Anna Farkas

Age- and size-specific patterns of heavy metals in the organs of freshwater fish Abramis brama L. populating a low-contaminated site.

Concentrations of cadmium, copper, mercury, lead and zinc were determined by atomic absorption spectrophotometry in the muscle, gill and liver of bream Abramis brama L. to study the relationship between the heavy metal load of fish and their age and size, and the seasonal variation of pollutant loads. Fish were collected from the Western basin of Lake Balaton (Hungary) in October 1999 and May 2000. The average metal concentrations of different organs varied in the following ranges: Cd 0.42-2.10; Cu 1.77-56.2; Hg 0.01-0.19; Pb 0.44-3.24; Zn 10.9-82.5 microg g(-1) dry weight. The highest Cd, Cu, Pb and Zn concentrations were detected in the gill or liver of fish, whereas the highest Hg concentrations were measured in the muscle. In the liver of bream for cadmium, copper and mercury the Pearson correlation analysis revealed positive associations related to age and size (length, net weight), as well as for the mercury load of all three investigated organs. In the muscle and gill the copper, lead and zinc concentrations, similarly to the lead and zinc concentrations of the liver, the associations related to age and size were negative. The correlations between the heavy metal concentrations of organs and the individual condition factors of fish samples proved to have opposite trends compared to those related to the age and size of fish. The seasonal variations in the heavy metal load of bream could be attributed rather to the seasonal change in the condition factor of fish than to variations in the pollutant load of the site.

Comparative study of cyanotoxins affecting cytoskeletal and chromatin structures in CHO-K1 cells

In this study we compared the effects of the two frequently occuring and most dangerous cyanobacterial toxins on the cellular organization of microfilaments, microtubules and on the chromatin structure in Chinese hamster ovary (CHO-K1) cells. These compounds are the widely known microcystin-LR (MC-LR) and cylindrospermopsin (CYN) classified as the highest-priority cyanotoxin. Toxic effects were tested in a concentration and time dependent manner. The hepatotoxic MC-LR did not cause significant cytotoxicity on CHO-K1 cells under 20 microM, but caused apoptotic changes at higher concentrations. Apoptotic shrinkage was associated with the shortening and loss of actin filaments and with a concentration dependent depolymerization of microtubules. No necrosis was observed over the concentration range (1-50 microM MC-LR) tested. Cylindrospermopsin did cause apoptosis at low concentrations (1-2 microM) and over short exposure periods (12h). Necrosis was observed at higher concentrations (5-10 microM) and following longer exposure periods (24 or 48h). Cyanotoxins also affected the chromatin structure. The condensation process was inhibited by MC-LR at a later stage and manifested as broken elongated prechromosomes. CYN inhibited chromatin condensation at the early fibrillary stage leading to blurred fluorescent images of apoptotic bodies and preventing the formation of metaphase chromosomes. Cylindrospermopsin exhibited a more pronounced toxic effect causing cytoskeletal and nuclear changes as well as apoptotic and necrotic alterations.

Molluscs in biological monitoring of water quality

Molluscs living in Lake Balaton accumulate persistent toxic substances, namely heavy metals, to a greater extent, than other organisms, and can serve as excellent passive biomonitors. Especially gills are good accumulators. Regular sampling showed that the level of Cd and Hg concentrations increased, while Pb contamination decreased during the past 20 years in mussels, corresponding probably to changes in pollution of the Lake. In functional, active monitoring various behavioral patterns of molluscs were employed. In mussels the periodicity of activity and rest (pumping activity vs. valve closure) is a sensitive indicator of unfavorable conditions and so of toxic substances. Low concentrations of inorganic and organic toxicants (heavy metals, PCBs, PAH compounds) cause reduction of the active and increase of the rest periods in a concentration dependent manner in a few hours. A second, suitable test for evaluating toxicity of chemicals is the measurement of the water flow through the exhalant siphon. Under the effect of toxicants the siphon activity, both the strength and duration of water flow changes characteristically within a few minutes. For both behavioral tests special techniques have been developed suitable for long duration recording, supported by mechano-electrical transduction and computerized data evaluation. In case of the pond snail (Lymnaea stagnalis L.) the behavior (positive/negative geotaxis and orientation) is disturbed in the presence of the above mentioned toxic chemicals. The execution and evaluation of the changes in the movement of the snail is based on video-recording and measurement of the direction and distance the animal performs in uncontaminated water and in the presence of the pollutants during the same period of time (0.5-4 h).

