Modifying effects of leaf litter extracts from invasive versus native tree species on copper-induced responses in Lemna minor.

Invasive plant species tend to migrate from their native habitats under favourable climatic conditions; therefore, trophic and other relationships in ecosystems are changing. To investigate the effect of natural organic matter derived from native Alnus glutinosa tree species and from invasive in Lithuania Acer negundo tree species on copper toxicity in Lemna minor, we analysed the dynamics of Cu binding in aqueous leaf litter extracts (LLE) and plant accumulation, morphophysiological parameters, and antioxidative response. The results revealed that A. glutinosa LLE contained polyphenols (49 mg pyrogallol acid equivalent (PAE)/g DM) and tannins (7.5 mg PAE/g DM), while A. negundo LLE contained only polyphenols (23 mg PAE/g DM). The ability of LLE to bind Cu increased rapidly over 1.5-3 h to 61% and 49% of the total Cu concentration (6.0 ± 0.9 mg/L), respectively for A. glutinosa (AG) and A. negundo (AN), then remained relatively stable until 48 h. At the same time, L. minor accumulated 384, 241 or 188 µg Cu/g FW when plants were exposed to Cu (100 µM CuSO4), Cu with 100 mg/L dissolved organic carbon (DOC) from either AG LLE or AN LLE, accordingly. Catalase (CAT) and guaiacol peroxidase (POD) played a dominant role in hydrogen peroxide scavenging when plants were exposed to Cu and 10 or 100 mg/L DOCAG mixtures in both the first (up to 6h) and the second (6-48 h) response phases. Due to functioning of oxidative stress enzymes, the levels of the lipid peroxidation product malondialdehyde (MDA) reduced in concentration-dependent manner, compared to Cu treatment. When combining Cu and DOCAN treatments, the most sensitive enzymes were POD, ascorbate peroxidase and glutathione reductase. Their activities collectively with CAT were sufficient to reduce MDA levels to Cu-induced in the initial, but not the second response phase. These data suggest that leaf litter extracts of different phenolic compositions elicited different antioxidant response profiles resulting in different reductions of Cu stress, thus effecting L. minor frond and root development observed after seven days. The complex data from this study may be useful in modelling the response of the aquatic ecosystem to a changing environment.

Accumulation of copper in the cell compartments of charophyte Nitellopsis obtusa after its exposure to copper oxide nanoparticle suspension.

Cu accumulation in the internodal cell of charophyte Nitellopsis obtusa or its compartments was investigated after 3-h-exposure to lethal effective concentrations (8-day LC50) of CuO nanoparticle (nCuO) suspension or CuSO4 solution, i.e. 100 mg/L nCuO or 3.18 mg Cu/L as CuSO4. In both cases, the major part of Cu accumulated in the cell walls. The presence of CuO NPs in the cell wall and within the cell was visualized by scanning electron microscope images as well as confirmed by energy dispersive X-ray spectrum data. Although a threefold higher intracellular concentration of Cu was found after treatment with nCuO suspension, 3.18 mg Cu/L as CuSO4 induced fast and substantial depolarization of cell membrane potential contrary to that of 100 mg/L nCuO. A delayed effect of nCuO on the survival of the cells was also observed. This suggests that internally accumulated Cu was far less active and further supports the hypothesis of delayed toxicity of internalized nCuO NPs to charophyte cells.

Ecotoxicity effects triggered in aquatic organisms by invasive Acer negundo and native Alnus glutinosa leaf leachates obtained in the process of aerobic decomposition.

The replacement of autochthonous tree species by invasive ones in coastal zones of freshwater bodies induces additional alteration of hydrochemical and microbiological characteristics due to decomposition of fallen leaves of non-indigenous species, which can lead to ecotoxic response of the littoral biota. Leaves of invasive to Lithuania boxelder maple (Acer negundo) and autochthonous black alder (Alnus glutinosa) lost more than half of biomass and released stable amount of DOC (60-70 mg/L) throughout 90-day mesocosm experiment under aerobic conditions. This, along with the relatively small BOD7 values detected after some variation within the first month confirms effective biodegradation by fungi and bacteria. The ambient water was more enriched with different forms of N and P by decomposing boxelder maple than by alder leaves. During the first month, both leachates were more toxic to charophyte (Nitellopsis obtusa) at mortality and membrane depolarization levels, while later to two crustacean species. Biomarker response, H(+)-ATPase activity in membrane preparations from N. obtusa, was stronger for A. negundo. Generally, boxelder maple leaf leachates were more toxic to tested hydrobionts and this coincides with previous study on leaves of the same pair of tree species conducted under microaerobic conditions (Krevs et al., 2013).

