exception of samples from the Szczecin Lagoon, in which the mean TBT concentration was about tenfold higher.

Persistent organic pollutants in sediment from the southern Baltic: risk assessment.

The concentrations of organochlorine pesticides, PCBs, and PCDD/Fs were measured in sediment samples collected from the Gulf of Gdansk, the Gdansk Deep, the Vistula Lagoon and the off-shore waters in the southern Baltic Sea. The determinations were conducted in the fraction <63 µm. The highest levels of pollutants were found in sediments from the Gulf of Gdansk and the Gdansk Deep. In these sediments, concentrations of pp'-DDT and its metabolites (pp'-DDE and pp'-DDD) ranged from 2 to 11 µg kg(-1) dw. Concentrations of HCB were between 0.1 and 1.0 µg kg(-1) dw. Concentrations of the sum of α-, ß-, γ-HCH were between 0.3 and 2.58 µg kg(-1) dw. Concentrations of marker PCBs ranged from about 2 to 11 µg kg(-1) dry weight. Total TEQ PCDD/F/dl-PCB values in the studied samples varied between 1 and 18 ng kg(-1) dw. The data were evaluated in terms of ecotoxicological criteria based on the environmental impact of the pollutant effects. Our research indicated that in the sediment from the southern Baltic Sea, the PCDD/F and PCBs occurred at concentrations which could be dangerous to marine organisms.

Marine and farmed fish on the Polish market: comparison of the nutritive value and human exposure to PCDD/Fs and other contaminants.

Chemical analyses were performed in nine fish species that are popular on the Polish market. These included Baltic fish (cod, herring, salmon), fish farmed in Poland (carp, trout), marine fish imported from China (Alaska pollock, sole), and farmed fish imported from Vietnam and China (sutchi catfish, tilapia). The nutritional composition (amino acid, micro- and macronutrients, fat-soluble vitamins - A(1), D(3), E) and certain contaminants (organochlorine pesticides, OCPs; indicator polychlorinated biphenyl, PCB(6); polychlorinated dibenzo-paradioxins and polychlorinated dibenzofurans, PCDD/Fs; dioxin-like polychlorinated biphenyls, dl-PCBs; organotin compounds, OCTs; dyes, malachite green and crystal violet; veterinary drug residues, nitrofurans and chloramphenicol; toxic metals, Cd, Pb, Hg) in the muscle tissues of fish were determined. It was confirmed that the fish species analyzed were excellent sources of amino acids, and were rich in phosphorous and selenium. Baltic Sea fish (salmon, herring), fish farmed in Poland (carp and trout), and tilapia were also rich in vitamin D(3). Traces of OCP, PCB(6), OCT, dyes, veterinary drug residues, and heavy metals were detected in concentrations which do not pose a threat to consumers at the current rate of fish consumption in Poland. However, the problem might arise from the content of PCDD/Fs and dl-PCBs in fatty Baltic fish. The fish species analyzed, differed in their nutritional values and degrees of contamination. We suggest that for optimum health and safety, it is advisable that consumers include a variety of different fish species in their diets.

Organochlorine pollutants in European eel (Anguilla anguilla L.) from Poland.

The purpose of the current paper is to report the levels of OC pesticides ([summation operator]HCHs, HCB, [summation operator]DDTs), marker PCBs, PCDD/Fs, and dl-PCBs in the muscle tissue of European eel, Anguilla anguilla (L.), collected in the Vistula and Szczecin lagoons (Poland), and in the local market. The results obtained were evaluated according to maximum limits adopted by EU, and some national legislatures. The intake of organochlorine contaminants from the consumption of eel from Polish waters is also estimated. The residues of tested contaminants ranged between: 0.6 and 6.0 ng g(-1)ww for [summation operator]HCHs; 9.8 and 273.9 ng g(-1)ww for [summation operator]DDTs; 0.4 and 23.8 ng g(-1)ww for HCB; 4.0 and 533.9 ng g(-1)ww [summation operator](7)PCBs; 0.72 and 8.14 pg-WHO-TEQ g(-1) ww for [summation operator]PCDD/F/DL-PCBs. The levels of OCPs, and PCDD/F/dl-PCBs in the muscle tissues of eels captured in the Vistula and Szczecin lagoons were compliant with European regulations.

