Biochemical responses of Lemna minor experimentally exposed to cadmium and zinc.

The effects of 5 µM cadmium (Cd), a non-essential toxic element and 25 and 50 µM zinc (Zn), an essential micronutrient, were investigated in aquatic plant Lemna minor L. after 4 and 7 days of exposure to each metal alone or to their combinations. Both metals showed tendency to accumulate with time, but when present in combination, they reduced uptake of each other. Cd treatment increased the lipid peroxidation and protein oxidation indicating appearance of oxidative stress. However, Zn supplementation in either concentration reduced values of both parameters, while exposure to Zn alone resulted in elevated level of lipid peroxidation and protein oxidation but only on the 7th day. Enhanced DNA damage, which was found on the 4th day in plants treated with Cd alone or in combination with Zn, was reduced on the 7th day in combined treatments. Higher catalase activity obtained in all treated plants on the 4th day of experiment was reduced in Zn-treated plants, but remained high in plants exposed to Cd alone or in combination with Zn after 7 days. Cd exposure resulted in higher peroxidase activity, while Zn addition prominently reduced peroxidase activity in the plants subjected to Cd stress. In conclusion, Cd induced more pronounced oxidative stress and DNA damage than Zn in applied concentrations. Combined treatments showed lower values of oxidative stress parameters--lipid peroxidation, protein oxidation and peroxidase activity as well as lower DNA damage, which indicates alleviating effect of Zn on oxidative stress in Cd-treated plants.

Effect of coal mining activities and related industry on composition, cytotoxicity and genotoxicity of surrounding soils.

Coal mining and related industries each leave their characteristic "metal fingerprint" in the surrounding soils. Although geochemical investigations of such soils most often indicate heavy contamination with certain metals and bioassays point to their cytotoxic and genotoxic effects, the majority of studies are based on only one of the mentioned approaches. Here, the presented study investigated the effect of coal mining activities and related industry on surrounding soils by means of both geochemical and biological tools. The multielement composition of soils and associated eluates were used for the assessment of soil contamination level and the element bioavailable fractions, respectively. For cytotoxicity and genotoxicity evaluation, shallot (Allium ascalonicum L.) roots were exposed to selected soil eluates. Root growth, frequency of mitosis, mitotic and chromosomal abnormalities in root meristem cells, level of lipid peroxidation, and DNA damage evaluated by a comet assay were scored as toxicity endpoints. The results point to significant differences in the composition of collected soils and a variety of factors that contribute not only to their total metal load but also to the observed cytotoxic and genotoxic effects; all of which emphasize the necessity of a multidisciplinary approach in assessing the impact of anthropogenic activities on the environment, especially in historical mining areas.

Effects of radiofrequency electromagnetic fields on seed germination and root meristematic cells of Allium cepa L.

The effects of exposure to radiofrequency electromagnetic fields (RF-EMFs) on seed germination, primary root growth as well as mitotic activity and mitotic aberrations in root meristematic cells were examined in Allium cepa L. cv. Srebrnjak Majski. Seeds were exposed for 2h to EMFs of 400 and 900MHz at field strengths of 10, 23, 41 and 120Vm(-1). The effect of longer exposure time (4h) and field modulation was investigated at 23Vm(-1) as well. Germination rate and root length did not change significantly after exposure to radiofrequency fields under any of the treatment conditions. At 900MHz, exposures to EMFs of higher field strengths (41 and 120Vm(-1)) or to modulated fields showed a significant increase of the mitotic index compared with corresponding controls, while the percentage of mitotic abnormalities increased after all exposure treatments. On the other hand, at 400MHz the mitotic index increased only after exposure to modulated EMF. At this frequency, compared with the control higher numbers of mitotic abnormalities were found after exposure to modulated EMF as well as after exposure to EMFs of higher strengths (41 and 120Vm(-1)). The types of aberration induced by the EMFs of both frequencies were quite similar, mainly consisting of lagging chromosomes, vagrants, disturbed anaphases and chromosome stickiness. Our results show that non-thermal exposure to the radiofrequency fields investigated here can induce mitotic aberrations in root meristematic cells of A. cepa. The observed effects were markedly dependent on the field frequencies applied as well as on field strength and modulation. Our findings also indicate that mitotic effects of RF-EMF could be due to impairment of the mitotic spindle.

Toxicity assessment of heavy metal mixtures by Lemna minor L.

