Combined biocidal action of silver nanoparticles and ions against Chlorococcales (S<Emphasis Type="Italic">cenedesmus quadricauda</Emphasis>, <Emphasis Type="Italic">Chlorella vulgaris)</Emphasis> and filamentous algae (<Emphasis Type="Italic">Klebsormidium sp.</Emphasis>)

Despite the extensive research, the mechanism of the antimicrobial and biocidal performance of silver nanoparticles has not been unequivocally elucidated yet. Our study was aimed at the investigation of the ability of silver nanoparticles to suppress the growth of three types of algae colonizing the wetted surfaces or submerged objects and the mechanism of their action. Silver nanoparticles exhibited a substantial toxicity towards Chlorococcales Scenedesmus quadricauda, Chlorella vulgaris, and filamentous algae Klebsormidium sp., which correlated with their particle size. The particles had very good stability against agglomeration even in the presence of multivalent cations. The concentration of silver ions in equilibrium with nanoparticles markedly depended on the particle size, achieving about 6 % and as low as about 0.1 % or even less for the particles 5 nm in size and for larger ones (40–70 nm), respectively. Even very limited proportion of small particles together with larger ones could substantially increase concentration of Ag ions in solution. The highest toxicity was found for the 5-nm-sized particles, being the smallest ones in this study. Their toxicity was even higher than that of silver ions at the same silver concentration. When compared as a function of the Ag⁺ concentration in equilibrium with 5-nm particles, the toxicity of ions was at least 17 times higher than that obtained by dissolving silver nitrite (if not taking into account the effect of nanoparticles themselves). The mechanism of the toxicity of silver nanoparticles was found complex with an important role played by the adsorption of silver nanoparticles and the ions released from the particles on the cell surface. This mechanism could be described as some sort of synergy between nanoparticles and ions. While our study clearly showed the presence of this synergy, its detailed explanation is experimentally highly demanding, requiring a close cooperation between materials scientists, physical chemists, and biologists.     

Combined bioremediation of atrazine-contaminated soil by <Emphasis Type="Italic">Pennisetum</Emphasis> and <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. strain DNS10

Strain DNS10 was isolated from the black soil collected from the northeast of China which had been cultivated with atrazine as the sole nitrogen source. Pennisetum is a common plant in Heilongjiang Province of China. The main objective of this paper was to evaluate the efficiency of plant–microbe joint interactions (Arthrobacter sp. DNS10 + Pennisetum) in atrazine degradation compared with single-strain and single-plant effects. Plant–microbe joint interactions degraded 98.10 % of the atrazine, while single strain and single plant only degraded 87.38 and 66.71 % after a 30-day experimental period, respectively. The results indicated that plant–microbe joint interactions had a better degradation effect. Meanwhile, we found that plant–microbe joint interactions showed a higher microbial diversity. The results of microbial diversity illustrated that the positive effects of cropping could improve soil microbial growth and activity. In addition, we planted atrazine-sensitive plants (soybean) in the soil after repair. The results showed that soybean growth in soil previously treated with the plant–microbe joint interactions treatment was better compared with other treatments after 20 days of growth. This was further proved that the soil is more conducive for crop cultivation. Hence, plant–microbe joint interactions are considered to be a potential tool in the remediation of atrazine-contaminated soil.     

Phytoextraction of lead-contaminated soil using vetivergrass (<Emphasis Type="Italic">Vetiveria zizanioides</Emphasis> L.), cogongrass (<Emphasis Type="Italic">Imperata cylindrica</Emphasis> L.) and carabaograss (<Emphasis Type="Italic">Paspalum conjugatum</Emphasis> L.)