Polybrominated diphenyl ethers (PBDEs) in selected fish species from the southern Baltic Sea.

This paper reports concentrations of seven polybrominated diphenyl ether (PBDE) congeners in selected fish species that were collected from the southern Baltic in the 2004-2006 period. Differences in concentrations of PBDEs among the fish species were observed. The mean summation Sigma(7)PBDE concentrations measured in herring (Clupeaharengus), sprat (Sprattus sprattus), and salmon (Salmo salar) samples were 1.2 ng g(-1) wet weight, 1.6 ng g(-1) wet weight, and 2.5 ng g(-1) wet weight, respectively. PBDE-47 was the prevalent congener in all the samples tested. PBDE concentrations in the herring samples tested in the current study were similar to those determined in herring sampled in the northern Baltic, but slightly lower in comparison with concentrations in fish from the Belgian North Sea. PBDE levels occurring in salmon sampled in the southern Baltic were lower than those measured in the northern and northeastern Baltic, but similar to levels determined in salmon from the central part of the Baltic Sea. The risk posed by consuming the fish tested in the current study was evaluated according to the Provisional Tolerable Weekly Intake (PTWI) value for PBDEs recommended by the European Food Safety Authority (EFSA).

Fish products available in Polish market--assessment of the nutritive value and human exposure to dioxins and other contaminants.

Chemical analyses were performed on one hundred and twenty of the most popular varieties of fish products (smoked fish, salted fish, and marinated fish) of the fish market in Poland. The contents of the nutritive substances of fish products (protein, micro- and macronutrients, vitamins A(1), D(3), E, and fatty acids) and the chosen contaminant (toxic metals--mercury, cadmium, lead, arsenic; dioxin/furans--PCDDFs; dioxin-like PCB--dl-PCBs; seven congeners of polybrominated diphenyl ethers--PBDEs; organochlorine pesticides--SigmaDDT, HCB, SigmaHCH and marker polychlorinated biphenyls--PCB(7)) levels were determined. It was confirmed that fish products are a good source of digestible proteins, iodine, selenium, and vitamin D(3). The fundamental nutritive benefit of processed fish lies in its highly beneficial fatty acid composition, which is what imparts them healthy nutritive qualities. The high content of long-chain polyunsaturated fatty acids (LC-PUFAs), which is not noted in other food products, is particularly important. The majority of contaminants studied were present in low levels. The possible threats, particularly in the case of pregnant/nursing women and young children, can pose the levels of dioxin/furans (PCDD/Fs) and dioxin-like (dl-PCBs) in smoked Baltic salmon and smoked sprat, elevated in a relation to particular requirements concerning the content of sum of PCDD/Fs and dl-PCBs in fish (8pg WHO-TEQg(-1)). The health benefits and risks stemming from consumption of fish products were determined according to the Provisional Tolerable Weekly Intake (PTWI) for chosen contaminants (Cd, Hg, As, PCDD/Fs+dl-PCB) and the quantity of ingredients that render a fish diet healthy based on data from the EFSA Journal [EFSA (European Food Safety Authority), 2005. Opinion of the scientific panel on contaminants in the food chain on a request from the European Parliament related to the safety assessment of wild and farmed fish. EFSA J. 236, 1-118]. In regard of high content of LC-PUFAa and other nutritive ingredients, fish products available in Polish market may be considered as healthy food. However, many authors point at contaminants (methylmercury, PCDD/Fs) occurring in fish and fish products as on potential health problem, and emphasize that the amount of that hazardous substances should be limited in human diet.

Study on the raw fish oil purification from PCDD/F and dl-PCB-industrial tests.

