Microtubule integrity and cell viability under metal (Cu, Ni and Cr) stress in the seagrass Cymodocea nodosa (2023)

Coastal macrophytes serve as bioindicators of coastal trace metal contamination. In this study, trace metal levels in India's seagrass and saltmarsh ecosystems were assessed for their suitability as bioindicators of metal contamination. Trace metal accumulation and bioindicator potential of both seagrasses and saltmarshes were found to be metal and species-specific. Higher concentrations of Cu, Fe, Mg and Mn were found in the tissues of seagrasses, while saltmarshes showed higher accumulation of Cd, Cr, Hg, Ni, Pb and Zn. The leaves of seagrasses are suitable bioindicator of metals in the water column, while the roots and rhizomes of saltmarshes/seagrasses are suitable bioindicators of metals in the sediment. This study proposes the development of a monitoring network using seagrasses and saltmarss as model organisms for short and long-term monitoring of coastal metal contamination. Determining the phytotoxic levels of trace metals in seagrasses and saltmarsh is important for monitoring plant die-offs and loss.

As an essential plant micronutrient, copper (Cu) is required as a component of several enzymes, but it can be highly toxic to plants when present in excess quantities. Nitrogen (N) application can help to alleviate the phytotoxic effects of heavy metals, including Cu, and different N forms significantly affect the uptake and accumulation of heavy metals in plants. The aim of this study was to determine the effects of different N forms, i.e., ammonium (NH₄⁺) and nitrate (NO₃⁻), on Cu detoxification in wheat seedlings. The inhibition of seedling growth under excess Cu was more obvious in wheat plants supplied with NO₃⁻ than in those supplied with NH₄⁺. This growth inhibition was directly induced by excess Cu accumulation and reduced absorption of other mineral nutrients by the plants. Compared with seedlings treated with NO₃⁻, those treated with NH₄⁺ showed a decrease in Cu-induced toxicity as a result of increased antioxidant capacity in the leaves and a lower redox potential in the rhizosphere. Furthermore, treatment with NH₄⁺ decreased the loss of mineral nutrients in wheat seedlings exposed to excess Cu. In conclusion, compared with supplying NO₃⁻, supplying NH₄⁺ to wheat seedlings under Cu stress improved their ability to maintain their nutritional and redox balance and increased their antioxidant capacity, thereby preventing a decline in photosynthesis. According to our results, NH₄⁺ is more effective than NO₃⁻ in reducing Cu phytotoxicity in wheat seedlings.

Antarctica has long been considered a continent free from anthropic interference. Unfortunately, recent evidence indicate that metal contamination has gone so far and that its effects are still unknown. For this reason, in the present work, the potential endocrine disrupting effect of two highly polluting metals, copper and cadmium, were examined in the Antarctic teleost Trematomus bernacchii. After a 10 days waterborne exposure, ovarian metal uptake was determined by atomic absorption; in parallel, classical histological approaches were adopted to determine the effects on oocyte morphology, carbohydrate composition and presence and localization of progesterone and estrogen receptors. Results show that both metals induce oocyte degeneration in about one third of the previtellogenic oocytes, no matter the stage of development. In apparently healthy oocytes, changes in cytoplasm, cortical alveoli and/or chorion carbohydrates composition are observed. Cadmium but not copper also induces significant changes in the localization of progesterone and beta-estrogen receptors, a result that well correlates with the observed increase in ovarian metals concentrations. In conclusion, the acute modifications detected are suggestive of a significantly impaired fecundity and of a marked endocrine disrupting effects of copper and cadmium in this teleost species.

