Transformation of phenylarsenic chemical warfare agents and their effect on bacterial communities in Baltic Sea sediment.

During the World Wars large quantities of phenylarsenic chemical warfare agents (CWAs) were dumped in the Baltic Sea. Many transformation products of these chemicals have been identified, but the pathways that produce the found chemicals has not been investigated. Here we studied the biotic and abiotic transformation of phenylarsenic CWAs under oxic and anoxic conditions and investigated how the sediment bacterial communities are affected by CWA exposure. By chemical analysis we were able to identify seventeen CWA-related phenylarsenicals, four of which (methylphenylarsinic acid (MPAA), phenylthioarsinic acid (PTAA), phenyldithioarsinic acid (PDTAA) and diphenyldithioarsinic acid (DPDTAA)) have not been reported for marine sediments before. For the first time PTAA was verified from environmental samples. We also observed equilibrium reactions between the found transformation products, which may explain the occurrence of the chemicals. 16S rRNA-analysis showed that bacterial communities in sediments are affected by exposure to phenylarsenic CWAs. We observed increases in the amounts of arsenic-resistant and sulphur-metabolising bacteria. Different transformation products were found in biotic and abiotic samples, which suggests that bacteria participate in the transformation of phenylarsenic CWAs. We propose that methylated phenylarsenicals are produced in microbial metabolism and that chemical reactions with microbially produced sulphur species form sulphur-containing transformation products.

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.

Exposure to leachates from post-consumer plastic and recycled rubber causes stress responses and mortality in a copepod Limnocalanus macrurus.

Effects of household post-consumer plastics and tyre rubber on a Baltic Sea copepod Limnocalanus macrurus were assessed. Fragments of commercial recycled low-density polyethylene vegetable bags and rubber originating from recycled car tyres were incubated in seawater, and the copepods were exposed to the filtrate of the water. L. macrurus experienced erratic swimming behaviour and increased mortality in the filtrate of unwashed vegetable bags, containing elevated concentrations of alcohols, organic acids and copper. Responses of the antioxidant defence system (ADS) were recorded in copepods exposed to rubber treatments containing high concentrations of zinc. Significant responses in the ADS enzymes indicate that reactive oxygen species (ROS) formation was exceeding the detoxification capacity of the ADS which may further lead to prolonged state of oxidative stress. Observed effects of exposure on the biochemical level coincide with impaired swimming activity of the copepods, indicating possible irreversible cellular responses leading to behavioural changes and mortality.

Metabolism and cytotoxicity of diphenylarsinic acid, a degradation product of sea-dumped chemical warfare agents, in a rainbow trout liver cell line RTL-W1.

Recent studies have found primary degradation products of phenylarsenic chemical warfare agents (CWAs) accumulating in fish tissues, while the potential effects of these dumped phenylarsenic CWAs, such as Clark I and II, in the Baltic Sea biota are poorly understood. In this study, the metabolism and cytotoxicity of diphenylarsinic acid (DPA), a primary degradation product of phenylarsenic CWA, was studied by incubating rainbow trout cell line RTL-W1 cells in media with 100 mg/L DPA. Previously undescribed metabolites were identified by ultra-high performance liquid chromatography-high resolution mass spectrometry (UPHLCHRMS). Moreover, the cytotoxicity of diphenylarsine glutathione conjugate (DPA-SG), the major metabolite of DPA, was studied. Cytotoxicity of the compounds was evaluated using the Neutral Red retention test (NRR), showing an IC50 value of 278 mg/L for DPA and 1.30 mg/L for DPA-SG, indicating that the glutathione (GSH) conjugate of DPA is more than two orders of magnitude toxic than DPA itself, suggesting that toxic properties of DPA are increased after conjugation with intracellular GSH leading enhanced toxicity after uptake. Results gained in this study give more detailed information for elucidating biological effects of dumped chemical munitions in marine environment. Moreover, the results help in assessing the environmental and health risks posed by marine munition continued presence and deterioration in the sea bottom.

Exposure to dissolved TNT causes multilevel biological effects in Baltic mussels (Mytilus spp.).

Baltic mussels (Mytilus spp.) were exposed to the explosive trinitrotoluene (TNT) for 96 h (0.31-10.0 mg/L) and 21 d (0.31-2.5 mg/L). Bioaccumulation of TNT and its degradation products (2- and 4-ADNT) as well as biological effects ranging from the gene and cellular levels to behaviour were investigated. Although no mortality occurred in the concentration range tested, uptake and metabolism of TNT and responses in antioxidant enzymes and histochemical biomarkers were observed already at the lowest concentrations. The characteristic shell closure behaviour of bivalves at trigger concentrations led to complex exposure patterns and non-linear responses to the exposure concentrations. Conclusively, exposure to TNT exerts biomarker reponses in mussels already at 0.31 mg/L while effects are recorded also after a prolonged exposure although no mortality occurs. Finally, more attention should be paid on shell closure of bivalves in exposure studies since it plays a marked role in definining toxicity threshold levels.

