Differential effects of pollution on adult and recruits of a canopy-forming alga: implications for population viability under low pollutant levels.

Marine macroalgal forests are highly productive and iconic ecosystems, which are seriously threatened by number of factors such as habitat destruction, overgrazing, ocean warming, and pollution. The effect of chronic, but low levels of pollutants on the long-term survival of the canopy-forming algae is not well understood. Here we test the effects of low concentrations (found in good quality water-bodies) of nitrates, heavy metals copper (Cu) and lead (Pb), and herbicides (glyphosate) on both adults and recruits of Carpodesmia crinita, a Mediterranean canopy forming macroalga. We show that although adult biomass, height and photosynthetic yield remain almost unaffected in all the assays, low Cu levels of 30 µg/L completely suppress adult fertility. In addition, all the assays have a strong and negative impact on the survival and growth of recruits; in particular, glyphosate concentrations above 1 µg/L almost totally inhibit their survival. These results suggest that the long-term viability of C. crinita may be severely compromised by low pollutant levels that are not affecting adult specimens. Our results provide important data for a better understanding of the present-day threats to marine canopy-forming macroalgae and for the design of future management actions aimed at preserving macroalgal forests.

The response of Pyropia haitanensis to inorganic arsenic under laboratory culture.

Up to now, complicated organoarsenicals were mainly identified in marine organisms, suggesting that these organisms play a critical role in arsenic biogeochemical cycling because of low phosphate and relatively high arsenic concentration in the marine environment. However, the response of marine macroalgae to inorganic arsenic remains unknown. In this study, Pyropia haitanensis were exposed to arsenate [As(V)] (0.1, 1, 10, 100 µM) or arsenite [As(III)] (0.1, 1, 10 µM) under laboratory conditions for 3 d. The species of water-soluble arsenic, the total concentration of lipid-soluble and cell residue arsenic of the algae cells was analyzed. As(V) was mainly transformed into oxo-arsenosugar-phosphate, with other arsenic compounds such as monomethylated, As(III), demethylated arsenic and oxo-arsenosugar-glycerol being likely the intermediates of arsenosugar synthesis. When high concentration of As(III) was toxic to P. haitanensis, As(III) entered into the cells and was transformed into less toxic organoarsenicals and As(V). Transcriptome results showed genes involved in DNA replication, mismatch repair, base excision repair, and nucleotide excision repair were up-regulated in the algae cells exposed to 10 µM As(V), and multiple genes involved in glutathione metabolism and photosynthetic were up-regulated by 1 µM As(III). A large number of ABC transporters were down-regulated by As(V) while ten genes related to ABC transporters were up-regulated by As(III), indicating that ABC transporters were involved in transporting As(III) to vacuoles in algae cells. These results indicated that P. haitanensis detoxifies inorganic arsenic via transforming them into organoarsenicals and enhancing the isolation of highly toxic As(III) in vacuoles.

Variation in microbial populations and antibiotic resistance genes in mariculture sediments in the present of the seaweed Ulva fasciata and under selective pressure of oxytetracycline.

The widely distributed seaweed Ulva fasciata has nutrient absorption abilities and can be used in the bioremediation of polluted maricultural environments. This study explored microbial community and antibiotic resistance gene (ARG) variation in mariculture sediments in response to different trace levels (10, 100, and 500 µg L-1) of oxytetracycline (OTC) and the presence of Ulva fasciata. The increase in OTC level promoted nutrient (NO3_-N and PO43--P) removal mainly due to Ulva fasciata adsorption. The abundances of the Euryarchaeota and Planctomycetes phyla in sediments were positively related to the increase in OTC stress, while a negative correlation occurred for the Proteobacteria phylum via metagenomic analysis. Compared with the control system, the increase rates of total ARGs were 3.90%, 7.36% and 13.42% at the OTC levels of 10, 100 and 500 µg L-1, respectively. OTC stress mainly favoured the collateral enrichment of non-corresponding polypeptide and MLS ARGs, mainly due to the enrichment of the phyla Planctomycetes and Euryarchaeota by the synergistic effect of OTC and nutrients. The results of quantitative PCR with tetracycline resistance genes (TRGs) (tetO, tetT, tetPB, tetW and otrA) and a horizontal transfer gene (intl1) demonstrated that all of genes had much higher gene numbers in sediments after 3 months of OTC stress than in those without OTC stress, which was strongly related to the variation in the phyla Bacteroidetes, Gemmatimonadetes and Acidobacteria. The significant correlation between intl1 and the target TRGs is indicative of the important role of the horizontal transfer of integron-resistant genes in the spread of TRGs.

