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.

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.

A case study on microlitter and chemical contaminants: Assessing biological effects in the southern coast of the Gulf of Finland (Baltic sea) using the mussel Mytilus trossulus as a bioindicator.

Chemical and microlitter (ML) pollution in three Estonian coastal areas (Baltic Sea) was investigated using mussels (Mytilus trossulus). Polycyclic aromatic hydrocarbons (PAH) in mussel tissues were observed in moderate levels with high bioaccumulation factors for the more hydrophilic and low molecular weight PAH (LMW PAH), namely anthracene and fluorene. Tissue concentrations of polybrominated diphenyl ethers (PBDE) and cadmium within mussel populations exceeded the Good Environmental Status thresholds by more than 200% and 60%, respectively. Multiple contamination at the Muuga Harbour site by tributyltin, high molecular weight PAH, including the highly toxic benzo[c]fluorene and PBDE, coincided with the inhibition of acetylcholinesterase activity and a lower condition index of the mussels. The metabolization and removal of bioaccumulated LMW PAH, reflected in the dominance of oxy-PAH such as anthracene-9,10-dione, is likely associated with the increased activity of glutathione S-transferase in caged mussels. Only a few microplastic particles were observed among the ML in mussel tissues, with coloured cellulose-based microfibers being the most prevalent. The average concentration of ML in mussels was significantly higher at the harbour area than at other sites. The integrated biomarker response index values allowed for the differentiation of pollution levels across studied locations representing high, intermediate, and low pollution levels within the studied area.

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.

Histopathological, histomorphometrical, and immunohistochemical biomarkers in flounder (Platichthys flesus) from the southern Baltic Sea.

Flounder (Platichthys flesus), collected in late fall of 2009 from four coastal sites in the southern Baltic Sea including the Gulf of Gdansk (GoG), were investigated for a suite of biomarkers of contaminant effects. The biomarkers included liver histopathologies, which were diagnosed and assessed using commonly applied lesion categories, the size and density of melano-macrophage aggregates (MMAs), expression of proliferating cell nuclear antigen (PCNA) and of cytochrome P450 1A (CYP1A) in liver as well as the size and density of MMAs and density of Perls'-positive cells in the spleen. The prevalence of liver lesions differed among the sites. Most frequently occurring were non-specific and early toxicopathic non-neoplastic lesions. Mean MMA size was in the range of 264-519 µm(2) and 717-2137 µm(2) in liver and spleen, respectively, and density was in the range of 6-13 and 15-26 MMAmm(-2), respectively. Mean density of PCNA-positive hepatocytes was in the range of 300-1281 cellsmm(-2). These histomorphometrical biomarkers correlated positively with the muscle Hg, Σ(7)PCB, and ΣDDT residues and negatively with the indices of general liver condition. They showed significant differences between the sites, which were in line with the spatial prevalence of liver lesions and CYP1A induction. Overall, the biomarker responses were more pronounced in the GoG sites in comparison to those outside the Gulf, which confirms some earlier results and broadens the knowledge of contaminant effects in the Polish coastal zone of the Baltic Sea.

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 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.

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.

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.

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.

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.

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.

Sand goby (Pomatoschistus minutus) males exposed to an endocrine disrupting chemical fail in nest and mate competition.

Endocrine disrupting chemicals (EDCs) are a widely studied group of chemicals that interfere with the endocrinology of organisms. So far, few studies have demonstrated the effect of EDCs on the reproductive behavior of aquatic wildlife. Here we show that sand goby males' (Pomatoschistus minutus) success in mating competition greatly decreases after an exposure for 7 to 24 days to 17alpha-ethinyl estradiol (EE2, measured concentration 4 ng L(-1)). The sand goby exhibits a polygynous mating system with male parental care, in which males compete for nest sites and females. The aim of this study was to test how EE2 exposure affects the ability of males to compete for breeding resources, i.e. nest sites and mates. First, EE2 exposed males competed over a nest site against a non-exposed, control male of the same size. Secondly, we examined male courtship behavior and female mate preferences for EE2 exposed males and similar-sized non-exposed, control males. In addition to the behavioral experiments we determined the zona radiata protein (Zrp) mRNA gene expression and measured morphometric indicators of sexual maturation. Our study revealed that EE2 treated males were not able to acquire or defend a nest site. Additionally, EE2 treated males spent significantly less time in active courtship and nest leading behavior than control males. As a result, females clearly preferred to mate with control males. However, we found no significant differences in Zrp mRNA expression or the morphometric indicators between treatments. Our study illustrates that exposure to this EDC can greatly reduce the chances of an individual reproducing successfully. Moreover, it demonstrates that severe behavioral effects can be seen before any effects are detectable at the molecular or morphometric level.

