Application of NMR-based techniques in aquatic toxicology: brief examples

Nuclear magnetic resonance spectroscopy (NMR) has been employed over many years for the elucidation of chemical structures. However, in more recent years it has been used to characterize sublethal actions of pollutants in aquatic organisms. For instance, in vivo NMR involves live, intact organisms or cell cultures and the application of chemical stressors to reveal toxic mechanisms in real time. Alternatively, NMR-based metabolomics involves rapid cessation of metabolic activity following chemical exposure (via liquid N(2)) to provide an assessment of metabolic actions via more traditional NMR analysis. Two examples are briefly presented to exemplify the power of NMR for assessing toxic actions in marine and freshwater organisms.     

Toxicological Effects Induced by Cadmium in Gills of Manila Clam Ruditapes philippinarum Using NMR‐Based Metabolomics

Cadmium (Cd) has become an important heavy metal contaminant in the sediment and seawater along the Bohai Sea and been of great ecological risk due to its toxic effects to marine organisms. In this work, the toxicological effects caused by environmentally relevant concentrations (10 and 40 µg L^?1) of Cd were studied in the gill tissues of Manila clam Ruditapes philippinarum after exposure for 24, 48, and 96 h. Both low (10 µg L^?1) and high (40 µg L^?1) doses of Cd caused the disturbances in energy metabolism and osmotic regulation and neurotoxicity based on the metabolic biomarkers such as succinate, alanine, branched chain amino acids, betaine, hypotaurine, and glutamate in clam gills after 24 h of exposure. However, the recovery of toxicological effects of Cd after exposure for 96 h was obviously observed in clam to Cd exposures. Overall, these results indicated that NMR‐based metabolomics was applicable to elucidate the toxicological effects of heavy metal contaminants in the marine bioindicator.     

Metabolism of a burrowing polychaete: Precaution needed when measuring toxic effects

The technique of direct calorimetry has been used to monitor the metabolic activity of the polychaete, Neanthes virens, an organism commonly used for testing the toxic effects of chemicals in the marine environment. In these tests Neanthes showed unpredictable alternating periods of hyperactivity and rest when deprived of sediment. If used in this abnormal state for toxicity tests, the results would represent the effects of the chemicals on abnormal organisms and may not, therefore, be realistic information for judging the chemical's harmful effects on it and other species in nature. In toxicity tests with heavy metals that have been reported in the scientific literature, the shorter survival time of test organisms without sediment, as compared to those in sediment, was probably the result of the abnormally high metabolic rates of the former group.     

Zur Toxizität einfacher und komplexer Cyanide gegenüber Wasserorganismen

The toxic effects of alkali cyanide, complex cyanide-containing heavy metal salts—as of iron, cadmium, copper, nickel and mercury—were investigated according to standard methods with the aid of the test organisms Poecilia reticulata (guppy) as well as the green alga Ankistrodesmus falcatus (β- bis α-mesosaprobic). Since various complex compounds can be photolytically decomposed, also discontinuous light-dark phases were used. As discussed equilibrium calculations have shown, inter alia also the pH-value is important in the fish organisms used with different reactions to ions and molecularly dissolved compounds. The results are summarized in tables and graphic representations, the substances investigated being classified into groups with “strong” and “high” toxic actions.     

Metabolomic Study on the Halophyte Suaeda salsa in the Yellow River Delta

Plant metabolomics has been well established and applied across multiple fields including medicine, biotechnology, and environmental sciences in the post‐genomic era. The Chenopodiaceae C₃ halophyte Suaeda salsa is the most important plant species in the vegetation of saline soil and even intertidal zone in the Yellow River Delta, which is economically consumed as food, widely used as a bioindicator of environmental stresses (salinity, drought, and pollution) and typically applied for the phyto‐remediation of degraded wetland. However, no global studies have been focused on the metabolic profile of this halophyte which is widely applied in environment related research areas. In metabolomics, the first crucial step is the preparation of plant samples. In this work, several strategies of metabolite extraction from this C₃ halophytes S. salsa were evaluated and the metabolic profile was characterized by NMR‐based metabolomics. Multiple replicates of plant tissues (approx. 250 mg whole fresh weight of leaves and stems) were homogenized by a high throughput automated Precellys 24 bead‐based homogenizer and then extracted using the following solvent systems of varying polarities: methanol, water, methanol/water, methanol/chloroform/water, and acetonitrile. The hydrophilic metabolites were analyzed using one‐dimensional proton NMR spectroscopy, and the subsequent NMR spectra were evaluated by principal components analysis (PCA). Each extraction protocol revealed unique metabolic profile from S. salsa. Overall, the quality of each extraction protocol was assessed based on the yield, reproducibility, ease, and speed, and we concluded that the solvent of methanol/water was preferable for the metabolite extraction due to its highest reproducibility, ease, and speed, and considerably high yield. Validation of methanol/water (1:1) solvent also showed a high degree of reproducibility with technical variation being considerably lower than biological variation in S. salsa samples. This demonstrated the quality and robustness of the methanol/water extraction method for NMR‐based metabolomics studies.     