This work aims to provide insight on the effects of chromium (Cr) uptake in seagrasses at environmentally relevant exposure concentrations; information on this issue can further increase the utility of marine angiosperms as indicators of trace metal contamination. Cr uptake kinetics, effects on physiological and structural cell traits, and the uptake-effects relationship in Halophila stipulacea leaves incubated in 0.01–10mgL⁻¹ (0.2–200μM) of Cr (VI) were examined. An initial uptake was followed by a decrease at 0.01–0.5mgL⁻¹ (0.2–10μM); at higher exposures, the fit of uptake data to a Michaelis-Menten-type equation was significant (p<0.001). The mean tissue concentration over the exposure period, and the highest tissue concentration attained both increased as exposure concentration (C_w) increased from 0.05 to 10mgL⁻¹ (1 to 200μM); uptake rate generally increased with C_w. Cytotoxic effects occurred even at 0.01mgL⁻¹ (0.2μM). Actin filaments (AFs) and endoplasmic reticulum (ER) were impacted in a dose- and time- dependent pattern, while microtubule integrity was not markedly affected even at the highest exposure. AF disruption in differentiating cells initiated at 0.01mgL⁻¹ (0.2μM), while ER disorganization did so at 0.1mgL⁻¹ (2μM); regression equations described the relationships of C_w vs. time required for effect onset, and of C_w vs. AF disturbance progress. Elevated levels of hydrogen peroxide (H₂O₂) were detected on the 2d. In addition, a significant reduction in epidermal cell viability appeared at 5–10 or 10mgL⁻¹ (100–200 or 200μM), depending on the leaf age. The lowest tissue concentration associated with effect onset was 31.05μgg⁻¹ dry wt (2d, 5mgL⁻¹); at lower exposures, higher tissue concentrations did not coincide with impact, implying that the uptake rate rather than the tissue concentration relates to the effect onset. The above suggest that (a) Cr (VI) may pose a high risk to seagrass meadows, (b) H. stipulacea can act as a bioindicator of ambient Cr (VI) bioavailability, mainly at elevated exposure concentrations, (c) AF and ER disturbances in seagrass leaf cells could be considered as sensitive biomarkers of Cr (VI)-induced stress, (d) regression equations describing the relationships C_w vs. time required for effect onset and C_w vs. effect progress increase the utility of AF and ER impairment as biomarkers, and (e) the time frame of the exposure should be considered in the interpretation of tissue residues. Our findings improve the utility of seagrasses as indicators of trace metal contamination and could be utilized in the overall effort for seagrass meadows conservation.

The organic pollutant bisphenol A (BPA) causes adverse effects on aquatic biota. The present study explored the toxicity mechanism of environmentally occurring BPA concentrations (0.03–3μgL⁻¹) on the seagrass Cymodocea nodosa intermediate leaf photosynthetic machinery. A "mosaic" type BPA effect pattern was observed, with "unaffected" and "affected”" leaf areas. In negatively affected leaf areas cells had a dark appearance and lost their chlorophyll auto-fluorescence, while hydrogen peroxide (H₂O₂) content increased time-dependently. In the "unaffected" leaf areas, cells exhibited increased phenolic compound production. At 1μgL⁻¹ of BPA exposure, there was no effect on the fraction of open reaction centers (q_P) compared to control and also no significant effect on the quantum yield of non-regulated non-photochemical energy loss in PSII (Φ_ΝΟ). However, a 3μgL⁻¹ BPA application resulted in a significant Φ_ΝΟ increase, even from the first exposure day. Ultrastructural observations revealed electronically dense damaged thylakoids in the plastids, while effects on Golgi dictyosomes and the endoplasmic reticulum were also observed at 3μgL⁻¹ BPA. The up-regulated H₂O₂ BPA-derived production seems to be a key factor causing both oxidative damages but probably also triggering retrograde signalling, conferring tolerance to BPA in the "unaffected" leaf areas.

Marine Pollution Bulletin, Volume 104, Issues 1–2, 2016, pp. 335-342

Trace elements concentrations (As, Ba, Cd, Cr, Cu, Ni, Pb, V and Zn) were found in samples of Halodule wrightii Aschers (Cymodoceaceae) seagrass from Todos os Santos Bay, Bahia, Brazil by inductively coupled plasma mass spectrometry (ICP-MS). This bay has been impacted by the presence of industrial activity that includes chemical and petrochemical plants as well as an oil refinery and harbor activities located in the north and northeastern area. Samples were collected at 4 stations to evaluate the distribution of element concentrations in this area and under different climatic conditions. The mean concentrations, in mgkg⁻¹, found in dry tissues for all the studied metals and stations were: As (1.08–5.42), Ba (3.72–32.0), Cd (0.135–1.68), Cr (1.15–10.4), Cu (2.23–13.4), Ni (1.95–9.87), Pb (0.873–5.18), V (3.39–22.4) and Zn (13.1–39.5). Statistical analysis (ANOVA) of the seagrass data showed significant inter-site differences for all elements examined except nickel and vanadium.