The discharge of untreated electroplating wastewaters directly into the environment is a certain source of heavy metals in surface waters. Even though heavy metal discharge is regulated by environmental laws many small-scale electroplating facilities do not apply adequate protective measures. Electroplating wastewaters contain large amounts of various heavy metals (the composition depending on the facility) and the pH value often bellow 2. Such pollution diminishes the biodiversity of aquatic ecosystems and also endangers human health. The aim of our study was to observe/measure the toxic effects induced by a mixture of seven heavy metals on a bioindicator species Lemna minor L. Since artificial laboratory metal mixtures cannot entirely predict behaviour of metal mixtures nor provide us with informations relating to the specific conditions in the realistic environment we have used an actual electroplating wastewater sample discharged from a small electroplating facility. In order to obtain three more samples with the same composition of heavy metals but at different concentrations, the original electroplating wastewater sample has undergone a purification process. The purification process used was developed by Orescanin et al. [Orescanin V, Mikelic L, Lulic S, Nad K, Rubcic M, Pavlovic G. Purification of electroplating wastewaters utilizing waste by-product ferrous sulphate and wood fly ash. J Environ Sci Health A 2004; 39 (9): 2437-2446.] in order to remove the heavy metals and adjust the pH value to acceptable values for discharge into the environment. Studies involving plants and multielemental waters are very rare because of the difficulty in explaining interactions of the combined toxicities. Regardless of the complexity in interpretation, Lemna bioassay can be efficiently used to assess combined effects of multimetal samples. Such realistic samples should not be avoided because they can provide us with a wide range of information which can help explain many different interactions of metals on plant growth and metabolism. In this study we have primarily evaluated classical toxicity endpoints (relative growth rate, Nfronds/Ncolonies ratio, dry to fresh weight ratio and frond area) and measured guaiacol peroxidase (GPX) activity as early indicator of oxidative stress. Also, we have measured metal accumulation in plants treated with waste ash water sample with EDXRF analysis and have used toxic unit (TU) approach to predict which metal will contribute the most to the general toxicity of the tested samples.

Toxicity of surfactants to green microalgae Pseudokirchneriella subcapitata and Scenedesmus subspicatus and to marine diatoms Phaeodactylum tricornutum and Skeletonema costatum.

Ecotoxicity of different commercial surfactants (six anionic, two amphoteric and one nonionic), essential constituents of cleansing hair products (shampoos), as well as ecotoxicity of eight shampoos containing different combinations of these surfactants, were tested in order to evaluate their possible toxic effects on microalgae. Specific objective of this research was to compare the sensitivity of selected freshwater and marine microalgae to these widely used surfactants and well-known pollutants in surface waters. Internationally validated methods (ISO standards) for the determination of toxic effects on the growth of planktonic freshwater green algae Pseudokirchneriella subcapitata and Scenedesmus subspicatus and marine diatoms Skeletonema costatum and Phaeodactylum tricornutum, were used. The obtained results showed that the concentrations of tested surfactants and shampoos, which resulted in 50% growth reduction of planktonic freshwater green algae, when compared to the controls without test substances (EC50), were in the range from 0.32 to 4.4 mg l(-1) for surfactants and from 2.1 to 8.5 mg l(-1) for shampoos expressed as active substance. Marine diatoms were significantly more sensitive to the tested surfactants than freshwater green algae (EC50 0.14-1.7 mg l(-1) for surfactants and 0.35-1.25 mg l(-1) for shampoos). According to the classification on the basis of environmental effects, the obtained results suggested that all tested surfactants can be classified as having toxic effects on freshwater green alga Pseudokirchneriella subcapitata. Some of them indicated that they have a very toxic effect on Scenedesmus subspicatus and marine diatoms Skeletonema costatum and Phaeodactylum tricornutum.

Assessment of acrylamide toxicity using a battery of standardised bioassays.

Acrylamide is a monomer widely used as an intermediate in the production of organic chemicals, e.g. polyacrylamides (PAMs). Since PAMs are low cost chemicals with applications in various industries and waste- and drinking water treatment, a certain amount of non-polymerised acrylamide is expected to end up in waterways. PAMs are non-toxic but acrylamide induces neurotoxic effects in humans and genotoxic, reproductive, and carcinogenic effects in laboratory animals. In order to evaluate the effect of acrylamide on freshwater organisms, bioassays were conducted on four species: algae Desmodesmus subspicatus and Pseudokirchneriella subcapitata, duckweed Lemna minor and water flea Daphnia magna according to ISO (International Organization for Standardisation) standardised methods. This approach ensures the evaluation of acrylamide toxicity on organisms with different levels of organisation and the comparability of results, and it examines the value of using a battery of low-cost standardised bioassays in the monitoring of pollution and contamination of aquatic ecosystems. These results showed that EC50 values were lower for Desmodesmus subspicatus and Pseudokirchneriella subcapitata than for Daphnia magna and Lemna minor, which suggests an increased sensitivity of algae to acrylamide. According to the toxic unit approach, the values estimated by the Lemna minor and Daphnia magna bioassays, classify acrylamide as slightly toxic (TU=0-1; Class 1). The results obtained from algal bioassays (Desmodesmus subspicatus and Pseudokirchneriella subcapitata) revealed the toxic effect of acrylamide (TU=1-10; Class 2) on these organisms.

