Occurrence of toxic blue-green algae in the Kucukcekmece lagoon (Istanbul, Turkey)

The concentration of microcystin (MC) in the Kucukcekmece Lagoon, Istanbul, Turkey, and the physicochemical and biological parameters of water quality were investigated from October 2000 to June 2003. Water samples were collected from surface waters at three sites. Most bloom samples were dominated by Microcystis aeruginosa. The major microcystin variants detected by HPLC-PDA were microcystin-YR and microcystin-LR. Microcystin concentration increased dramatically from early summer to early autumn and thereafter tended to decrease. The toxin concentration found in the filtered samples from surface waters varied between 0.06 and 24.2 microg L(-1) microcystin-LR equivalents. Each year extensive fish mortality was recorded between mid-June and early October, coinciding with heavy algal blooms. A comparison of the conditions associated with cyanotoxin episodes in 2000, 2001, and 2002 showed that the microcystin increase was related to temperature, high concentration of dissolved nutrients, high light intensity (PAR). The highest MC concentrations were recorded at temperatures between 24 degrees C and 28.5 degrees C. Field data showed that the highest MC concentration (>3 microg L(-1)) and the highest cyanobacterial biomass (>30 mg L(-1)) corresponded to a total nitrogen:total phosphorus ratio greater than 7:1. The highest concentrations of M. aeruginosa biomass (173 mg L(-1)) and MC (24.2 microg L(-1) MC-LR equiv.) and the highest salinity (8.8%) were measured concurrently in the lagoon. To our knowledge, this is the first evidence of cyanobacterial toxins in the Kucukcekmece Lagoon.     

Effects of cyanobacterial crude extracts from Planktothrix agardhii on embryo-larval development of medaka fish, Oryzias latipes.

Embryonic toxicity from exposure to microcystins, cyclic hepatotoxic heptapeptides from cyanobacteria, receives increasing attention as a public human health biohazard. Using a microinjection technology, we have introduced cyanobacterial extracts from Planktothrix agardhii directly into the vitellus of late neurula embryos (stage 19) of medaka (Oryzias latipes). Microinjection (2 nL) of P. agardhii PMC 75.02 extract containing microcystins (MC) resulted in a dose-dependent mortality of embryos. Survival rates were reduced up to 81% with extract concentrations of 10 mg mL(-1) (EC(50)=7.8 microg mL(-1)). On the other hand, injection of P. agardhii PMC 87.02 extract in which no microcystin could be detected resulted in much less embryonal toxicity (EC(50)=460 microg mL(-1)). In addition, advanced embryonic hatching processing was limited with PMC 75.02 crude extract and less obvious than had been described with pure MC-LR injections. In agreement with the known hepatotoxic effects of microcystin, embryos injected with PMC 75.02 extract consistently displayed hepatobiliary abnormalities. Loss of glycogen content of the hepatocytes and hepatic haemorrhage were evidenced in surviving post-hatching juveniles. Thus, the methodology presented in this paper should be a valuable tool to analyse the effects of crude extracts of cyanobacterial toxins on the development of aquatic vertebrate embryos.     

Microcystins associated with Microcystis dominated blooms in the Southwest wetlands, Western Australia

Potentially toxic cyanobacterial blooms are becoming common in the freshwater wetlands on the Swan Coastal Plain, Western Australia. During summer the dominant bloom-causing species belong to the genera Microcystis and Anabaena and to a lesser extent Aphanizomenon and Nodularia. Although toxic cyanobacteria have been recorded in the Swan-Canning and Peel-Harvey estuaries in Western Australia, very little is known about the blooms in the surrounding freshwater lakes. In this study, a total of 32 natural bloom samples representing 13 lakes were analyzed by HPLC for microcystin (MC)-LR, -RR, and -YR. Twenty-eight samples proved to be toxic. The highest total microcystin concentration ranged from 1645 to 8428.6 microg L(-1), and the lowest concentrations were less than 10 microg L(-1) with some below the detection limit (< 0.05 microg L(-1)). MC-LR (100%) was the predominant microcystin, followed by MC-YR (71.4%) and MC-RR (60.7%). The presence of a Nodularia spumigena bloom in the freshwater Lake Yangebup was associated with the detection of nodularins (1664 microg L(-1)). This is the first study to demonstrate the presence of microcystins and nodularins in urban lakes on the Swan Coastal Plain, Western Australia.     