Acute aquatic toxicity of arsenic-based chemical warfare agents to Daphnia magna.

Sea dumping of chemical warfare (CW) took place worldwide during the 20th century. Submerged CW included metal bombs and casings that have been exposed for 50-100 years of corrosion and are now known to be leaking. Therefore, the arsenic-based chemical warfare agents (CWAs), pose a potential threat to the marine ecosystems. The aim of this research was to support a need for real-data measurements for accurate risk assessments and categorization of threats originating from submerged CWAs. This has been achieved by providing a broad insight into arsenic-based CWAs acute toxicity in aquatic ecosystems. Standard tests were performed to provide a solid foundation for acute aquatic toxicity threshold estimations of CWA: Lewisite, Adamsite, Clark I, phenyldichloroarsine (PDCA), CWA-related compounds: TPA, arsenic trichloride and four arsenic-based CWA degradation products. Despite their low solubility, during the 48 h exposure, all CWA caused highly negative effects on Daphnia magna. PDCA was very toxic with 48 h D. magna LC50 at 0.36 µg × L-1 and Lewisite with EC50 at 3.2 µg × L-1. Concentrations at which no immobilization effects were observed were slightly above the analytical Limits of Detection (LOD) and Quantification (LOQ). More water-soluble CWA degradation products showed no effects at concentrations up to 100 mg × L-1.

Detection of chemical warfare agent related phenylarsenic compounds and multibiomarker responses in cod (Gadus morhua) from munition dumpsites.

Recently, sea-dumped chemical weapons (CWs) containing toxic chemical warfare agents (CWAs) have raised international attention. It is well known that CWAs are leaking from corroded munitions causing a risk to the surrounding marine environment, while the impact on marine biota is still unknown. In this study, cod (Gadus morhua) was used as a model species to study the possible bioaccumulation of phenylarsenic CWAs and their negative effects at multiple levels of biological organization on fish living in the vicinity of a major CWs dumpsite in the Bornholm Basin in the Baltic Sea. In total, 14% of the cod muscle samples collected close to the main dumpsite contained trace levels of phenylarsenic CWAs. However, most of the biomarkers measured did not show clear differences between this area compared with a lesser contaminated reference area. On the other hand, significant changes in some biomarkers were observed in individuals containing trace levels of CWA-related chemicals. The results gained in this study have significant importance for environmental risk assessment and for evaluating the risk of CWA contamination for human seafood consumers.

Contaminated by war: A brief history of sea-dumping of munitions.

Munitions introduced to the sea during military activities, including naval combat and mine warfare represent only a fraction of military material present in seas and oceans. Huge amounts of obsolete conventional munitions and chemical munitions were dumped to the sea until 1975, when London convention put a stop of sea dumping. Such munitions are a threat for maritime workers, but also for environment. Corroding shells release toxic degradation products to sediments and bottom water, and unlike other contaminants, they cannot be reduced by land measures. Only removal of source can reduce the contamination. Much work has been done in the last decade, and mechanisms of toxicity and bioaccumulation are being recognized, as well as transport and spreading mechanisms. The full assessment of the risk associated with munitions now depends on broad application of developed techniques.

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.

Influence of season-depending ecological variables on biomarker baseline levels in mussels (Mytilus trossulus) from two Baltic Sea subregions.

For reliable mussel monitoring programmes based on biomarkers, regionally relevant reference values and their natural variability need to be known. The Baltic Sea exhibits high inter-regional and seasonal variability in physical factors such as salinity, temperature and primary production. The aim of this pilot study is to depict the effects of season-related environmental factors in a selected battery of biomarkers in two environmentally different subregions of the Baltic Sea to help establishing reference data for biochemical, cellular and tissue-level biomarkers. In order to achieve that, mussels were collected from reference sites in Kiel (Germany) and Tvärminne (Finland) during three seasons: summer and autumn 2016, and spring 2017. Finally, in order to characterize the ecological situation, analysis of the chemical tissue burden was performed and chlorophyll­a and particulate organic carbon concentration and temperature changes were analyzed at each sampling locality using satellite remote sensing images. An integrated biomarker response index was performed to summarize the biomarker responses of each locality and season. The biochemical endpoints showed seasonal variability regulated by temperature, food supply and reproductive cycle, while among the cellular endpoints only lipofuscin accumulation and lysosomal structural changes showed slight seasonal variation. Seasonal changes in tissue level biomarkers were observed only at the northern Baltic Sea site Tvärminne, dictated by the demanding energetic trade-off caused by reproduction. In conclusion, the characterization of the ecological variables and physico-chemical conditions at each site, is crucial to perform a reliable assessment of the effects of a hypothetical pollution scenario in the Baltic Sea. Moreover, reference levels of biomarkers and their responses to natural environmental conditions must be established.