鄱阳湖水体沉积物中Zn、Cd对底栖生物的毒性效应及基准验证

以泥鳅（Misgurnus anguillicaudatus）、铜锈环棱螺（Bellamya aeruginosa）、河蚬（Corbicula fluminea）为受试生物,以死亡率、渗血率、体质量变化、行动迟钝率、挖洞率为测试指标,探究沉积物中Zn和Cd对底栖生物的21 d慢性毒性效应,并对鄱阳湖Zn和Cd的沉积物基准值进行了验证.结果表明,沉积物中Cd对底栖生物的毒性效应强于Zn,沉积物中Zn、Cd对泥鳅、铜锈环棱螺、河蚬的LC₅₀（半数致死浓度）分别为644和61 mg/kg、751和54 mg/kg、652和81 mg/kg,EC₅₀（半数效应浓度）值分别为526和49 mg/kg、589和35 mg/kg、474和45 mg/kg.经验证,沉积物Zn和Cd的TEL（临界效应浓度）、PEL（必然效应浓度）值比较合理,具有一定适用性,即当Zn、Cd的加标含量低于TEL值时,Q_TEL<1,不具有毒性;当加标含量高于PEL值时,Q_PEL>1,毒性明显.     

Status of butyltin pollution along the coasts of western Japan in 2001, 11 years after partial restrictions on the usage of tributyltin

Restrictions on the use of tributyltin (TBT) in aquaculture and on boats in coastal regions, except for ocean-going vessels, have been in place in Japan since 1990 due to the strong toxic effects of TBT on marine organisms. However, TBT pollution along the Japanese coasts has been reported after this legislation was enacted. In order to elucidate the present status of contamination by butyltin (BT) compounds, we measured the levels of BTs [monobutyltin (MBT), dibutyltin (DBT) and TBT] in seawater and Caprella spp. samples obtained from the western part of Seto Inland Sea, Uwa Sea and Uranouchi Bay in western Japan during March to September, 2001. Butyltins were detected in more than 90% of the seawater samples (n = 59), with average concentrations of 8.2 ± 9.2 (SD) ng MBT L⁻¹, 3.3 ± 3.0 ng DBT L⁻¹ and 9.0 ± 7.0 ng TBT L⁻¹. Among 41 stations situated on coastal lines, a sufficient number of Caprella organisms for chemical analysis could be collected from only 16 stations. The butyltin concentrations in seawater and Caprella samples from Uwa Sea and Uranouchi Bay, in which the dominant industry in both waters is aquaculture, showed significantly higher than or no significant differences from those samples from the western part of Seto Inland Sea, a major heavy-industry area in Japan. As the TBT concentration in seawater increased, the number of Caprella organisms collected decreased. The mean TBT concentration among the seawater samples was above the estimated lowest observable effect concentration (LOEC) that reduces the survival rate of Caprella danilevskii. Thus, the present study indicates that TBT is still a potential ecological hazard to the survival of marine invertebrates inhabiting coasts along western Japan, even 11 years after the partial ban on TBT usage was implemented.     

Peak and integral seismic parameters of L��Aquila 2009 ground motions: observed versus code provision values

The M _w 6.3 L’Aquila earthquake of April 6, 2009 hit a wide area of the Abruzzo region (Central Italy). The epicentre of the main shock was very close to the urban centre of L’Aquila, the regional capital, with an epicentral distance less than 10 km. It was the strongest earthquake ever recorded in Italy which has provided ground motion recordings from accelerometric stations located in close proximity to the epicentre. Because of this, several remarkable results can be achieved by analysing the strong motion recorded signals in terms of peak (PGA, PGV and PGD) and integral (Housner Intensity, I _H) seismic parameters. Additionally, an alternative time-domain representation of recorded signals has been used to furnish a rapid comparison of traces recorded at different stations and along different directions. Some comparisons between the response spectra derived from the recordings and the elastic demand spectra provided in the new seismic Italian code have also been performed. PGA recorded values are very high and generally higher than code values for seismic actions with return period T _R = 475 years. In some cases, this also happens for seismic actions with T _R up to 2,475 years. With regard to I _H, recorded values are generally higher for T _R = 475 years, whilst they are remarkably lower for T _R = 2,475 years. Accurate analyses have been carried out in the article to better understand the above differences and their significance and implications.     

