Aquatic Macrophyte Ceratophyllum Demersum Research Articles

Estimation of background concentrations of macro- and trace elements in an aquatic plant as a basis for the passive biomonitoring of pollution

Chemical pollution was indicated as a global environmental problem since elevated concentrations of toxic substances were recorded in almost all ecosystems worldwide. Trace elements, released to environment due to industrial, agricultural and urban activities, are of special concern due to their non-degradability, persistence, bioaccumulation in organisms and potential toxicity. Reliable methods for assessing the level of pollution are essential for proper monitoring and control of pollution. A useful tool for this purpose is the geochemical background (GB), which enables to differentiate between unpolluted and polluted areas as well as calculate pollution indices. The study presents the first attempt to estimate the background values for aquatic plants using cosmopolitan submerged aquatic macrophyte Ceratophyllum demersum as a model species. Water and plant samples were collected from 117 water bodies. Contents of 15 elements (As, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Pb, V, Zn) were determined using atomic absorption spectrometry and flame photometry. Four methods were tested for estimation of the background concentrations: Median ± 2Median Absolute Deviation, Iterative 2σ technique, Tukey box-plot, Grouping of data with 60 % coefficient of variation (CV). Wide ranges of element concentrations in water and various values of Contamination Factor indicated to a variety of natural and anthropogenic impacts in the studied area, which confirmed that the database covered a real environmental variability. Very different estimates of background concentrations were obtained depending on the method. The highest background values were usually given by Me±2MAD method. Grouping of data with 60 % CV was most exigent in defining a site as undisturbed, therefore this method was recommended as the most suitable for estimation of the background values for C. demersum. Pollution Load Index validated the use of estimated background concentrations as reliable for bioindication of pollution in aquatic reservoirs.

Artificial intelligence-based approaches to evaluate and optimize phytoremediation potential of in vitro regenerated aquatic macrophyte Ceratophyllum demersum L.

Water bodies or aquatic ecosystem are susceptible to heavy metal accumulation and can adversely affect the environment and human health especially in underdeveloped nations. Phytoremediation techniques of water bodies using aquatic plants or macrophytes are well established and are recognized as eco-friendly world over. Phytoremediation of heavy metals and other pollutants in aquatic environments can be achieved by using Ceratophyllum demersum L. - a well-known floating macrophyte. In vitro regenerated plants of C. demersum (7.5g/L) were exposed to 24, 72, and 120h to 0, 0.5, 1.0, 2.0, and 4.0mg/L of cadmium (CdSO4·8H2O) in water. Results revealed significantly different relationship in terms of Cd in water, Cd uptake by plants, bioconcentration factor (BCF), and Cd removal (%) from water. The study showed that Cd uptake by plants and BCF values increased significantly with exposure time. The highest BCF value (3776.50) was recorded for plant samples exposed to 2mg/L Cd for 72h. Application of all Cd concentrations and various exposure duration yielded Cd removal (%) between the ranges of 93.8 and 98.7%. These results were predicted through artificial intelligence-based models, namely, random forest (RF), extreme gradient boosting (XGBoost), and multilayer perceptron (MLP). The tested models predicted the results accurately, and the attained results were further validated via three different performance metrics. The optimal regression coefficient (R2) for the models was recorded as 0.7970 (Cd water, mg/L), 0.9661 (Cd plants, mg/kg), 0.9797 bioconcentration factor (BCF), and 0.9996 (Cd removal, %), respectively. These achieved results suggest that in vitro regenerated C. demersum can be efficaciously used for phytoremediation of Cd-contaminated aquatic environments. Likewise, the proposed modeling of phytoremediation studies can further be employed more comprehensively in future studies aimed at data prediction and optimization.

Allelopathic effects of the aquatic macrophyte Ceratophyllum demersum L. on phytoplankton species: contrasting effects between cyanobacteria and chlorophytes

