Field Of Ecotoxicology Research Articles

Proteomics in aquatic amphipods: Can it be used to determine mechanisms of toxicity and interspecies responses after exposure to atrazine?

Proteomics has gained popularity in the field of ecotoxicology as a holistic tool for unraveling novel mechanisms of toxicity and elucidating subtle effects of contaminant exposure. The holoarctic amphipod Diporeia spp. is declining at precipitous rates in the Great Lakes, and we are evaluating the use of the well-studied amphipod model Hyalella azteca as a surrogate for Diporeia spp. This article presents proteomics data from both amphipod species exposed to atrazine (ATZ) and one of its metabolites, desethylatrazine (DEA; 3 and 30 µg/L for 21 and 42 d). We used a proteomics approach to determine whether these two species of amphipods responded similarly to the same chemicals and to understand better the mechanisms of toxicity of ATZ and DEA in aquatic invertebrates. We observed disruption in energy production and mitochondrial function as well as hormesis in exposed organisms. In addition, we identified a two proteins (GAPDH and HSP 90 kDa) that have been linked to hormonal disruptions, suggesting potential endocrine disruption. Finally, we found that H. azteca and Diporeia spp. responded with similar proteomic profiles after ATZ and DEA exposure, suggesting that H. azteca may be used as a surrogate model organism for Diporeia spp.

Transcriptomic analysis of Caenorhabditis elegans exposed to nickel (II) acetate using microarray

Nickel can be exposed to human population from air, food, and water sources by which may produce abnormal consequences in organs including kidneys, liver, cardiovascular, immune, and reproductive systems. Recognition of nickel biomarkers for the toxicity assessment is of prime importance. Caenorhabditis elegans (C. elegans) is well known animal model for genetic studies which serves as a living biosensor in ecotoxicological researches. Our study is the first evidence demonstrating changes of genomic expression in C. elegans in response to nickel (II) acetate using nematode-specific microarray. We have found 23 genes as a nickel responsive genes that were differentially expressed (>2 fold) following 24 hours exposure to nickel acetate. The analysis of gene ontology revealed molecular components involved in detoxification, carcinogenesis, and oxidative stress defense in the C. elegans exposed to the nickel. In addition, using the comparative toxicogenomics database, we have deduced the nickel-affected molecular mechanisms including histone modification and its stability. In conclusion, our investigation would be able to present precise route for understanding important roles of several nickel responsive genes in nickel toxicity. Furthermore, our discovery also provides significant biomarkers in response to nickel acetate. This might be virtue for monitoring and evaluating hazard risk in the field of ecotoxicology.

Characterizing the Embryonic Transcriptome of the Snail Ilyanassa

The snail Ilyanassa obsoleta is a useful model for a variety of investigations in the fields of developmental biology, cell biology, larval ecology, ecotoxicology, parasitology, and chemical ecology. To enhance such studies, we have carried out two cDNA sequencing projects to characterize the mRNA transcripts that are present during development of this embryo. These efforts have generated 480 megabases of new sequence, which have been assembled into transcript contigs and represent thousands of newly identified Ilyanassa genes. We identified the orthologs of 182 transcription factors in these data, focusing on families that are likely to be sequence-specific transcriptional regulators. To demonstrate the utility of identifying and examining such transcripts, we describe the expression pattern during organogenesis for IoOnecut, an Ilyanassa ortholog of the HNF6/onecut family of transcription factors.

Intraspecific competition delays recovery of population structure

Ecotoxicological field studies have shown that total abundance and biomass often recover shortly after pulsed toxicant stress. In contrast, population structure showed comparatively long-term alterations before reaching pre-treatment conditions. We investigated two mechanisms that may explain the prolonged recovery of population structure: latent toxicant effects on life-history traits on the individual level and competition on the population level. To test these hypotheses we exposed populations of Daphnia magna to a pulse of the pyrethroid Fenvalerate. For several generations the populations were kept at two different degrees of competition: strong competition at carrying capacity and reduced competition maintained by simulated predation. After disturbance due to Fenvalerate exposure, biomass recovered after 14–17 days. In contrast, size structure characterised by a lack of large and dominance of small organisms recovered after 43 days in populations with strong competition. Size structure recovered twice faster in populations with reduced competition. We explain this as follows: due to toxicant induced mortality, food availability and consequently birth rate increased and populations were dominated by small individuals. In populations without predation, these cohorts grew and eventually exerted high intraspecific competition that (i) stopped further growth of juveniles and (ii) increased mortality of adults. These demographic processes were mainly responsible for the prolonged recovery of size structure. In contrast, for populations with predation, the regular harvest of individuals reduced competition. Juveniles developed continuously, allowing a fast recovery of size structure in these dynamic populations. In risk assessment the duration for populations to recover from (toxicant) stress, is crucial for the determination of ecological acceptable effects. We conclude that competition needs to be considered in order to understand and predict recovery of size structure.

