Toxins in mussels (Mytilus galloprovincialis) associated with diarrhetic shellfish poisoning episodes in China

More than 200 people in China suffered illness with symptoms of diarrhetic shellfish poisoning (DSP) following consumption of mussels (Mytilus galloprovincialis). The event occurred in the cities of Ningbo and Ningde near the East China Sea in May, 2011. LC-MS/MS analysis showed that high concentrations of okadaic acid, dinophysistoxin-1, and their acyl esters were responsible for the incidents. The total concentration was more than 40 times the EU regulatory limit of 160 μg OA eq./kg. Pectentoxin-2 and its seco-acids were also present in the mussels. Additionally, yessotoxins were found to be responsible for positive mouse bioassay results on scallop (Patinopecten yessoensis) and oyster (Crassostrea talienwhanensis) samples collected from the North Yellow Sea in June, 2010.This work shows that high levels of lipophilic toxins can accumulate in shellfish from the Chinese coast and it emphasises that adequate chemical analytical methodologies are needed for monitoring purposes. Further research is required to broaden the knowledge on the occurrence of lipophilic toxins in Chinese shellfish.     

First report on toxicity assessment of the Lessepsian migrant pufferfish Lagocephalus sceleratus (Gmelin, 1789) from European waters (Aegean Sea, Greece)

According to the current European Union legislative requirements (Regulation 853/2004/EC; Regulation 854/2004/EC, poisonous fish of the family Tetraodontidae and products derived from them must not be placed on the European markets. Following the increased publicity regarding the presence of the pufferfish species Lagocephalus sceleratus in Greek waters, this study was undertaken in order to confirm its toxicity and assess the risk of poisoning in case of accidental consumption. Acidic extracts from tissues of L. sceleratus specimens of different sizes were examined by means of the official mouse bioassay for tetrodotoxin, while some of the extracts were also tested for the presence of Paralytic Shellfish Poisoning (PSP) toxins with a commercial ELISA kit. Toxicity in mice, with symptomatology indicative of tetrodotoxin, was confirmed in a number of samples and indicated a correlation with fish size. Toxicity of certain tissues (liver, gonads, gastrointestinal tract) in larger individuals, expressed as μg/g tetrodotoxin equivalents, was largely above levels required to cause death in human adults. On the other hand, all tested extracts provided a positive reaction in the ELISA test for PSP toxins. This constitutes the first report for presence of toxicity in L. sceleratus caught in European coastal waters.     

Assimilation,Accumulation, and Metabolism of Dinophysistoxins (DTXs) and Pectenotoxins (PTXs) in the Several Tissues of Japanese Scallop Patinopecten yessoensis

Japanese scallops, Patinopecten yessoensis, were fed with the toxic dinoflagellate Dinophysis fortii to elucidate the relative magnitude of assimilation, accumulation, and metabolism of diarrhetic shellfish toxins (DSTs) and pectenotoxins (PTXs). Three individual scallops were separately exposed to cultured D. fortii for four days. The average cell number of D. fortii assimilated by each individual scallop was 7.7 × 10⁵. Dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2) and their metabolites were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) and the toxin content in individual tissues (digestive gland, adductor muscle, gill, gonad, mantle, and the others), feces and the seawater medium were quantified. Toxins were almost exclusively accumulated in the digestive gland with only low levels being detected in the gills, mantles, gonads, and adductor muscles. DTX1 and PTX2 were the dominant toxins in the D. fortii cells fed to the scallops, whereas the dominant toxins detected in the digestive gland of scallops were PTX6 and esterified acyl-O-DTX1 (DTX3). In other tissues PTX2 was the dominant toxin observed. The ratio of accumulated to assimilated toxins was 21%–39% and 7%–23% for PTXs and DTXs respectively. Approximately 54%–75% of PTX2 and 52%–70% of DTX1 assimilated by the scallops was directly excreted into the seawater mainly without metabolic transformation.     