Diversity of hepatotoxic microcystins and bioactive anabaenopeptins in cyanobacterial blooms from Greek freshwaters

Microcystins (MCs) and anabaenopeptins from 26 cyanobacterial bloom samples dominated mainly by the genus Microcystis and collected from seven Greek freshwaters were identified and quantified by high-performance liquid chromatography coupled to a diode array detector. All the samples analyzed contained microcystins; in 27% of the samples anabaenopeptins were detected but not anabaenopeptilide (A). In each sample 1-7 microcystins and up to two anabaenopeptins (anabaenopeptins A and B) were identified. MC-RR and MC-LR were the predominant microcystins, followed by MC-YR. MC-LA and demethylated variants of MC-LR and MC-RR also were present but were not abundant. Total content of microcystin and anabaenopeptin varied from 40 to 2565 microg g(-1) freeze-dried material (mean 674.5 microg g(-1)) and from undetectable to 48 microg g(-1) freeze-dried material (mean 6.2 microg g(-1)), respectively. Qualitative and quantitative variation in the microcystins in the samples indicates there may be geographical trends in the distribution of microcystins. This study reports for the first time (1) the widespread occurrence of several different microcystins in Greek freshwaters and (2) quantitative data on the anabaenopeptins produced in natural cyanobacterial populations.     

The influence of microcystin-LR and hepatotoxic cyanobacterial extract on the water plant Spirodela oligorrhiza

The eutrophication of the Sulejów Reservoir dam in Poland is related to toxicity from cyanobacterial blooms. The main species responsible for hepatotoxic bloom formation is Microcystis aeruginosa. The aim of this study was to evaluate the influence of toxic cyanobacterial extract on the growth and morphology of the water plant Spirodela oligorrhiza, compared with commercial-grade microcystin-LR (MC-LR). It was found that after 96 h of incubation the highest concentration of cyanobacterial extract, containing 0.344 mg MC-LR/L, reduced the number of fronds by about 50% in comparison with the control. The extract effected a reduction in the frond mass and a decrease in chlorophyll (a + b) concentration. A reduction in the number of fronds was also observed after the first 24 h of incubation in the presence of 0.2 and 0.1 microg/L of commercial-grade MC-LR. Changes in activity of constitutive acid phosphatase and RNase after 7 days of incubation with commercial-grade MC-LR were observed. The results confirm the toxicity of cyanobacterial hepatotoxins to Spirodela oligorrhiza, which can be used as a sensitive bioindicator.     

Microcystin concentrations and genetic diversity of Microcystis in the lower Great Lakes

The resurgence of Microcystis blooms in the lower Great Lakes region is of great concern to public and ecosystem health due to the potential for these colonial cyanobacteria to produce hepatotoxic microcystins. A survey of Microcystis cell densities and microcystin concentrations during August 2004 showed particularly high concentrations of both cells and toxin in the nearshore regions of Saginaw Bay (Lake Huron) and western Lake Erie, often exceeding the World Health Organization's recommended drinking water limit of 1 microg L(-1). The dominant congener of microcystin in both basins was microcystin-LR (MC-LR), whereas the second most abundant congeners, accounting for up to 20-25% of the total microcystin concentrations, were MC-LA in Saginaw Bay and MC-RR in western Lake Erie. Multiplex PCR assays of Microcystis colonies isolated from these two regions showed that a much greater percentage of the Microcystis colonies from Saginaw Bay carried the mcyB gene necessary for microcystin production, in comparison with those from western Lake Erie. The mcyB genotypes sequenced separated into two distinct phylogenetic clusters, with Microcystis originating from Lake Erie predominantly in one branch and from Saginaw Bay present in both branches. These results indicate that the genetic composition of the bloom could impact the concentrations and congeners of microcystin produced and that the cell count methods currently being used to gauge public health threats posed by Microcystis blooms may not sufficiently assess actual bloom toxicity.     