Background, Aims and Scope The global problem concerning contamination of the environment as a consequence of human activities is increasing. Most of the environmental contaminants are chemical by-products and heavy metals such as lead (Pb). Lead released into the environment makes its way into the air, soil and water. Lead contributes to a variety of health effects such as decline in mental, cognitive and physical health of the individual. An alternative way of reducing Pb concentration from the soil is through phytoremediation. Phytoremediation is an alternative method that uses plants to clean up a contaminated area. The objectives of this study were: (1) to determine the survival rate and vegetative characteristics of three grass species such as vetivergrass, cogongrass and carabaograss grown in soils with different Pb levels; and (2) to determine and compare the ability of the three grass species as potential phytoremediators in terms of Pb accumulation by plants. Methods The three test plants: vetivergrass (Vetiveria zizanioides L.); cogongrass (Imperata cylindrica L.); and carabaograss (Paspalum conjugatum L.) were grown in individual plastic bags containing soils with 75 mg kg⁻¹ (37.5 kg ha⁻¹) and 150 mg kg⁻¹ (75 kg ha⁻¹) of Pb, respectively. The Pb contents of the test plants and the soil were analyzed before and after experimental treatments using an atomic absorption spectrophotometer. This study was laid out following a 3 × 2 factorial experiment in a completely randomized design. Results On the vegetative characteristics of the test plants, vetivergrass registered the highest whole plant dry matter weight (33.85–39.39 Mg ha⁻¹). Carabaograss had the lowest herbage mass production of 4.12 Mg ha⁻¹ and 5.72 Mg ha⁻¹ from soils added with 75 and 150 mg Pb kg⁻¹, respectively. Vetivergrass also had the highest percent plant survival which meant it best tolerated the Pb contamination in soils. Vetivergrass registered the highest rate of Pb absorption (10.16 ± 2.81 mg kg⁻¹). This was followed by cogongrass (2.34 ± 0.52 mg kg⁻¹) and carabaograss with a mean Pb level of 0.49 ± 0.56 mg kg⁻¹. Levels of Pb among the three grasses (shoots + roots) did not vary significantly with the amount of Pb added (75 and 150 mg kg⁻¹) to the soil. Discussion Vetivergrass yielded the highest biomass; it also has the greatest amount of Pb absorbed (roots + shoots). This can be attributed to the highly extensive root system of vetivergrass with the presence of an enormous amount of root hairs. Extensive root system denotes more contact to nutrients in soils, therefore more likelihood of nutrient absorption and Pb uptake. The efficiency of plants as phytoremediators could be correlated with the plants’ total biomass. This implies that the higher the biomass, the greater the Pb uptake. Plants characteristically exhibit remarkable capacity to absorb what they need and exclude what they do not need. Some plants utilize exclusion mechanisms, where there is a reduced uptake by the roots or a restricted transport of the metals from root to shoots. Combination of high metal accumulation and high biomass production results in the most metal removal from the soil. Conclusions The present study indicated that vetivergrass possessed many beneficial characteristics to uptake Pb from contaminated soil. It was the most tolerant and could grow in soil contaminated with high Pb concentration. Cogongrass and carabaograss are also potential phytoremediators since they can absorb small amount of Pb in soils, although cogongrass is more tolerant to Pb-contaminated soil compared with carabaograss. The important implication of our findings is that vetivergrass can be used for phytoextraction on sites contaminated with high levels of heavy metals; particularly Pb. Recommendations and Perspectives High levels of Pb in localized areas are still a concern especially in urban areas with high levels of traffic, near Pb smelters, battery plants, or industrial facilities that burn fuel ending up in water and soils. The grasses used in the study, and particularly vetivergrass, can be used to phytoremediate urban soil with various contaminations by planting these grasses in lawns and public parks. ESS-Submission Editor: Dr. Willie Peijnenburg (wjgm.peijnenburg@rivm.nl)     

<Emphasis Type="Italic">Ailanthus Altissima</Emphasis> and <Emphasis Type="Italic">Phragmites Australis</Emphasis> for chromium removal from a contaminated soil

The comparative effectiveness for hexavalent chromium removal from irrigation water, using two selected plant species (Phragmites australis and Ailanthus altissima) planted in soil contaminated with hexavalent chromium, has been studied in the present work. Total chromium removal from water was ranging from 55 % (Phragmites) to 61 % (Ailanthus). After 360 days, the contaminated soil dropped from 70 (initial) to 36 and 41 mg Cr/kg (dry soil), for Phragmites and Ailanthus, respectively. Phragmites accumulated the highest amount of chromium in the roots (1910 mg Cr/kg_{(dry tissue)}), compared with 358 mg Cr/kg_{(dry tissue)} for Ailanthus roots. Most of chromium was found in trivalent form in all plant tissues. Ailanthus had the lowest affinity for Cr^VI reduction in the root tissues. Phragmites indicated the highest chromium translocation potential, from roots to stems. Both plant species showed good potentialities to be used in phytoremediation installations for chromium removal.     