Geochemical survey of Levante Bay,Vulcano Island (Italy), a natural laboratory for the study of ocean acidification

Shallow submarine gas vents in Levante Bay, Vulcano Island (Italy), emit around 3.6t CO₂ per day providing a natural laboratory for the study of biogeochemical processes related to seabed CO₂ leaks and ocean acidification. The main physico-chemical parameters (T, pH and Eh) were measured at more than 70 stations with 40 seawater samples were collected for chemical analyses. The main gas vent area had high concentrations of dissolved hydrothermal gases, low pH and negative redox values all of which returned to normal seawater values at distances of about 400 m from the main vents. Much of the bay around the vents is corrosive to calcium carbonate; the north shore has a gradient in seawater carbonate chemistry that is well suited to studies of the effects of long-term increases in CO₂ levels. This shoreline lacks toxic compounds (such as H₂S) and has a gradient in carbonate saturation states.     

藻类对重金属的耐性与解毒机理

重金属是一类具有潜在危害的重要污染物,越来越多的重金属被排入水体,对水生生态环境构成严重威胁.藻类在长期响应重金属胁迫过程中,建立起一系列的适应机制.藻类通过控制重金属的吸收、富集、转运与解毒,使不同细胞组分中的重金属维持在正常浓度范围内.这些保护机制主要包括藻细胞的某些胞外组分与重金属结合,从而减少重金属进入胞内;在重金属诱导下藻细胞可合成金属结合蛋白或多肽;重金属诱导藻细胞合成一些代谢物使其免受伤害或修复由重金属胁迫造成的损伤;藻细胞通过液泡区室化作用使重金属远离代谢;藻细胞对重金属具有排斥与排出作用.     

Biocides in Relation to Water Pollution Part I: Bioassay Studies on the Effects of a few Biocides on Fresh Water Fish,Channa gachua

• - In the present paper the toxicity of four biocides, endrin, thiodon, malathion and phosvel, has been measured by performing static bioassay experiments with Channa gachua as test fish to establish the median Tolerance Limits (TL_m and TL₅₀).
• - The results of bioassay experiments indicate that the fish is harmed even at a very low concentration of these biocides. The values of TL_m are 0.0076, 0.0073, 0.00695 and 0.0068 for endrin; 0.01120, 0.01088, 0.01069 and 0.01064 for thiodon; 9.20, 8.10, 7.90 and 7.60 for malathion and 32.90, 32.10, 31.65 and 31.15 mg/l for phosvel for the time interval of 24, 48, 76 and 96 hours, respectively.
• - As indicated by TL_m's, endrin is most toxic and phosvel least. Thiodon and malathion came in a descending order between endrin and phosvel.
• - During bioassay studies, the effect of some variables, like temerature, pH and fish-size, on TL_m's has also been observed. They gave the following results.
• a) An increase in temperature increased the toxicity of all the biocides.
• b) An increase in pH decreased the TL_m as such, making the biocides more toxic to Channa gachua.
• c) For larger fishes, the biocide solutions were less toxic, but the same solutions were more toxic to fishes of a smaller size.
• - The safe harmless concentrations of endrin, thiodon, malathion and phosvel are - 0.00289 mg/l, 0.00337 mg/l, 1.88365 mg/l and 9.16736 mg/l, respectively.

     

Toxicity of oil-sinking agents

It may be necessary to immobilize an oil spill of more than 10,000 tons in European waters by sinking with sand. The possibility of damage to marine organisms from the sinking agents used has been investigated and it is concluded that toxic effects are unlikely to result.     

A practical ranking system to compare toxicity of anti-fouling paints

The toxicity of a number of new anti-fouling paints, claimed to function by physical means and not by leakage of toxic substances, have been tested on two common organisms in the Baltic Sea, i.e., the red macro alga Ceramium tenuicorne and the copepod Nitocra spinipes. In order to compare the toxicity between the paints a ranking system was developed based on the EC₅₀- and LC₅₀-values. The results showed a wide span in toxicity with the most toxic paints ranked 160 times more toxic than the ones ranked least toxic.