The results of tests for the purification of fish oils with activated carbon for industrial use are presented. The optimum parameters for the process of purification (granulation of the activated carbon, its dosage, the oil temperature, and the duration of mixing the oil with activated carbon) were previously established for the laboratory scale. The optimization of the process consisted of selecting purification parameters that would allow for maximum reduction of the toxic polychlorinated dibenzo-para-dioxins and polychlorinated dibenzofurans (PCDD/Fs) content, while retaining the favorable high fatty acid content [C20:5 n-3, eicosapentaenoic acid (EPA) and C22:6 n-3, docosahexaenoic acid (DHA)]. The use of that optimum parameters in industrial conditions confirmed the satisfactory results obtained in laboratory tests. Five types of oil derived from various Baltic fish were purified. Reduction in the PCDD/Fs content was 77.0-93.6% on average, whereas in the dioxin-like polychlorinated biphenyls (dl-PCBs)-it was 42.7-50.5% on average, with insignificant changes in the total amount of EPA and DHA content. Furthermore, a significant reduction in the content of arsenic was noted (by about 62% on average), with insignificant changes in the content of organochlorine pesticides (OCPs), and marker polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), cadmium, lead, and mercury. Purification provided fish oil having standardized parameters that allow for its use as feed additives, whilst retaining its favorable fatty acid content.

Investigation of PCDD/Fs and dl-PCBs in fish from the southern Baltic Sea during the 2002-2006 period.

Concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (dl-PCBs) in the southern Baltic herring (Clupea harengus), sprat (Sprattus sprattus), and salmon (Salmo salar) are reported. The significant interspecies and season-specific differences in PCDD/Fs and dl-PCBs concentrations were observed. The contribution of dl-PCBs to the total toxicity equivalents (TEQs) ranged between 50% and 70%, dependently on fish species. In all samples, concentrations of PCDF were higher in comparison with that of PCDD. The permissible limits of the content of PCDD/Fs and dl-PCBs (8 pg g(-1) fresh weight for the sum of PCDD/Fs and dl-PCBs, and 4 pg g(-1) fresh weight for the PCDD/Fs) were exceeded mainly in salmon samples (in 80% of salmon samples studied). The elevated levels of PCDD/F and dl-PCB were observed only in two of seventy two herring samples, and in ten of 62 sprat samples. The multiple regression analysis revealed that fish lipid content, and concentration of PCB 153, used simultaneously, might be useful in the prediction of TEQ values of fish samples. These two variables explained more than 80% of total variance. For all fish species studied, the correlation coefficients obtained with the multiple regression analysis were higher than that obtained with the regression analysis involving total TEQ values, and only one independent variable: fish lipid content.

PCBs in fish from the southern Baltic Sea: levels, bioaccumulation features, and temporal trends during the period from 1997 to 2006.

Levels of seven marker polychlorinated biphenyls (PCBs) have been determined in five species of Baltic fish collected during 1997-2006. Downward time trends in the concentrations of heavier congeners of PCBs in different Baltic fish, with the exception of cod, have been observed between 1997 and 2001. In case of sprat and herring samples, the statistical significance of the time trends of the PCBs: 101, 118, 153, 138 and 180 concentrations has been proved. Species-specific bioaccumulation of PCBs has been indicated, and the lowest and highest levels of PCBs (expressed on the basis of lipid weight) have been observed in sprat and salmon samples, respectively. PCB profiles have been found to be similar in all the fish species tested. Sampling location has not been a crucial factor for the observed levels of various PCBs. In some fish species, PCB concentrations are negatively correlated with the fat content but have no relation with the fish length.

Organochlorine pesticides in fish from the southern Baltic Sea: levels, bioaccumulation features and temporal trends during the 1995-2006 period.

The levels of HCH isomers, HCB and summation DDTs were determined in five species of Baltic fish collected during 1995-2006. Some downward time trends of HCH isomer, pp'-DDE and pp'-DDD concentrations in Baltic fish were observed; in contrast, HCB and DDT concentrations did not exhibit any obvious trend. Concentrations of summation HCHs expressed on a lipid weight basis were very similar in all species studied. In contrast, concentrations of summation DDTs varied in tested species. The most abundant HCH isomer was beta-HCH and among DDT-related compounds, pp'-DDE prevailed. In our investigations the sampling sites were not a crucial factor for organochlorine pesticide concentrations and patterns observed in fish, but the occurrence of several inter-species differences in the bioaccumulation features of OC pesticides were observed. For cod there was a positive correlation between fish size (length) and summation DDT concentration. In herring, sprat and salmon samples, summation DDT concentrations were negatively correlated with fat content.

