Monitoring toxic microalgae Ostreopsis (dinoflagellate) species in coastal waters of the Mediterranean Sea using molecular PCR-based assay combined with light microscopy

A molecular PCR-based assay was developed and applied to macrophyte and seawater samples containing mixed microphytobenthic and phytoplanktonic assemblages, respectively, in order to detect toxic Ostreopsis species in Mediterranean Sea. The specificity and sensitivity of the molecular PCR assay were assessed with both plasmidic and genomic DNA of the target genus or species using taxon-specific primers in the presence of background macrophyte DNA. The PCR molecular technique allowed rapid detection of the Ostreopsis cells, even at abundances undetectable within the resolution limit of the microscopy technique. Species-specific identification of Ostreopsis was determined only by PCR-based assay, due to the inherent difficulty of morphological identification in field samples. In the monitoring of the toxic Ostreopsis blooms PCR-based methods proved to be effective tools complementary to microscopy for rapid and specific detection of Ostreopsis and other toxic dinoflagellates in marine coastal environments.     

A comparison of seven methods for the inverse modelling of groundwater flow. Application to the characterisation of well catchments

Inverse modelling is a key step in groundwater-related hydrological studies. Several inversion techniques were developed during the last decades, but hardly any comparison between them was presented. We compare seven modern inverse methods for groundwater flow: the Regularised Pilot Points Method (both the estimation, RPPM-CE, and the Monte Carlo (MC) simulation variants, RPPM-CS), the MC variant of the Representer Method (RM), the Sequential Self-Calibration Method (SSC), the Moment Equations Method (MEM), the Zonation Method (ZM) and a non-iterative Semi-Analytical Method (SAM). These methods are applied to a two-dimensional synthetic example, depicting steady-state groundwater flow around a pumping well. Their relative performance is assessed in terms of their ability to characterise the log-transmissivity and hydraulic head fields and to predict the extent of the well catchment, both for a mildly and a strongly heterogeneous transmissivity field. The main conclusions drawn from the comparison are: (1) MC-based methods (RPPM-CS, SSC and RM) yield very similar results, regardless the degree of heterogeneity and despite they use different parameterisation schemes and objective functions; (2) statistical moments of the target quantities provided by MEM and RPPM-CE are similar to those of MC-based methods; (3) ZM and SAM are negatively affected by strong heterogeneity; and (4) in general, observed differences between the performances of all methods are not very large. MC-based inverse methods need considerably more CPU time than the other tested approaches. An advantage of MC-based methods is that they allow computing the posterior probability distribution of the target quantities, which can be directly fed to probabilistic risk-assessment procedures.     

微囊藻群体总RNA提取方法的比较

微囊藻群体富含多糖类物质是影响微囊藻RNA提取的关键因素.为了获得高质量的微囊藻群体总RNA,对比分析4种针对多糖含量较高的方法——方法 1 PGTX-bead法、方法 2 CTAB-bead法、方法 3 Fast RNAPro Blue Kit和方法 4RNeasy Mini Kit对微囊藻群体总RNA的提取效果.采用琼脂糖凝胶电泳检测微囊藻群体RNA的完整性,Nanodrop ND1000分光光度计检测RNA纯度及浓度,并采用q PCR检测DNA污染情况.结果表明,4种方法都能从微囊藻群体中提取获得RNA,并在去除DNA后都可以进行RT-PCR等后续实验.方法 1 PGTX-bead提取的RNA产量最高,纯度好,DNA污染小,成本低,适合从微囊藻群体中大量提取RNA;方法 2 CTAB-bead提取的RNA样品产量也较高,但DNA污染严重,适合需要同时提取DNA和RNA的样本;方法 3 Fast RNAPro Blue Kit和方法 4 RNeasy Mini Kit提取的RNA产量都较低,但方法 4操作简单,耗时短,所检测目的基因的相对表达量较高,更适合从少量的微囊藻群体中提取总RNA.     