Effects of cyanobacterial biomass and purified microcystins on malformations in Xenopus laevis: teratogenesis assay (FETAX)

Xenopus laevis (African clawed frog) embryos in a 96-h teratogenesis assay (FETAX) were exposed to 0-250 microg/L and 500 microg/L of purified microcystin-LR (MCYST-LR) for the estimation of lethality, as well as to equivalent concentrations of biomass containing MCYST-LR (natural water bloom dominated by Microcystis aeruginosa) and biomass without MCYST-LR (bloom dominated by Microcystis wesenbergii). The highest tested concentrations of purified MCYST-LR caused up to 30% lethality after a 96-h exposure, corresponding to a LC(25) of 380 microg/L. Cyanobacterial biomass containing MCYST-LR caused significant lethality up to 50% at the highest tested concentrations (300 mg/L, i.e., 250 microg/L of MCYST-LR). The estimated 96-h LC(25) values varied from 125 mg/L (biomass containing MCYST-LR) up to 232 mg/L (biomass without MCYST-LR). A statistically significant increase in the number of malformed embryos was observed after exposure to cyanobacterial samples. Purified MCYST-LR at and above 25 microg/L significantly increased the number of malformations, with 53% of surviving embryos malformed in the highest tested concentration, 250 microg/L (EC(25) = 27 microg/L). Exposure to the highest concentration of MCYST-LR containing biomass resulted in more than 60% of the embryos being malformed and an EC(25) of 52 mg/L (i.e., 43 microg of MCYST-LR/L). Cyanobacterial biomass with no natural microcystin also induced substantial malformations-about 50% aberrant embryos at the highest concentration, 300 mg/L (EC(25) = 75 mg/L). External additions of purified MCYST-LR to the biomass that was originally without microcystins resulted in a slight additional increase in the rate of malformations (80% at the highest concentration, 300 mg of biomass plus 250 microg of MCYST-LR per liter). A comparison of lethality and effects on malformations (teratogenic index, TI = LC(25)/EC(25)) showed that all samples had significant teratogenic potential in the FETAX assay (TI(MCYST-LR) = 14; TI for biomass with and without microcystin ranged between 2.4 and 3.1, respectively). We conclude that cyanobacterial water blooms can significantly alter the normal development of amphibian embryos.     

Dose-dependent effects of extracted microcystins on embryonic development, larval growth and histopathological changes of southern catfish (Silurus meridionalis)

A laboratory toxic experiment was conducted to examine dose-dependent effects of extracted microcystins (MCs) on embryonic development, larval growth and histopathological changes of southern catfish (Silurus meridionalis). Fertilized eggs were incubated in solutions with four concentrations of MCs (0, 1, 10, 100 microg MC-L Req l(-1)). Higher MCs retarded egg development (2-10h delays) and larval growth, reduced hatching rate (up to 45%), and caused high malformation rate (up to 15%) and hepatocytes damage (characterized by disorganization of cell structure and a loss of adherence between hepatocytes, cellular degeneration with vacuolar hepatocytes and marginal nuclei, even hepatocellular necrosis). A 10 microg MC-L Req l(-1) is close to a high concentration in natural cyanobacterial blooms, suggesting a possible existence of such toxic effects in eutrophic waters.     

The accumulation of cylindrospermopsin from the cyanobacterium Cylindrospermopsis raciborskii in tissues of the Redclaw crayfish Cherax quadricarinatus.

Redclaw crayfish, Cherax quadricarinatus harvested from an aquaculture pond infested by a bloom of the cyanobacterium Cylindrospermopsis raciborskii (order: Nostocales), were shown to accumulate the toxic alkaloid cylindrospermopsin. Pond water samples collected during the bloom contained 589 microg l(-1) of the toxin (93% in the cyanobacterial cells, 7% in the water). Crayfish from the pond contained cylindrospermopsin at concentrations of 4.3 microg g freeze dried hepatopancreas tissue and 0.9 microg g freeze dried muscle tissue. Trichomes of C. raciborskii were observed in gut contents of crayfish harvested during the cyanobacterial bloom, indicating that the most likely mechanism for accumulation of the toxin was by ingestion of cyanobacterial cells. Crayfish subjected to an extract of harvested bloom material under laboratory conditions for a period of 14 days were also found to accumulate cylindrospermopsin, indicating that this toxin is also absorbed into the tissues by direct uptake of the toxin in solution.     