Characterisation of Mytilus edulis hemocyte subpopulations by single cell time-lapse motility imaging

In bivalve molluscs, defence against pathogens mainly relies on fast tissue infiltration by immunocompetent hemocytes that migrate from circulating hemolymph to sites of infection, in order to deliver, in situ, an effective immune response. In the present work, we have investigated dynamics of hemocyte subpopulations motility by combining flow cytometry coupled to Coulter-type cell volume determination, Hoffman modulation contrast microscopy, time-lapse imaging and off-line analysis of cell shape changes. Our results revealed fast modifications of hemocyte aspect in vitro, with bidirectional transitions from spread outlines to condensed cell body morphologies, in the minute range. Amoeboid or non-amoeboid types of locomotion were observed, depending on the cell shapes and on the cell subtypes, with velocities reaching up to 30 μm min −1. Correlations between motion profiles, Hemacolor staining and flow cytometry analysis on living cells help to propose a functional mussel hemocyte classification including the motile properties of these cells. In particular, basophils were shown to be involved in dynamic hemocyte–hemocyte interactions and in the constitution of aggregation cores. Physiological implications, in terms of immune response in organisms devoid of endothelium-closed vascular system, and potential applications of hemocyte motility studies for the development and the interpretation of experiments involving hemocytes in the field of marine ecotoxicology are discussed.

Evaluation of chrono-amperometric signal detection for the analysis of genotoxicity by a whole cell biosensor

Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alternative to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl β- d-galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the standard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.

Considerations for research on the environmental fate and effects of nanoparticles

Considerations for research on the environmental fate and effects of nanoparticles

Metallothionein response in earthworms Lampito mauritii (Kinberg) exposed to fly ash

Among pollutants, the coal fly ash occupies a significant position in industrial wastes. The fly ash matrix is a complex mixture of various organic (polyhalogenated compounds) and inorganic (Si, Al, Fe, As, Cd, Bi, Hg, etc.) chemicals. The application of fly ash for agricultural purposes and as landfills may lead to the contamination of the land with some of the toxic chemical compounds present in fly ash. Thus prior to the application of fly ash for developmental activities, it requires bio-monitoring and risk characterization. In order to achieve this objective adult Lampito mauritii were exposed to different proportions of fly ash in soil for 30 d and the concentrations of metallothionein in earthworm were assessed. The results revealed that up to 50% of fly ash amendment does not apparently harm the earthworm in respect of their survival and growth. A significant increase in tissue metallothionein level was recorded in L. mauritii exposed to fly ash amended soil without tissue metal accumulation indicating that metallothionein is involved in scavenging of free radicals and reactive oxygen species metabolites. It is concluded that this biochemical response observed in L. mauritii exposed to fly ash amended soil could be used in ecotoxicological field monitoring.

First REP-LECOTOX Workshop—ecotoxicogenomics: the challenge of integrating genomics/proteomics/metabolomics into aquatic and terrestrial ecotoxicology