Also, TBT, irgarol and diuron, which have been used as active ingredients in traditional anti-fouling paints, were used to evaluate the sensitivity of the two test organisms. The results showed that the test organisms were equally sensitive to the substances as similar organisms in earlier studies.

In conclusion, the ranking system presented in this study permits ranking and comparison of total toxicity of complex mixtures.     

Long-term variation in mercury concentrations in estuarine organisms with changes in releases into Lavaca Bay,Texas

Mercury releases into Lavaca Bay resulted in public health notices and a fishing closure for crabs and finfish. Mercury containing wastewater discharges were halted in 1970, but sediments were contaminated with mercury. The sediments are a source of mercury to the benthic food web via methylation. Mercury in water column feeding organisms has decreased markedly. Concentrations in benthic food web organisms remain elevated. Human consumption concerns for oysters have ended, but a closure for fishing for crabs and finfish continues. Monitoring of mercury in organisms since 1977 shows a gradual downward trend in mercury in crabs and finfish, but this was not sufficient to remove the closure. Continued releases of mercury, man's activities, and resuspension of sediments keep mercury in the sediment's biologically active zone. This enables the uptake of mercury via the benthic food web. If releases are ended, it will take years for the mercury to decrease to concentrations not of concern for human consumption. If more remediation to reduce the mercury concentrations in the sediments takes place, decrease of mercury in the organisms might occur more quickly.     

Current empirical ground-motion prediction equations for Europe and their application to Eurocode 8

The first ground-motion prediction equation derived from European and Middle Eastern strong-motion data was published more than 30 years ago; since then strong-motion networks and the resulting databank of accelerograms in the region have expanded significantly. Many equations for the prediction of peak ground-motion parameters and response spectral ordinates have been published in recent years both for the entire Euro-Mediterranean and Middle Eastern region as well as for individual countries within this region. Comparisons among empirical ground-motion models for these parameters, developed using large regional datasets, do not support the hypothesis of there being significant differences in earthquake ground-motions from one area of crustal seismicity to another. However, there are certain regions within Europe—affected by different tectonic regimes—for which the existing pan-European equations may not be applicable. The most recent European equations make it possible to now implement overdue modifications to the presentation of seismic design actions in Eurocode 8 that allow an improved approximation to the target uniform hazard spectrum (UHS). Using these recent equations, this study outlines a new approach via which an approximation to the UHS may be constructed using hazard maps calculated for peak ground velocity and the corner period T _D in addition to the maps for peak ground acceleration that underpin the current stipulations of Eurocode 8.     

Long-term modification of Arctic lake ecosystems: Reference condition, degradation under toxic impacts and recovery (case study Imandra Lakes, Russia)

In this study, published data on Lake Imandra, north-west Russia, have been synthesised to investigate trends in lake contamination and recovery due to changing inputs of heavy metals and nutrients over time. Records of water chemistry, phytoplankton, zooplankton and fish communities have been used to determine the status of aquatic ecosystem health in three distinct phases of Lake Imandra's recent history. Firstly, background (reference) conditions within the lake have been established to determine lake conditions prior to anthropogenic influences. Secondly, a period of ecosystem degradation due to anthropogenic inputs of toxic metals and nutrients has been described. Finally, evidence of lake recovery due to recent decreases of toxic metals and nutrients has been explored. Pollution of Lake Imandra began in the 1930s, reaching a peak in the 1980s. Increases in heavy metal and nutrient inputs transformed the typical Arctic ecosystem. During the contamination phase, there was a decrease in Arctic species and in biodiversity. During the last 10 years, pollution has decreased and the lake has been recolonised by Arctic water species. Ecosystem recovery is indicated by a change of predominant species, an increase in the individual mass of organisms and an increase in the biodiversity index of plankton communities. In accordance with Odum's ecosystem succession theory, this paper demonstrates that the ecosystem has transformed to a more stable condition with new defining parameters. This illustrates that the recovery of Arctic ecosystems towards pre-industrial reference conditions after a reduction in anthropogenic stresses occur, although a complete return to background conditions may not be achievable. Having determined the status of current ecosystem health within Lake Imandra, the effect of global warming on the recovery process is discussed. Climate warming in Arctic regions is likely to move the ecosystem towards a predominance of eurybiontic species in the community structure. These organisms have the ability to tolerate a wider range of environmental conditions than typical Arctic inhabitants and will gain advantages in development. This indicates that the full recovery of Arctic ecosystems in a warming climate may not be possible.     