Growth and photosynthetic responses of Lemna minor L. exposed to cadmium in combination with zinc or copper.

Metals have a variety of negative outcomes on plants, essential components of any ecosystem. The effects of CdCl2 (5 µmol L-1), ZnCl2 (25 or 50 µmol L-1), and CuCl2 (2.5 or 5 µmol L-1) and combinations of CdCl2 with either ZnCl2 or CuCl2 on the growth, photosynthetic pigments, and photosystem II (PSII) efficiency of duckweed (Lemna minor L.) were investigated. All of the treatments caused growth inhibition and remarkable metal accumulation in plant tissue after 4 and 7 days. In the combined treatments, the accumulation of each metal applied was lesser in comparison to treatments with single metals. After 4 days, all of the treatments generally diminished chlorophyll a content and decreased the maximum quantum yield (Fv/Fm) and effective quantum yield (ΔF/F'm) of PSII. However, after 7 days of exposure to a combination of Cd and Zn, pigment content and PSII activity recovered to control levels. A higher concentration of Cu (5 µmol L-1) as well as Cd in combination with Cu had a prolonged inhibitory effect on photosynthetic features. Our results suggest that growth inhibition was due to the toxic effect of absolute metal quantity in plant tissue. Zn counteracted Cd uptake, as seen from the recovery of pigment content and PSII efficiency in plants exposed for 7 days to the Cd and Zn combination. Cu-induced oxidative stress led to a prolonged inhibitory effect in plants treated both with a higher concentration of Cu (5 µmol L-1) and simultaneously with Cd and Cu. Our findings could contribute to general knowledge on anthropogenic and environmental contaminants that endanger plant communities and significantly disrupt the sensitive balance of an ecosystem by influencing photosynthetic mechanisms.

Xenobiotics removal from polluted water by a multifunctional constructed wetland.

Removal efficiencies on xenobiotics from polluted water in a twin-shaped constructed wetland consisting of a vertical flow chamber with the crop plant Colocasia esculenta L. Schott and a reverse vertical flow one with Ischaemum aristatum var. glaucum Honda, were assessed by chemical analysis and bioassays. After a four-month period of application, removal efficiencies of the applied pesticides parathion and omethoate were 100% with no detectable parathion and omethoate in the effluent. For the applied herbicides, the decontamination was less efficient with removal efficiencies of 36% and 0% for 4-chloro-2-methyl-phenoxyacetic acid and dicamba, respectively. As shown by toxicity assay with duckweed Lemna minor L., growth retardation may occur if the water treated for herbicide removal is used in irrigation of sensitive cultivars in agriculture or horticulture. In contrast to I. aristatum var. glaucum Honda, the crop C. esculenta L. Schott has a high yield in biomass production as a valuable source of renewable energy.

Effect of copper on the toxicity and genotoxicity of cadmium in duckweed (lemna minor L.).

We investigated interactions between copper (in the concentrations of 2.5 µmol L-1 and 5 µmol L-1) and cadmium (5 µmol L-1) in common duckweed (Lemna minor L.) by exposing it to either metal or to their combinations for four or seven days. Their uptake increased with time, but it was lower in plants treated with combinations of metals than in plants treated with either metal given alone. In separate treatments, either metal increased malondialdehyde (MDA) level and catalase and peroxidase activity. Both induced DNA damage, but copper did it only after 7 days of treatment. On day 4, the combination of cadmium and 5 µmol L-1 copper additionally increased MDA as well as catalase and peroxidase activity. In contrast, on day 7, MDA dropped in plants treated with combinations of metals, and especially with 2.5 µmol L-1 copper plus cadmium. In these plants, catalase activity was higher than in copper treated plants. Peroxidase activity increased after treatment with cadmium and 2.5 µmol L-1 copper but decreased in plants treated with cadmium and 5 µmol L-1 copper. Compared to copper alone, combinations of metals enhanced DNA damage after 4 days of treatment but it dropped on day 7. In conclusion, either metal given alone was toxic/genotoxic and caused oxidative stress. On day 4 of combined treatment, the higher copper concentration was more toxic than either metal alone. In contrast, on day 7 of combined treatment, the lower copper concentration showed lower oxidative and DNA damage. These complex interactions can not be explained by simple antagonism and/or synergism. Further studies should go in that direction.