The Workshop “Ecotoxicogenomics: The challenge of integrating genomics/proteomics/metabolomics into aquatic and terrestrial ecotoxicology” held in Novi Sad, Serbia, at UNSFS, Department of Biology and Ecology, organized as a part of the Project INCO-CT-2006-043559-REP-LECOTOX, brought together experts in the field of genomics, transcriptomics, proteomics, metabolomics, and bioinformatics. Five plenary lectures and seven lectures on the specific themes, as well as discussion afterwards, gave to participants a comprehensive insight into the possibility of application of omic technologies in the field of ecotoxicology. The workshop was attended by approximately 50 participants mostly from Romania, Bulgaria, Croatia, Macedonia, Montenegro, Bosnia and Herzegovina, and Serbia (Fig. 1). The Workshop program was prepared by the Scientific Committee: J.K. Chipman, S. Scholz, K. Hilscherova, and R. Kovacevic. In the first keynote lecture “Overview of ecotoxicogenomics,” Prof. J.K. Chipman (School of Biosciences, University of Birmingham, UK) stressed that a major advantage of functional genomics technologies, which enable measurements of thousands of transcripts, proteins, and metabolites, is their “open” nature that does not require prior assumptions about the choice of biomarkers, thus, being particularly valuable to assess mechanisms of action and the effect of mixtures of chemicals where unknown biological targets may be involved. He also pointed out that attention needs to be given to distinguishing between compensatory, adaptive, and toxic responses and to discovering patterns of change that are diagnostic and predictive. Many interesting examples of “omic” responses were shown together with discussion and proposals for use of such data in ecological monitoring and risk assessment. In the second keynote lecture “Environmental transcriptomics—predicting adverse and long-term effects,” Dr. Stefan Scholz (Department for Cell Toxicology, UFZ, Leipzig, Germany) showed that information obtained by whole genome analysis can be useful if they are linked to appropriate applications, such as the identification of mechanisms of action or biomarkers for biomonitoring and the prediction of (long-term) adverse effects. His presentation highlighted two examples demonstrating applications in ecotoxicology and/or regulatory testing: the first one was the application of gene expression as a marker for long-term adverse effects of endocrine disrupting chemicals, while the second highlighted example described the potential application of gene expression markers for the prediction of chronic fish toxicity by extending the fish embryo test from acute to chronic toxicity test. In the third keynote lecture “Environmental proteomics,” Dr. Thomas Knigge (Laboratoire d’Ecotoxicologie— Milieux Aquatiques, Universite du Havre, France) pointed out that the proteomic profile represents a link between effects at the molecular and whole organism level, since proteins are the first functional level directly or indirectly affected by toxicants. Showing the results of several different studies using gel and non-gel-based proteomics, he illustrated some of the practical and conceptual difficulties when applying proteomics to ecotoxicological studies. He stressed the needs for sophisticated bioinformatics and Environ Sci Pollut Res (2009) 16 (Suppl 1):S130–S131 DOI 10.1007/s11356-009-0128-2

Behavioral Ecotoxicology: Mechanisms, Effects, Applications, and Biomonitoring

We introduce the second series of articles in the emerging field of behavioral ecotoxicology published in the Journal of Human and Ecological Risk Assessment (HERA). We highlight the key points of ...

Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola)

BackgroundGenomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation.ResultsEight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase (SDHA) and eukaryotic transcription initiation factor 1A (ETIF) genes were found the most stable over the different treatments, while for O. cincta, the beta actin (ACTb) and tyrosine 3-monooxygenase (YWHAZ) genes were the most stable.ConclusionWe present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements.

Meeting the Challenges of Aquatic Vertebrate Ecotoxicology

The field of ecotoxicology uses biomarkers to assess the health of populations of sentinel organisms and to determine risk associated with environmental chemicals. The tools of modern biology are being used to develop promising new suites of biomarkers that must be rigorously tested and validated within a comprehensive mechanistic understanding of how toxic chemicals in the environment influence basic physiology and behavior. The zebrafish is a well-established laboratory model organism with a well-equipped molecular toolbox for basic biology and biomedicine with logical applications in ecotoxicology. As a model organism for ecotoxicology, the zebrafish can be used to develop mechanistic models of gene-environment interactions that will provide a foundation for the development of genomic resources in other fish species. Integration of mechanistic molecular data from multiple fish species will lead to the development of integrated dynamic models that will enable better diagnosis and treatment of environmen...

Hemocytes/coelomocytes DNA content in five marine invertebrates: cell cycles and genome sizes

The hemocytes/coelomocytes DNA content in five selected marine invertebrates (sea mouse Aphrodita aculeata, spiny crab Maja crispata, sea star Echinaster sepositus, sea urchin Paracentrotus lividus, and tunicate Phallusia mammillata) was investigated by flow cytometry. The cell cycle analyses identified sea mouse coelomocytes as proliferating cells and revealed that spiny crab hemocytes and sea urchin coelomocytes complete their division in the hemolymph and coelom, respectively. The genome sizes of sea mouse and spiny crab are reported for the first time. The diploid DNA content (2C) in sea mouse A. aculeate was 1.24 pg, spiny crab M. crispata 7.76 pg, red starfish E. sepositus 1.52 pg and sea urchin P. lividus 1.08 pg. The mean diploid DNA content in tunicate P. mammillata was 0.11 pg with a high interindividual variability (45%). The presented results provide a useful database for future studies in the field of invertebrate physiology, ecotoxicology, biodiversity, species conservation and phylogeny.