Increased activity on Ebeko Volcano, Paramushir I., North Kurils in 2005–2006

In recent years the gas hydrothermal activity of Ebeko Volcano has been characterized by a constant heat flow and a constant chemical composition of water and fumarole gases. The volcano’s activity suddenly increased on January 27, 2005. Two high-output steam-gas jets 5 m across formed in the Active Funnel of the North Crater. A new high-output, high-temperature, fumarole field originated in the funnel in July 2005. Changes have been recorded in the chemical composition of fumarole gases. Ratios between individual components of the gas were calculated, with the ratios being geochemical precursors of eruptions for the volcano under study. Based on these results and on the very fact of increased fumarole activity on Ebeko, an intermediate-term forecast of explosive eruptions has been developed. Atmospheric contamination by a toxic gas (hydrogen sulfide) is described. It was previously thought that toxic gases pose a short-lived threat only to the population of the town of Severo-Kuril’sk because of frequent strong winds prevailing there. It is shown that the presence of a snow cover during increased activity on Ebeko sharply enhances the hazard of contamination by toxic gases, the hazard being constant both on the volcano itself and in the Severo-Kuril’sk area.     

Review on toxicity testing with marine macroalgae and the need for method standardization--exemplified with copper and phenol

Toxic effects on macroalgae have been compiled. Eighty-two articles have been found in literature during 1959-2000. A total of 120 substances were investigated using 65 different macroalgae species. About one-third of the tested compounds were organic substances (33%), another third metal-organic substances (35%), and the last third were oils (14%), metals (8%), detergents (7.5%) and other inorganic chemicals (2.5%). Half of the substances were only tested once on a single species. Likewise, toxicity data has only been reported for one chemical tested on a single occasion for about half of the 65 species. Thus little is known about the toxic effects on marine macroalgae. The objectives of the previous studies undertaken varied and therefore the toxicity data was presented in numerous ways, e.g. using different exposure times, temperature, light intensity, light regime, salinity, and nutrients, which makes a direct comparison of the data difficult. This review also shows that many stages in the lifecycle of macroalgae are often more sensitive to toxic substances than other aquatic organisms. Consequently, tests using macroalgae may discover toxicity earlier, which would in turn also protect the fauna. If toxic compounds have a negative affect on the distribution and growth of structurally and functionally dominating macroalgae, there may indirectly be a large and harmful influence on the whole marine coastal ecosystem. For this reason tests on macroalgae should be included in control programs along the coasts.     

Treatment of Domestic sewage in lagoons

Summary At Fuglebjerg, County of Soroe, South Zeeland (about 1000 inhabitants) a sewage treatment plant, consisting of three lagoons in series, has been installed in an empty gravel pit. The plant has been in use since May 1952 and has produced a most satisfactory effluent. Two times a year, chemical, bacteriological, and biological examinations have been performed, besides occasional more restricted investigations. The bacteriological examinations have chiefly taken into consideration, the reduction in counts of faecal bacteria, of whichE. coli is diminished considerably,Str. faecalis, and especiallyCl. welchii much less. The special value of the two last mentioned organisms as indicators of the efficiency of purification and the condition in the receiving streams is stressed. As regards the change in microflora in the course of treatment, a variation takes place from polysaprobic micro-communities characterized bybacteria andflagellates via α-mesosaprobic zones (flagellates andblue-green algae) to β-mesosaprobic conditions dominated bydiatoms.     

Dibenzofurans a greater global pollutant than dioxins ? : Evidence from analyses of open ocean killer whale

Three specimens of killer whales (Orcinus orca), an open ocean carnivore, were analysed for extremely toxic polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) to understand their long-range distribution patterns. Several PCDF congeners, including the highly toxic 2,3,7,8-tetra- and 2,3,4,7,8-penta-CDFs were identified in the blubber of those specimens. The PCDF isomeric pattern in killer whale is more complex than the reported patterns in humans and birds, indicating the weaker metabolic potency of killer whales for these toxic compounds. High levels of PCBs (about 400 mg kg⁻¹) have also been detected in those specimens. The 2,3,7,8-substituted PCDF congeners identified in commercial PCBs were also found in killer whale, indicating PCBs as the possible source. Isomer-specific and trace level determinations of PCDD in killer whale, revealed no detectable quantities. The detection of comparatively high levels ( > 300 ng kg⁻¹) of PCDFs and undetection of PCDDs in open ocean killer whales suggest that PCDFs are more ubiquitous than PCDDs.