Pollution patterns and their effects on biota within lotic and lentic freshwater ecosystems: How well contamination and response indicators correspond?

Aquatic environments are often severely polluted with chemical substances of anthropogenic origin, which can pose a potential threat to aquatic organisms and human health. In this study, patterns and sources of heavy metals (HMs, 6 metals) and polycyclic aromatic hydrocarbons (PAHs, 16 hydrocarbons), contamination indicators, environmental genotoxicity measures and metrics of ecological status in lotic and lentic ecosystems were collated for the first time. Chemical analysis has confirmed previously reported long-term contamination at certain study sites. The sediments of Lake Talksa, located in a city and characterized by exclusive anthropogenic pressure, exhibited the highest levels of contamination by both HMs and PAHs. Through positive matrix factorization (PMF) analysis, vehicle and industrial emissions were identified as the primary sources of HMs and PAHs. Our results revealed that frequencies of genotoxic aberrations were higher in river sites compared to lakes, with the highest genotoxic risk observed in the Nemunas River below industrial cities Alytus and Kaunas. Surprisingly, even the severely contaminated Lake Talksa showed only a "moderate" grade of genotoxic risk, highlighting the potential for adaptation of biota to long-term contamination especially in lentic ecosystems. The ecological quality status assessed by macroinvertebrate metrics, which may be sensitive to observed high biological contamination, appeared to be unrelated to contamination patterns. Consequently, to obtain the robust information on anthropogenic contamination and its effects, a combination of various assessment methods and metrics should be employed.

Impact of copper and zinc mixture on haematological parameters of rainbow trout (Oncorhynchus mykiss): acute exposure and recovery.

Significant changes in composition of rainbow trout Oncorhynchus mykiss blood cells types were induced after 4-days exposure with mixture of Cu2+ and Zn2+ at 0.25, 0.125 and 0.06 parts of LC50 in comparison to control group. The highest concentration of metal mixture (0.25 of LC50) significantly induced elevation of the number of monocytes and poly-segmented neutrophils. Treatment with 0.125 parts of LC50 concentration increased the number of thrombocytes, monocytes and non-segmented neutrophils. The most diluted mixture resulted in significant induction of thrombocytes, monocytes, non- and poly segmented neutrophils. Analysis of leucocyte cell types in the O. mykiss blood samples after 4-days of exposure at all applied mixture parts showed signs of monocytosis and neutrophilia. Comparison of different types of leucocytes' percentages (leukogram) in fish after 4-days exposure to metal mixture and after 4, 8, and 12-days recovery periods showed that, values of neutrophils even after the 12-days recovery period at all tested parts of LC50, and monocytes after exposure with the highest (0.25) used part of LC50 were not restored to control group levels. Depuration and recovery processes in treated fish are concentration and recovery period dependent.

Contamination and genotoxicity biomarker responses in bivalve mussels from the major Lithuanian rivers.

European inland waters are under continuous threat of anthropogenic pollution. Determination of background level of biomarker response and subsequent classification of the impact increases the applicability of results. In the current study, we evaluate the range of chemical contamination by measuring the concentrations of metals, polycyclic aromatic hydrocarbons and polychlorinated biphenyls, and the levels of environmental genotoxicity by using the micronuclei and nuclear bud tests in bivalve mussels of the major Lithuanian rivers. Second, we aimed to evaluate the association between chemical contamination and genotoxicity biomarker responses. Finally, we set to determine the background level of genotoxic effects. Such value (summed frequency of MN and NB) was assessed-6‰. On that basis, we develop a scale of potential genotoxic impact and perform ranging sites into five categories. The results clearly indicate the existence of significant differences in the levels of chemical pollution and genotoxicity in different sites. Increased levels of studied parameters were assessed at the areas affected by municipal and industrial effluents, road runoff, combustion products, and in the area contaminated by accidental spillage. On the other hand, downstream decrease of contamination level, presumably associated with biological degradation and photochemical oxidation, were also observed. Genotoxicity parameters were associated with PAH and metal concentrations measured in mussel tissues as well as in sediments. Results also indicate that in situ genotoxicity assessment performed in the areas affected by long-term contamination of municipal origin might be not sufficiently precise. Study highlights the necessity to combine genotoxicity assessment with chemical analysis. Environ. Mol. Mutagen. 61:338-354, 2020. © 2019 Wiley Periodicals, Inc.

Induction of nuclear abnormalities in herring (Clupea harengus membras), flounder (Platichthys flesus), and Atlantic cod (Gadus morhua) collected from the southern part of the Gotland Basin-the Baltic Sea (2010-2017).