First evidence of azaspiracids (AZAs): A family of lipophilic polyether marine toxins in scallops (Argopecten purpuratus) and mussels (Mytilus chilensis) collected in two regions of Chile

Azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 following the consumption by consumers of intoxicated shellfish (Mytilus edulis). These azaspiracids have now been identified in mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) from two Chilean locations. This is the first report of the occurrence of azaspiracid toxins in these species (Mytilus chilensis and Argopecten purpuratus) from Chile. The areas studied were Bahía Inglesa (III Region, 27° SL) and Chiloé Archipelago, both important scallop and mussels farming areas. Separation of azaspiracid (AZA1), azaspiracid isomer (AZA6) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and toxins were identified using a turbo electrospray ionisation (ESI) source, to a triple quadrupole mass spectrometer.In mussels, AZA1 was the predominant toxin in mussel hepatopancreas with AZA2, AZA3 and AZA6 present in approximate equivalent amounts in the remaining tissues, 20-30% of the AZA1 level. AZA2 predominated in the scallop samples with the toxin almost entirely present in the hepatopancreas (digestive gland). AZA1 was only observed in some of the scallop samples and was present at 12-15% of the AZA2 levels.Whilst the levels of AZAs in Chilean samples are below the EU regulatory limit of 160 μg/kg, it is significant that this toxin is present in Pacific Ocean species. Consequently measures should be taken by regulatory authorities to implement regular seafood monitoring to ensure safety of harvested product.     

Tetrodotoxin and Its Analogues in Cephalothrix cf. simula (Nemertea: Palaeonemertea) from the Sea of Japan (Peter the Great Gulf): Intrabody Distribution and Secretions

Some nemertean species from the genus Cephalothrix accumulate tetrodotoxin (TTX) in extremely high concentrations. The current study is the first to provide high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) data on tetrodotoxin and its analogues (TTXs) profile and concentration in different regions and organs of Cephalothrix cf. simula, and its secretions produced in response to stimulation. Different specimens of C. cf. simula possessed 7–11 analogues, including nine previously found in this species and two new for nemerteans—4,9-anhydro-8-epi-5,6,11-trideoxyTTX and 1-hydroxy-8-epi-5,6,11-trideoxyTTX. The study of the toxins’ distribution in different regions and organs of nemerteans revealed the same qualitative composition of TTXs throughout the body but differences in the total concentration of the toxins. The total concentration of TTXs was highest in the anterior region of the body and decreased towards the posterior; the ratio of the analogues also differed between regions. The data obtained suggest a pathway of TTXs uptake in C. cf. simula and the role of toxins in the life activity of nemerteans.     

First episode of shellfish contamination by palytoxin-like compounds from Ostreopsis species (Aegean Sea, Greece).

In order to investigate the toxicity of Ostreopsis species present in Greek coastal waters, cultures of Ostreopsis sp. and Ostreopsis ovata, mixed Ostreopsis field populations and shellfish collected from coastal waters of North Aegean Sea during late summer and autumn periods of 2004, 2005 and 2006 were examined by both mouse bioassay (MBA) and hemolysis neutralization assay (HNA). MBA testing was based on two different extraction protocols, while HNA also included the use of ouabain, a known palytoxin (PLT) antagonist. Results indicated the presence of a compound in both Ostreopsis cells and shellfish tissues, which was strongly toxic to mice. This compound exhibited characteristic symptomatology in mice (death, numbness, waddling gait and blindness) to that of PLT, as well as delayed hemolytic activity, which was neutralized by ouabain. HNA indicated that Ostreopsis cells contained a PLT-like compound (putative PLT, p-PLT) at concentrations ranging between 0.4 and 0.9 pg/cell, whereas concentration in shellfish tissues was estimated to range from about 33.3 to 97.0 microg p-PLT/kg tissue. To our knowledge, this is the first report of p-PLT contamination of shellfish by natural Ostreopsis species populations in European coastal waters and possibly globally, and also the first evidence on Ostreopsis cells' toxicity in the Eastern Mediterranean Sea.     