Comparison of Enterococcus density estimates in marine beach and bay samples by real-time polymerase chain reaction, membrane filtration and defined substrate testing

Currently, densities of Enterococcus in marine bathing beach samples are performed using conventional methods which require 24 h to obtain results. Real-time PCR methods are available which can measure results in as little as 3 h. The purpose of this study was to evaluate a more rapid test method for the determination of bacterial contamination in marine bathing beaches to better protect human health. The geometric mean of Enterococcus densities using Enterolert® defined substrate testing and membrane filtration ranged from 5.2 to 150 MPN or CFU/100 mL and corresponding qPCR results ranged from 6.6 to 1785 CCE/100 mL. The regression analysis of these results showed a positive correlation between qPCR and conventional tests with an overall correlation (r) of 0.71. qPCR was found to provide accurate and sensitive estimate of Enterococcus densities and has the potential to be used as a rapid test method for the quantification of Enterococcus in marine waters.     

Potential use of dissolved cyanobacterial DNA for monitoring toxic Microcystis cyanobacteria in filtered water

Toxic and non-toxic Microcystis sp. are morphologically indistinguishable cyanobacteria that are increasingly posing health problems in fresh water systems by producing odours and/or toxins. Toxic Microcystis sp. produces toxicologically stable water soluble toxic compounds called microcystins (MCs) that have been associated with cases of aquatic life and wildlife poisoning and kills including some cases of human illnesses/deaths around the world. Thus, the need for rapid detection of toxic Microcystis sp. in surface water is imperatively a necessity for early mitigation purposes. Genomic DNA from potentially toxic Microcystis sp. comprises of ten microcystin synthetase (mcy) genes of which six major ones are directly involved in MCs biosynthesis. In Polymerase Chain Reaction (PCR) methodsmcy genes can be amplified from intracellular/extracellular genomic DNA using PCR primers. However, little is known about the limitations of sourcing genomic DNA templates from extracellular DNA dissolved in water. In this work, filtered water (0.45 μM) from a Microcystis infested Dam (South Africa) was re-filtered on 0.22 μM syringe filters followed by genomic DNA isolation and purification from micro-filtrates (9 mL). Six major mcy genes (mcyABCDEG) from the isolated DNA were amplified using newly designed as well as existing primers identified from literature. PCR products were separated by gel electrophoresis and visualized after staining with ethidium bromide. The limitation of using dissolved DNA for amplification of mcy genes was qualitatively studied by establishing the relationship between input DNA concentrations (10.0–0.001 ng/μL) and the formation of respective PCR products. The amplification of mcyA gene using new primers with as little as 0.001 ng/μL of DNA was possible. Other mcy gene sensitivities reached 0.1 ng/μL DNA dilution limits. These results demonstrated that with appropriately optimized PCR conditions the method can provide accurate cost-effective tools for rapid detection of toxic Microcystis sp. in water giving early information for water quality monitoring against MC producing cyanobacteria.     

Hypoxia does not promote naphthalene bioaccumulation in the brown shrimp,Penaeus aztecus

Since increased ventilation is known to be a common strategy used by aquatic animals to cope with hypoxia, we tested in present study the hypothesis that hypoxia can promote the bioaccumulation of naphthalene, a representative polycyclic aromatic hydrocarbon (PAH), in Penaeus aztecus, a penaeid shrimp subject to hypoxia and PAH contamination in the northern Gulf of Mexico. For each of the two naphthalene concentrations, five groups of shrimps were, respectively, subjected to five different conditions, namely, clean seawater under normoxia, seawater containing acetone under normoxia and hypoxia, and seawater containing 10 or 250 μg/L naphthalene under normoxia and hypoxia. Our results suggest that hypoxia does not significantly alter naphthalene bioaccumulation in either the gills or the hepatopancreas of P. aztecus. The absence of a promoting effect of hypoxia on naphthalene bioaccumulation is attributed to the increased disposition of naphthalene when the shrimps are subjected to hypoxia.     

