János Győri

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.

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.

Study on Soil Mobility of Two Neonicotinoid Insecticides

Movement of two neonicotinoid insecticide active ingredients, clothianidin (CLO) and thiamethoxam (TMX), was investigated in different soil types (sand, clay, or loam) and in pumice. Elution profiles were determined to explore differences in binding capacity. Soil characterized by high organic matter content retained the ingredients, whereas high clay content resulted in long release of compounds. Decrease in concentration was strongly influenced by soil types: both CLO and TMX were retained in loam and clay soils and showed ready elution through sandy soil and pumice. Elution capability of the active ingredients in sandy soil correlated with their water solubility, indicating approximately 30% higher rapidity for TMX than for CLO. Soil organic carbon-water partitioning coefficients () determined were in good agreement with literature values with somewhat lower value for CLO in sandy soil and substantially higher values for TMX in clay soil. High mobility of these neonicotinoid active ingredients in given soil types urges stronger precautionary approach taken during their application.

Neurotoxic effects evoked by cyanobacterial extracts suggest multiple receptors involved in electrophysiological responses of molluscan (CNS, heart) models

The responses of the snail central neurons (Helix pomatia, Lymnaea stagnalis) and the isolated Helix heart were characterized evoked by cyanobacterial extracts (Cylindrospermopsis raciborskii ACT strains) isolated from Lake Balaton (Hungary). The nicotinergic acetylcholine (ACh) receptors in the CNS (both excitatory and inhibitory) were blocked by the extracts of ACT 9502 and ACT 9505 strains and the anatoxin- a (homoanatoxin-a) producing reference strain of Oscillatoria sp. (PCC 6506), similar to the inhibitory effects of the pure anatoxin-a. The enhancement of the ACh responses by the ACT 9504 extract suggests additional, probably acetylcholine esterase inhibitory mechanisms. On the isolated Helix heart the crude ACT 9505 and PCC 6506 extracts evoked frequency increase and transient twitch contraction, opposite to the ACh evoked heart relaxation. Anatoxin-a similarly contracted the heart but did not increase its contration frequency. These data suggest the involvement of some non-cholinergic mechanisms, acting very likely by direct modulation of the electrical or contractile system of the isolated heart. Diversity of the effects evoked by the cyanobacterial extracts in the CNS and heart suggest pharmacologically different neuroactive components among the secondary metabolites of the cyanobacteria acting on both (anatoxin-a like) cholinergic and (unidentified) non-cholinergic receptors.

The terrestrial snail, Helix pomatia , adapts to environmental conditions by the modulation of central arousal

The osmotic stimulation which is able to change the behavioral state of the animal are most effective during rainy weather while they are less effective during dry weather conditions. In isolated CNS preparations from aestivated animals the highest firing activity and serotonin sensitivity of the serotonergic (RPas) heart modulator neurons are recorded during rainy weather and the lowest parameters are observed in dry conditions. In aestivated animals the serotonin (5HT) content in both the CNS and the foot is higher than the dopamine (DA) content during rainy weather, while in dry weather the DA level is higher than the 5HT. The inactivation-reactivation process is accompanied by decreasing both the DA and 5HT levels in the CNS and increasing them in the peripheral organs. Our results suggest that aestivated animals adapt to (favorable and unfavorable) environmental conditions by modulating their central arousal state through changing the levels and distribution of monoamines (5HT, DA) in their body.

Interspecies comparison of selected pollution biomarkers in dreissenid spp. inhabiting pristine and moderately polluted sites

Stress biomarkers, which can outline impacts of contaminants in aquatic biota at the biochemical level, are increasingly used as early warning tools in environmental monitoring. Reliable biomarker based assessment schemes, however, request appropriate knowledge of baseline levels of selected endpoints, and the potential influence of a range of natural influencing factors (both abiotic and biotic) as well. In this study, we examined the interspecies variability of various biomarkers (metallothioneins (MT), ethoxyresorufin-O-deethylase activity (EROD), lipid peroxidation (LPO), DNA strand breaks (DNA_sb), vitellogenin-like proteins (Vtg)) in Dreissena polymorpha and Dreissena bugensis inhabiting either pristine- or moderately impacted sites of Lake Balaton (Hungary). Levels of all biomarkers considered revealed low interspecies variability in the two dreissenid species at all sampling sites, with consistently higher (but statistically insignificant) values in Dreissena polymorpha. Levels of all biomarkers varied within the two investigated seasons, with significant influence of the reproduction cycle particularly on the levels of metallothioneins and vitellogenin-like proteins. Each biomarker considered was elevated by October, with significantly higher values in the mussels inhabiting harbours. Insignificant spatial and temporal variability in the general health indicators (condition index, total protein content) of dreissenids was observed, which, in parallel with evident rise in biomarker levels, apparently suggest that the anthropogenic impacts in harbours affect mussel fitness yet at sub organismal level. Our data might serve useful basis for future environmental monitoring surveys, especially in habitats where the progressive replacement of Dreissena polymorpha by Dreissena bugensis is taking place, as the interspecies variability in susceptibility to chemical stress of the two species is well comparable.