Effect of allelochemicals on photosynthetic and antioxidant defense system of Ulva prolifera.

Ulva prolifera is a macroalgae that forms massive blooms, negatively impacting natural communities, aquaculture operations and recreation. The effects of the natural products, eugenol, ß-myrcene, citral and nonanoic acid on the growth rate, antioxidative defense system and photosynthesis of Ulva prolifera were investigated as a possible control strategy for this harmful taxon. Negative effects on growth were observed with all four chemicals, due to the excessive production of reactive oxygen species and oxidative damage to the thalli. However, the response of U. prolifera under the four chemicals stress was different at the cellular level. ß-myrcene, the most effective compound in terms of growth inhibition, induced oxidative stress as shown by the damage of total antioxidant capacity (T-AOC) and the downregulation of the glutathione-ascorbate (GSH-ASA) cycle which inhibited the antioxidative system. This chemical also inhibited photosynthesis and photoprotection mechanisms in U. prolifera, resulting in growth limitation. In contrast, U. prolifera was less affected by the second tested chemical, eugenol, and showed no significant change on photosynthetic efficiency in the presence of the chemical. The inhibition effects of the third and fourth tested chemicals, nonanoic acid and citralon, on growth and on the antioxidant defense system in U. prolifera were inferior. These results provide a potential avenue for controlling green tides in the future.

Optimal number of Fucus vesiculosus samples to differentiate between sites affected by distinct levels of heavy metal contamination.

The presence of trace elements in marine habitats is a serious environmental problem which increasingly affects ecosystem and human health. The use of macroalgae as contamination biomonitors represents a valuable alternative approach to traditional physicochemical methods. The present study was carried out to determine the optimal number of samples of Fucus vesiculosus needed to detect statistically significant differences in the mean concentrations of Al, As, Cd, Co Cr, Cu, Fe, Hg, Ni, Pb, Zn, N and δ15N between two sampling sites affected by different levels of contamination. For this purpose, we plotted the density distributions of the concentrations of the different elements and examined the local variability at three sites. For sites with mean concentrations differing by more than 30 %, 20 samples were sufficient to detect significant differences for all of the elements, except Cr. The proposed methodology could be used in other studies in the absence of specific research on each species and region.

Purification and Evaluation of N-benzyl Cinnamamide from Red Seaweed Gracilaria fisheri as an Inhibitor of Vibrio harveyi AI-2 Quorum Sensing.

Previously, we reported that the ethanol extract from red seaweed Gracilaria fisheri effectively decreased biofilm formation of Vibrio harveyi. In this study, the anti-biofilm active compounds in the ethanol extract were isolated and their structures identified. The anti-biofilm fractionation assay for minimum inhibitory concentration (MIC) produced two fractions which possessed maximal inhibitory activities toward the biofilm formation of V. harveyi strains 1114 and BAA 1116. Following chromatographic separation of the bioactive fractions, two pure compounds were isolated, and their structures were elucidated using FTIR, NMR, and HR-TOF-MS. The compounds were N-benzyl cinnamamide and α-resorcylic acid. The in vitro activity assay demonstrated that both compounds inhibited the biofilm formation of V. harveyi and possessed the anti-quorum sensing activity by interfering with the bioluminescence of the bacteria. However, the N-benzyl cinnamamide was more potent than α-resorcylic acid with a 10-fold lesser MIC. The present study reveals the beneficial property of the N-benzyl cinnamamide from the ethanol extract as a lead anti-microbial drug against V. harveyi.

Efficient enzymatic saccharification of macroalgal biomass using a specific thermostable GH 12 endoglucanase from Aspergillus terreus JL1.