Tetrodotoxins in French Bivalve Mollusks—Analytical Methodology,Environmental Dynamics and Screening of Bacterial Strain Collections

Tetrodotoxins (TTXs) are potentially lethal paralytic toxins that have been identified in European shellfish over recent years. Risk assessment has suggested comparatively low levels (44 µg TTX-equivalent/kg) but stresses the lack of data on occurrence. Both bacteria and dinoflagellates were suggested as possible biogenic sources, either from an endogenous or exogenous origin. We thus investigated TTXs in (i) 98 shellfish samples and (ii) 122 bacterial strains, isolated from French environments. We optimized a method based on mass spectrometry, using a single extraction step followed by ultrafiltration without Solid Phase Extraction and matrix-matched calibration for both shellfish and bacterial matrix. Limits of detection and quantification were 6.3 and 12.5 µg/kg for shellfish and 5.0 and 10 µg/kg for bacterial matrix, respectively. Even though bacterial matrix resulted in signal enhancement, no TTX analog was detected in any strain. Bivalves (either Crassostrea gigas or Ruditapes philippinarum) were surveyed in six French production areas over 2.5–3 month periods (2018–2019). Concentrations of TTX ranged from ‘not detected’ to a maximum of 32 µg/kg (Bay of Brest, 17 June 2019), with events lasting 2 weeks at maximum. While these results are in line with previous studies, they provide new data of TTX occurrence and confirm that the link between bacteria, bivalves and TTX is complex.     

Protein markers of algal toxin contamination in shellfish

Filter-feeding bivalve molluscs are often contaminated by algal toxins. We have probed whether proteomic analysis of extracts from the digestive gland (DG) of mussels could be employed to identify biomarkers of contamination due to okadaic acid-group toxins. The protein extracts were obtained from 18 separate mussel samples and were analyzed by two-dimensional gel electrophoresis. When samples were divided into four different classes based on the content of OA-group toxins in the starting material, we found that two proteins varied as a function of OA contamination. By BLAST analysis, the two proteins were identified as a component of photosystem II and a subunit of NADH dehydrogenase. The analysis of peptide homologies showed that the peptide of photosystem II we detected in extracts from the DG of mussels contaminated by OA-group toxins is identical to its counterpart in Dinophysis algae, which are the producers of this group of toxins. We concluded that proteomic analysis can be used for the detection and identification of biomarkers of biotoxin contamination in shellfish, including both proteins expressed by the toxin producers and components that participate to the tissue response to the exogenous bioactive contaminant.     

Study of Adsorption and Flocculation Properties of Natural Clays to Remove Prorocentrum lima

High accumulations of phytoplankton species that produce toxins are referred to as harmful algal blooms (HABs). HABs represent one of the most important sources of contamination in marine environments, as well as a serious threat to public health, fisheries, aquaculture-based industries, and tourism. Therefore, methods effectively controlling HABs with minimal impact on marine ecology are required. Marine dinoflagellates of the genera Dinophysis and Prorocentrum are representative producers of okadaic acid (OA) and dinophysistoxins responsible for the diarrhetic shellfish poisoning (DSP) which is a human intoxication caused by the consumption of shellfish that bioaccumulate those toxins. In this work we explore the use of natural clay for removing Prorocentrum lima. We evaluate the adsorption properties of clays in seawater containing the dinoflagellates. The experimental results confirmed the cell removal through the flocculation of algal and mineral particles leading to the formation of aggregates, which rapidly settle and further entrain cells during their descent. Moreover, the microscopy images of the samples enable one to observe the clays in aggregates of two or more cells where the mineral particles were bound to the outer membranes of the dinoflagellates. Therefore, this preliminary data offers promising results to use these clays for the mitigation of HABs.     

Azaspiracid poisoning (AZP) toxins in shellfish: Toxicological and health considerations

It has been almost a decade since a previously unknown human toxic syndrome, azaspiracid poisoning (AZP), emerged as the cause of severe gastrointestinal illness in humans after the consumption of mussels (Mytilus edulis). Structural studies indicated that these toxins, azaspiracids, were of a new unprecedented class containing novel structural features. It is now known that the prevalent azaspiracids in mussels are AZA1, AZA2 and AZA3, which differ from each other in their degree of methylation. Several hydroxylated and carboxylated analogues of the main azaspiracids have also been identified, presumed to be metabolites of the main toxins. Since its first discovery in Irish mussels, the development of facile sensitive and selective LC-MS/MS methods has resulted in the discovery of AZA in other countries and in other species. Mice studies indicate that this toxin class can cause serious tissue injury, especially to the small intestine, and chronic exposure may increase the likelihood of the development of lung tumours. Studies also show that tissue recovery is very slow following exposure. These observations suggest that AZA is more dangerous than the other known classes of shellfish toxins. Consequently, in order to protect human consumers, proper risk assessment and regulatory control of shellfish and other affected species is of the utmost importance.     