Eight nuclear abnormalities of genotoxicity and cytotoxicity were studied in peripheral blood erythrocytes of herring (Clupea harengus membras), flounder (Platichthys flesus), and Atlantic cod (Gadus morhua) sampled (2010-2017) from the Polish and the Lithuanian Exclusive Economic Zones (EEZ) in the Baltic Sea. At all study stations, total genotoxicity (∑Gentox) was found to be higher than total cytotoxicity (∑Cytox). A significant time-related decrease in genotoxicity was detected in the Lithuanian EEZ (2015-2017), while in the Polish EEZ (2014-2016), the opposite tendency was revealed. The highest ∑Gentox and ∑Cytox values recorded in fish sampled at the study stations located relatively close to each other clearly indicate an increased environmental genotoxicity and cytotoxicity pressure for fish in these areas. Exceptionally high and high-level genotoxicity risks to herring followed by those to flounder and cod were determined at a higher percentage of the stations studied.

Environmental genotoxicity and cytotoxicity levels in fish from the North Sea offshore region and Atlantic coastal waters.

In the framework of the ICON project, environmental genotoxicity and cytotoxicity levels were assessed in blood erythrocytes of dab (Limanda limanda) and haddock (Melanogrammus aeglefinus) collected at 25 stations in the North Sea and near the coast of Iceland in August-October 2008. Micronuclei, nuclear buds and bi-nucleated cells with nucleoplasmic bridges were assessed as environmental genotoxicity biomarkers, and the frequency of fragmented-apoptotic and bi-nucleated erythrocytes were assessed as environmental cytotoxicity biomarkers. The lowest frequencies of genotoxic and cytotoxic abnormalities were detected in fish from the Icelandic study stations. The highest frequencies of abnormalities were recorded in dab from the Dogger Bank and the German Bight, in haddock from the Egersund Bank and from an area off the Firth of Forth (North Sea). In fish from the Icelandic reference area, frequencies of genotoxicity and cytotoxicity responses were significantly lower than in fish from most areas of the North Sea.

Biomarkers of environmental contaminants in the coastal waters of Estonia (Baltic Sea): effects on eelpouts (Zoarces viviparus).

The eastern Baltic Sea near the Estonian coast is heavily navigated by numerous cargo ships and oil tankers. Hundreds of accidents and oil spills happen yearly in this area. Yet, there is a lack of data concerning the distribution and effects of the environmental contaminants, especially polycyclic aromatic hydrocarbons (PAHs). Different parts of the Baltic Sea have different levels of contamination; therefore a wide range of monitoring stations in coastal areas in the Gulf of Finland and Gulf of Riga were chosen. The aim of the present research was to document the responses of chosen biomarkers of environmental contaminants in different unstudied areas of the Estonian coastal sea. During 2009 and 2010 we measured PAH metabolites, EROD activities, geno- and cytotoxicity, histology, parasites and other biomarkers from the eelpout (Zoarces viviparus), a resident benthic fish species. The results showed that fish from the Gulf of Riga emitted lower levels of fluorescence in fixed wavelength analyses (representing equivalents of PAH metabolites in bile and urine), and consistently, showed less geno- and cytotoxicity and parasite infection, higher liver somatic index (LSI) and a higher condition factor (CF) than fish inhabiting areas close to the Baltic proper and in the Gulf of Finland. The results point to the effect of long-range contaminant transportation, whether atmospheric or hydrodynamic, and also to the intensive shipping activity in international routes. This study fills the gap of knowledge in this area that has persisted until now. Nevertheless, more studies in this area on the different groups of contaminants are necessary, to specify the factors that are responsible for observed biological effects.

Risk of environmental genotoxicity in the Baltic Sea over the period of 2009-2011 assessed by micronuclei frequencies in blood erythrocytes of flounder (Platichthys flesus), herring (Clupea harengus) and eelpout (Zoarces viviparus).

Environmental genotoxicity was investigated at 82 locations encompassing different regions of the Baltic Sea. Micronuclei (MN) analysis was performed in erythrocytes of 1892 specimens of flounder Platichthys flesus, herring Clupea harengus and eelpout Zoarces viviparus, three of the most common native fish species of the Baltic Sea collected in 2009-2011. MN background levels in fish were determined using data obtained in 2001-2011 from 107 Baltic sites. Extremely high genotoxicity risk zones were found for flounder at 11 stations out of 16 in 2009 and 33 stations of 41 in 2010-2011, for herring, at 5 of 18 stations in 2009 and 20 of 43 stations in 2010-2011, in eelpout only at one out of 29 stations. The sampling stations were restricted mainly to the southern and eastern Baltic Sea offshore zones and in most of them, MN frequencies in flounder and herring significantly exceeded the reference and background levels of micronuclei. This is a first attempt to evaluate the background MN responses, as well as low, high and extremely high genotoxicity risk levels for native fish species.