Vulnerability of biomarkers in the indigenous mollusk Anodonta cygnea to spontaneous pollution in a transition country

The aim of this study was to estimate the sensitivity of biomarkers of stress and exposure in the bivalve mollusk Anodonta cygnea to spontaneous anthropogenic activities typical for the Western Ukraine. Three sites were examined during spring, summer and autumn: an agricultural site (A); the cooling pond of nuclear power plant (N) and a forestry close to the municipal water inlet (F). Common temporal changes of a battery of biochemical markers in the gills and hemolymph and morphological characteristics were shown by discriminant functional analysis. Classification trees built on the basis of the screened biomarkers demonstrated persistent peculiarities at each site: genotoxicity (nuclear abnormalities) at site A and endocrine disruption (high levels of vitellogenin-like proteins (Vtg-LP) in hemolymph) at site F. Interim local effects were best characterized by metallothionein (MT) concentrations, lipid peroxidation (LPO), activities of glutathione S-transferase and lactate dehydrogenase, and the conditional index of the gills. In autumn, the mollusks from the three sites revealed the highest differences in pollution status: the activation of antioxidant defense and cholinesterase were typical for site A, highest levels of MT related to high levels of Cu and Cd in the water at site B, and a steep increase in the level of Vtg-LP and the decrease of lysosomal membrane stability were recorded at the site selected as reference (F). The biomarker alterations recorded at site F were later related to an emergency event at the municipal dump located nearby. Thus, our case study demonstrated the reliability of using biomarkers of exposure to assess both long-term and accidental environmental pollution loads.

The ecotoxicological evaluation of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) employing a battery of bioassays and chemical screening

Ecotoxicity of four Cylindrospermopsis raciborskii strains (ACT 9502, ACT 9503, ACT 9504, ACT 9505) isolated from Lake Balaton (Hungary) was evaluated in four aquatic bioassays including the Thamnocephalus platyurus acute lethality test; Daphnia magna acute immobilization assay; D. magna feeding inhibition assay and Danio rerio embryo developmental toxicity assay, assisted by chemical screening for known toxins by HPLC-MS. For reference, we analyzed in parallel the toxin content and toxic effects of two previously characterized toxin-producing strains: the Australian cylindrospermopsin producer AQS C. raciborskii and the anatoxins producer Oscillatoria sp. PCC 6506. Bioassays were used to evaluate the overall toxicity of the hydrophilic bioactive metabolites pool synthesized by the selected cyanobacteria. Chemical screening has proven that the ACT C. raciborskii extracts investigated did not contained cylindrospermopsins and anatoxins. The relative toxicity of the ACT C. raciborskii aqueous extracts observed in each bioassay was comparable to the effects recorded for the anatoxins producer PCC 6506 strain while toxicity values (EC50/LC50) calculated for the AQS extract were in general one order of magnitude lower. Concerning sublethal effects of ACT C. raciborskii extracts to the D. rerio embryogenesis, the general morphological abnormality observed was a significant retardation of development. Overall, our results suggest that C. raciborskii populating Lake Balaton produce metabolites with significant bioactive potencies. Therefore, continued investigation of these unknown compounds is required.