Ecotoxicological effects evoked in hydrophytes by leachates of invasive Acer negundo and autochthonous Alnus glutinosa fallen off leaves during their microbial decomposition.

Throughout 90-day biodegradation under microaerobic conditions, invasive to Lithuania species boxelder maple (Acer negundo) leaves lost 1.5-fold more biomass than that of autochthonous black alder (Alnus glutinosa), releasing higher contents of N(tot), ammonium and generating higher BOD(7). Boxelder maple leaf leachates were characterized by higher total bacterial numbers and colony numbers of heterotrophic and cellulose-decomposing bacteria than those of black alder. The higher toxicity of A. negundo aqueous extracts and leachates to charophyte cell (Nitellopsis obtusa), the inhabitant of clean lakes, were manifested at mortality and membrane depolarization levels, while the effect on H(+)-ATPase activity in membrane preparations from the same algae was stronger in case of A. glutinosa. Duckweed (Lemna minor), a bioindicator of eutrophic waters, was more sensitive to leaf leachates of A. glutinosa. Fallen leaves and leaf litter leachates from invasive and native species of trees, which enter water body, affect differently microbial biodestruction and aquatic vegetation in freshwater systems.

Toxicity of copper oxide nanoparticle suspensions to aquatic biota.

Toxicity effects induced by nanosuspensions of CuO (<50 nm; Sigma-Aldrich) on macrophytic algae cells of Nitellopsis obtusa (96-h median lethal concentration [LC50]), microphytic algae Chlorella (30-min median inhibitory concentration [IC50]), shrimp Thamnocephalus platyurus (24-h LC50), and rotifer Brachionus calyciflorus (24-h LC50) were investigated. No substantial differences between the effects of nonsonicated and sonicated nCuO suspensions were observed. The particle size distribution analysis accomplished by the laser diffraction technique at suspension concentration from 3 to 100 mg/L revealed rapid (within 5 min) reagglomeration of the particles after the sonication. The observed adverse effects on N. obtusa cells may be attributed to nanoparticles per se, but not to ionic Cu, because neither chemical analysis nor biological testing (algae survival in the supernatants of suspensions) confirmed the presence of cupric ions in toxic amounts. Contrary to ionic Cu form, nCuO delayed the initial phase of N. obtusa cell membrane depolarization. Lethality tests with rewash demonstrated that the least used 5-min exposure in 100 mg/L nCuO sonicated suspension induced 70% mortality in charophyte cells after 8 d, whereas the rewash after a short exposure to a noticeably toxic concentration of Cu(2+) prevented cell mortality. The obtained data suggested the possible influence of a thick charophyte cell wall on the dynamics of nanotoxicity effects.

Response of the charophyte Nitellopsis obtusa to heavy metals at the cellular, cell membrane, and enzyme levels.

The responses of the freshwater macroalga Nitellopsis obtusa to heavy metal (HM) salts of Hg, Cd, Co, Cu, Cr, and Ni were assessed at different levels: whole-cell mortality (96-h LC(50)), in vivo cell membrane (45-min depolarization of resting potential, EC(50)), and enzyme in plasma membrane preparations (K+, Mg2+-specific H+-ATPase inhibition, IC(50)). To measure ATPase activity, a novel procedure for isolation of plasma membrane-enriched vesicles from charophyte cells was developed. The short-term ATPase inhibition assay (IC(50) from 6.0 x 10(-7) to 4.6 x 10(-4) M) was slightly more sensitive than the cell mortality test (LC(50) from 1.1 x 10(-6) to 2.6 x 10(-3) M), and the electrophysiological test with the end point of 45-min depolarization of resting potential was characterized by less sensitivity for HMs (EC(50) from 1.1 x 10(-4) to 2.2 x 10(-2) M). The variability of IC(50) values assessed for HMs in the ATPase assays was close to that of LC(50) values in the mortality tests (CVs from 33.5 to 83.5 and from 12.4% to 57.7%, respectively), whereas the EC(50) values in the electrophysiological tests were characterized by CVs generally below 30%. All three end points identified two separate HM groups according to their toxicity to N. obtusa: Co, Ni, and Cr comprised a group of less toxic metals, whereas Hg, Cu, and Cd comprised a group of more toxic metals. However, the adverse effects within each group were discriminated differently. For example, the maximum difference between the highest and lowest LC(50) for the group of less toxic metals in the long-term mortality test was approximately 60% of the response range, whereas the corresponding difference in IC(50) values in the ATPase assay was 30%. In contrast, the LC(50) values of the more toxic metals occupied only 10% of the response range, whereas the IC(50) values were spread over 70%. Further investigation should be done of the underlying mechanism or mechanisms responsible for the observed differences in the dynamic range of a particular end point of the groups of toxicants of varying strength.