Environmental genotoxicity and cytotoxicity studies in mussels before and after an oil spill at the marine oil terminal in the Baltic Sea.

Environmental genotoxicity and cytotoxicity effects in the gills of mussels Mytilus edulis, from the Baltic Sea areas close to the Butinge oil terminal (Lithuania) before and after accidental oil spill in 31 January 2008 were studied. Mussels from the oil spillage zones were collected in 12 days, in 3 and 6 months after the spill to determine the effects of the spill. Mussels sampled in 2006-2007 were used for the assessment of the background levels of genotoxicity and cytotoxicity in the Butinge oil terminal area. Comparison of the responses in M. edulis before and after the oil spill revealed significant elevation of frequencies of micronuclei (MN), nuclear buds (NB) and fragmented-apoptotic (FA) cells. Environmental genotoxicity and cytotoxicity levels in mussels from the Palanga site before the accident (in June 2007) served as a reference. Six months after the accident, in July 2008, 5.6-fold increase of MN, 2.9-fold elevation of NB, and 8.8-fold elevation of FA cells were observed in mussels from the same site.

Environmental genotoxicity and cytotoxicity in the offshore zones of the Baltic and the North Seas.

Micronuclei (MN), nuclear buds (NB) and fragmented-apoptotic cells (FA) were analyzed in mature peripheral blood and immature cephalic kidney erythrocytes of flounder (Platichthys flesus), dab (Limanda limanda) and cod (Gadus morhua) from 12 offshore sites in the Baltic Sea (479 specimens) and 11 sites in the North Sea (291 specimens), which were collected during three research vessel cruises in December 2002, 2003 and in September 2004. The highest levels of environmental genotoxicity (frequencies of MN up to 0.5 per thousand, NB - up to 0.75 per thousand) and cytotoxicity (FA - up to 0.53 per thousand) were observed in flatfishes from areas close to oil and gas platforms in the North Sea and in zones related to the extensive shipping and potentially influenced by contamination from large European Rivers (Elbe, Vistula, Oder). In dab from the offshore zones of the North Sea, the levels of nuclear abnormalities were higher as compared to those in dab from the Baltic Sea. Responses in immature kidney erythrocytes were higher than in mature erythrocytes from peripheral blood. MN frequency lower than 0.05 per thousand (the Baltic Sea) and lower than 0.1 per thousand (the North Sea) could be suspected as a reference level in the peripheral blood erythrocytes of flatfish.

Biomarker responses in flounder Platichthys flesus from the Polish coastal area of the Baltic Sea and applications in biomonitoring.

The objective of this study was to investigate the pattern of enzymatic activities, environmental genotoxicity and cytotoxicity in flounder, Platichthys flesus, from the Polish coastal area of the Baltic Sea. Fish were sampled in different contaminated sites in the Gulf of Gdansk and in a reference area outside the gulf. The activities of acetylcholinesterase (AChE), glutathione S: -transferase (GST), catalase (CAT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), were studied, as well as the frequency of micronuclei, nuclear buds and fragmented-apoptotic cells. A higher mean activity level of muscular AChE and a lower activity level of hepatic GST were evident in samples taken from the reference site, relative to those found in the gulf. Modeled CAT activity (in both liver and gill tissue), blood plasma LDH and CK activities were all significantly higher in flounder collected at locations within the Gulf of Gdansk than at the reference site. No statistically significant alterations were observed in the activities of ALT and AST in the blood plasma of flounder in this study. Fish collected from a location at the mouth of the Vistula River showed the highest hepatic GST and CAT, the highest gill CAT activity, and the highest frequency of blood micronuclei, nuclear buds and fragmented-apoptotic cell inductions, as well as the lowest level of blood plasma CK. The present study confirms that compared to fish from the reference area, flounder from the Gulf of Gdansk clearly demonstrate a different enzyme activity, genotoxicity and cytotoxicity biomarker response pattern.

Biomarker responses in flounder (Platichthys flesus) and mussel (Mytilus edulis) in the Klaipeda-Butinge area (Baltic Sea).