Uptake and tissue distribution of C4-C7 alkylphenols in Atlantic cod (Gadus morhua): relevance for biomonitoring of produced water discharges from oil production

The sensitivity of different tissues for assessment of chronic low-dose environmental exposure of fish to alkylphenols (APs) was investigated. We exposed Atlantic cod (Gadus morhua) in the laboratory to tritium labelled 4-tert-butylphenol, 4n-pentylphenol, 4n-hexylphenol, and 4n-heptylphenol via seawater (8 ng/l) and via contaminated feed (5 μg/kg fish per day). Measurements of different fish tissues during eight days of exposure and eight subsequent days of recovery revealed that APs administered via spiked seawater were readily taken up whereas the uptake was far less efficient when APs were administered in spiked feed. AP residues were mainly located in the bile fluid whereas the concentrations in liver were very low, indicating a rapid excretion and the liver-bile axis to be the major route of elimination. The biological half-life of APs in the exposed cod was short, between 10 and 20 h. Our study shows that in connection with biomonitoring of AP exposure in fish, assessment of AP metabolites in bile fluid is a more sensitive tool than detection of parent AP levels in liver or other internal tissues.     

Use of a harpacticoid copepod in toxicity tests

The harpacticoid copepod Nitocra spinipes has been tested for acute toxicity of 12 metal chlorides in brackish water. Their order of toxicity, expressed as 96 h LC₅₀, was in good agreement with other investigations performed in freshwater and seawater. The 96 h LC₅₀-values were of intermediate levels compared to these two environments. The organochlorines p,p′-DDE and p,p′-DDE methyl sulphone were tested for effects on reproduction and mortality during two weeks, and it was found that p,p′-DDE was the most toxic. It is concluded from the investigation that N. spinipes is a suitable toxicity test organism in brackish water.     

Monitoring polycyclic aromatic hydrocarbons in the Northeast Aegean Sea using Posidonia oceanica seagrass and synthetic passive samplers

The concentrations of 22 polyaromatic hydrocarbons (PAH) in Posidonia oceanica seagrass, sediments, and seawater from the Alexandroupolis Gulf in the Aegean Sea, were investigated from 2007 to 2011. Temporal trends of total PAH contents in P. oceanica and sediments were similar. PAH levels in seawater, sediments, and seagrasses generally decreased with increasing distance from Alexandroupolis Port. Leaves and sheaths of P. oceanica had higher PAH levels than roots and rhizomes. P. oceanica accumulates PAHs and has good potential as a bioindicator of spatiotemporal pollution trends. PAH concentrations were also examined using in situ passive seawater sampling and were compared to results of passive sampling in the laboratory using local sediments and seawater. Levels of high molecular weight PAHs assessed using passive samplers confirmed the decreasing gradient of pollution away from Alexandroupolis Port. Passive sampling also proved useful for investigating sources of PAHs in P. oceanica meadows.     

Induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis

Biomarkers are generally applied to detect pollution in environmental monitoring. Such biological responses should accurately reflect the stress over time in a quantitative manner. As such, the initial and maximum responses induced by stress, as well as adaptation and recovery of these biomarkers, need to be fully understood or else erroneous false-negative or false-positive may be arrived. However, most of the biomarker studies only provided information on initially induced responses under different concentrations of toxicants, while biological adaptation and recovery were poorly known. In this study, the time required for induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis upon exposure to benzo[a]pyrene was investigated over a period of 62 days. Maximum induction occurred on day 6 when lysosomal integrity was significantly reduced by 51%, and no further change or adaptation was detected thereafter. When mussels were depurated in clean seawater after 18 days of exposure to benzo[a]pyrene, a gradual recovery was observed, with lysosomal integrity returning to its background level and showing a complete recovery after 20 days of depuration. Lysosomal integrity was significantly correlated with the body burden concentrations of benzo[a]pyrene and condition index of the mussels. The relatively fast induction (6 days) and recovery (20 days) without apparent adaptation suggested that lysosomal integrity in P. viridis can serve as a good biomarker in biomonitoring, as its response is not likely to generate both false-negative and false-positive results.     

Impact of elevated levels of CO2 on animal mediated ecosystem function: The modification of sediment nutrient fluxes by burrowing urchins

A mesocosm experiment was conducted to quantify the relationships between the presence and body size of two burrowing heart urchins (Brissopsis lyrifera and Echinocardium cordatum) and rates of sediment nutrient flux. Furthermore, the impact of seawater acidification on these relationships was determined during this 40-day exposure experiment. Using carbon dioxide (CO₂) gas, seawater was acidified to pH_NBS 7.6, 7.2 or 6.8. Control treatments were maintained in natural seawater (pH ≈ 8.0). Under normocapnic conditions, burrowing urchins were seen to reduce the sediment uptake of nitrite or nitrate whilst enhancing the release of silicate and phosphate. In acidified (hypercapnic) treatments, the biological control of biogeochemical cycles by urchins was significantly affected, probably through the combined impacts of high CO₂ on nitrifying bacteria, benthic algae and urchin behaviour. This study highlights the importance of considering biological interactions when predicting the consequences of seawater acidification on ecosystem function.     