Disruption of sexual selection in sand gobies (Pomatoschistus minutus) by 17alpha-ethinyl estradiol, an endocrine disruptor.

In aquatic environments, endocrine disrupting chemicals (EDCs) that interfere with the reproductive physiology of males form a threat to the reproduction of populations. This is often manifested as decreased sexual performance or sterility among males. We show that exposure to EDCs can directly affect the mating system of a marine fish, the sand goby (Pomatoschistus minutus). We exposed males for 1 to 4 weeks to two different concentrations (5 ng L(-1) and 24 ng L(-1)) of 17alpha-ethinyl estradiol (EE2); a synthetic compound mimicking estrogen and a water control. The sand goby exhibits a polygynous mating system, in which male mating success is typically skewed towards the largest males, resulting in strong sexual selection for increased male size. Our experiment shows that when males have been exposed to EE2, male size has a smaller effect on mating success, resulting in weaker sexual selection on male size as compared to the control. There was an interaction between treatment and exposure time on the expression of vitellogenin and zona radiata protein mRNAs. Males exposed to high EE2 reached much higher expression levels than males exposed to low EE2. Of the somatic markers, the hepatosomatic index was lower in males exposed to high EE2 than in the low EE2 and control males. Our results suggest that exposure to EDCs can have effects on the mating system before physiological changes are observable. These effects can be of profound nature as they interfere with sexual selection, and may in the long run lead to the loss of traits maintained through sexual selection.

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.

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.

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.

DNA damage, acetylcholinesterase activity and lysosomal stability in native and transplanted mussels (Mytilus edulis) in areas close to coastal chemical dumping sites in Denmark.

Biomarkers of genotoxicity (DNA damage, measured as tail moment in the Comet assay), neurotoxicity (acetylcholinesterase inhibition, AChE) and general stress (lysosomal membrane stability, LMS) were studied in native and transplanted blue mussels (Mytilus edulis) in coastal areas of western Denmark potentially affected by anthropogenic pollution originating from chemical dumping sites. The results indicate responses to pollution in all the biomarkers applied at the suspected areas, but the results were not consistent. Seasonal fluctuations in exposure situations at the study sites make interpretation of chemical pollution complex, as seen especially in the variability in results on DNA damage, and also in regard to AChE activity. These investigations further stress the importance of understanding the effects of natural factors (salinity, temperature, water levels, rain and storm events) in correct interpretation of the biomarker data obtained. In addition, adaptation of populations to local contamination may play a role in some of the response patterns observed.

Accumulation and depuration of cyanobacterial toxin nodularin and biomarker responses in the mussel Mytilus edulis.

Blue mussels (Mytilus edulis) were exposed to an extract made of natural cyanobacterial mixture containing toxic cyanobacterium Nodularia spumigena (70-110 microg nodularin l(-1), 24-h exposure followed by 144-h depuration period in clean water). Toxin concentration increased from initial 400 to 1100 mg kg(-1) after 24-h exposure, measured by liquid chromatography/mass spectrometry (LC/MS). Acetylcholinesterase activity (AChE), a biomarker of direct neurotoxic effects, showed inhibition after 12 and 24h exposure but returned to control level during the depuration period. Catalase (CAT) activity, an indicator of oxidative stress, showed significantly elevated levels in exposed mussels but only 72 h after the end of the exposure. No change in the activity of glutathione-S-transferase (GST) involved in conjugation reactions could be observed. A gradual yet incomplete elimination of nodularin (from 1100 to 600 mg kg(-1)) was observed during the depuration period, and the tissue levels were 30% lower in clean water after 24 h. The observed increase in oxidative stress indicated by elevated CAT activity is likely connected to detoxification reactions leading to the production of reactive oxygen species, including an apparent time lag in this specific enzymatic defence response. That no change in GST activity was observed suggests that this enzyme is not significantly involved in the detoxification process of nodularin-containing cyanobacterial extract in M. edulis.