Acute,chronic and biochemical effects of chlorothalonil on <Emphasis Type="Italic">Agalychnis callidryas</Emphasis>, <Emphasis Type="Italic">Isthmohyla pseudopuma</Emphasis> and <Emphasis Type="Italic">Smilisca baudinii</Emphasis> tadpoles

Declines of amphibian populations have been a worldwide issue of concern for the scientific community during the last several decades. Efforts are being carried out to elucidate factors related to this phenomenon. Among these factors, pathogens, climate change, and environmental pollution have been suggested as possible causes. Regarding environmental pollutants, some pesticides are persistent in the environment and capable of being transported long distances from their release point. In Costa Rica, some pesticides have been detected in protected areas, at locations where amphibian populations have declined. Information about toxicity of pesticides used in Costa Rican agriculture to amphibians is still scarce, particularly for native species.Toxicity tests with chlorothalonil, a fungicide intensively used in Costa Rica, were carried out exposing tadpoles of three Costa Rican native species: Agalychnis callidryas, Isthmohyla pseudopuma, and Smilisca baudinii in order to evaluate acute and chronic toxicity as well as the biomarkers cholinesterase activity (ChE), glutathione-S transferase activity (GST), and lipid peroxidation (LPO).96-h LC₅₀: 26.6 (18.9–35.8) μg/L to A. callidryas, 25.5 (21.3–29.7) μg/L to I pseudopuma and 32.3 (26.3–39.7) μg/L to S. baudinii were determined for chlorothalonil. These three species of anurans are among the most sensitive to chlorothalonil according to the literature. Besides, GST was induced in S. baudinii after exposure to sub-lethal concentrations of chlorothalonil while evisceration occurred in S. baudinii and A. callidryas tadpoles exposed to lethal concentrations of the fungicide. Chronic exposure to sub-lethal concentrations accelerated development in S. baudinii and caused lesions in tail of S. baudinii and I. pseudopuma tadpoles. Our results demonstrate that chlorothalonil is highly toxic to native amphibian species and that low concentrations can cause biochemical responses related to phase II of biotransformation and effects on development.     

Response of denitrification genes <Emphasis Type="Italic">nirS</Emphasis>, <Emphasis Type="Italic">nirK</Emphasis>, and <Emphasis Type="Italic">nosZ</Emphasis> to irrigation water quality in a Chinese agricultural soil

Purpose  

Denitrification is an important biochemical process in global nitrogen cycle, with a potent greenhouse gas product N₂O. Wastewater irrigation can result in the changes of soil properties and microbial communities of agricultural soils. The purpose of this study was to examine how the soil denitrification genes responded to different irrigation regimes.     

Exposure reduction of seed treatments through dehusking behaviour of the wood mouse <Emphasis Type="Bold">(</Emphasis><Emphasis Type="Italic">Apodemus sylvaticus</Emphasis><Emphasis Type="Bold">)</Emphasis>

Background, aim and scope  

Seed treatments are widely used on cereals and other annual crops throughout Europe. Most of the formulated pesticide is found on the outside of the seed, the husk. Risk assessments of seed treatments are especially needed for granivorous mice living in the agricultural landscape e.g. for registration using the guidance for risk assessment for birds and mammals (EFSA 2009). The dehusking of seeds before consumption is a known behaviour of these mammals, but so far, no quantitative data on the reduction of exposure of seed treatments by dehusking were published. Therefore, we aimed at providing a first quantitative estimate of this behaviour-related exposure reduction for the wood mouse (Apodemus sylvaticus) with different seed types.     

