Biomarker responses in mussels (Mytilus trossulus) from the Baltic Sea exposed to water-accommodated fraction of crude oil and a dispersant at different salinities.

Oil spills pose significant environmental risks, particularly in cold seas. In the Baltic Sea, the low salinity (from 0 to 2 up to 18) affects the behaviour of the spilled oil as well as the efficiency and ecological impacts of oil spill response methods such as mechanical collection and the use of dispersants. In the present study, mussels (Mytilus trossulus) were exposed under winter conditions (5 °C) to the water-accommodated fraction (WAF) of Naphthenic North Atlantic crude oil prepared by mechanical dispersion or to the chemically enhanced fraction (CEWAF) obtained using the dispersant Finasol OSR 51 at salinities of 5.6 and 15.0. Especially at the lower salinity, high bioaccumulation of polycyclic aromatic hydrocarbons was recorded in mussels in the CEWAF treatments, accompanied by increased biomarker responses. In the WAF treatments these impacts were less evident. Thus, the use of dispersants in the Baltic Sea still needs to be carefully considered.

Cytogenetic damage in native Baltic Sea fish species: environmental risks associated with chemical munition dumping in the Gotland Basin of the Baltic Sea.

This study represents the first attempt to assess genotoxicity and cytotoxicity effects in herring (Clupea harengus membras), flounder (Platichthys flesus), and cod (Gadus morhua callarias) caught at 47 study stations, located close to chemical munition dumpsites in the Gotland Basin, the Baltic Sea. Herring sampled from stations located in the center of chemical munition dumpsites exhibited the highest levels of micronuclei (MN) and total genotoxicity (ΣGentox), which is defined as the sum of frequencies of such nuclear abnormalities as micronuclei, nuclear buds, nuclear buds on the filament, and bi-nucleated erythrocytes with nucleoplasmic bridges. Exceptionally high and high ΣGentox risks were determined for flounder (89.47%), herring (79.31%), and cod (50%) caught at the stations located close to the chemical munition dumpsites.

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.

Biological effects of dumped chemical weapons in the Baltic Sea: A multi-biomarker study using caged mussels at the Bornholm main dumping site.

After World War II, thousands of tons of highly toxic chemical warfare agents (CWA) were deposited in the Baltic Sea, the main dumping site locating in the Bornholm Basin. In the present study, Baltic mussels (Mytilus trossulus) were transplanted in the area in cages at two hotspot sites and a reference site at the depths of 35 and 65 m for 2.5 months to study bioaccumulation and biological effects of CWA possibly leaking from the corroding warfare materials. No traces of degradation products of the measured phenylarsenic CWA could be detected in the tissues of mussels. Nevertheless, several biochemical and histochemical biomarkers, geno- and cytotoxicity indicators, and bioenergetic parameters showed significant responses. The Integrated Biomarker Index calculated from the single biomarkers also showed a higher total response at the two hotspot areas compared to the reference site. Although no direct evidence could be obtained confirming the responses being caused specifically by exposure to CWA, the field exposure experiment showed unambiguously that organisms in this sea area are confronting environmental stress affecting negatively their health and this is likely related to chemical contamination, which is possibly connected to the sea-dumped CWA.

Biomarker responses and accumulation of polycyclic aromatic hydrocarbons in Mytilus trossulus and Gammarus oceanicus during exposure to crude oil.