Inactivation of the gene katA or sodA affects the transient entry into the viable but non-culturable response of Staphylococcus aureus in natural seawater at low temperature

We have investigated the fate of Staphylococcus aureus by starving the cells and maintaining them in natural seawater at 22 and 4 °C. At 22 °C, cells developed a long-term survival state where about 0.037% of the initial population remained culturable over more than 7 months, whereas at 4 °C, bacteria lost culturability and transiently entered into the viable but non-culturable state (VBNC). However, after 22 days of entry into the VBNC state, the number of viable cells detected via the direct viable count method decreased significantly. We show here that mutational inactivation of catalase (KatA) or superoxide dismutase (SodA) rendered strains hypersensitive to seawater stress at 4 °C and consequently, part of the seawater lethality on S. aureus at low temperature is mediated by reactive oxygen species (ROS) during microcosm-survival process. Shifting the temperature from 4 to 22 °C of totally non-culturable wild-type cells induced a partial recovery of the population. However, deficiencies in catalase or superoxide dismutase prevent resuscitation ability.     

Hafnium and neodymium isotopes in seawater and in ferromanganese crusts: The “element perspective”

Hafnium and Nd isotopes are increasingly used as paleoceanographic proxies. Comparing the “mantle–crust array” and the “seawater array” in plots of ε_Hf vs. ε_Nd, it has been observed that for a given ε_Nd value the corresponding ε_Hf value is higher for seawater than it is for terrestrial rocks. While this difference had initially been explained by significant hydrothermal input of mantle Hf into seawater, the currently favoured explanation is incongruent weathering of continental rocks producing radiogenic riverine Hf input.We here address this topic from the perspective of the behaviour of these two elements in seawater and in ferromanganese (Fe–Mn) crusts. We distinguish between a “truly dissolved” and a “dissolved” Hf and Nd pool, the latter being comprised of truly dissolved and colloid-bound (“colloidal”) Hf and Nd. While there exists a hydrothermal pathway for colloid-bound dissolved mantle Hf into the oceans, there is, in marked contrast to Nd, no important riverine pathway for colloidal or truly dissolved continental Hf. Owing to their respective chemical speciation in seawater, there exists truly dissolved Nd in the ocean, while the amount of truly dissolved Hf is insignificant.Neodymium is in exchange equilibrium between local seawater and both, the hydrous Fe and Mn oxides hydrogenetic Fe–Mn crusts are composed of. Due to continuous ad- and desorption there is continuous isotopic re-equilibration and the isotopic composition of Nd in a Fe–Mn crust reflects that of truly dissolved Nd in local ambient seawater. In contrast, Hf is only associated with the hydrous Fe oxides on which it forms surface precipitates that do not exchange with seawater. Due to this lack of isotopic re-equilibration, the isotopic composition of Hf in a Fe–Mn crust is the average of that of all the Hf scavenged during the lifetime of the hydrous Fe oxide particles. Since the Hf-bearing hydrous Fe oxides in a Fe–Mn crust do not form from local ambient seawater at the crust's growth site but are advected as colloids or fine particles, their Hf isotopic composition depends on the origin and migration pathway of these colloids. Hence, while Nd isotopes in Fe–Mn crusts provide reliable information on truly dissolved Nd in local ambient seawater, Hf isotopes rather indicate the origin and pathway of hydrous Fe oxide colloids, and might differ from truly dissolved Hf in local ambient seawater. This may explain the occasional decoupling of Nd and Hf isotopes in Fe–Mn crusts and supports the notion of a significant hydrothermal mantle signal of Hf in seawater.     