A feasibility study into the provision of Paralytic Shellfish Toxins laboratory reference materials by mass culture of Alexandrium and shellfish feeding experiments

As the official control laboratory for biotoxin testing in England, Wales and Scotland, Cefas employs two approaches for the detection of Paralytic Shellfish Poisons (PSP) in bivalve shellfish: a qualitative HPLC method for oysters, whole king scallops and cockles (with PSP bioassay confirmation of positive HPLC samples) with subsequent quantitation of positive samples by mouse bioassay and a quantitative HPLC method for mussels (no PSP bioassay confirmation required). To aid the validation of the quantitative HPLC method for native oysters, Pacific oysters, cockles and king scallops and ultimately remove the need for the PSP bioassay for these species, appropriate contaminated shellfish matrices were required. As it was not possible to obtain naturally contaminated material for these species, shellfish were contaminated in-house through feeding experiments with high concentrations of Alexandrium species. A number of feeding experiments with two Alexandrium strains were performed successfully. The contaminated shellfish materials generated contained a number of different profiles of PSP toxins.This work has demonstrated the feasibility of these methods for the production of laboratory reference materials in a variety of bivalve shellfish species. Based on this study laboratory reference material production via these methods is now undertaken routinely within Cefas. By running two concurrent feeding trials per year for each species, enough laboratory reference material is produced for approximately 1 year of the programme. This removes the necessity for natural contaminated material which is not always available for reference material production. Additionally, such materials enable both the comparative testing of different PSP methodologies and the ongoing generation of long-term precision data for the HPLC method.     

First U.S. report of shellfish harvesting closures due to confirmed okadaic acid in Texas Gulf coast oysters

Between March 7 and April 12, 2008, several bay systems on the east (Gulf of Mexico) coast of Texas, USA were closed to the harvesting of oysters (Crassostrea virginica) due to the presence of the DSP (Diarrheic Shellfish Poisoning) toxin okadaic acid in excess of the 20 μg/100 g tissue FDA regulatory guidance level. This was the first shellfish harvesting closure due to the confirmed presence of DSP toxins in US history. Light microscopic cell counts were performed on water samples collected from numerous sampling sites along the Texas Gulf coast where shellfish harvesting occurs. Ultra performance liquid chromatography, electrospray ionization, selected reaction monitoring, mass spectrometry (UPLC/ESI/SRM/MS) was used to detect DSP toxins in oysters. The closures were associated with an extensive bloom of the dinoflagellate Dinophysis cf. ovum. Only okadaic acid (OA) and OA acyl esters were found in shellfish tissues (max. OA eq. levels 47 μg/100 g tissue). OA was also confirmed in a bloom water sample. No illnesses were reported associated with this event. DSP toxins now add to a growing list of phycotoxins, which include those responsible for PSP (paralytic shellfish poisoning), NSP (neurotoxic shellfish poisoning), and ASP (amnesic shellfish poisoning) which must now be monitored for in US coastal waters where shellfish are harvested.     

Combined Effects of Temperature and Toxic Algal Abundance on Paralytic Shellfish Toxic Accumulation,Tissue Distribution and Elimination Dynamics in Mussels Mytilus coruscus

This study assessed the impact of increasing seawater surface temperature (SST) and toxic algal abundance (TAA) on the accumulation, tissue distribution and elimination dynamics of paralytic shellfish toxins (PSTs) in mussels. Mytilus coruscus were fed with the PSTs-producing dinoflagellate A. catenella under four simulated environment conditions. The maximum PSTs concentration was determined to be 3548 µg STX eq.kg⁻¹, which was four times higher than the EU regulatory limit. The increasing SST caused a significant decline in PSTs levels in mussels with rapid elimination rates, whereas high TAA increased the PSTs concentration. As a result, the PSTs toxicity levels decreased under the combined condition. Additionally, toxin burdens were assessed within shellfish tissues, with the highest levels quantified in the hepatopancreas. It is noteworthy that the toxin burden shifted towards the mantle from gill, muscle and gonad at the 17th day. Moreover, variability of PSTs was measured, and was associated with changes in each environmental factor. Hence, this study primarily illustrates the combined effects of SST and TAA on PSTs toxicity, showing that increasing environmental temperature is of benefit to lower PSTs toxicity with rapid elimination rates.     