Marine Pollution Bulletin, Volume 109, Issue 1, 2016, pp. 325-333

To investigate the potential influences of anthropogenic pollutants, we evaluated the responses of the intertidal seagrass Zostera japonica to three heavy metals: copper (Cu), lead (Pb), and cadmium (Cd). Z. japonica was exposed to various concentrations of Cu, Pb_, and Cd (0, 0.5, 5, 50μM) over seven days. The effects were then analyzed using the antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GPX), and lipid peroxidation measured using malondialdehyde (MDA) as proxy. Metal accumulation in the above-ground tissues and phenotypic changes were also investigated. Our results revealed that heavy metal concentration increased in seagrass exposed to high levels of metals. Z. japonica has great potential for metal accumulation and a suitable candidate for the decontamination of moderately Cu contaminated bodies of water and can also potentially enhanced efforts of environmental decontamination, either through phytoextraction abilities or by functioning as an indicator for monitoring programs that use SOD, CAT, GPX, POD and MDA as biomarkers.

Marine Pollution Bulletin, Volume 116, Issues 1–2, 2017, pp. 196-203

This study investigated the trace element bioaccumulation capacity of the Mediterranean seagrass Posidonia oceanica, and its suitability as a bioindicator of contamination in water and sediments. Results showed that P. oceanica leaves accumulate higher concentrations of Ni and Zn. Since P. oceanica regenerates its leaves periodically, the higher concentrations in aerial organs may suggest a “removal” strategy according to which P. oceanica accumulates greater concentrations of trace elements in its temporary organs. In turn, P. oceanica seems to adopt an exclusion strategy for toxic non-essential elements (As, Cr, Pb). Results showed also that P. oceanica organs are correlated with As, Cd, Cu, Ni, and Zn concentrations in sediments. No significant relationship was found between P. oceanica and water. This study showed that P. oceanica may adopt different tolerance strategies compared to mainland-rooted macrophytes, and its possible use as a bioindicator of trace elements in sediments should be considered.

Ecotoxicology and Environmental Safety, Volume 189, 2020, Article 109925

Information on silver nanoparticle (AgNP) phytotoxicity on seagrasses is provided for the first time. Toxic effects of environmentally relevant AgNP concentrations on Halophila stipulacea were assessed to identify sensitive biomarkers, to determine threshold effect concentrations and to evaluate potential risks. Potential alterations in the cytoskeleton, endoplasmic reticulum, cell ultrastructure and viability, oxidative stress parameters and elongation in H. stipulacea leaves exposed to AgNP concentrations ranging from 0.0002 to 0.2mgL⁻¹ for 8 days were examined. The first signs of actin filament (AF) response in differentiating cells, exhibiting disorientation and slight bundling, were observed on the 4th day at 0.0002mgL⁻¹, while at the end of the experiment and at the higher concentrations, AFs were extremely bundled. Endoplasmic reticulum was affected in meristematic and differentiating cells; massive aggregations and loss of the “grainy” structure were observed, initially on the 6th day at 0.002mgL⁻¹. Effects on microtubules were detected on the last day at 0.2mgL⁻¹. An increase in H₂O₂ levels on the 4th and/or 6th day even at 0.0002mgL⁻¹ was followed by a decrease on, or up to the last day. On the 6th day at the lowest concentration, elevated malondialdehyde content, and superoxide dismutase and peroxidase activity were detected, indicating oxidative damage and antioxidant defense mechanism activation. Dead epidermal cells mainly occurred at 0.02 and 0.2mgL⁻¹, while no dead vein cells were detected. A significant inhibition in leaf elongation was observed only at 0.2mgL⁻¹. Therefore, AF disturbance in differentiating leaf cells, being a susceptible response parameter, could be regarded as an early warning indicator of risk posed by AgNPs to H. stipulacea meadows, while most of the remaining parameters examined also constitute useful biomarkers. The lowest observed effect concentration (0.0002mgL⁻¹), being within the range of environmentally relevant AgNPs concentrations, suggests the possibility of negative impacts of AgNPs on seagrass health. A risk quotient of 1.33 was calculated, indicating that AgNPs may pose a significant potential risk to the coastal environment. The data presented highlight the importance of future research to further investigate the seagrass-AgNP interactions, stress the need for a refinement of the environmental risk assessment of AgNPs and could be utilized for the design of biomonitoring programs for rational management of the coastal environment.