Polychlorinated biphenyls in farmed and wild <Emphasis Type="Italic">Onchorhynchus kisutch</Emphasis> and <Emphasis Type="Italic">Onchorhynchus mykiss</Emphasis> from the Chilean Patagonia

Purpose  

This paper analyses the presence of polychlorinated biphenyls (PCBs) in escaped: farmed and wild salmons in southern Chile, analysing their concentrations and congener profiles in two species (Oncorhynchus kisutch and Oncorhynchus mykiss).     

Capturing forest dependency in the central Himalayan region: Variations between Oak (<Emphasis Type="Italic">Quercus</Emphasis> spp.) and Pine (<Emphasis Type="Italic">Pinus</Emphasis> spp.) dominated forest landscapes

Our study explores the nexus between forests and local communities through participatory assessments and household surveys in the central Himalayan region. Forest dependency was compared among villages surrounded by oak-dominated forests (n = 8) and pine-dominated forests (n = 9). Both quantitative and qualitative analyses indicate variations in the degree of dependency based on proximity to nearest forest type. Households near oak-dominated forests were more dependent on forests (83.8%) compared to households near pine-dominated forests (69.1%). Forest dependency is mainly subsistence-oriented for meeting basic household requirements. Livestock population, cultivated land per household, and non-usage of alternative fuels are the major explanatory drivers of forest dependency. Our findings can help decision and policy makers to establish nested governance mechanisms encouraging prioritized site-specific conservation options among forest-adjacent households. Additionally, income diversification with respect to alternate livelihood sources, institutional reforms, and infrastructure facilities can reduce forest dependency, thereby, allowing sustainable forest management.     

Toxicity of cobalt-complexed cyanide to <Emphasis Type="Italic">Oncorhynchus mykiss,Daphnia magna</Emphasis>, and <Emphasis Type="Italic">Ceriodaphnia dubia</Emphasis>

Background Cobalt cyanide complexes often result when ore is treated with cyanide solutions to extract gold and other metals. These have recently been discovered in low but significant concentrations in effluents from gold leach operations. This study was conducted to determine the potential toxicity of cobalt-cyanide complexes to freshwater organisms and the extent to which ultraviolet radiation (UV) potentiates this toxicity. Tests were also conducted to determine if humic acids or if adaptation to UV influenced sensitivity to the cyanide complexes. Methods Rainbow trout (Oncorhynchus mykiss), Daphnia magna, and Ceriodaphnia dubia were exposed to potassium hexacyanocobaltate in the presence and absence of UV radiation, in the presence and absence of humic acids. Cyano-cobalt exposures were also conducted with C. dubia from cultures adapted to elevated UV. Results With an LC50 concentration of 0.38 mg/L, cyanocobalt was over a 1000 times more toxic to rainbow trout in the presence of UV at a low, environmentally relevant irradiance level (4 μW/cm² as UVB) than exposure to this compound in the absence of UV with an LC50 of 112.9 mg/L. Toxicity was immediately apparent, with mortality occurring within an hour of the onset of exposure at the highest concentration. Fish were unaffected by exposure to UV alone. Weak-acid dissociable cyanide concentrations were observed in irradiated aqueous solutions of cyanocobaltate within hours of UV exposure and persisted in the presence of UV for at least 96 hours, whereas negligible concentrations were observed in the absence of UV. The presence of humic acids significantly diminished cyanocobalt toxicity to D. magna and reduced mortality from UV exposure. Humic acids did not significantly influence survival among C. dubia. C. dubia from UV-adapted populations were less sensitive to metallocyanide compounds than organisms from unadapted populations. Conclusions The results indicate that metallocyanide complexes may pose a hazard to aquatic life through photochemically induced processes. Factors that decrease UV exposure such as dissolved organic carbon or increased pigmentation would diminish toxicity.     