Effects of rotenone and other mitochondrial complex I inhibitors on the brine shrimp Artemia

(Artemia) nauplii was used to asses the toxicity of rotenone, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), MP+ (1-methyl-4-phenylpyridinium) and the effect of L-DOPA co-treatment with rotenone. Rotenone had a dose dependent effect on mortality (LC₅₀: 0.37 ± 0.04 μM mean ± S E, n = 24), while MPTP and MP+ proved to be toxic in millimolar range (LC₅₀: 0.21 ± 0.09 mM and 0.20 ± 0.08 mM, respectively, n = 4). L-DOPA (50-200 μM) co-treatment increased the survival of the rotenone-treated animals (LC₅₀: 0.51 ± 0.23 μM, 1.03 ± 0.66 μM, and 0.76 ± 0.52 μM, respectively). In the whole body tissue homogenates of Artemia, sublethal (up to 0.3 μM) concentrations of rotenone increased the glutathione S-transferase (GST) activity by up to 50 about percent (LC₅₀: 53.3 ± 6.8 nM/min/mg protein, against 34.7 ± 3.6 nM/min/mg protein, n = 4). Nauplii treated in 100 mM L-DOPA and rotenone together showed further increase of GST activity all across the range of rotenone concentrations. These results on Artemia nauplii show similarities with other animal models, when complex I inhibitors were tested. Biochemical measurements suggest a protective role of L-DOPA by increasing the GST activity as part of the intracellular defences during toxin-evoked oxidative stress.

Occurrence of neonicotinoids in guttation liquid of maize – soil mobility and cross-contamination

ABSTRACT Movement of clothianidin (CLO) and thiamethoxam (TMX) applied as maize seed dressing or pre-emergence spray application was determined in different soil types. Uptake of these neonicotinoids in plants emerging from coated or non-coated seeds was characterised via guttation liquid measurements. Applied in spray at a recommended dosage of 16.5 µmol/L, TMX and CLO occurred in the guttation liquid at peak concentrations of 1.82 and 1.63 mg/L, respectively, in plants in sandy soil and at 20–40% lower levels in plants in clay or loam soils. Peak emergence was substantially higher (above 100 mg/L) in the guttation liquid of plants emerging from neonicotinoid-coated seeds at dosages of 4.87 and 2.07 µmol per seed for CLO and TMX, respectively, strongly influenced by soil type. CLO and TMX levels in plants emerging from non-coated seeds in the proximity of neonicotinoid-coated seeds were demonstrated: cross-contamination may occur by uptake through soil from neighbouring plants. CLO appeared in the guttation liquid of plants emerging from non-coated seeds in proximity to coated ones at a peak concentration (approximately 53 mg/L) in 15 days corresponding to one-fifth of the levels in plants emerging from CLO-coated seeds; CLO concentrations gradually decreased to the same levels (6.6 ± 0.3 mg/L) after 24 days. A similar trend was observed for non-coated seeds near TMX-coated ones, with a gradual increase of TMX in 17–18 days, the levels in the guttation liquid of plants emerged from non-coated seeds (approximately 123 mg/L) corresponding to two-thirds of the levels in plants emerging from TMX-coated seeds. TMX concentrations dropped to the same levels in plants emerging from non-coated and coated seeds by day 20, and CLO occurred as a metabolite. To our knowledge, this is the first scientific record of neonicotinoid levels in guttation liquid of plants emerged from non-coated maize seeds.

Contamination of the guttation liquid of two common weeds with neonicotinoids from coated maize seeds planted in close proximity

Neonicotinoid uptake by maize plants emerged from coated seeds and by two common weeds grown in close proximity to coated seeds has been studied. Uptake of thiamethoxam (TMX) and clothianidin (CLO) have been characterized via guttation liquid measurements. The creeping thistle (Cirsium arvense), a well-known maize weed, as well as red poppy or Flanders poppy (Papaver rhoeas) were chosen as model species. The results confirmed that cross-contamination may occur by uptake of the neonicotinoid AIs through soil from neighbouring plants that emerged from coated seeds. Although the levels of these neonicotinoids were substantially lower in the guttation liquid of the weeds than in that of maize plants emerged from coated seeds, the compounds were detected up to 36th day after planting of the maize seeds. The highest peak concentrations of TMX were around 150 and 21 mg L-1, while similar data for CLO were around 70 and 21 mg L-1 for maize and creeping thistle, respectively. Mostly due to its higher guttation intensity significantly lower values were determined for red poppy (0.740 mg L-1).