Stranded green macroalgae represents an important and renewable biomass that remains under valorized despite the numerous environmental problems generated by their accumulation in coastal regions. This work describes the isolation of a filamentous thermophile fungus identified as Aspergillus terreus JL1 that produces an efficient cellulolytic activity for green macroalgae saccharification. The characterization of the endoglucanase activity obtained after submerged fermentation showed a differential induction depending on the carbon source used with a unique isoform released when Ulva lactuca was used as inducer. The crude extract obtained hydrolyzed efficiently the untreated algal biomass (70.5%) compared to other cellulolytic extracts. The unique endoglucanase released was then purified to homogeneity (Yield: 49.6%; Specific activity: 30.1 U/mg; Purification fold: 4.36) and characterized biochemically. Its peptidic sequence was then determined and showed its belonging to the GH12. The described enzyme represents a promising biotechnological tool for algal biomass conversion.

Physiological damages of Sargassum cymosum and Hypnea pseudomusciformis exposed to trace metals from mining tailing.

The damages of Mariana's mining mud in the physiology of the brown algae Sargassum cymosum and its main epiphytic, the red algae Hypnea pseudomusciformis, were evaluated by controlled essays. Seaweeds were exposed to presence or absence of mud, isolated or in biological association, for 5 and 15 days. Measured parameters were growth rates, biochemical descriptors, and the chemical investigation of concentration and metal profile of the mud dissolved in seawater. Results showed that the highest values for metals were Al > Fe > Mn > Zn in both exposure periods. The mud also affected the growth rate with lethality in both isolated and associative treatments with H. pseudomusciformis after 15 days. According to our redundancy analysis (RDA), the profile and concentration of all metallic elements can induce different physiological responses of the organisms. We were able to observe a higher physiological adaptive ability of S. cymosum against the long-term presence of metals by the synthesis of phenolic compounds, while the deviation of metabolic routes in H. pseudomusciformis can be addressed as the main responsible for its lethality. Moreover, the presence of Hypnea in associative treatments reduces Sargassum's detoxification ability. The present results reinforce the importance of biological interaction studies in a context of physiological resilience against mining mud pollution and mutual influences of species over the individual ability to avoid oxidative stress.

Evaluation of macroalgae and amphipods as bioindicators of petroleum hydrocarbons input into the marine environment.

The brown alga Sargassum furcatum and three families of amphipods (Ampithoidae, Caprellidae and Hyalidae) associated to that algae were evaluated as bioindicators of petroleum hydrocarbons input into the marine environment of São Sebastião Channel, in southeastern region of Brazil. The n-alkanes pattern were mainly associated with the natural composition of the macroalgae and amphipods, although some indicatives of petroleum hydrocarbons such as unresolved complex mixture and the no predominance of odd over even n-alkanes have been observed in some samples. Total PAHs ranged from 33.4 to 2010 ng g-1 dry weight with the predominance of low molecular weight PAHs, mostly of naphthalene and alkyl-naphthalenes, which also suggested petroleum input. Even in low concentration, Sargassum furcatum and amphipods species studied seems to be good indicators of the introduction of petroleum hydrocarbons.

Hazardous effects of silver nanoparticles for primary producers in transitional water systems: The case of the seaweed Ulva rigida C. Agardh.

The acute toxicity of citrate capped silver nanoparticles (AgNP) and silver nitrate was evaluated on the marine macroalga Ulva rigida C. Agardh (1823). Silver bioaccumulation, ultrastructural chloroplast damages verified by TEM microscopy, inhibition of primary production, neutral lipid production and oxidative stress were observed after 24 h of exposure to AgNP. The toxic effects of silver nitrate in artificial seawater started from a concentration of 0.05 ppm and was more toxic than AgNP that produced effects from a concentration of 0.1 ppm. However only AgNP induced lipid peroxidation in U. rigida. The addition of natural organic and inorganic ligands, represented by transparent exopolymer particles (TEP) and clay, drastically reduced AgNP acute toxicity in a ratio AgNP:ligand of 1:100 and 1:200, respectively. The findings suggest a marked toxicity of Ag on marine macroalgae which however should be mitigated by the high natural ligand concentrations of the transitional environments.