Toxicity of sediment cores from Yangtze River estuary to zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos

Toxicity evaluation is an important segment in sediment quality monitoring in order to protect aquatic organisms and human health. The purpose of this study is to assess the toxicity of sediments from three sediment cores in Yangtze River Estuary, China, using the zebrafish (Danio rerio) embryo tests. Fertilized zebrafish eggs were exposed to both whole sediments and sediment organic extracts prepared from collected sediments, in order to provide a comprehensive and realistic insight into the bioavailable toxicity potential of the sediments. As end points, development parameters (mortality, hatching rate, and abnormality) in the developing embryos were recorded during the 96-h exposure. The results showed that some samples increased mortality, inhibited the hatching of embryos, and induced morphological abnormalities. The embryonic toxicities presented serrated changes and irregular distribution with depth, which may be related to hydrodynamic effect and unstable environmental input. However, lethal and sub-lethal effects were more significant at the sub-surface sediments (10～40 cm), which indicated that the pollution is more serious in recent decades.     

Patterns of volatile metabolites and nonvolatile trichothecenes produced by isolates of <Emphasis Type="Italic">Stachybotrys,Fusarium, Trichoderma,Trichothecium</Emphasis> and <Emphasis Type="Italic">Memnoniella</Emphasis>

We reported previously that trichodiene, a volatile trichothecene derivative, was produced by a Stachybotrys isolate, also known to produce highly cytotoxic, non-volatile, macrocyclic trichothecenes (satrotoxins). We investigated the relationship between the production of trichodiene and various non-volatile trichothecenes for several molds. Volatile metabolites were concentrated by adsorption on Tenax TA and analyzed by GC/MS, while non-volatile metabolites were separated by HPLC, derivatized and analyzed by GC/MS. Stachybotrys chartarum isolates producing macrocyclic trichothecenes secreted significantly larger amounts of trichodiene and other sesquiterpenes than isolates which only produced simple trichothecenes. The amounts of secreted trichodiene were relatively small in all cases. With the exception of Memnoniella, which excreted small amounts of sesquiterpenes, the other isolates produced varying amounts of sesquiterpenes, including trichodiene, as well as simple tricothecenes, no detectable trichodiene, but large amounts of griseofulvin derivatives. In Stachybotrys there is apparently a correlation between trichodiene and macrocyclic trichothecene production. In the remaining isolates, there was no simple relationship between trichodiene and non-volatile trichothecene synthesis. Trichodiene is produced in larger amounts by Stachybotrys isolates, which also produce satratoxins, but it will be difficult to utilize this metabolite to detect toxic isolates in buildings due to the relatively small amounts excreted.     

Acute toxicity of polybrominated diphenyl ethers (PBDEs) for turbot (<Emphasis Type="Italic">Psetta maxima</Emphasis>) early life stages (ELS)

Background, aim and scope  

The environmental presence of polybrominated diphenyl ethers (PBDEs), among which BDE-47 and BDE-99 are particularly abundant, makes toxicity data necessary to assess the hazard risk posed by PBDE to aquatic organisms. This study examines the effects of BDE-47 and BDE-99 on embryo-larval stages of the marine flatfish turbot.     

Side effects of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. kurstaki on the hymenopterous parasitic wasp <Emphasis Type="Italic">Trichogramma chilonis</Emphasis>

Most of the detrimental effects of using conventional insecticides to control crop pests are now well identified and are nowadays major arguments for replacing such compounds by the use of biological control agents. In this respect, the bacterium Bacillus thuringiensis var. kurstaki and Trichogramma (Hymenoptera: Trichogrammatidae) parasitic wasp species are both effective against lepidopterous pests and can actually be used concomitantly. In this work, we studied the potential side effects of B. thuringiensis var. kurstaki on Trichogramma chilonis females. We first evidenced an acute toxicity of B. thuringiensis on T. chilonis. Then, after ingestion of B. thuringiensis at sublethal doses, we focused on life history traits of T. chilonis such as longevity, reproductive success and the time spent on host eggs patches. The reproductive success of T. chilonis was not modified by B. thuringiensis while a significant effect was observed on longevity and the time spent on host eggs patches. The physiological and ecological meanings of the results obtained are discussed.     