Toxic cyanobacteria and microcystin concentrations in a public water supply reservoir in the Brazilian Amazonia region.

Toxic cyanobacteria in public water supply reservoirs represent a serious health risk as they can release potent cyanotoxins into the water. In the present study we analyzed surface water collected from the Utinga Reservoir, the main source of drinking water for the city of Belem-PA, in order to characterize the reservoir's cyanobacterial biota and to determine the toxicity of these organisms and the concentration of microcystins in raw and treated water. The cyanobacterial biota included potentially toxic genera such as Aphanizomenon, Microcystis, Nostoc, Oscillatoria, Planktothrix and Radiocystis. Mouse bioassays revealed a 43.6% frequency of hepatotoxic strains, including 30.8% Microcystis viridis strains and 12.8% Radiocystis fernandoi strains, with the lowest LD(100) (ip) of 45 and 75 mg kg(-1) body weight, respectively. Subacute hepatotoxicity was observed for Aphanizomenon cf. gracile and Coelomoron pusillum strains. HPLC analysis confirmed the production of microcystins at maximum concentrations of 4.22 microg mg(-1) dry weight for M. viridis and 2.47 microg mg(-1) for R. fernandoi. Microcystins at concentrations of up to 1.25 microg L(-1) detected in raw water by ELISA, together with a cyanobacterial density of 20,000 cells mL(-1), represents the first report of a bloom of cyanobacteria for an Amazonian water body.     

Organ distribution and bioaccumulation of microcystins in freshwater fish at different trophic levels from the eutrophic Lake Chaohu, China

This article reports the organ distribution and bioaccumulation of hepatotoxic microcystins (MCs) in freshwater fishes at different trophic levels from the large, shallow, eutrophic Lake Chaohu in September 2003, when there were heavy surface blooms of toxic cyanobacteria. Among all fish, intestines and blood had the highest average content of MC-RR + MC-LR (22.0 and 14.5 microg g(-1) DW, respectively), followed by liver, bile, and kidney (7.77, 6.32, and 5.81 microg g(-1) DW, respectively), whereas muscle had the least (1.81 microg g(-1) DW). MC content in muscle was highest in carnivorous fish (Culter ilishaeformis, 2.22 microg g(-1) DW) and omnivorous fish (Carassius auratus, 1.96 microg g(-1) DW) and was lowest in phytoplanktivorous fish (Hypophthalmichthys molitrix, 1.65 microg g(-1) DW) and herbivorous fish (Parabramis pekinensis 0.660 microg g(-1) DW). However, the amount of MC in the gut of H. molitrix (137 microg g(-1) DW) was more than 20 times that in the other fish (<6.50 microg g(-1) DW). The MCs showed a tendency to accumulate up the food chain, and piscivorous fish at the top of the food chain were at high risk of exposure to MCs in Lake Chaohu. Our study is the first to report MC concentrations in the bile and blood of wild fish. One hundred grams of fish muscle would contain 2.64-49.7 microg of MC-LR equivalent, or about 1.3-25 times the recommended tolerable daily intake of MC-LR by humans, indicating that fish are already severely contaminated by MCs and that the local authorities should warn the public of the risk of poisoning by eating the contaminated fish.     

Stage sensitivity of medaka (Oryzias latipes) eggs and embryos to permethrin

The effects of exposure to permethrin on gametes, fertilization and embryonic development were examined in medaka (Oryzias latipes). Following range finding (25, 50, 100, 200 or 300 microg/l) and duration of exposure (0, 120, 144, 168, 192, 216, or 240-h) assays, the relative sensitivity was studied when initiation of exposure to permethrin (100 microg/l, for 192-h) occurred at one of four different stages, i.e., unfertilized egg (0-h), late morula (5-h), early neurula (24-h), and early organogenesis (40-h). The later exposure interval proved the most sensitive. Also, differences were observed in rates of recovery in larvae initially affected following the earliest exposure treatment (0-h, gametes prior to fertilization). Permethrin (100 microg/l) did not affect fertilization success and no lethal effects were observed in embryos. Sublethal effects were primarily observed at hatch. Toxicity endpoints in larvae included: delayed swim bladder inflation; inability of hatchling to respond to stimulus; uncoordinated movements, myoskeletal defects and transient enlargement of gall bladder. These changes were characteristic for all hatchlings exposed to nominal concentrations of 50 microg/l. While certain of the above alterations were reversed within 72-h after hatching, lack of swim bladder inflation and inability to respond to stimuli were two features that persisted with significant incidences. Based on persistence of sublethal effects, results from this work indicate the importance of exposures to gametes and to embryos prior to water hardening. The approach taken herein may better reflect environmental risk conditions than assays limited to exposure of embryonated eggs.     