During the EU project BEEP a battery of biomarkers was applied in flounder (Platichthys flesus) and the blue mussel (Mytilus edulis) collected at three locations off the Lithuanian coast (Baltic Sea) in June and September 2001 and 2002. The elevated biomarker responses in specimens sampled in September 2001 were apparently related to the extensive dredging activities in the Klaipeda port area and subsequent dumping of contaminated sediments. High concentrations of organic pollutants (organochlorines and PBDEs) were also measured in the tissues of both indicator species. In addition, response levels of genotoxicity, cytotoxicity, immunotoxicity as well as concentrations of PAH metabolites in the bile of flounder showed elevations in 2002 after an oil spill in the Butinge oil terminal in November 2001. In flounder, biomarker measurements 10 months after the spill indicated recovery processes but in mussels a high level of genotoxicity could still be observed 22 months later. The present study illustrates the usefulness of the multi-biomarker approach in the detection of biological effects of pollution in this region of the Baltic Sea.

Investigation of micronuclei and other nuclear abnormalities in peripheral blood and kidney of marine fish treated with crude oil.

The induction of micronuclei and other nuclear abnormalities (nuclear buds, bi-nucleated and fragmented-apoptotic cells) was analyzed in the erythrocytes of peripheral blood and cephalic kidney of turbot (Scophthalmus maximus) and Atlantic cod (Gadus morua), treated with crude oil (Statfjord B, Norway) and with nonylphenol. Significant increase in MN was observed in turbot kidney and blood after exposure to 30 ppb of nonylphenol, 0.5 ppm of oil, and after co-exposure to 0.5 ppm of oil spiked with additional mixture of alkylphenols and PAHs (P varied between 0.0054 and <0.0001). The induction of micronuclei was observed only in cod kidney after exposure to spiked oil (P=0.0317). Significant inter-specific differences after the exposure to 0.5 ppm of oil (P=0.0385) and after treatment with spiked oil (P=0.0067) were observed. In turbot cephalic kidney, the elevated levels of bi-nucleated cells were observed in all treatment groups (P values varied in a range from 0.05 to 0.0025) while the increase in cells with nuclear buds was noted after the exposure to 0.5 ppm of oil (P=0.05). The fragmented-apoptotic cells appeared after the exposure to nonylphenol (P=0.0039) and to spiked oil (P<0.0001). In turbot blood, only the significant induction in nuclear buds was detected. Statistically significant inter-tissue differences were found only in the induction of fragmented-apoptotic cells after the exposure to nonylphenol and to spiked oil.

Cytogenetic and cytotoxic effects in gill cells of the blue mussel Mytilus spp. from different zones of the Baltic Sea.

Frequency of micronuclei (MN) and other nuclear abnormalities (nuclear buds, bi-nucleated and fragmented-apoptotic cells) was analysed in gill cells of the blue mussel (Mytilus spp.) from selected coastal sites in the Baltic Sea--Kvädöfjärden (Sweden), Klaipeda-Butinge zone (Lithuania), Gulf of Gdansk (Poland) and Wismar Bay (Germany). Samples were collected from 650 specimens during bi-annual sampling campaigns in 2001 and 2002. The lowest frequency of MN (0.37 MN/1000 cells) was found in blue mussels from the reference site (Kvädöfjärden). The highest MN values (up to 6.7 MN/1000 cells) were registered in blue mussels from the Gulf of Gdansk in autumn 2001 and 2002, and at Wismar Bay in spring 2001 (up to 5.06 MN/1000 cells). Gradients of MN incidences were observed when comparing the three studied locations in Wismar Bay, and at the Lithuanian coast before the crude oil spill in the Butinge oil terminal. Moreover, significant seasonal and inter-location differences in the responses were documented (P<0.0001). Nuclear abnormalities were observed most frequently in blue mussels from the Gulf of Gdansk.

Analysis of micronuclei in blue mussels and fish from the Baltic and North Seas.

Micronuclei (MN) were analyzed in erythrocytes of flounder (Platichthys flesus) and wrasse (Symphodus melops) and in gill cells of blue mussels (Mytilus edulis). The organisms were collected from three study stations in the Baltic Sea and from seven stations in the North Sea (Karmsund area, Norway) 4 times. The statistically significant differences obtained were related to the season, sex of the fish, and sampling locality. Higher MN frequencies were found in fish and mussels collected from the most polluted study stations in the North Sea. The same tendency could be described in the Baltic Sea; however, it was masked by the recent oil spill from the Butinge oil terminal. Our results showing higher MN frequencies in presumably what were the most polluted study locations suggest that MN tests in fish and mussels may be used for the detection of genotoxic effects in a marine environment. The endpoint is well characterized and can be easily recognized, and the technique is convenient to use in field samplings following standard procedures and protocols.