Antioxidant defense gene analysis in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Trifolium repens</Emphasis> exposed to Cd and/or Pb

This study focused on the expression analysis of antioxidant defense genes in Brassica oleracea and in Trifolium repens. Plants were exposed for 3, 10, and 56 days in microcosms to a field-collected suburban soil spiked by low concentrations of cadmium and/or lead. In both species, metal accumulations and expression levels of genes encoding proteins involved and/or related to antioxidant defense systems (glutathione transferases, peroxidases, catalases, metallothioneins) were quantified in leaves in order to better understand the detoxification processes involved following exposure to metals. It appeared that strongest gene expression variations in T. repens were observed when plants are exposed to Cd (metallothionein and ascorbate peroxidase upregulations) whereas strongest variations in B. oleracea were observed in case of Cd/Pb co-exposures (metallothionein, glutathione transferase, and peroxidase upregulations). Results also suggest that there is a benefit to use complementary species in order to better apprehend the biological effects in ecotoxicology.     

Chlorpyrifos degradation by the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. strain PUPCCC 64

Background, aim, and scope  

Indiscriminate use of insecticides leads to environmental problems and poses a great threat to beneficial microorganisms. The aim of the present work was to study chlorpyrifos degradation by a rice field cyanobacterium Synechocystis sp. strain PUPCCC 64 so that the organism is able to reduce insecticide pollution in situ.     

Bacterial community composition characterization of a lead-contaminated <Emphasis Type="Italic">Microcoleus</Emphasis> sp. consortium

Introduction  

A Microcoleus sp. consortium, obtained from the Ebro delta microbial mat, was maintained under different conditions including uncontaminated, lead-contaminated, and acidic conditions.     

How the tapeworm <Emphasis Type="Italic">Hymenolepis diminuta</Emphasis> affects zinc and cadmium accumulation in a host fed a hyperaccumulating plant (<Emphasis Type="Italic">Arabidopsis halleri</Emphasis>)

The effects of plant-bound zinc (Zn) and cadmium (Cd) on element uptake and their interactions in a parasite-host system were investigated in a model experiment. Male Wistar rats were divided into four groups (C, P, TC and TP). Groups TC and TP were infected with the rat tapeworm Hymenolepis diminuta. Groups C and TC were fed a standard rodent mixture (ST-1) and received 10.5 mg of Zn per week, while groups P and TP were fed a mixture supplemented with the Zn- and Cd-hyperaccumulating plant Arabidopsis halleri at a dosage of 236 mg Zn/week and 3.0 mg Cd/week. Rats were euthanized after 6 weeks, and Cd and Zn levels were determined in rat and tapeworm tissue. The results indicate that tapeworm presence did have an effect on Cd and Zn concentrations in the host tissue; the majority of tissues in infected rats had statistically significant lower Zn and Cd concentrations than did uninfected rats. Tapeworms accumulated more zinc and cadmium than did the majority of host tissues. This important finding confirms the ability of tapeworms to accumulate certain elements (heavy metals) from the host body to their own body tissues. Thus, tapeworms can decrease heavy metal concentrations in host tissues.     

Remediation potential of caffeine,oxybenzone, and triclosan by the salt marsh plants <Emphasis Type="Italic">Spartina maritima</Emphasis> and <Emphasis Type="Italic">Halimione portulacoides</Emphasis>

Pharmaceuticals and personal care products (PPCPs) have attracted increasing concern during the last decade because of their widespread uses and continuous release to the aquatic environment. This work aimed to study the distribution of caffeine (CAF), oxybenzone (MBPh), and triclosan (TCS) when they arrive in salt marsh areas and to assess their remediation potential by two different species of salt marsh plants: Spartina maritima and Halimione portulacoides. Experiments were carried out in the laboratory either in hydroponics (sediment elutriate) or in sediment soaked in elutriate, for 10 days. Controls without plants were also carried out. CAF, MBPh, and TCS were added to the media. In unvegetated sediment soaked in elutriate, CAF was mainly in the liquid phase (83%), whereas MBPh and TCS were in the solid phase (90% and 56%, respectively); the highest remediation was achieved for TCS (40%) and mainly attributed to bioremediation. The presence of plants in sediment soaked in elutriate-enhanced PPCPs remediation, decreasing CAF and TCS levels between approximately 20-30% and MBPh by 40%.. Plant uptake, adsorption to plant roots/sediments, and bio/rhizoremediation are strong hypothesis to explain the decrease of contaminants either in water or sediment fractions, according to PPCPs characteristics.     