Enzymatic methods for the determination of pollution in seawater using salt resistant alkaline phosphatase from eggs of the sea urchin Strongylocentrotus intermedius

A new salt resistant alkaline phosphatase from eggs of the sea urchin Strongylocentrotus intermedius (StAP) has been shown to have a unique property to hydrolyze substrate in seawater without loss of enzymatic activity. The enzyme has pH optimum at 8.0–8.5. Model experiments showed various concentrations of copper, zinc, cadmium and lead added to seawater or a standard buffer mixture to inhibit completely the enzyme activity at the concentrations of 15–150 μg/l. StAP sensitivity to the presence in seawater of metals, pesticides, detergents and oil products appears to be considerably less. Samples of seawater taken from aquatic areas of the Troitsy Bay of the Peter the Great Bay, Japan Sea have been shown to inhibit the enzyme activity; the same was shown for the samples of fresh waters. The phosphatase inhibition assay developed proved to be highly sensitive, technically easy-to use allowing to test a great number of samples.     

Using qPCR Assays to Predict Rates of Cometabolism of TCE in Aerobic Groundwater

Monitored natural attenuation (MNA) is commonly used as a remedy for trichloroethene (TCE) in anaerobic groundwater; however, MNA has not been applied to TCE contamination in aerobic groundwater. Under aerobic conditions, bacteria initiate the degradation of many organic substances with oxygenase enzymes. Several of these enzymes are known to degrade TCE through a fortuitous reaction known as cometabolism. There are commercially available qPCR assays that can determine the number of gene copies of these enzymes. If the qPCR assay could be used to predict the first-order rate constant for cometabolism of TCE, the qPCR assay could be used to screen sites to determine whether MNA was a plausible remedy for TCE contamination. This study reevaluated data from water samples that were collected from 19 wells on five sites in Minnesota, New York, and Utah. Data had previously been published on the rate constant for cometabolism of TCE in the water samples as determined by a ¹⁴C-assay and the abundance of gene copies for five enzymes that cometabolize TCE as determined using a qPCR assay. The Michaelis-Menten (Haldane) kinetic parameters for cometabolism of TCE and the abundance of DNA for the five oxygenase enzymes were used to predict the rate constant for cometabolism of TCE. The predicted rate constants were evaluated and validated by comparing them to the rate constants derived from the ¹⁴C-assay. For predicted rate constants greater than 0.003 per year, the predicted rate constants agreed with the measured rate constants within a factor of three. The qPCR assay serves as a convenient screening tool to determine whether MNA is a plausible remedy for an aerobic plume of TCE.     

P/Ca in coral skeleton as a geochemical proxy for seawater phosphorus variation in Daya Bay, northern South China Sea

The P/Ca ratio in coral skeletons is considered to be a direct proxy for the nutrient P in seawater. We examined the reliability of this proxy by analyzing P/Ca in a Porites coral collected from a eutrophic area in the northern South China Sea. P concentrations were significantly higher compared to previously reported values from pristine and open seas, corresponding to the elevated nutrients from the study site. We compared coral P/Ca against recent in-situ records of seawater P concentrations. Our results show that P/Ca was primarily a function of TP sw rather than PO₄sw, and that the signal of skeletal P included not only phosphate, but also organic phosphorus. Besides the form of skeletal P, sub-sampling and analytical procedures and the distinctive nutrient regime were the most reasonable explanations for our results. We suggest that total P in coral skeletons may be an efficient proxy for seawater P variations and associated phytoplankton dynamics in eutrophic environments.     