Abstract Aim To assess the allelopathic effects of the submerged macrophyte Ceratophyllum demersum on four strains of phytoplankton species: two cyanobacteria (Microcystis aeruginosa - microcystin producing and M. panniformis - non-microcystin producing), and two chlorophytes (Ankistrodesmus falcatus and Raphidocelis subcapitata). Methods A coexistence experiment between C. demersum and the four strains was carried out for six days, with eight treatments and three replicates. The strains were cultivated in ASM1 culture medium, under controlled laboratory conditions. Two treatments were assigned for each strain, one with 6 g.L-1 of the macrophyte, and the control without the plant. Biomasses and growth rates of the strains were evaluated every two days, which were compared through the T-test and two-way ANOVA, respectively. Results The results varied among the strains, with toxic M. aeruginosa being intensely inhibited by C. demersum, with a decrease of 99.5% in its biomass (p<0.001), while non-toxic M. panniformis was less affected by the allelochemicals, with a reduction of 86.2% (p<0.001). Ankistrodesmus falcatus delayed its growth when in coexistence with the macrophyte, decreasing its biomass in 50.4% (p<0.01), while R. subcapitata was not altered (p>0.05). In coexistence with C. demersum, M. aeruginosa exhibited the lowest growth rates (-0.65 d-1), followed by M. panniformis (-0.15 d-1), A. falcatus (0.19 d-1), and R. subcapitata (0.34 d-1), with significant differences between all strains (p<0.001). Microcystis aeruginosa presented higher inhibition rates than M. panniformis (p<0.001), as well as, A. falcatus was more inhibited than R. subcapitata (p<0.05). Conclusions The presence of microcystins could influence the allelopathic responses of C. demersum, that may release more allelochemicals in coexistence with toxic strains of M. aeruginosa. Accordingly, C. demersum can be used in biomanipulation strategies to control toxic and non-toxic cyanobacterial blooms, without damaging other phytoplankton species, like chlorophytes.

Expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in Escherichia coli and Arabidopsis enhances heavy metal(loid)s accumulation

Phytochelatin synthase (PCS) gene encoding key enzyme for heavy metal detoxification and accumulation has been characterised from different sources and used to develop a technology for bioremediation. Past efforts provided limited success and contradictory results. Therefore, functional characterisation of PCS gene from new sources into different target systems is considered as an important task in the area of bioremediation. Earlier, we isolated and functionally characterised PCS gene from an aquatic macrophyte Ceratophyllum demersum L., a metal accumulator aquatic plant. Expression of this gene, CdPCS1, in tobacco enhanced PC synthesis and metal accumulation of transgenic tobacco plants. In the present study, we have expressed CdPCS1 in more diverse systems, Escherichia coli and Arabidopsis, and studied growth and metal accumulation of transgenic organisms. The expression of CdPCS1 in E. coli offered tolerance against cadmium as well as higher accumulation accompanied with PCS1 activity. The expression of CdPCS1 in Arabidopsis showed a significant enhanced accumulation of heavy metal(loid)s in aerial parts without significant difference in growth parameters in comparison to wild-type Arabidopsis plants. Our study suggests that CdPCS1 can be utilised for enhancing bioremediation potential of different organisms using biotechnological approaches.

Effects of nanomolar copper on water plants—Comparison of biochemical and biophysical mechanisms of deficiency and sublethal toxicity under environmentally relevant conditions

Toxicity and deficiency of essential trace elements like Cu are major global problems. Here, environmentally relevant sub-micromolar concentrations of Cu (supplied as CuSO4) and simulations of natural light- and temperature cycles were applied to the aquatic macrophyte Ceratophyllum demersum. Growth was optimal at 10nM Cu, while PSII activity (Fv/Fm) was maximal around 2nM Cu. Damage to the PSII reaction centre was the first target of Cu toxicity, followed by disturbed regulation of heat dissipation (NPQ). Only after that, electron transport through PSII (ΦPSII) was inhibited, and finally chlorophylls decreased. Copper accumulation in the plants was stable until 10nM Cu in solution, but strongly increased at higher concentrations. The vein was the main storage site for Cu up to physiological concentrations (10nM). At toxic levels it was also sequestered to the epidermis and mesophyll until export from the vein became inhibited, accompanied by inhibition of Zn uptake. Copper deficiency led to a complete stop of growth at “0”nM Cu after 6 weeks. This was accompanied by high starch accumulation although electron flow through PSII (ΦPSII) decreased from 2 weeks, followed by decrease in pigments and increase of non photochemical quenching (NPQ). Release of Cu from the plants below 10nM Cu supply in the nutrient solution indicated lack of high-affinity Cu transporters, and on the tissue level copper deficiency led to a re-distribution of zinc.

Arsenic accumulation by aquatic macrophyte coontail (Ceratophyllum demersum L.) exposed to arsenite, and the effect of iron on the uptake of arsenite and arsenate

In this study, the aquatic macrophyte Ceratophyllum demersum L. (coontail or hornwort) was tested for its efficiency of arsenic (As) uptake under laboratory conditions. Our results revealed that the solution pH had a significant effect on As accumulation by C. demersum (p<0.001). The accumulation was highest at pH 5 and decreased as pH values increased. Plants that were exposed to various concentrations of arsenite (As(III)) for 24 and 48h, exhibited tolerance and toxic responses, respectively. As accumulation by C. demersum depended on the concentrations of As(III) and the duration of exposure (p<0.001). At 40μM after 24h, plants accumulated 227.5μg Asg−1 dw and showed no visible symptoms of toxicity. However, after 48h, As level reached 302.4μgg−1 dw and biomass production decreased significantly. Toxic effects were evident by plant necrosis and negative biomass production, leading to a decrease in the amount of accumulated As. Also, the addition of iron (Fe) into the nutrient solutions (0.18mM) had contrasting effects on the uptake of 2 As species – the uptake of As(III) was enhanced by the presence of Fe, but the uptake of arsenate (As(V)) was considerably inhibited.