Heavy metal resistance in algae and its application for metal nanoparticle synthesis.

The ungenerous release of metals from different industrial, agricultural, and anthropogenic sources has resulted in heavy metal pollution. Metals with a density larger than 5 g cm-3 have been termed as heavy metals and have been stated to be potentially toxic to human and animals. Algae are known to be pioneer organisms with the potential to grow under extreme conditions including heavy metal-polluted sites. They have evolved efficient defense strategies to combat the toxic effects exerted by heavy metal ions. Most of the algal strains are reported to accumulate elevated metal ion concentration in cellular organelles. With respect to that, this review focuses on understanding the various strategies used by algal system for heavy metal resistance. Additionally, the application of this metal resistance in biosynthesis of metal nanoparticles and metal oxide nanoparticles has been investigated in details. We thereby conclude that algae serve as an excellent system for understanding metal uptake and accumulation. This thereby assists in the design and development of low-cost approaches for large-scale synthesis of nanoparticles and bioremediation approach, providing ample opportunities for future work.

Identification of contaminants of concern in the upper Tagus river basin (central Spain). Part 2: Spatio-temporal analysis and ecological risk assessment.

This study provides a description of the water quality status in the tributaries of the upper Tagus River and a preliminary risk assessment for freshwater organisms. A wide range of physico-chemical parameters, nutrients, metals and organic contaminants (20 pesticides, and 32 point source chemicals, mainly pharmaceuticals) were monitored during spring, summer and autumn of 2016. Monitoring of organic contaminants was performed using conventional grab sampling and passive samples (POCIS). The variation of the different groups of parameters as regards to land use and sampling season was investigated. The prioritization of organic and inorganic contaminants was based on the toxic unit (TU) approach, using toxicity data for algae, invertebrates and fish. Finally, the compliance with the Environmental Quality Standards (EQS) set as part of the Water Framework Directive (WFD) was evaluated for the listed substances. This study shows that the land use characteristics had a large influence on the spatial distribution of the contaminants and other water quality parameters, while temporal trends were only significant for physico-chemical parameters, and marginally significant for insecticides. Acute toxicity is likely to occur for some metals (copper and zinc) in the most impacted sites (TU values close to or above 1). Low acute toxicity was determined for organic contaminants (individual compounds and mixtures) on the basis of grab samples. However, the assessment performed with POCIS samples identified diuron, chlorpyrifos and imidacloprid as potentially hazardous compounds. Several contaminant mixtures that may cause chronic toxicity and that should be considered in future regional chemical monitoring plans were identified. Our study also shows that some metals and pesticides exceeded the WFD regulatory thresholds and that only 30% of the sampled sites had a good chemical status. Further research is needed to identify chemical emission sources and to design proper abatement options in the Tagus river basin.

Phytoremediation potential of Ulva ohnoi (Chlorophyta): Influence of temperature and salinity on the uptake efficiency and toxicity of cadmium.

Ulva ohnoi is a green macroalga with fast growth and high rates of nitrogen and phosphorus absorption. Recently, this species has been recorded in several places with record green tide formation in some of them. Using molecular tools, we herein report the first occurrence of this species in Brazil and demonstrate its potential for phytoremediation in typical environmental concentrations of Cd (0.625-15 µg L-1). Similarly, the effects of physicochemical parameters (salinity and temperature) on the toxicity and uptake efficiency of this species were evaluated. Molecular analysis of two sequences (1141 bp) obtained corroborates another 34 sequences for U. ohnoi obtained from GenBank. The addition of Cd in the medium affected photosynthetic parameters and reduced growth rate. U. ohnoi showed resistance to Cd when cultivated at 18 °C, S15 and 18-25 °C, S35, at concentrations between 0.625 and 2.5 µg. L-1 of Cd; yet, positive growth rate was maintained. Dose-dependent accumulation was observed in all combinations of factors used with a maximum value of 4.20 µg Cd per gram of dry seaweed at 15 µg. L-1 of Cd at 18 °C and S35. Maximum value of the concentration factor was 81.3 ±â€¯1.1% of Cd added at the concentration of 0.625 µg. L-1 to S15 and 18 °C. Our results demonstrate the potential of using U. ohnoi in the phytoremediation of Cd in saltwater or brackish water.