Marine Pollution Bulletin, Volume 157, 2020, Article 111305

Metals such as Cd, Pb, Cu and Zn were analysed in five seagrass species from five locations of the Palk Bay coast of south-eastern India. The maximum Cd concentration (2.83±0.08μgg⁻¹) was recorded in Syringodium isoetifolium on the Mallipattinam coast in the post-monsoon season, and the Cu (29.54±1.03μgg⁻¹) in Enhalus acoroides of the Thondi coast during monsoon season. The maximum concentrations of Pb (3.12±0.14μgg⁻¹) and Zn (52.66±0.88μgg⁻¹) were observed in S. isoetifolium and Cymodoceae rotundata on the Devipattinam and Karangadu coasts, respectively, during the monsoon season. The metal concentrations obtained in different seagrass species varied significantly among species, and no clear monsoon season had a clear effect on the accumulation of heavy metals in the investigated seagrass species. S. isoetifolium has higher capacity for Cd and Pb accumulation than other tested seagrass species.

Aquatic Toxicology, Volume 207, 2019, pp. 91-100

Copper (Cu) is an essential micronutrient for plants and as such is vital to many metabolic processes. Nevertheless, when present at elevated concentrations, Cu can exert toxic effects on plants by disrupting protein functions and promoting oxidative stress. Due to their proximity to the urbanised estuaries, seagrasses are vulnerable to chemical contamination via industrial runoff, waste discharges and leachates. Zostera muelleri is a common seagrass species that forms habitats in the intertidal areas along the temperate coast of Australia. Previous studies have shown the detrimental effects of Cu exposure on photosynthetic efficiency of Z. muelleri. The present study focuses on the impacts of sublethal Cu exposure on the physiological and molecular responses. By means of a single addition, plants were exposed to 250 and 500 μg Cu L⁻¹ (corresponding to 3.9 and 7.8 μM, respectively) as well as uncontaminated artificial seawater (control) for 7 days. Chlorophyll fluorescence parameters, measured as the effective quantum yield ($\varphi$PSII), the maximum quantum yield (Fv/Fm) and non-photochemical quenching (NPQ) were assessed daily, while Cu accumulation in leaf tissue, total reactive oxygen species (ROS) and the expression of genes involved in antioxidant activities and trace metal binding were determined after 1, 3 and 7 days of exposure. Z. muelleri accumulated Cu in the leaf tissue in a concentration-dependent manner and the bioaccumulation was saturated by day 3. Cu exposure resulted in an acute suppression of $\varphi$PSII and Fv/Fm. These two parameters also showed a concentration- and time-dependent decline. NPQ increased sharply during the first few days before subsequently decreasing towards the end of the experiment. Cu accumulation induced oxidative stress in Z. muelleri as an elevated level of ROS was detected on day 7. Lower Cu concentration promoted an up-regulation of genes encoding Cu/Zn-superoxide dismutase (Cu/Zn-sod), ascorbate peroxidase (apx), catalase (cat) and glutathione peroxidase (gpx), whereas no significant change was detected with higher Cu concentration. Exposure to Cu at any concentration failed to induce regulation in the expression level of genes encoding metallothionein type 2 (mt2), metallothionein type 3 (mt3) and cytochrome c oxidase copper chaperone (cox17). It is concluded that chlorophyll fluorescence parameters provide timely probe of the status of photosynthetic machinery under Cu stress. In addition, when exposed to a moderate level of Cu, Z. muelleri mitigates any induced oxidative stress by up-regulating transcripts coding for antioxidant enzymes.