Geographically distinct Ceratophyllum demersum populations differ in growth, photosynthetic responses and phenotypic plasticity to nitrogen availability.

Two geographically distinct populations of the submerged aquatic macrophyte Ceratophyllum demersum L. were compared after acclimation to five different nitrogen concentrations (0.005, 0.02, 0.05, 0.1 and 0.2mM N) in a common garden setup. The two populations were an apparent invasive population from New Zealand (NZ) and a noninvasive population from Denmark (DK). The populations were compared with a focus on both morphological and physiological traits. The NZ population had higher relative growth rates (RGRs) and photosynthesis rates (Pmax) (range: RGR, 0.06-0.08 per day; Pmax, 200-395µmolO2g-1 dry mass (DM) h-1) compared with the Danish population (range: RGR, 0.02-0.05 per day; Pmax, 88-169µmol O2 g-1 DM h-1). The larger, faster-growing NZ population also showed higher plasticity than the DK population in response to nitrogen in traits important for growth. Hence, the observed differences in growth behaviour between the two populations are a result of genetic differences and differences in their level of plasticity. Here, we show that two populations of the same species from similar climates but different geographical areas can differ in several ecophysiological traits after growth in a common garden setup.

β-N-Methylamino- l-alanine (BMAA) uptake by the aquatic macrophyte Ceratophyllum demersum

Free-living freshwater cyanobacteria contain BMAA in both free cellular and protein-associated forms. Free BMAA released on bloom collapse or during cellular turnover creates a potential source of the non-proteinogenic amino acid for bioaccumulation and biomagnification in aquatic ecosystems. Uptake of free amino acids is well documented in macrophytes and the potential for aquatic macrophytes to bioaccumulate BMAA therefore poses a potential threat where such macrophytes constitute a food source in an ecosystem. BMAA uptake and accumulation by the aquatic macrophyte Ceratophyllum demersum was therefore investigated. Rapid uptake of significant amounts of BMAA was observed in C. demersum. Both free and protein-associated BMAA were observed with protein association following accumulation of free BMAA. The protein association suggests potential biomaccumulation by aquatic macrophytes and offers a possibility of phytoremediation for BMAA removal.

The aquatic macrophyte Ceratophyllum demersum immobilizes Au nanoparticles after their addition to water

The aquatic macrophyte Ceratophyllum demersum immobilizes Au nanoparticles after their addition to water

Effects of leaf extracts on glutathione reductase expression, hydrogen peroxide and glutathione contents in the aquatic macrophyte Ceratophyllum demersum

Due to senescence and anthropogenic activities, leaves fall into aquatic systems or in their proximity. They decompose and release chemicals that may influence aquatic biota by imposing oxidative stress. It is known that allochthonous leaf litter alone contributes 30% of total dissolved organic carbon (DOC) in streams. We investigated changes in H2O2 and glutathione contents in the coontail, Ceratophyllum demersum, in response to crude extracts from oak (Quercus robur) and reed (Phragmites australis). Glutathione reductase (GR) expression was assessed by semiquantitative RT-PCR. H2O2 showed a dose-dependent increase that tends to plateau at high DOC concentration in both extracts. In parallel, total glutathione (GSH + GSSG) increased, whereas glutathione/glutathione disulphide (GSH/GSSG) ratio decreased. We isolated, for the first time, a partial GR sequence from C. demersum and it showed significant homology (79 – 85%) to GR of other plants. Transcriptional changes in GR after exposure to oak extracts occurred at high DOC concentration (10 mg/L). Reed extracts induced a downregulation of GR at 4 h. Taken together, our data provide evidence that in C. demersum, the regulation of GR expression and redox state of antioxidant (glutathione) pools are, at least in part, influenced by leaf extract-induced H2O2 production. These results suggest that the mechanism of protection against stress posed by the two leaf extracts can be mediated by antioxidant redox responses and regulated GR expression. We suggest that GR could serve as an important biomarker gene in aquatic ecosystems since it was modulated at an environmentally realisticDOC concentration.