Coupled Application of Antioxidant Defense Response and Embryo Development in Amphipod Crustaceans in the Assessment of Sediment Toxicity.

Survival rate, frequency of malformed embryos, and antioxidant defense system responses in the benthic amphipod Gmelinoides fasciatus from the Baltic Sea were measured to examine the effects of toxic sediments, and to assess the usefulness of these endpoints in sediment toxicity biotesting. A highly contaminated sediment sample from the Baltic Sea was diluted with sediment from a clean site to come up with a series of 5 test sediments with dilutions from 1:32 to 1:1024, and the reference sediment. The 1:32 dilution of the test sediment was analyzed for organotins (2862 µg tin [Sn] kg dry wt -1 ), polycyclic aromatic hydrocarbons (6064 µg kg dry wt -1 ), and selected trace metals (e.g., copper 352 mg kg dry wt -1 ). The survival rate of G. fasciatus (10-d toxicity test) was 100% in the reference and 1:1024 treatments, and began to decline from the 1:256 dilution onward. In a 28-d experiment, various types of morphological malformations were observed in 11 to 80% of the amphipod embryos in the 1:64, 1:128, and 1:256 dilutions, with only <5% in the reference treatment. Also, elevated activities in the antioxidant defense system enzymes glutathione S-transferase and catalase were observed in amphipods exposed to the contaminated sediments compared with the reference treatment, with responses at lower contamination levels compared with the appearance of malformations in the embryos. The results obtained illustrate the effectiveness of the combined application of embryonic malformations and antioxidant defense system biomarkers in amphipods in the assessment of sediment toxicity, and potentially also of sublethal effects of chemical contamination in aquatic ecosystems. Environ Toxicol Chem 2019;38:2020-2031. © 2019 SETAC.

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.

An integrated assessment of pollution and biological effects in flounder, mussels and sediment in the southern Baltic Sea coastal area.

Organic and metal contaminants and biological effects were investigated in flounder, mussels, and sediments in the southern Baltic Sea coastal area in order to assess environmental quality status in that area. Four sites were selected, including two within the Gulf of Gdansk (GoG). In biota and sediment at each site, DDTs dominated over PCBs and PBDEs were the least abundant among organic contaminants. Their concentrations decreased progressively outward from GoG. Among metal contaminants, the levels of Hg, Pb, and Cd were elevated in GoG. Biomarkers in flounder, EROD activity and DNA SB, showed moderate positive correlations with organic and metal contaminants. In flounder, the integrated biomarker index (IBR/n) presented a spatial trend coherent with chemical pollution index (CPI), but there was no clear spatial correspondence between IBR/n and CPI in mussels nor between sediment toxicity index (STI) and sediment CPI. The integrated assessment of contaminant and biological effect data against available assessment criteria indicated that in biota, the contaminant assessment thresholds were most often exceeded by CB-118, heptachlor, PBDE, and Hg (in the GoG sediments by p,p'-DDT, Hg and Cd), while of the biological determinants, the threshold was breeched by AChE activity in mussels in GoG. Applying the ICES/OSPAR traffic-light approach showed that of the 50 parameters assessed at each site, there were 18% of determinants in the red color category in the two GoG sites and 8% of determinants in the two sites outside GoG, which indicated that none of the four investigated sites attained good environmental status (GES).

An integrated chemical-biological study using caged mussels (Mytilus trossulus) along a pollution gradient in the Archipelago Sea (SW Finland, Baltic Sea).

Mussels (Mytilus trossulus) were caged along a known pollution gradient in the inner Archipelago Sea (northern Baltic Sea) and retrieved after 71 and 121 d for the measurement of selected chemical contaminants in tissues and biological endpoints including biochemical biomarkers and growth. Additional samples were collected during the growth season from a native mussel population at an alleged reference site. Elevated concentrations of numerous contaminants (e.g., PAH) were observed in spring, apparently due to the loss of tissue mass during the winter, while also the levels of many biomarkers (e.g., glutathione S-transferase activity) were elevated. Spatial and temporal changes in the accumulation of contaminants and biological parameters were observed with some of them (e.g., growth) linked to seasonal changes in environmental factors. The results underline the importance of understanding the effects of seasonal natural factors on the growth dynamics and general condition of mussels when assessing tissue concentrations of contaminants and biological effects.