Neonicotinoid insecticides inhibit cholinergic neurotransmission in a molluscan (Lymnaea stagnalis) nervous system

Neonicotinoids are highly potent and selective systemic insecticides, but their widespread use also has a growing impact on non-target animals and contaminates the environment, including surface waters. We tested the neonicotinoid insecticides commercially available in Hungary (acetamiprid, Mospilan; imidacloprid, Kohinor; thiamethoxam, Actara; clothianidin, Apacs; thiacloprid, Calypso) on cholinergic synapses that exist between the VD4 and RPeD1 neurons in the central nervous system of the pond snail Lymnaea stagnalis. In the concentration range used (0.01-1 mg/ml), neither chemical acted as an acetylcholine (ACh) agonist; instead, both displayed antagonist activity, inhibiting the cholinergic excitatory components of the VD4-RPeD1 connection. Thiacloprid (0.01 mg/ml) blocked almost 90% of excitatory postsynaptic potentials (EPSPs), while the less effective thiamethoxam (0.1 mg/ml) reduced the synaptic responses by about 15%. The ACh-evoked membrane responses of the RPeD1 neuron were similarly inhibited by the neonicotinoids, confirming that the same ACh receptor (AChR) target was involved. We conclude that neonicotinoids act on nicotinergic acetylcholine receptors (nAChRs) in the snail CNS. This has been established previously in the insect CNS; however, our data indicate differences in the background mechanism or the nAChR binding site in the snail. Here, we provide the first results concerning neonicotinoid-related toxic effects on the neuronal connections in the molluscan nervous system. Aquatic animals, including molluscs, are at direct risk while facing contaminated surface waters, and snails may provide a suitable model for further studies of the behavioral/neuronal consequences of intoxication by neonicotinoids.

Crustaceans as biological indicators of heavy metal pollution in Lake Balaton (Hungary)

Trace metals (Cd, Cu, Pb, Zn) were measured in mixed zooplankton samples collected from the open water of Lake Balaton (Hungary) in order to assess spatial and seasonal changes in the heavy metal load of different sites, during 1996–2000.Samplings were performed usually twice a year in different seasons (spring, summer and autumn). The heavy metal concentrations of zooplankton biomass were determined by atomic absorption spectrophotometry. Additionally, for some of the sampling sites – Western- and Eastern basins – the correlations between the heavy metal load of Crustaceans and the element concentrations of the water recorded monthly by the Transdanubian Environmental Protection Agency were also analyzed.The average metal concentrations in zooplankton varied in the following ranges: Cd: 0.25 – 3.91; Cu: 5.9 – 26.4; Pb: 1.59 – 12.84; Zn: 37.6 – 180.5 mg kg−1 dry weight. Significant spatial and seasonal variations with occasionally outstanding differences in the heavy metal load of samples could be observed during the investigated period. The heavy metal load of the Crustacea plankton biomass in summer and autumn proved to be usually higher than in spring. During the investigated period an overall significant increase in the cadmium, copper and lead load of the Crustacea plankton populating the lake could be observed. Pearson correlation analysis performed between the heavy metal concentration of Crustacea plankton biomass and that of the water in the two outlying basins of the lake revealed strong relationship for Cd, Cu and Zn, while for lead the correlation proved to be not significant.

Diversity of the molecular responses to separate wastewater effluents in freshwater mussels

The environmental safety of pharmaceutical and personal care products (PPCPs) requires a crucial examination. The aim of this study was to evaluate the responses of biomarkers of stress and toxicity in freshwater mussels to the effect of commonly found PPCPs in wastewater. We treated male mussels Unio tumidus, from an undisturbed site with ibuprofen (IBU, 250 ng L(-1)), triclosan (TCS, 500 ng L(-1)), or estrone (E1, 100 ng L(-1)) for 14 days. Untreated mussels from this site (C) and mussels inhabiting a polluted area (P) were also examined after a similar time of being kept in the laboratory. The consequences of chronic exposure of the mussels in the P-group were reflected in elevated concentrations of oxyradicals (1.4 times), oxidized glutathione (4.3 times), lipofuscin (2.2 times), and DNA-strand breaks in the digestive gland (DG) in comparison to the C-group, higher levels of caspase-3 activity in the DG, and vitellogenin-like proteins in gonads among all studied groups. Exposed mussels demonstrated some common responses with mussels in the P-group: elevated levels of lactate/pyruvate ratio, lipofuscin (IBU and E1), DNA fragmentation (TCS and E1), and caspase-3 activity (TCS and E1). Exposed to PPCPs mussels also showed elevation of ethoxyresorufin-O-deethylase and/or glutathione-S-transferase activity in the DG and a decrease in lysosomal stability in hemocytes (TCS and E1). The TCS group was distinguished by having the highest level of DNA-fragmentation and the lowest concentrations of total glutathione, oxyradicals, lipofuscin, pyruvate, and lactate, reflecting total metabolic depression. These results show that selected PPCPs at low concentrations alter a variety of physiological processes in this animal model system.