In the brackish water Baltic Sea, oil pollution is an ever-present and significant environmental threat mainly due to the continuously increasing volume of oil transport in the area. In this study, effects of exposure to crude oil on two common Baltic Sea species, the mussel Mytilus trossulus and the amphipod Gammarus oceanicus, were investigated. The species were exposed for various time periods (M. trossulus 4, 7, and 14 days, G. oceanicus 4 and 11 days) to three oil concentrations (0.003, 0.04, and 0.30 mg L-1 based on water measurements, nominally aimed at 0.015, 0.120, and 0.750 mg L-1) obtained by mechanical dispersion (oil droplets). Biological effects of oil exposure were examined using a battery of biomarkers consisting of enzymes of the antioxidant defense system (ADS), lipid peroxidation, phase II detoxification (glutathione S-transferase), neurotoxicity (acetylcholinesterase inhibition), and geno- and cytotoxicity (micronuclei and other nuclear deformities). In mussels, the results on biomarker responses were examined in connection with data on the tissue accumulation of polycyclic aromatic hydrocarbons (PAH). In M. trossulus, during the first 4 days of exposure the accumulation of all PAHs in the two highest exposure concentrations was high and was thereafter reduced significantly. Significant increase in ADS responses was observed in M. trossulus at 4 and 7 days of exposure. At day 14, significantly elevated levels of geno- and cytotoxicity were detected in mussels. In G. oceanicus, the ADS responses followed a similar pattern to those recorded in M. trossulus at day 4; however, in G. oceanicus, the elevated ADS response was still maintained at day 11. Conclusively, the results obtained show marked biomarker responses in both study species under conceivable, environmentally realistic oil-in-seawater concentrations during an oil spill, and in mussels, they are related to the observed tissue accumulation of oil-derived compounds.

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.

Toxic effects of chemical warfare agent mixtures on the mussel Mytilus trossulus in the Baltic Sea: A laboratory exposure study.

Baltic blue mussels (Mytilus trossulus) were implemented to assess potential toxicity, health impairments and bioaccumulation of dumped chemical warfare agents on marine benthic organisms. Mussels were collected from a pristine cultivation side and exposed under laboratory conditions to different mixtures of chemical warfare agents (CWAs) related phenyl arsenic compounds, Clark I and Adamsite as well as chloroacetophenone. Using a multi-biomarker approach, mussels were assessed thereafter for effects at different organisational levels ranging from geno-to cytotoxic effects, differences in enzyme kinetics and immunological responses. In an integrated approach, chemical analysis of water and tissue of the test organisms was performed in parallel. The results show clearly that exposed mussels bioaccumulate the oxidized forms of chemical warfare agents Clark I, Adamsite (DAox and DMox) and, to a certain extent, also chloroacetophenone into their tissues. Adverse effects in the test organisms at subcellular and functional level, including cytotoxic, immunotoxic and oxidative stress effects were visible. These acute effects occurred even at the lowest test concentration.

Genotoxic, cytotoxic, and neurotoxic responses in Anodonta cygnea after complex metal mixture treatment.

Environmental effects associated with the release of various metals even at maximum permissible concentrations (MPC) to the aquatic ecosystems are evident. In the present work, time-dependent increase in accumulated metals amount in gills of Anodonta cygnea after exposure to complex metal (Zn 0.1, Cu 0.01, Ni 0.01, Cr 0.01, Pb 0.005, and Cd 0.005 mg/L, MPC accepted for the inland waters in EU) mixture at various time points (1, 2, 4, 7, 14, and 28 days) was investigated. Statistically significant increase of Cu and Cd was determined in mussel's gills after 7-day exposure, in comparison to control group; moreover, significantly elevated concentration of Cu was measured and after 14-day treatment (in comparison to control and pre-exposure group). Concentrations of five (Cu, Ni, Cr, Pb, and Cd) out of 6 investigated metals were statistically increased in gills tissue after 28-day treatment. Moreover, complex metal mixture has demonstrated tissue- and time-dependent genotoxicity (∑Gentox) and cytotoxicity (∑Cytox) responses in mussels. After 4-day exposure, there were found the highest ∑Gentox levels in gills cells and haemocytes. Two-day treatment of mussels resulted in the highest and statistically significant induction of ∑Cytox level (in gills). Furthermore, after short-term (4 days) exposure, statistically significant inhibition of AChE activity in hemolymph of metal mixture-exposed mussels, in comparison to control and pre-exposure group, was found. Comparison of investigated responses in different tissue of A. cygnea discloses new information about metal mixture (at MPC) impacts at different treatment time. According to the obtained geno- and cytotoxicity data, it is suggested that gills are more sensitive tissue. Environmentally relevant trace metal concentrations when existing in mixture are able to cause adverse effects in A. cygnea; therefore, biological effects at different levels of organism are expected as a realistic scenario.