Neodymium isotopic variations in seawater

New data for the direct measurement of the isotopic composition of neodymium in Atlantic Ocean seawater are compared with previous measurements of Pacific Ocean seawater and ferromanganese sediments from major ocean basins. Data for Atlantic seawater are in excellent agreement with Nd isotopic measurements made on Atlantic ferromanganese sediments and are distinctly different from the observed compositions of Pacific samples. These results clearly demonstrate the existence of distinctive differences in the isotopic composition of Nd in the waters of the major ocean basins and are characteristic of the ocean basin sampled. The average ε_Nd(0) values for the major oceans as determined by data from seawater and ferromanganese sediments are as follows: Atlantic Ocean,ε_Nd(0) ? ?12 ± 2; Indian Ocean,ε_Nd(0) ? ?8 ± 2; Pacific Ocean,ε_Nd(0) ? ?3 ± 2. These values are considerably less than ε_Nd(0) value sources with oceanic mantle affinities indicating that the REE in the oceans are dominated by continental sources. The difference in the absolute abundance of¹⁴³Nd between the Pacific and Atlantic Oceans corresponds to ～10⁶ atoms¹⁴³Nd per gram of seawater. The correspondence between the¹⁴³Nd/¹⁴⁴Nd in seawater and in the associated sediments suggests the possible application of this approach to paleo-oceanography.Distinctive differences in ε_Nd(0) values are observed in the Atlantic Ocean between deep-ocean water associated with North Atlantic Deep Water and near-surface water. This suggests that North Atlantic Deep Water may be relatively well mixed with respect to Nd isotopic composition whereas near-surface water may be quite heterogeneous, reflecting different sources for surface waters relative to deep water. This suggests that it may be possible to distinguish the sources of water masses within an ocean basin on the basis of Nd isotopic composition.The Nd isotopic variations in seawater are used to relate the residence time of Nd and mixing rates between the oceans.     

Chemistry of solutions from the 13°N East Pacific Rise hydrothermal site

Ten samples were recovered by the submersible “Cyana” submersible from two groups of hydrothermal vents located 2600 m deep along the East Pacific Rise at 13°N. The maximum measured temperature was 317°C and minimum pH 3.8. A systematic determination of major and trace elements has been carried out and mixing lines between a high-temperature component (HTC) and seawater are observed. The water chemistry of the HTC slightly differs for several elements at the two sites. This HTC is deprived of SO₄ and Mg and is greatly enriched in most other species. Maximum concentrations are (in units per kg):Cl = 0.72mol; Br = 1.1mmol; Na = 0.55mol; K = 29mmol; Rb = 14 μmol; Ca = 52mmol; Sr = 170 μmol; Mn = 750 μmol; Fe = 1mmol; Al = 15 μmol; Si = 21mmol. For many elements, the magnitude of the anomaly relative to seawater does not compare with the results obtained from the Galapagos or East Pacific Rise 21°N. The enrichment of cations relative to seawater is likely related to the huge Cl excess through charge balance. TheBr/Cl ratio is close to that for seawater. However, it is not clear whether the Cl excess is due to gas release or basalt hydration (formation of amphibole chlorite or epidote).P-T dependence of SiO₂ solubility suggests that water-rock interaction last occurred at a depth in excess of 1 km below the sea floor. A mixing line of⁸⁷Sr/⁸⁶Sr vs. Mg/Sr demonstrates that the HTCs have a nearly identical⁸⁷Sr/⁸⁶Sr ratio of 0.7041 for both sites. A water/rock ratio of about 5 is inferred, which differs from the 1.5 value obtained at 21°N.     

Benthic foraminifera show some resilience to ocean acidification in the northern Gulf of California,Mexico

Extensive CO₂ vents have been discovered in the Wagner Basin, northern Gulf of California, where they create large areas with lowered seawater pH. Such areas are suitable for investigations of long-term biological effects of ocean acidification and effects of CO₂ leakage from subsea carbon capture storage. Here, we show responses of benthic foraminifera to seawater pH gradients at 74–207 m water depth. Living (rose Bengal stained) benthic foraminifera included Nonionella basispinata, Epistominella bradyana and Bulimina marginata. Studies on foraminifera at CO₂ vents in the Mediterranean and off Papua New Guinea have shown dramatic long-term effects of acidified seawater. We found living calcareous benthic foraminifera in low pH conditions in the northern Gulf of California, although there was an impoverished species assemblage and evidence of post-mortem test dissolution.     

Spectral Reflectance Analysis of the Caribbean Sea

Reflectance spectral curves were analysed by a derivative method. Derivative reflectance spectra revealed concealed peaks of both reflectance and absorption curves of Caribbean seawater and elements contained within it. Reflectance curves showed a predominant blue colour (400–500 nm) characteristic of Caribbean oligotrophic waters, conspicuous peaks result from the optical properties of chlorophyll a and seawater. Reflectance curves had a similar spectral response. This paper analyses reflectance spectra of surface seawater at 31 stations in the Caribbean Sea during the summer of 2001.