The mussel caging approach in assessing biological effects of wastewater treatment plant discharges in the Gulf of Finland (Baltic Sea).

Biological effects of wastewater treatment plant (WWTP) effluents were investigated in Baltic mussels (Mytilus trossulus) caged for one month 800m and 1100m from the WWTP discharge site and at a reference site 4km away. Significant antioxidant, genotoxic and lysosomal responses were observed close to the point of the WWTP discharge. Passive samplers (POCIS) attached to the cages indicated markedly higher water concentrations of various pharmaceuticals at the two most impacted sites. Modeling the dispersal of a hypothetical passive tracer compound from the WWTP discharge site revealed differing frequencies and timing of the exposure periods at different caging sites. The study demonstrated for the first time the effectiveness of the mussel caging approach in combination with passive samplers and the application of passive tracer modeling to examine the true exposure patterns at point source sites such as WWTP pipe discharges in the Baltic Sea.

Effects of 17α-ethinyl estradiol exposure on estrogen receptors α and ß and vitellogenins A, B and C mRNA expression in the liver of sand goby (Pomatoschistus minutus).

This study aims to characterize the estrogen receptor (er) in sand goby (Pomatoschistus minutus) and determine the temporal effects of 17α-ethinyl estradiol (EE2) on erα and vitellogenin (vtg) gene expression in males. Two partial cDNA sequences (erα and erß1) are presented showing conserved structural features with ers of other species. Transcript levels for both ers were low in control fish but EE2 exposure (11 ng/L, for 29 days) increased both to a pattern similar to vitellogenic females. The relative expression of three vtg genes (vtga, vtgb and vtgc) along with erα was determined in control and male fish exposed to EE2 (11 ng/L) at multiple time-points over 29 days. All four transcripts were significantly induced due to exposure and expression rose during the time course with distinct temporal patterns and vtga reached a substantially higher level at the end of the time course coinciding with rapid elevation in erα expression.

Development of tools for integrated monitoring and assessment of hazardous substances and their biological effects in the Baltic Sea.

The need to develop biological effects monitoring to facilitate a reliable assessment of hazardous substances has been emphasized in the Baltic Sea Action Plan of the Helsinki Commission. An integrated chemical-biological approach is vitally important for the understanding and proper assessment of anthropogenic pressures and their effects on the Baltic Sea. Such an approach is also necessary for prudent management aiming at safeguarding the sustainable use of ecosystem goods and Services. The BEAST project (Biological Effects of Anthropogenic Chemical Stress: Tools for the Assessment of Ecosystem Health) set out to address this topic within the BONUS Programme. BEAST generated a large amount of quality-assured data on several biological effects parameters (biomarkers) in various marine species in different sub-regions of the Baltic Sea. New indicators (biological response measurement methods) and management tools (integrated indices) with regard to the integrated monitoring approach were suggested.

Cumulative effects of exposure to cyanobacteria bloom extracts and benzo[a]pyrene on antioxidant defence biomarkers in Gammarus oceanicus (Crustacea: Amphipoda).

The Baltic Sea suffers from extensive blooms of the toxic cyanobacteria Nodularia spumigena that produces nodularin toxin (NOD). Additionally, intensification of oil transportation and related activities in the area increase the risk of oil spills. The current experiment was designed to mimic a situation where an oil spill occurs during a cyanobacterial bloom by exposing the amphipod Gammarus oceanicus to a NOD-rich cyanobacterial extract and the polycyclic aromatic hydrocarbon compound benzo[a]pyrene (B[a]P), a common constituent of oil. The activity of the antioxidant enzymes glutathione S-transferase (GST), glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) were examined after 48 and 96 h of exposure. Exposure to low and high levels of the NOD-rich extract produced a time-dependent activation of GST, GPx and SOD. CAT levels were elevated only by high NOD treatment. Also the toxicity of B[a]P was indicated by significantly elevated antioxidant response. In the combined exposures treatment-dependent additive increases in the activity of GPx and SOD were observed as well as inhibitory (antagonistic) effects on GST, CAT and GPx. Rapid concentration-dependent accumulation of NOD by G. oceanicus was observed. The addition of B[a]P reduced the accumulation of NOD and resulted in different biomarker response patterns compared to single exposures demonstrating the effects of mixture toxicity.