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 N₂) 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.     

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.     

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.     

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.     

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.     

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.     

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.     

New biocide-free anti-fouling paints are toxic

A number of new anti-fouling paints claimed to be more friendly to the environment, have entered the market since prohibition of biocide containing paints have been enforced in many areas. Leakage waters from five new anti-fouling paints were tested for toxic effects to the macro algae Ceramium tenuicorne and Ceramium strictum and to the crustacean Nitocra spinipes. A banned copper and irgarol 1051 containing anti-fouling paint was used as reference. Five of the six paints tested were toxic to all or some of the organisms after two weeks of leakage with EC50 ranging from 0.08 to around 2% leakage water and LC50 ranging from 1.1% to 88%. The toxicity of leakage water from these paints was still high after 16 weeks. We conclude that these paints contain substances toxic to common organisms in the coastal Baltic ecosystem. A silicone based paint did not exhibit toxic effects to the two organisms. We recommend that biological tests should be used to identify the most harmful products before they are released on the market.     

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.     

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.     

Developing methods for bioidentification of xenobiotics for water quality assessment

The role of bioassay in the pollution control system for water bodies is considered. Studies of the mechanisms of toxic impact of various chemical compounds on aquatic organisms were used to develop a bioindication method to give an integral estimate of water toxicity and determine some classes of chemical pollutants. The results of application of such methods to assessing water quality in Lake Ladoga are given.     

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.     

Potential for photoenhanced toxicity of spilled oil in Prince William Sound and Gulf of Alaska waters

Photoenhanced toxicity is the increase in the toxicity of a chemical in the presence of ultraviolet light (UV) compared to a standard laboratory test conducted with fluorescent lighting (minimal UV). Oil products, weathered oil, and specific polycyclic aromatic compounds present in oil are 2 to greater than 1000 times more toxic in the presence of UV. The photoenhanced toxicity of oil to fish and aquatic invertebrates appears to occur through a process of photosensitization, rather than photomodification of the aqueous phase oil. In photosensitization, the bioaccumulated chemical transfers light energy to other molecules causing toxicity through tissue damage rather than a narcosis mechanism. The available evidence indicates that phototoxic components of oil are specific 3-5 ring polycyclic aromatic hydrocarbons (PAHs) and heterocycles. Determinants of photoenhanced toxicity include the extent of oil bioaccumulation in aquatic organisms and the spectra and intensity of UV exposure. No studies have specifically investigated the photoenhanced toxicity of spilled oil in Alaska waters. Although there are substantial uncertainties, the results of this evaluation indicate there is potential for photoenhanced toxicity of spilled oil in Prince William Sound and the Gulf of Alaska. The potential hazard of photoenhanced toxicity may be greatest for embryo and larval stages of aquatic organisms that are relatively translucent to UV and inhabit the photic zone of the water column and intertidal areas. Photoenhanced toxicity should be considered in oil spill response because the spatial and temporal extent of injury to aquatic organisms may be underestimated if based on standard laboratory bioassays and existing toxicity databases. Additionally, the choice of counter measures and oil removal operations may influence the degree of photoenhanced toxicity.     

Sediment quality in the Guadalquivir estuary: lethal effects associated with the Aznalcóllar mining spill

Monitoring from 1998 to 2001 has assessed the impact of the Aznalcóllar mining spill on the sediment quality in the Guadalquivir estuary. Chemical analysis has been completed with biological effects measured in different organisms. The toxicity of sediments obtained from dilutions of toxic mud and from environmental stations affected by the accidental spill was tested using the amphipod Ampelisca brevicornis and the clam Scrobicularia plana.The results obtained show that amphipods are more sensitive to the accidental spill than the clams. A dilution of clean sediment by more than 1.8% of toxic mud produced 100% mortality of amphipods. In GR2 station is detected toxicity to amphipods but not to clams. The rest of the environmental stations show no toxicity. Toxicity to amphipods in GR2 station decreased along time (from 50% to 60% of mortality in 1998 to 10 to 15% in 2001) and it can be associated with a recovery of the areas impacted by the accidental spill.     

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.     

The toxicity of ammonium molybdate to marine invertebrates

Molybdenum occurs naturally in seawater as molybdate ion, but little is known of its effects, at elevated concentrations, upon marine organisms. The toxicity of ammonium molybdate, a constituent of some industrial wastes, to marine invertebrates has now been tested. It proves to be not very toxic.     

Toxicity identification in metal plating effluent: implications in establishing effluent discharge limits using bioassays in Korea

Because of complexity and diversity of toxicants in effluent, chemical analysis alone gives very limited information on identifying toxic chemicals to test organisms. Toxicity identification evaluation (TIE) techniques have been widely used to identify toxicants in various samples including industrial wastewater as well as natural waters. In response to new regulation for effluent discharge in Korea, which will be effective from 2011, a necessity of studies emerges that investigates toxicity levels in industrial effluents. This work was a preliminary study examining toxicity levels in effluent from one metal plating factory using Daphnia magna (48 h immobility) and identifying toxicity-causing substances. Toxicity tests showed variability on different sampling occasions and the results of TIE methods indicated that both organic compounds and metals contributed to the observed toxicity in metal plating effluent. Further studies are necessary to help reduce effluent toxicity especially from direct dischargers, who will have to comply with the new regulation.