Biotransformation and antioxidant response in Ceratophyllum demersum experimentally exposed to 1,2- and 1,4-dichlorobenzene

We report the effects of 1,2- and 1,4-dichlorobenzene (1,2-DCB and 1,4-DCB) on the aquatic macrophyte Ceratophyllum demersum. We evaluated the response of the antioxidant system through the assay of glutathione reductase (GR), guaiacol peroxidase (POD) and glutathione peroxidase (GPx). Additionally, the effect of DCBs on the detoxication system by measuring the activity of glutathione- S-transferase (GST) was evaluated. C. demersum showed elevated GST activities when exposed to 10 and 20 mg l −1 1,2-DCB, and at 10 mg l −1 for 1,4-DCB. These results show that glutathione conjugation take place at relatively high concentrations of both isomers. Significantly increased activities of POD were also detected in C. demersum exposed to concentrations above 5 mg l −1 of the corresponding isomer. The GR activity was enhanced in plants exposed to 1,2-DCB (5 mg l −1) and 1,4-DCB (10 mg l −1). GPx was also significantly increased in exposures to the corresponding isomer, each at a concentration of 10 mg l −1. However, plants exposed to low doses of 1,4-DCB (1 mg l −1) showed significantly decreased activities of both enzymes GR and GPx. Consequently, it is clear that the exposure of the aquatic macrophyte C. demersum to DCBs is able to cause an activation of the antioxidant system, showing an isomer specific pattern, which suggests that the defence system of this plant is playing an important role in scavenging ROS, helping to protect the organism against adverse oxidative effects generated by the prooxidant action of the tested xenobiotics. Furthermore, increased GST activities give indirect evidence on the conjugation of either DCBs or the corresponding metabolites during phase II of detoxication, which supports the elimination process of toxic metabolites from cells of C. demersum.

Phragmites australis and Quercus robur leaf extracts affect antioxidative system and photosynthesis of Ceratophyllum demersum

During senescence, leaves are deposited on aquatic bodies and decay under water releasing chemical substances that might exert physiological stress to aquatic organisms. Leaf litter alone contributes 30% of the total dissolved organic carbon (DOC) in streams. We investigated the impact of leaves extract from Phragmites australis and Quercus robur on the antioxidative system and photosynthetic rate of the aquatic macrophyte Ceratophyllum demersum exposed for 24 h. Rate of photosynthetic oxygen release and antioxidant enzyme activity (glutathione S-transferases, glutathione reductases and peroxidases) as well as lipid peroxidation in C. demersum were measured. Significant ( P<0.01) elevations of antioxidative enzyme activity in C. demersum which tends to plateau at high DOC concentrations were observed. There was no detectable effect on lipid peroxidation. A significant dose-dependent reduction in photosynthetic oxygen production was measured.

Effects of 3-chlorobiphenyl on photosynthetic oxygen production, glutathione content and detoxication enzymes in the aquatic macrophyte Ceratophyllum demersum

Organic contaminants of environmental concern such as polychlorinated biphenyls have dispersed widely throughout the ecosystems and accumulate in living organisms, and a variety of adverse biological effects have been reported. In this study, we investigated the effects of 3-chlorobiphenyl in the aquatic macrophyte Ceratophyllum demersum and the capacity of its detoxication system. After 24 h of exposure to various concentrations of 3-chlorobiphenyl, the total glutathione content (tGSH) was determined and the dose–response curves for glutathione reductase (GR) and microsomal/cytosolic glutathione S-transferases (m- and c-GSTs, respectively) were established. C. demersum showed a decrease of photosynthesis after exposure to 3-chlorobiphenyl, although only significantly at 5 μg l −1. At 0.005 and 0.05 μg l −1 the GR, m-GST and c-GST activities were significantly increased and concomitantly a non-significant effect on total GSH was observed. At 0.5 μg l −1, GR as well as c-GST were still significantly induced, while at 5 μg l −1 none of the enzymes were activated. These results show that detoxication through glutathione conjugation takes place at low concentrations of 3-chlorobiphenyl, while concentrations in the order of parts per billion cause the inactivation of the enzymatic systems evaluated, enough to place C. demersum in an important physiological stress condition.