Effects of microcystin-LR on development of medaka fish embryos (Oryzias latipes).

Chronic and subchronic toxicity from exposure to microcystins, cyclic hepatotoxic heptapeptides from cyanobacteria, receives increasing attention as a public human health biohazard. So far, the effects of microcystin on fish have been studied mainly in adults, rather than during early life stages. Limitations of direct ambient exposure experiments to fish egg have resulted from the difficult access of microcystin through the egg chorion. Using a microinjection technology, we have introduced microcystin-LR (MC-LR) directly into one-cell stage embryos or into the vitellus of late neurula embryos (stage 19) or into the vitellus of stage 25 embryos of medaka (Oryzias latipes) at the onset of the liver anlage. Microinjection (100 pl; stage 1 or 2 nl; stage 19 or 25) of MC-LR resulted in a dose dependent mortality of embryos. Survival rates were reduced up to 90% with microcystin concentrations of 10 or 1 microg/ml (corresponding to 1-20 pg or 0.1-2 pg of toxin injected), injected either at stages 1, 19 or 25. Also, a dose dependent advanced embryonic hatching processing was observed; hatching being brought forward from 2 or 3 days compared to controls in most of the microcystin injected groups. In agreement with the known hepatotoxic effects of microcystin, injected embryos consistently displayed hepatobiliary abnormalities such as liver hypertrophy and hepatic hemorrhage, also evidenced in post-hatching juveniles. Thus, the methodology presented in this paper should be valuable tool to analyze the effects of toxins on the development of aquatic vertebrate embryos.     

The influence of microcystin-LR on fish phagocytic cells

The in vitro influence of microcystin-LR (MC-LR) on rainbow trout phagocytic cell functions was studied. The cells isolated from the fish blood were exposed on MC-LR at the concentrations of 1, 5, 10, 20 microg/mL. The viability of the cells after 2, 4 and 24 hour incubation with MC-LR was studied as well as phagocytic cell ability measured as zymozan particle phagocytosis and metabolic activity measured as respiratory burst activity. The toxin caused time- and dose-dependent viability decrease, the strongest after cell exposure on 10 and 20 microg/mL and observed only after 24 hours incubation when 1 microg/mL was applied. The phagocytic ability was elevated in the presence of MC-LR at the dose of 5 microg/mL. MC-LR had also modulatory influence on respiratory burst activity. While the two lower doses caused the stimulation of the parameter, the two higher ones diminished superoxide anion production in the triggered cells. The results show the potency of the toxin to influence the fish phagocytic cell functions.     

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in two freshwater shrimps, Palaemon modestus and Macrobrachium nipponensis, from a large shallow, eutrophic lake of the subtropical China.

So far no information is available on microcystin (MC) contents in shrimps, prawns or crayfish from natural freshwaters. Tissue distributions and seasonal dynamics of the hepatotoxic MC-LR and -RR in two freshwater shrimps, Palaemon modestus and Macrobrachium nipponensis were studied monthly (during June-November, 2003) in a Chinese lake containing toxic cyanobacterial blooms. The shrimps P. modestus and M. nipponensis accumulated high MCs not only in the hepatopancreas (mean 4.29 and 0.53 microg g(-1) DW, respectively) but also in the gonad (mean 1.17 and 0.48 microg g(-1) DW, respectively), and the crayfish Procambarus clarkii accumulated as much as 0.93 microg g(-1) DW in the gonad. This indicates that gonads of these invertebrates are the second important target organ of MCs. P. modestus apparently accumulated more MCs in their organs than M. nipponensis, which might be a reflection of their difference in trophic niche. Eggs of the shrimps accumulated 8.4% (M. nipponensis, 0.27 microg g(-1) DW) and 29.0% (P. modestus, 2.34 microg g(-1) DW) of total toxin burden, indicating that MCs had been transferred into offspring from their adults. Among the shrimp muscle samples analyzed, 31% were above the provisional WHO TDI level, suggesting the risk of consuming shrimps in Lake Chaohu.     