Antioxidative responses of duckweed (<Emphasis Type="Italic">Lemna minor</Emphasis> L.) to short-term copper exposure

Goal, Scope and Background

Elevated concentrations of copper in the environment result in accumulation of the metal in plants and cause an increase in reactive oxidative species (ROS). The first response to elevated amounts of ROS is increased levels of enzymatic and non-enzymatic antioxidants that reduce oxidative stress. The aim of our study was to evaluate the early stages of antioxidative responses to the low copper concentrations usually present in moderately polluted environments. In addition, some other parameters were examined to evaluate the effect of copper on plants.

Methods

Duckweed (Lemna minor L.) was exposed to different concentrations of copper sulphate for up to 24 hours. Glutathione concentration and enzymatic activities of catalase, guaiacol peroxidase and glutathione reductase were measured spectrophotometrically. Additionally, delayed and prompt chlorophyll fluorescence was measured by luminometry and fluorometry, respectively. The accumulation of copper in plants exposed for 24 hours to various concentrations of copper sulphate was measured by flame atomic absorption spectrophotometry.

Results

The treatment of plants with copper sulphate resulted in an immediate decrease of the glutathione pool, which was replenished after 24 hours at CuSO₄ concentrations lower than 2 μM. Higher CuSO₄ concentrations caused a decrease of reduced glutathione. The responses of the antioxidant enzymes glutathione reductase, guaiacol peroxidase and catalase to CuSO₄ differed during the first six hours of exposure, but their enzyme activities all increased after 24 hours of exposure. All these enzymes displayed biphasic activity curves with maximum values between 0.5 μM and 1 μM CuSO₄. The response of guaiacol peroxidase was the most pronounced and statistically significantly specific and that of catalase the least. Delayed chlorophyll fluorescence decreased after exposure to 1 μM CuSO₄, but no significant effect on maximum quantum yield of photosystem II (Fv/Fm) was observed. L. minor accumulated relatively high concentrations of copper. The accumulation rate was higher at lower concentrations of copper in the test medium (up to 2 μM CuSO₄) than at concentrations above 2 μM CuSO₄.

Discussion

One of the most pronounced antioxidative responses to copper exposure was modified levels of oxidized and reduced forms of glutathione. The decrease of the glutathione pool is most probably coupled with induced production of phytochelatins. Antioxidative enzymes showed the biphasic enzyme activity characteristic of stress response. Guaiacol peroxidase exhibited the greatest significant increase of activity, even at higher CuSO₄ concentrations at which the activity of catalase and glutathione reductase dropped. The intensity of delayed chlorophyll fluorescence decreased, indicating reduced photosynthesis of plants under stress. All the measured parameters showed that plants respond to even low copper concentrations very soon after exposure. The accumulation rate of copper in duckweed tissues indicates that L. minor is an accumulator species.

Conclusions

The synchronized and prompt inducibility of antioxidants indicates their involvement in a general plant defence strategy for coping with metal-induced oxidative stress. Glutathione concentration and guaiacol peroxidase activity were found to be the most sensitive of the early indicators of exposure to copper concentrations present in polluted water bodies.

Recommendation and Perspectives

The experimental design of the present study allowed us to compare the sensitivity of various methods and parameters for detecting plant responses to heavy metal-induced oxidative stress. The level of glutathione and the enzyme activities of guaiacol peroxidase and glutathione reductase could be used as a rapidly determined early warning system in toxicity studies.