Environmental genotoxicity and cytotoxicity levels in herring (Clupea harengus), flounder (Platichthys flesus) and cod (Gadus morhua) inhabiting the Gdansk Basin of the Baltic Sea.

Frequencies of eight nuclear abnormalities (NAs) reflecting environmental genotoxicity and cytotoxicity, were examined in 739 specimens of herring (Clupea harengus), flounder (Platichthys flesus) and cod (Gadus morhua) collected between 2009 and 2017 at 50 study stations located in the Gulf of Gdansk and the southern part of the Gdansk Deep (the Baltic Sea). The highest levels of geno-cytotoxicity were recorded in fish caught at stations located in close proximity to chemical and conventional munitions dumping sites or in zones polluted by Chemical Warfare Agents (CWAs). The values of NAs were significantly higher (up to 51-fold compared with the reference level) in herring caught at seven stations and in cod from one station located close to the munitions dumping sites. Exceptionally high total genotoxicity (∑Gtox) risk was found for flounder collected from 18 (72.0%) stations, herring caught at 12 (38.7%) stations and cod caught at four (17.4%) studied stations.

Environmental genotoxicity and risk assessment in the Gulf of Riga (Baltic Sea) using fish, bivalves, and crustaceans.

Environmental genotoxicity in the Gulf of Riga was assessed using different bioindicators (fish, clams, and isopods) collected from 14 study stations. Comparison of genotoxicity responses (micronuclei (MN) and nuclear buds (NB)) in blood erythrocytes of herring (Clupea harengus), eelpout (Zoarces viviparous), and flounder (Platichthys flesus) revealed the species- and site-specific differences. For the first time, the analysis of genotoxicity was carried out in gill cells of isopods Saduria entomon. The highest inductions of MN and NB in gill cells of investigated S. entomon and clams (Macoma balthica) were evaluated in specimens from station 111A (offshore zone). In fish, the highest incidences of MN were measured in eelpout and in herring collected in the southern part of Gulf of Riga (station GOR3/41S). Moreover, in the southern coastal area, the assessment of genotoxicity risk (according to micronuclei levels) indicated exceptionally high risk for flounder, eelpout, and clams.

Analysis of nuclear abnormalities in erythrocytes of rainbow trout (Oncorhynchus mykiss) treated with Cu and Zn and after 4-, 8-, and 12-day depuration (post-treatment recovery).

The induction of micronuclei (MN), nuclear buds (NB), bi-nucleated erythrocytes with nucleoplasmic bridge (BNb), vacuolated (VacNuc), blebbed (BL), 8-shaped nuclei, bi-nucleated (BN) and fragmented-apoptotic (FA) erythrocytes was analysed in the peripheral blood, cephalic kidney and liver of rainbow trout Oncorhynchus mykiss after 4-day treatment with copper (Cu) and zinc (Zn) mixture solutions and in 4-, 8- and 12-day depuration process. Fish (three treatment and one control group, N=40) were exposed to 0.0625, 0.125 and 0.25 fractions of 96-h LC50, respectively under semi-static conditions. Exposure of O. mykiss to Cu and Zn induced significant increase of MN (in blood in all test groups; in liver 0.125, 0.25 and in kidney 0.25 groups, respectively), NB and BL (in blood and kidney 0.25 group), 8-shaped (in blood 0.25; in liver 0.125, 0.25 and in kidney all test groups, respectively) and VacNuc (in liver and kidney 0.0625 and 0.125 groups). After 4-day recovery, significantly elevated levels of MN (in blood 0.0625, 0.125; in liver and kidney 0.125 group, respectively) and 8-shaped (in kidney-0.0625 group) were observed in fish. Significant recovery was observed in 0.0625 group after 12-day depuration, estimating the formation of MN in erythrocytes of blood, of 8-shaped nuclei erythrocytes in liver and kidney (after 8-, 12-day and 8-day recovery, respectively). Significant decrease of MN in blood (after 8- and 12-day recovery), in liver (after 8-day recovery), of NB in blood and kidney (after 8-day recovery) and of 8-shaped nuclei erythrocytes in blood (after 8 and 12-day recovery), kidney and liver (after 8-day recovery) was determined in 0.25 group. Changes in gross morphometric indices and biological parameters were observed. The binary metal mixture did not induce FA erythrocytes in any tissue at any test concentration.