Investigation of the toxin profile of Greek mussels Mytilus galloprovincialis by liquid chromatography-mass spectrometry.

Samples of Mytilus galloprovincialis were harvested from five different locations in Thermaikos gulf, Greece after harmful algae bloom. All of the mussel samples were found positive by mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins. Liquid chromatography (LC) coupled with mass spectrometry (MS) was used to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and yessotoxins (YTXs). In order to investigate the presence of okadaic acid esters, alkaline hydrolysis was performed for all the samples, and LC-MS analyses were carried out on the samples before and after hydrolysis. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) analyses were also carried out to investigate the presence of domoic acid and paralytic shellfish poisoning (PSP) toxins at trace levels. All of the samples were found to be contaminated only with okadaic acid at levels 0.10-0.20 microg/g.     

Tetrodotoxin and Its Analogues in the Pufferfish Arothron hispidus and A. nigropunctatus from the Solomon Islands: A Comparison of Their Toxin Profiles with the Same Species from Okinawa,Japan

Pufferfish poisoning has not been well documented in the South Pacific, although fish and other seafood are sources of protein in these island nations. In this study, tetrodotoxin (TTX) and its analogues in each organ of the pufferfish Arothron hispidus and A. nigropunctatus collected in the Solomon Islands were investigated using high resolution LC-MS. The toxin profiles of the same two species of pufferfish from Okinawa, Japan were also examined for comparison. TTXs concentrations were higher in the skin of both species from both regions, and relatively lower in the liver, ovary, testis, stomach, intestine, and flesh. Due to higher TTX concentrations (51.0 and 28.7 µg/g at highest) detected in the skin of the two species from the Solomon Islands (saxitoxin was <0.02 µg/g), these species should be banned from consumption. Similar results were obtained from fish collected in Okinawa, Japan: TTX in the skin of A. hispidus and A. nigropunctatus were 12.7 and 255 µg/g, respectively, at highest, and saxitoxin was also detected in the skin (2.80 µg/g at highest) and ovary of A. hispidus. TTX, 5,6,11-trideoxyTTX (with its 4-epi form), and its anhydro forms were the most abundant, and 11-oxoTTX was commonly detected in the skin.     

Paralytic Shellfish Poisoning (PSP) in Mussels from the Eastern Cantabrian Sea: Toxicity,Toxin Profile,and Co-Occurrence with Cyclic Imines

In the late autumn of 2018 and 2019, some samples taken by the official monitoring systems of Cantabria and the Basque Country were found to be paralytic shellfish poisoning (PSP)-positive using a mouse bioassay. To confirm the presence of PSP toxins and to obtain their profile, these samples were analyzed using an optimized version of the Official Method AOAC 2005.06 and using LC–MS/MS (HILIC). The presence of some PSP toxins (PSTs) in that geographical area (~600 km of coast) was confirmed for the first time. The estimated toxicities ranged from 170 to 983 µg STXdiHCl eq.·kg⁻¹ for the AOAC 2005.06 method and from 150 to 1094 µg STXdiHCl eq.·kg⁻¹ for the LC–MS/MS method, with a good correlation between both methods (r² = 0.94). Most samples contained STX, GTX2,3, and GTX1,4, and some also had NEO and dcGTX2. All of the PSP-positive samples also contained gymnodimine A, with the concentrations of the two groups of toxins being significantly correlated. The PSP toxin profiles suggest that a species of the genus Alexandrium was likely the causative agent. The presence of gymnodimine A suggests that A. ostenfeldii could be involved, but the contribution of a mixture of Alexandrium species cannot be ruled out.     