Interaction of amphotericin B and its low toxic derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester, with fungal, mammalian and bacterial cells measured by the energy transfer method.

Amphotericin B (AMB) derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) retains the broad antifungal spectrum and potency of the parent antibiotic, whereas its toxicity towards mammalian cells is reduced by about two orders of magnitude. The purpose of this work was to find out whether the differences observed in the toxicity of MFAME and native AMB are due to the differential drugs affinity to fungal and mammalian cell membranes. Comparative studies on AMB and MFAME biological activity and their affinity to fungal, mammalian and bacterial cells were performed. The interaction of AMB and MFAME with cells have been studied by fluorescence method based on the energy transfer between membrane fluorescent probe (donor) and the polyenic chromophore of the antibiotic (acceptor) simultaneously present in the cell membrane. The amount of the antibiotic bound to cells was indicated by the extent of fluorescence quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) or 1,6-diphenyl-1,3,5-hexatriene (DPH) by polyenic chromophore of the antibiotic. The results obtained indicate that binding extent and characteristics for both antibiotics are comparable in the three types of cells studied. Dramatically lower toxicity of MFAME as compared to AMB towards mammalian cells is not related to the antibiotic-cell affinity, but rather to different consequences of these interactions for cells, reflected in membrane permeabilization. MFAME is definitely less effective than parent AMB in the permeabilizing species formation in mammalian cell membrane.

Comparative in vitro studies on liposomal formulations of amphotericin B and its derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME).

N-Methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) is a semisynthetic derivative of the antifungal antibiotic amphotericin B (AMB). In contrast to the parent antibiotic, the derivative is characterised by low toxicity to mammalian cells and good solubility in water of its salts. Comparative studies on biological properties of free MFAME, AMB and their liposomal formulations were performed. To obtain liposomal forms, the antibiotics were incorporated into small unilamellar vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and DMPC:cholesterol or ergosterol, 8:2 molar ratio. The effectivity of the liposomal and free forms of AMB and MFAME were compared by determination of fungistatic and fungicidal activity against Candida albicans ATCC 10261, potassium release from erythrocytes, and haemolysis. The results obtained indicate that in contrast to AMB, incorporation of MFAME into liposomes did not further improve its selective toxicity. Studies on the antagonistic effect of ergosterol and cholesterol on the antifungal activity of the antibiotics indicated that sterol interference was definitely less pronounced in the case of MFAME than in the case of AMB.

Facilitated diffusion of glucosamine-6-phosphate synthase inhibitors enhances their antifungal activity.

N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) and 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) are strong inhibitors of the essential fungal enzyme, glucosamine-6-phosphate synthase, but their antifungal activity is poor, due to slow penetration of these agents through the cytoplasmic membrane. In the present studies we have exploited the possibility of enhancement of ADGP and FMDP antifungal activity by improving their transport properties. It has been found that membrane-permeabilising polyene macrolides amphotericin B (AMB) and its N-methyl-N-fructosyl methyl ester derivative (MF-AME), at subinhibitory concentrations, facilitate diffusion of ADGP through the fungal cell membrane, thus allowing a decrease of its minimal inhibitory concentration (MIC). Synergistic effects have been observed for combinations of ADGP with AMB or MF-AME. Fractional inhibitory concentration (FIC) indexes, determined against a number of Candida spp., have been in the 0.18-0.81 range. Weak antifungal synergistic effects have been found for combinations of FMDP with AMB or MF-AME. ADGP can be easily encapsulated into unilamellar lipid vesicles. Liposomal preparations of ADGP demonstrated stronger antifungal activity against some fungal strains than free ADGP.