Environmental genotoxicity assessment along the transport routes of chemical munitions leading to the dumping areas in the Baltic Sea.

The frequencies of micronuclei (MN), nuclear buds (NB) and nuclear buds on filament (NBf) were examined in 660 specimens of herring (Clupea harengus) collected in 2009-2014 at 65 study stations located mainly along the chemical munition transport routes in the Baltic Sea. The frequency of nuclear abnormalities was strongly increased in herring caught at four stations located close to chemical munition dumping sites, or CWAs - substances (chemical warfare agents) in sediments. Significant increase of MN, NB and NBf was observed in fish caught November 2010-2013 compared to 2009. The most significantly increased genotoxicity responses were recorded in fish caught at stations along CW (chemical weapons) transport routes, close to the Bornholm CW dumping area, in zones with CWAs in sediments and with oil-gas platforms.

Assessing the environmental genotoxicity risk in the Baltic Sea: frequencies of nuclear buds in blood erythrocytes of three native fish species.

Nuclear bud (NB) formation was investigated in blood erythrocytes of 1892 flounder Platichthys flesus, herring Clupea harengus, and eelpout Zoarces viviparus specimens that were collected at 82 sites representing different regions of the Baltic Sea in 2009-2011. This is the first attempt to evaluate the baseline levels of NB and rank the genotoxicity risk for native fish species. NB levels were compared to the previously published micronuclei (MN) data from the same individual fish specimens in order to compare the two methods of genotoxicity assessment and investigate the relationship between MN as the cytogenetic measure of genotoxicity and the DNA damage reflecting NB. In 2009-2011, elevated NB levels in 89.4% of flounder sampling groups indicated high and extremely high genotoxicity risk levels. Herring and eelpout sampling groups showed elevated levels of NB, 74.6 and 45.7%, respectively. In general, herring and eelpout NB measure was more sensitive as the genotoxicity biomarker than MN.

Environmental genotoxicity and cytotoxicity in flounder (Platichthys flesus), herring (Clupea harengus) and Atlantic cod (Gadus morhua) from chemical munitions dumping zones in the southern Baltic Sea.

The data on environmental genotoxicity and cytotoxicity levels as well as on genotoxicity risk in flounder (Platichthys flesus), herring (Clupea harengus) and cod (Gadus morhua) collected in 2010-2012 at 42 stations located in chemical munitions dumping areas of the southern Baltic Sea are presented. The frequency of micronuclei, nuclear buds and nucleoplasmic bridges in erythrocytes was used as genotoxicity endpoint and the induction of fragmented-apoptotic, bi-nucleated and 8-shaped erythrocytes as cytotoxicity endpoint. The most significantly increased geno-cytotoxicity levels were determined in fish collected near known chemical munitions dumpsites. Extremely high genotoxicity risk for flounder were identified at 21 out of 24 stations, for herring at 29 out of 31 and for cod at 5 out of 10 stations studied. The reference level of genotoxicity was not recorded at any of the stations revealing that in the sampling area fish were affected generally.