Promotion of oxidative stress in the aquatic macrophyte Ceratophyllum demersum during biotransformation of the cyanobacterial toxin microcystin-LR

Cyanobacterial toxins have been shown to have adverse effects on mammals, birds and fish and are therefore being increasingly recognised as a potent stress and health hazard factor in aquatic ecosystems. Microcystins, which are cyclic heptapeptides and a main group of the cyanotoxins, are mainly retained within the producer-cells during cyanobacterial bloom development. However, these toxins are released into the surrounding medium by senescence and lysis of the blooms. The released toxins could then come into contact with a wide range of aquatic organisms including invertebrates, fish and aquatic plants. In many organisms, biotransformation of the toxins will take place via several glutathione-related conjugate. During the biotransformation process in which the toxin and the toxin conjugate are broken down, the formation of reactive oxygen species might occur. These reactive oxygen species activate several antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and also influence the glutathione-ascorbate cycle. Aim of this study was to investigate formation of the glutathione-conjugate, activation of glutathione S-transferases and the elevation of several antioxidant enzymes giving evidence for the promotion of oxidative stress by microcystins. During exposure of Ceratophyllum demersum to the cyanobacterial toxin microcystin-LR in an concentration of 5.0 μg/L, an elevation of microsomal and cytosolic glutathione S-transferase was measured, showing the beginning formation of the glutathione-toxin conjugate. The superoxide dismutase as well as in parallel the hydrogen peroxide level increased giving evidence for oxidative stress in the aquatic plant. Other reactive oxygen detoxifiying enzymes were also elevated and the glutathione pool, expressed in reduced glutathione and glutathione disulfide concentration was changed accordingly.

Accumulation of free amino acids and humic substances in a freshwater modular system and their ecological significance

1. Methods for measuring concentrations of total and individual free amino acids (TFAA, FAA), other amino compounds and humic substances (HS) in media containing the following three treatments and the control are described: (i) the non‐axenic aquatic macrophyte Ceratophyllum demersum alone; (ii) the pulmonate snail, Biomphalaria glabrata alone; (iii) the snail, plant and the epiphytic bacteria and algae together as a four‐component modular system; (iv) control without organisms.2. TFAA accumulated to give asymptotic values of 2.5 μm, 10 μm and 15 μm in treatments (i), (ii) and (iii), respectively, by the end of the 30‐day incubation period. The mass‐specific accumulation rate in the treatment with the snail alone [89.4 nmol g–1 (wet weight minus shell) day–1] was approximately ten times that with the plant alone [8.4 nmol g–1 (wet mass) day–1]. No FAA or HS could be detected at any time in (iv).3. On the second day of incubation the concentrations of TFAA and of some individual amino acids were significantly higher in the treatment containing the snail and plant together than the sum of the concentrations in the treatments with the plant and snail alone, presumably due to an increase in the snails’ metabolic rate and ‘sloppy feeding’.4. The differences in the relative abundance of amino compounds accumulating in media conditioned by snails alone (e.g. much larger proportions of ammonia, ethanolamine and phosphoserine than in plant‐conditioned media) and plants alone (e.g. larger proportions of asparagine and glutamine than in snail‐conditioned media) suggest that snails and plants may derive mutual benefits by exchanging amino compounds.5. The accumulation patterns of the more recalcitrant HS differ from those of the amino acids in two respects. First, the HS concentrations continued to increase throughout the 30‐day incubation period in all three treatments. Second, most of the HS in the module originates from the plant as both the concentrations and mass‐specific accumulation rates were significantly higher in the treatment with the plant alone [2.6 mg l–1, 9.09 μg g–1 (wet mass) day–1, respectively] than in the treatment with the snail alone [0.75 mg l–1 and 7.7 μg g–1 (wet mass) day–1, respectively].6. The possible reasons for the differences in the accumulation patterns of FAA and HS in the three treatments are discussed. Evidence is also given in support of the testable hypothesis that the four components of the module derive several mutual benefits, including those arising from the release of dissolved organic matter, such as FAA and HS, by living organisms.

Note: Colonization and invasion of leaves of the aquatic macrophyteCeratophyllum demersum L. by epiphytic bacteria

The colonization of leaves of the aquatic macrophyteCeratophyllum demersum L. by epiphytic bacteria, and the hypothesis that bacterial invasion causes leaf senescence, was studied using transmission and scanning electron microscopy and light microscopy. Population densities of epiphytic bacterial communities onCeratophyllum leaves were positively correlated with leaf age. Initial settlement of bacteria on young leaves appeared to favour the boundaries between epidermal cells. On older leaves, large populations of bacteria were present over the whole surface. One third of senescentCeratophyllum leaves examined by transmission electron microscopy showed signs of bacterial invasion. Of these, up to 54% of the leaf's epidermal cells contained bacteria. Areas of cell wall degradation were associated with invasive bacteria in senescent leaves. In healthy, nonsenescent leaves, no bacterial invasion was observed. These results suggest that epiphytic bacteria did not cause leaf senescence but probably colonized the internal tissues of leaves once senescence had occurred.