Freshwater fishes, fisheries, and habitat prospects of Nepal

This paper deals with the status of fish, fisheries, and habitats in Nepal. Being a landlocked country, it has only freshwater habitats covering an area of 745,000 hectares (5% of the total area) that includes rivers, lakes, ponds, wetlands, reservoirs, and irrigated rice fields. It has a diverse fish species totaling 200 fish species, of which 191 are indigenous and nine exotic. Thirty-four fish species were categorized as threatened (IUCN categories), requiring due attention for the conservation. Legal protection is recommended for ten fish species in the endangered and vulnerable categories. Fish harvests are mainly based on the subsistence fish farming, and from capture fisheries. National fish production in the year 2000/01 was 33,270 metric tons. Lowland areas are most suitable for aquaculture, whereas hill streams have a great attraction for sport fishing. Some destructive fishing methods are in use in capture fishery, e.g., electric fishing, explosives and poisons. In Nepal, some of the major fish habitats are in protected conservation areas, e.g., national parks and World Ramsar Sites. However, proper consideration has not been given for fish habitat management. The national plan includes a fisheries/aquaculture sub sector mainly for supplying animal protein, and for generating self-employment and income of small-scale farmers. The number of people involved in the capture fisheries has increased and included 142,000 men and 223,000 women in the year 2000/01. At the same time, commercial trout farming in the private sector has increased. Long-term perspectives in the fields of aquatic ecology, genetics, biotechnology and ecotoxicology are essential to enhance the fish and fisheries science in the country. [Supplementary material is available for this article. Go to the publisher's online edition of Aquatic Ecosystem Health & Management for the following free supplemental resource: an appendix containing a checklist of freshwater fishes of Nepal.]

Analysis of environmental stress response on the proteome level

Thousands of man-made chemicals are annually released into the environment by agriculture, transport, industries, and other human activities. In general, chemical analysis of environmental samples used to assess the pollution status of a specific ecosystem is complicated by the complexity of the mixture, and in some cases by the very low toxicity thresholds of chemicals present. In that sense, a proteomics approach, capable of detecting subtle changes in the level and structure of individual proteins within the whole proteome in response to the altered surroundings, has obvious applications in the field of ecotoxicology. In addition to identifying new protein biomarkers, it can also help to provide an insight into underlying mechanisms of toxicity. Despite being a comparatively new field with a number of caveats, proteomics applications have spread from microorganisms and plants to invertebrates and vertebrates, gradually becoming an established technology used in environmental research. This review article highlights recent advances in the field of environmental proteomics, mainly focusing on experimental approaches with a potential to understand toxic modes of action and to identify novel ecotoxicological biomarkers.

Ecotoxicological applications and significance of the comet assay

Application of the single-cell gel electrophoresis or comet assay has revolutionized the field of genetic ecotoxicology or eco-genotoxicology. It is a rapid, sensitive and relatively inexpensive method providing the opportunity to study DNA damage (including oxidative damage), repair and cell death (apoptosis) in different cell types without prior knowledge of karyotype and cell turnover rate. The assay has, however, often attracted criticism for its lack of ecotoxicological relevance. In addition, in contrast to genetic toxicology where rapid technical progress has been made to improve cell- and tissue-specific adoption of the assay, only limited advancement has been made to transfer the methodologies to ecotoxicological studies. While reviewing the recent information available in the literature and underscoring the importance of induced genetic damage in natural species, the aims of this article are to (i) highlight and judiciously analyse the ecotoxicological relevance of the assay; (ii) attempt to correlate the comet response with other relevant biological responses or biomarkers; (iii) identify the technical challenges and various factors affecting its application in order to make it reliable, reproducible and robust; (iv) critically compare the technical developments in genetic toxicology and genetic ecotoxicology and (v) evaluate the future developments with respect to applications of the assay. It is suggested that while complementing other ecotoxicological parameters and further improving the methodologies, the comet assay will continue to play an important role in genetic ecotoxicology to determine induced genetic damage, which has significant consequences for short- and long-term survival of the natural or wild species. Information obtained through integrated studies using simultaneous applications of multiple biomarkers on different wild organisms could also provide an holistic dimension of toxicological impact of environmental contaminants for the protection of human health.