Occurrence and elimination of cyanobacterial toxins in two Australian drinking water treatment plants.

In Australian freshwaters, Anabaena circinalis, Microcystis spp. and Cylindrospermopsis raciborskii are the dominant toxic cyanobacteria. Many of these surface waters are used as drinking water resources. Therefore, the National Health and Medical Research Council of Australia set a guideline for MC-LR toxicity equivalents of 1.3 microg/l drinking water. However, due to lack of adequate data, no guideline values for paralytic shellfish poisons (PSPs) (e.g. saxitoxins) or cylindrospermopsin (CYN) have been set. In this spot check, the concentration of microcystins (MCs), PSPs and CYN were determined by ADDA-ELISA, cPPA, HPLC-DAD and/or HPLC-MS/MS, respectively, in two water treatment plants in Queensland/Australia and compared to phytoplankton data collected by Queensland Health, Brisbane. Depending on the predominant cyanobacterial species in a bloom, concentrations of up to 8.0, 17.0 and 1.3 microg/l were found for MCs, PSPs and CYN, respectively. However, only traces (<1.0 microg/l) of these toxins were detected in final water (final product of the drinking water treatment plant) and tap water (household sample). Despite the low concentrations of toxins detected in drinking water, a further reduction of cyanobacterial toxins is recommended to guarantee public safety.     

Accumulation of microcystins in various organs of the freshwater snail Sinotaia histrica and three fishes in a temperate lake, the eutrophic Lake Suwa, Japan.

So far, there has been only one study to examine microcystin (MC) contents in various organs of snails in a subtropical Chinese lake. In this study, tissue distribution and seasonal dynamics of MC-RR and -LR were investigated in various organs of a freshwater snail (Sinotaia histrica) in a temperate eutrophic lake, Lake Suwa, Japan. Accumulation of microcystins in some fish was also investigated. There was marked temporal variation in the MC content of various organs of the snail. The digestive tract had the highest MC content (mean 9.03 microg g(-1) DW and range 3.74-23.2 microg g(-1) DW), followed by the gonad (mean 6.90 microg g(-1) DW and range 0.07-22.7 microg g(-1) DW) and hepatopancreas (mean 5.38 microg g(-1) DW and range 1.08-8.79 microg g(-1) DW), whereas the foot had the least (mean 2.48 microg g(-1) DW and range 0.04-4.45 microg g(-1) DW). The disappearance of MC-LR in the hepatopancreas indicated that S. histrica is able to depurate MC-LR efficiently. MC-RR was detected in the muscle of three species of fish, with the highest content in Carassius auratus (79.4 microg kg(-1) BW). Because of substantial MC accumulation in these edible aquatic animals in Lake Suwa, it is recommended that regular monitoring of MCs should be undertaken in both cyanobacteria and aquatic animals.     

Identification of microcystins in waters used for daily life by people who live on Tai Lake during a serious cyanobacteria dominated bloom with risk analysis to human health

Tai Lake is the third largest freshwater lake in China with annual cyanobacteria blooms. Microcystins produced by these blooms have serious health risks for populations surrounding the lake, especially for people living on Tai Lake, because they usually drink raw lake water after a simple alum treatment. This study presents data on the detection and identification of microcystins in waters used for daily life by people living on Tai Lake, during the cyanobacterial blooming in July 2007. The health risks from drinking these microcystin-polluted waters were also calculated. The main microcystins detected by high-performance liquid chromatography-electrospray ionization mass spectrometry in the water samples collected from two parts of Tai Lake (Wuli Lake and Meiliang Bay) were MC-LR (4.33-12.27 microg/L), MC-RR (8.36-16.91 microg/L) and MC-YR (1.41-5.57 microg/L). Risk assessment showed that the drinking water simply treated by alum was not safe. The lowest calculated hazards ratios in all water samples was 6.4, which indicated that the risk of microcystins exposure from drinking water was over six times higher than the tolerable daily intake (TDI) recommended by The World Health Organization (WHO). Further studies should be conducted to elucidate the relationships between the epidemiology of people living on Tai Lake and microcystins exposure from drinking water.     