Long-term effects of the fungicide pyrimethanil on aquatic primary producers in macrophyte-dominated outdoor mesocosms in two European ecoregions.

Even though empirical data supporting sound ecoregion-specific ecotoxicological evaluations are still scarce, the differences of environmental (including climatic) conditions in specific ecoregions are already currently being regulated for environmental risk assessment of pesticides in Europe. To shed new light on the ecotoxicological effects of pesticides on aquatic communities across ecoregions, the model pollutant pyrimethanil (fungicide) was tested in an outdoor mesocosm study with macrophyte-dominated communities in the European "South" (Portugal) and "Centre" (Germany) regulatory zones. Phytoplankton indicators monitored over 12 months indicated a low risk of the fungicide (0.73 or 0.77 mg pyrimethanil L-1, single application) to phytoplankton functioning; as expected since exposure simulated worst-case scenarios. However, the growth of key structural macroalgae and macrophytes was affected by the fungicide and negative effects occurred, especially in the Central zone experiment. Such effects were not detected earlier than approximately nine months post single pyrimethanil application. The presence or absence of such extremely long-lasting/delayed pyrimethanil effects depended on species, competitive situation, and ecoregion-specific physico-chemical environment. The present findings suggest that a better understanding of both direct and indirect effects of fungicide pollution on aquatic flora in two European ecoregions helps to consolidate the environmental risk assessment of pesticides in specific regulatory zones.

Rhamnan sulfate extracted from Monostroma nitidum attenuates blood coagulation and inflammation of vascular endothelial cells.

Rhamnan sulfate (RS) is a polysaccharide with a rhamnose backbone isolated from Monostroma nitidum. Like heparin, it exerts anticoagulant activity in the presence of antithrombin. Endothelial cells facilitate the crosstalk between blood coagulation and vascular inflammation. In this study, we compared the effect of RS with that of heparin on blood coagulation and vascular endothelial cells in the presence or absence of inflammatory factors, using human umbilical vein endothelial cells. We found that RS significantly enhances inhibition of thrombin and factor Xa in the presence of antithrombin as well as heparin, and that RS inhibits tissue factor expression and von Willebrand factor release from the endothelial cells treated with or without lipopolysaccharide, tumor necrosis factor-α, or thrombin. Heparin did not show any effects on endothelial cell inflammation. Our findings suggest that RS, like heparin, is an antithrombin-dependent anticoagulant and, unlike heparin, is a potent anti-inflammatory agent acting on vascular endothelial cells.

The index of ideality of correlation: improvement of models for toxicity to algae.

Toxicity to algae is important characteristic of substances from ecologic point of view. The CORAL software ( http://www.insilico.eu/coral ) gives possibility to build up model of toxicity to algae using data on the molecular architecture and experimental toxicity, without additional data on physicochemical and/or biochemical parameters. Considerable improvement of the model is observed in the case of using the index of ideality of correlation (IIC) in the role of additional criterion of predictive potential. The IIC is calculated with using of the correlation coefficient between experimental and calculated values of endpoint for the calibration set, with taking into account the positive and negative dispersions between experimental and calculated values. The best model calculated with use the IIC is characterized (the validation set) by n = 50, r2 = 0.947, RMSE = 0.401 whereas, model calculated without use the IIC is characterized by n = 50, r2 = 0.805, and RMSE = 0.539. The suggested models are built up in accordance to five OECD principles.

The impact of elevated atmospheric CO2 on cadmium toxicity in Pyropia haitanensis (Rhodophyta).