The effects of humic substances on copper toxicity to Ceriodaphnia silvestrii Daday (Crustacea, Cladocera)

A major question in the field of ecotoxicology is how DOM affects copper accumulation and toxicity in planktonic organisms; copper acute toxicity and bioaccumulation in Ceriodaphnia silvestrii were investigated in the presence and absence of humic substances (HS) under controlled laboratory conditions. Copper was determined as free Cu2+ ions in the media and total copper in the animals; metal ion buffers were used for ion selective electrode calibration, extending the lower detection limit to 10(-11) mol l(-1). Groups of 20 adult females of similar sizes were exposed (24 h) to a range of nominal copper concentrations. Based on total added copper, LC50 was 4.4 x 10(-8) mol l(-1) without HS, whereas with 20 mg l(-1) HS, it was 25 times higher (1.1 x 10(-6) mol l(-1)). Based on free Cu2+ ions LC50 was statistically similar either with (2.8 x 10(-8) mol l(-1)) or without HS (3.3 x 10(-8 )mol l(-1)). The present results showed that natural DOM reduced copper toxicity and that free Cu2+ ions correlates to the bioavailable fraction to zooplankton. Nevertheless, copper bioaccumulation by C. silvestrii was similar either in the presence or absence of humic substances, suggesting that C. silvestrii regulates its body copper content up to 3.0 x 10(-8) mol l(-1) free Cu2+ ions in the media. The organisms were not able to deal with higher free Cu2+ ions concentrations in the media.

Workshop on rotifers in ecotoxicology

The aim of the workshop on rotifers in ecotoxicology was to stimulate discussions on new developments in the field. Discussions about the use of biomolecular tools indicate that gene expression analysis with rotifers should be available in the next few years. Such analyses will be a great asset as they enable ecotoxicologists to study molecular mechanisms of toxicity. Rotifers also appear as useful tools in the risk assessment of pharmaceuticals and their metabolites that find their way into aquatic ecosystems because their sensitivity to some of these substances is higher than that of cladocerans and algae. The nature and extent of the impact of potential endocrine disruptors on aquatic invertebrates is another poorly resolved issue for which rotifers are a promising tool. Indeed, rotifers seem to be particularly sensitive to androgenic and anti-antiandrogenic substances, whereas copepods and cladocerans are typically more affected by estrogens and juvenile hormone-like compounds. Besides their usefulness in these emerging fields of aquatic ecotoxicology, it was emphasized that research with rotifers on basic issues like, e.g., toxicant interference with predation, competition, or interspecific and interclonal variation in ecotoxicological tests is still needed.

Transcriptomics and proteomics. Applications to ecotoxicology

Researchers from Europe and the USA met at the Joint Research Center (JRC) of the European Commission to discuss how to integrate gene and protein expression analyses with bioinformatic tools in the field of ecotoxicology and how this new approach could be translated in improved risk assessment procedures. The measurements of gene and/or protein expression levels, upon exposure to a chemical or a stressor, can be used to develop robust molecular biomarkers that will allow the early detection of environmental stress, study long-term exposure and infer the mechanism of action. These molecular biomarkers should be linked to phenotypic end points of exposure such as adverse effects in growth and reproduction in single organisms and populations. At environmentally realistic exposure levels there could be “non-linear” dose-response curves, which should be accounted for in the experimental design and in the analyses of microarray and proteomic data. The application of gene and protein expression profiling in ecotoxicology will have a significant impact on the ecotoxicology field in the near future and international collaborations will play an important role in accelerating the application of those techniques.

Linkage of biochemical responses to population‐level effects: A case study with vitellogenin in the fathead minnow (Pimephales promelas)

A challenge in the field of ecotoxicology is the linkage of alterations at molecular and biochemical levels of organization to adverse outcomes in individuals and populations. In the present study, a predictive relationship between plasma vitellogenin (VTG) concentration and fecundity in female fathead minnows (Pimephales promelas) was derived from 21-d laboratory toxicity tests with five chemicals (17beta-trenbolone, 17alpha-trenbolone, prochloraz, fenarimol, and fadrozole) that inhibit VTG production through different mechanisms. Because VTG is key to egg production in female oviparous animals, changes in the lipoprotein could, theoretically, serve as an indicator of reproductive success. Regression of fecundity versus VTG concentration from the various studies yielded a highly significant linear model (fecundity = -0.042 + 0.95 x VTG, p < 0.01, r2 = 0.88). This relationship was integrated into a population model to translate changes in VTG concentrations of female fathead minnows to alterations in population growth. The model predicted relatively profound effects on population size of fish experiencing moderate decreases in vitellogenesis. For example, a fathead minnow population at a carrying capacity exposed to a chemical stressor that causes a 25% decrease in VTG concentration in females from baseline values would exhibit a 34.6% projected decrease in size after two years of exposure and reach an equilibrium population size that was only 30.2% of the preexposed population. Overall, the current study provides an example of how changes in a biomarker (VTG concentration) can be quantitatively translated into adverse effects at the individual and population levels.