A convenient and cost-effective method for monitoring marine algal toxins with passive samplers

Passive sampling disks were developed based on the method of MacKenzie, L., Beuzenberg, V., Holland, P., McNabb, P., Selwood, A. [2004. Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves. Toxicon 44, 901-918] and protocols were formulated for recovering toxins from the adsorbent resin via elution from small columns. The disks were used in field studies to monitor in situ toxin dynamics during mixed algal blooms at Flødevigen in Norway. Examples are given from time-integrated sampling using the disks followed by extraction and high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis for azaspiracids, okadaic acid analogues, pectenotoxins, yessotoxins and spirolides. Profiles of accumulated toxins in the disks and toxin profiles in blue mussels (Mytilus edulis) were compared with the relative abundance of toxin-producing algal species. Results obtained showed that passive sampling disks correlate with the toxin profiles in shellfish. The passive sampling disks were cheap to produce and convenient to use and, when combined with HPLC-MS or enzyme-linked immunosorbent assay (ELISA) analysis, provide detailed time-averaged information on the profile of lipophilic toxin analogues in the water. Passive sampling is therefore a useful tool for monitoring the exposure of shellfish to the toxigenic algae of concern in northern Europe.     

Complex toxin profile of Mytilus galloprovincialis from the Adriatic sea revealed by LC–MS

This paper reports on the determination of toxin profile of mussels (Mytilus galloprovincialis) collected in November–December 2003 along the Emilia Romagna coasts (Italy) when a high concentration of Alexandrium ostenfeldii cells was detected in seawater. Detailed liquid chromatography–mass spectrometry (LC–MS) analyses were performed on the crude extracts in both selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes. They revealed that M. galloprovincialis had accumulated the three major spirolides produced by the alga, namely 13-desMethyl spirolide C, 13,19-didesMethyl spirolide C and 27-hydroxy-13,19-didesMethyl spirolide C, which fully accounted for toxicity of lipophilic extracts shown in mouse bioassay. Interestingly, yessotoxin (YTX) and its analogues were still present in mussel polar extracts but YTX itself was not the major toxin contained in mussels. The presence of pectenotoxin-2 seco acid (PTX-2sa) and its putative epimer was also assessed. The presence of azaspiracids, never reported from the Adriatic sea, as well as of diarrhetic shellfish poisoning toxins (okadaic acid, dinophysistoxins and OA esters) and domoic acid, long known as contaminants of Adriatic mussels, was also investigated.     

Toxicity of French strains of the dinoflagellate Prorocentrum minimum experimental and natural contaminations of mussels

Mediterranean strains of Prorocentrum minimum do not appear to have the same toxic component as Japanese strains since they showed no cytotoxicity for hepatocytes in culture. However, their toxic components, which appear to block calcium channels, were detectable by the immobilisation test on Diptera larvae. A bio-accumulation experiment in the laboratory showed that the toxins could accumulate in nearly equivalent amounts in the hepatopancreas and meat of cultured mussels. The same toxicity was found in natural samples collected in a period of bloom of P. minimum. These results suggest that P. minimum could be responsible for shellfish toxicity in the natural environment and thus present a risk for human health.     

Dynamics of glutathione-S-transferases in Mytilus galloprovincialis exposed to toxic Microcystis aeruginosa cells, extracts and pure toxins

Molluscs and especially bivalves are able to accumulate dinoflagelates, diatoms and cyanobacteria toxins, and, being vectors for these toxins, transfer them along food chains. The data obtained from laboratory experiments showed that bivalve molluscs are resistant to cyanobacteria toxins. In this work, we wanted to test if Mytilus galloprovincialis organs react to microcystins and other cyanobacteria compounds by inducing or decreasing its GST activity. Acclimated mussels M. galloprovincialis were exposed to the toxic Microcystis aeruginosa M13 strain. Exposure of mussels to toxins was done in three ways: living Microcystis cells, crude Microcystis extracts and pure toxins. The measurement of soluble and microsomal GST activity in the different mussel organs was done by using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dichloro-1-nitrobenzene (DCNB). Analysis of the GST activity of the control mussels using CDNB as a substrate showed that cytosolic activity is much more significant than microsomal. Intact M. aeruginosa cells did not induce any significant response from the mussels, showing that these animals are quite resistant to the cyanobacteria if they are intact. On the other hand, cell extracts caused an important effect in the gut, in the gills and in the labial palps, although in different ways. There was an increase in GST activity in the gut and gills of mussels exposed to Microcystis extracts, showing a response of this detoxication pathway, but in the labial palps a severe reduction in GST activity occurred. Pure MC LR+YR induced an increase in GST activity in all organs but the labial palps. The results showed that other substances apart from microcystins may cause stress to mussels and affect detoxication enzymes such as GST.