Assimilation and depuration of microcystin-LR by the zebra mussel, Dreissena polymorpha

Zebra mussels (Dreissena polymorpha) are an important component of the foodweb of shallow lakes in the Netherlands, amongst others in Lake IJsselmeer, an international important wetland. Large numbers of ducks feed on these mussels in autumn and winter. The mussels are filter feeders and are exposed to high densities of cyanobacteria in summer and autumn. Mussels and cyanobacteria both thrive in Lake IJsselmeer. Apparently the mussels are somehow protected against accumulation of harmful quantities of cyanobacterial toxins. In this study, we investigated the assimilation of the cyanobacterial toxin microcystin-LR (MC-LR) in zebra mussels when fed the toxic cyanobacterium Microcystis aeruginosa as sole food or in a mixture with the eustigmatophyte Nannochloropsis limnetica. After 3 weeks of assimilation we studied the depuration of MC-LR during 3 weeks when the food of the mussels was free of cyanobacteria. These assimilation/depuration experiments were combined with grazing experiments, using the same food treatments. Microcystins were analyzed using liquid chromatography-mass spectrometry (LC-MS); in addition, covalently bound MC were analyzed using the MMPB method. The mussels showed higher clearance rates on Microcystis than on Nannochloropsis. No selective rejection of either phytoplankton species was observed in the excretion products of the mussels. Zebra mussels fed Microcystis as single food, assimilated microcystin-LR relatively fast, and after 1 week the maximum value of free unbound microcystin assimilation (ca. 11 microg g DW(-1)) was attained. For mussels, fed with the mixed food, a maximum of only 3.9 microg g DW(-1) was recorded after 3 weeks. Covalently bound MC never reached high values, with a maximum of approximately 62% of free MC in the 2nd week of the experiment. In the depuration period microcystin decreased rapidly to low values and after 3 weeks only very low amounts of microcystin were detectable. The amount of toxin that accumulated in the mussels would appear to be high enough to cause (liver) damage in diving ducks. However, death by exposure to microcystin seems unlikely. Mussels seem efficient in minimizing the assimilation of microcystin. If it were not for this, mass mortalities of ducks in shallow lakes in the Netherlands would presumably occur on a much more widespread scale than is currently observed.     

Cyanobacterial Toxins and Peptides in Lake Vegoritis,Greece

Cyanotoxins (CTs) produced by cyanobacteria in surface freshwater are a major threat for public health and aquatic ecosystems. Cyanobacteria can also produce a wide variety of other understudied bioactive metabolites such as oligopeptides microginins (MGs), aeruginosins (AERs), aeruginosamides (AEGs) and anabaenopeptins (APs). This study reports on the co-occurrence of CTs and cyanopeptides (CPs) in Lake Vegoritis, Greece and presents their variant-specific profiles obtained during 3-years of monitoring (2018–2020). Fifteen CTs (cylindrospermopsin (CYN), anatoxin (ATX), nodularin (NOD), and 12 microcystins (MCs)) and ten CPs (3 APs, 4 MGs, 2 AERs and aeruginosamide (AEG A)) were targeted using an extended and validated LC-MS/MS protocol for the simultaneous determination of multi-class CTs and CPs. Results showed the presence of MCs (MC-LR, MC-RR, MC-YR, dmMC-LR, dmMC-RR, MC-HtyR, and MC-HilR) and CYN at concentrations of <1 μg/L, with MC-LR (79%) and CYN (71%) being the most frequently occurring. Anabaenopeptins B (AP B) and F (AP F) were detected in almost all samples and microginin T1 (MG T1) was the most abundant CP, reaching 47.0 μg/L. This is the first report of the co-occurrence of CTs and CPs in Lake Vegoritis, which is used for irrigation, fishing and recreational activities. The findings support the need for further investigations of the occurrence of CTs and the less studied cyanobacterial metabolites in lakes, to promote risk assessment with relevance to human exposure.