Cadmium is one of the major heavy metal pollutions in coastal waters, and it is well known that cadmium at trace concentration is toxic to macroalgae. Change in marine carbonate system and ocean acidification caused by elevated atmospheric CO2 also alter physiological characteristics of macroalgae. However, less research is focused on the combined impacts of elevated CO2 and cadmium pollution on the growth and physiology in macroalgae. In this study, the maricultivated macroalga Pyropia haitanensis (Rhodophyta) was cultured at three levels of Cd2+ (control, 4 and 12 mg L-1) and two concentrations of CO2, the ambient CO2 (AC, 410 ppm) and elevated CO2 (HC, 1100 ppm). The results showed that 12 mg L-1 Cd2+ significantly suppressed the relative growth rate and superoxide dismutase activity in AC-grown P. haitanensis, while such inhibition extents by Cd2+ were alleviated in HC-grown algae. Cd2+ had no effects on efficiency of electron transport (α) and maximum electron transport rate (ETRmax), but α was increased by elevated CO2. Cd2+ dramatically suppressed the maximum net photosynthesis oxygen evolution rate (NPRm) and the minimum saturation irradiance (Ik) when the algal thalli were grown at AC, while such suppression of NPRm by Cd2+ was much decreased when the thalli were grown at HC. Collectively, our results suggested that elevated CO2 would alleviate Cd2+ toxicity on P. haitanensis.

Enrichment and physiological responses of dechlorane plus on juvenile marine macroalgae (Ulva pertusa).

Dechlorane Plus (DP), a chlorinated flame retardant, is increasingly reported in aquatic ecosystems worldwide. But little information is available regarding the toxicity of DP in marine organisms, especially in macroalgae. The objective of this study was to investigate effects of DP exposure on photosynthesis, oxidative stress and its enrichment in juvenile marine macroalgae (Ulva pertusa). Following 21- day uptake and 21- day depuration (10-8 mol/L), algae accumulated 1.18 times of DP compared to the initial concentration. Anti-DP was prone to accumulate in juvenile macroalgae. The enrichment of DP affected the physiological responses in algae. After 1, 7 and 14 days DP exposure (10-8, 10-7 and 10-6 mol/L), antioxidant enzymes (SOD and CAT) activities and MDA content changed in a dose and time depended manner. Chlorophyll fluorescence parameters, including Fv/Fm, ΦPSII and ETR decreased with the increasing DP concentration. It indicated that DP leads to a low rate of light energy utilization in algae which may ascribe to the oxidative damage induced by DP enrichment. Present study provides insight into the toxicological effects of DP on marine macroalgae, which is useful for risk assessment of DP in intertidal zone ecosystems.

Changes in the Structural Features of Organic Matter Extracted from Compost by the Seawater during a 90-day Period under Anaerobic Conditions, and Its Effect on Oogenesis of Brown Macroalga.

Seawater-extractable organic matter (SWEOM) has a potential to serve as an Fe complexing agent in an Fe-fertilizer intended to restore and preserve macroalgal forests. To better understand the restoration technique, structural alterations in SWEOM that occurred during a 90-day period of incubation in seawater were determined. Up to 82% of the total eluted SWEOM occurred during the initial 30 days; a small amount of SWEOM then continued to elute during the next 60 days. Spectroscopic analyses showed that the high-molecular-weight SWEOM fraction (HMW) altered in aliphatic-rich materials, while the low-molecular-weight fraction (LMW) became significantly enriched in aromatic structures. The structural alterations in the HMW and LMW could be caused by the action of anaerobic microorganisms. In addition, approximately 30 and 60% of the gametophytes were converted to eggs in the presence of HMW and LMW with Fe, respectively.

Ecophysiological and metabolic responses to interactive exposure to nutrients and copper excess in the brown macroalga Cystoseira tamariscifolia.

Global scenarios evidence that contamination due to anthropogenic activities occur at different spatial-temporal scales, being important stressors: eutrophication, due to increased nutrient inputs; and metal pollution, mostly derived from industrial activities. In this study, we investigated ecophysiological and metabolic responses to copper and nutrient excess in the brown macroalga Cystoseira tamariscifolia. Whole plants were incubated in an indoor system under control conditions, two levels of nominal copper (0.5 and 2.0µM), and two levels of nutrient supply for two weeks. Maximal quantum yield (Fv/Fm) and maximal electron transport rate (ETRmax) increased under copper exposure. Photosynthetic pigments and phenolic compounds (PC) increased under the highest copper levels. The intra-cellular copper content increased under high copper exposure in both nutrient conditions. C. tamariscifolia from the Atlantic displayed efficient metal exclusion mechanisms, since most of the total copper accumulated by the cell was bound to the cell wall.