Pharmacological studies confirm neurotoxic metabolite(s) produced by the bloom‐forming Cylindrospermopsis raciborskii in Hungary

A rapid cyanobacterial bloom of Cylindrospermopsis raciborskii (3.2 × 104 filaments/mL) was detected early November, 2012, in the Fancsika pond (East Hungary). The strong discoloration of water was accompanied by a substantial fish mortality (even dead cats were seen on the site), raising the possibility of some toxic metabolites in the water produced by the bloom‐forming cyanobacteria (C. raciborskii). The potential neuronal targets of the toxic substances in the bloom sample were studied on identified neurons (RPas) in the central nervous system of Helix pomatia. The effects of the crude aqueous extracts of the Fancsika bloom sample (FBS) and the laboratory isolate of C. raciborskii from the pond (FLI) were compared with reference samples: C. raciborskii ACT 9505 (isolated in 1995 from Lake Balaton, Hungary), the cylindrospermopsin producer AQS, and the neurotoxin (anatoxin‐a, homoanatoxin‐a) producer Oscillatoria sp. (PCC 6506) strains. Electrophysiological tests showed that both FBS and FLI samples as well the ACT 9505 extracts modulate the acetylcholine receptors (AChRs) of the neurons, evoking ACh agonist effects, then inhibiting the ACh‐evoked neuronal responses. Dose–response data suggested about the same range of toxicity of FBS and FLI samples (EC50 = 0.397 mg/mL and 0.917 mg/mL, respectively) and ACT 9505 extracts (EC50 = 0.734 mg/mL). The extract of the neurotoxin‐producing PCC 6506 strain, however, proved to be the strongest inhibitor of the ACh responses on the same neurons (EC50 = 0.073 mg/mL). The presented results demonstrated an anatoxin‐a‐like cholinergic inhibitory effects of cyanobacterial extracts (both the environmental FBS sample, and the laboratory isolate, FLI) by some (yet unidentified) toxic components in the matrix of secondary metabolites. Previous pharmacological studies of cyanobacterial samples collected in other locations (Balaton, West Hungary) resulted in similar conclusions; therefore, we cannot exclude that this chemotype of C. raciborskii which produce anatoxin‐a like neuroactive substances is more widely distributed in this region.

Seasonal and size-related variation of subcellular biomarkers in quagga mussels (Dreissena bugensis) inhabiting sites affected by moderate contamination with complex mixtures of pollutants

The size-related differences in subcellular biomarker responses were assessed in Dreissena bugensis mussels inhabiting harbours moderately affected by pollution with complex mixtures of heavy metals and polycyclic aromatic hydrocarbons (PAHs). Adult D. bugensis samples were collected from three harbours of Lake Balaton (Hungary) characterized by moderate shipping activity, and as reference site, from a highly protected remote area of the lake. Biomarkers of exposure (metallothioneins (MTs), ethoxyresorufin-o-deethylase (EROD)), oxidative stress (lipid peroxidation (LPO), DNA strand breaks (DNAsb)) and possible endocrine disruption (vitellogenin-like proteins (VTG)) were analysed in whole-tissue homogenates of differently sized groups of mussels in relation to environmental parameters and priority pollutants (heavy metals and polycyclic aromatic hydrocarbons). Integrated biomarker response (IBR) indices were calculated for biomarker responses gained through in situ measurements to signalize critical sites and to better distinguish natural tendencies from biological effects of contaminants. Biomarker responses showed close positive correlation in case of MT, EROD, LPO, and DNAsb and negative correlation with VTG levels with mussel shell length in autumn, when higher levels of biomarkers appeared, possibly due to natural lifecycle changes of animals.