Evaluation of phenanthrene toxicity on earthworm (Eisenia fetida): An ecotoxicoproteomics approach

The goal of this study was to identify promising new biomarkers of phenanthrene by identifying differentially expressed proteins in Eisenia fetida after exposure to phenanthrene. Extracts of earthworm epithelium collected at days 2, 7, 14, and 28 after phenanthrene exposure were analyzed by two dimensional electrophoresis (2-DE) and quantitative image analysis. Comparing the intensity of protein spots, 36 upregulated proteins and 45 downregulated proteins were found. Some of the downregulated and upregulated proteins were verified by MALDI-TOF/TOF-MS and database searching. Downregulated proteins in response to phenanthrene exposure were involved in glycolysis, energy metabolism, chaperones, proteolysis, protein folding and electron transport. In contrast, oxidation reduction, oxygen transport, defense systems response to pollutant, protein biosynthesis and fatty acid biosynthesis were upregulated in phenanthrene-treated E. fetida. In addition, ATP synthase b subunit, lysenin-related protein 2, lombricine kinase, glyceraldehyde 3-phosphate dehydrogenase, actinbinding protein, and extracellular globin-4 seem to be potential biomarkers since these biomarker were able to low levels (2.5 mg kg⁻¹) of phenanthrene. Our study provides a functional profile of the phenanthrene-responsive proteins in earthworms. The variable levels and trends in these spots could play a potential role as novel biomarkers for monitoring the levels of phenanthrene contamination in soil ecosystems.     

Application of biomarkers to assess the condition of European Marine Sites

A series of European Marine Sites has been designated as Special Areas of Conservation (SAC) in England. The aim of this study was to develop a practical methodology to assess the condition of SACs by applying a suite of biomarkers. Biomarkers were applied to the blue mussel Mytilus edulis and the shore crab Carcinus maenas from the Fal and Helford SAC (Cornwall). Individual biomarkers provided useful diagnostic information on the activity of certain classes of contaminants and an integrated Biomarker Response Index (BRI) was used to achieve a more holistic understanding of the condition of the SAC. The BRI indicated that the general health of both organisms was impacted in the upper part of the SAC (Fal Estuary) which correlated well with known chemical hotspots and sources of contamination. The BRI allows a pragmatic way to prioritise SAC sites that may require further investigative studies.     

Effects of organic contaminants in reactive oxygen species, protein carbonylation and DNA damage on digestive gland and haemolymph of land snails

The present study focused on early responses of land snails Eobania vermiculata to organic environmental contaminants, by investigating the use of a newly-established method for the measurement of protein carbonylation as a new biomarker of terrestrial pollution, as well as by measuring the ROS production and the DNA damage. Land snails were exposed to different concentrations of chlorpyrifos, parathion-methyl or PAHs in vivo or in vitro in the laboratory. The susceptibility of exposed snails was increased in relation to oxidative stress induced by contaminants tested. A statistically significant increase in ROS production, protein carbonylation and DNA damage was revealed in the snails treated with pollutants, compared to the untreated ones. The results indicated the effectiveness of measuring ROS production and DNA damage and reinforce the application of the present ELISA method in organic terrestrial pollution biomonitoring studies.     

Influence of anthropogenic stress on fitness and behaviour of a key-species of estuarine ecosystems, the ragworm Nereis diversicolor

Fitness, (biometric measurements, reproduction) and behaviour that are ecologically relevant biomarkers in assessing the quality of estuarine sediments were studied by comparing the responses of the polychaete worm Nereis diversicolor - a key species in estuaries - along a pollution gradient. Intersite differences were shown for all the measured parameters: size-weight relationships, energy reserves as glycogen and lipids, sexual maturation patterns, total number of oocytes per female, total and relative fecundity, burrowing behaviour. The physiological and behavioural status of N. diversicolor was consistently disturbed in the larger, most contaminated estuaries (Loire and Seine, Fr.) compared to reference sites (Bay of Bourgneuf, Goyen estuary, Fr.). Many classes of potentially toxic chemicals present in these estuaries most likely contribute to these impairments but food availability may act as a confounding factor, interfering with the potential impact of contaminants.     

Multiparametric approach for assessing environmental quality variations in West African aquatic ecosystems using the black-chinned tilapia (Sarotherodon melanotheron) as a sentinel species

The study highlights the potential of the black-chinned tilapia to be used as a sentinel to assess environmental contaminants based on the use of a set of biomarkers. The usefulness of fish species as sentinels for assessing aquatic environment contamination was tested using a set of biomarkers in Senegalese environments characterized by multi-pollution sources. The black-chinned tilapia (Sarotherodon melanotheron) was selected as a sentinel because of its abundance, wide distribution in all coastal aquatic ecosystems and physiological properties. The potential influence of confounding factors such as salinity on biomarker in the tilapia has been examined. Individuals were sampled during two seasons (dry and wet) in eight sites characterized by various degrees of anthropogenic contamination and different salinities (from 0 to 102?psu). Biomarkers??including growth rate (GR), condition factor (CF), biotransformation enzymes such as 7-ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST), lipid peroxidation (TBARS) and acetylcholinesterase (AChE)??were measured. Chemical contaminant [polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs)] levels showed different sources of contamination with relatively high concentrations of PAHs in the Hann Bay and Foundiougne locations. The most sensitive biomarker present in different sites according to the principal component analysis is EROD. Few variations of the AChE activity and TBARS levels were found. No clear relationship was found between biomarker responses and salinity, but GR and CF were lower in hypersaline conditions. Tilapia is responsive to environmental contaminants such as PAHs, OCPs and PCBs. The S. melanotheron multiparametric approach showed a better discrimination of sites.     

Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers

In this study, the effects of three widespread heavy metals, As(III), As(V) and Cd, and their binary mixtures on the proteomic profile in D. magna were examined to screen novel protein biomarkers using the two-dimensional gel electrophoresis method (2DE). Ten 20d daphnia were exposed to the LC20 concentrations for each of a total of 8 treatments, including the control, As(III), As(V), Cd, [As(III)+As(V)], [As(III)+Cd], [As(V)+Cd], and [As(III), As(V), Cd], for 24 h before protein isolation. Three replicates were performed for each treatment. These protein samples were employed for 2DE experiments with a pH gradient gel strip from pH 3 to pH 10. The protein spots were detected by a silver staining process and their intensities were analyzed by Progenesis software to discover the differentially expressed proteins (DEPs) in response to each heavy metal. A total of 117 differentially expressed proteins (DEPs) were found in daphnia responding to the 8 treatments and mapped onto a 2D proteome map, which provides some information of the molecular weight (MW) and pI value for each protein. All of these DEPs are considered as potential candidates for protein biomarkers in D. magna for detecting heavy metals in the aquatic ecosystem. Comparing the proteomic results among these treatments suggested that exposing D. magna to binary mixtures of heavy metals may result in some complex interactive molecular responses within them, rather than just the simple sum of the proteomic profiles of the individual chemicals, (As(III), As(V), and Cd).     

Effects of black carbon on bioturbation-induced benthic fluxes of polychlorinated biphenyls

It is unknown whether carbonaceous geosorbents, such as black carbon (BC) affect bioturbation by benthic invertebrates, thereby possibly affecting sediment-water exchange of sediment-bound contaminants. Here, we assess the effects of oil soot on polychlorinated biphenyl (PCB) mass transfer from sediment to overlying water, for sediments with and without tubificid oligochaeta as bioturbators. PCB levels were so low that toxicity to the oligochaeta played no role, whereas soot levels and binding affinity of PCBs to soot were so low that pore water PCB concentrations were not significantly affected by binding of PCBs to soot. This setup left direct effects of BC on bioturbation activity as the only explanation for any observed effects on mass transfer. Mass transfer coefficients (K_L) for benthic boundary layer transport were measured by a novel flux method using Empore™ disks as a sink for PCBs in the overlying water. For the PCBs studied (logK_ow 5.2-8.2), K_L values ranged from 0.2 to 2 cm × d⁻¹ in systems without tubificids. Systems with tubificids showed K_L values that were a factor of 10-25 higher. However, in the presence of oil soot, tubificids did not cause an increase in mass transfer coefficients. This suggests that at BC levels as encountered under field conditions, the mechanism for reduction of sediment-water transfer of contaminants may be twofold: (a) reduced mass transfer due to strong binding of the contaminants to BC, and (b) reduced mass transfer of contaminants due to a decrease in bioturbation activity.     

Assessment of typical pollutants in waterborne by combining active biomonitoring and integrated biomarkers response

Organic pollutants, heavy metals and pharmaceuticals are continuously dispersed into the environment and have become a relevant environmental emerging concern. In this study, a situ assay to assess ecotoxicity of mixed pollutants was carried out in three typical sites with different priority contaminations in Guangzhou, China. Chemical analysis of organic pollutants, metals and quinolones in three exposure sites were determined by GC-ECD/MS, ICP-AES and HPLC, as well as, a combination of biomarkers including: ethoxyresorufin O-deethylase (EROD); aminopyrine N-demethylase (APND); erythromycin N-demethylase (ERND); glutathione S-transferase (GST); malondialdehyde (MDA); CYP1A; and P-glycoprotein (P-gp) mRNA expressions were evaluated in Mugilogobius abei. Results of chemical analysis in sediment samples revealed that the dominant chemicals were organic pollutants and heavy metals in Huadi River while quinolones in the pond. Bioassays indicated that differences among sites were in relation to some specific biomarkers. EROD and GST activities significantly increased after 72 h in situ exposure, but no difference was observed among the exposure sites. APND, ERND and MDA exhibited dissimilar change patterns for different priority pollutants. CYP1A and P-gp mRNA expressions were significantly induced at all exposure sites, whilst P-gp activity was typical for S2 with the highest levels of quinolones. The molecular biomarkers seemed to be more susceptible than enzyme activities. These assays confirmed the usefulness of applying a large array of various combined biomarkers at different levels, in assessing the toxic effects of mixed pollutants in a natural aquatic environment.     

Biochemical and lysosomal biomarkers in the mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> from the Mar Piccolo of Taranto (Ionian Sea,Southern Italy)

Biomarkers are internationally recognized as useful tools in marine coastal biomonitoring, in particular, as early-warning signals at the level of individual organisms to assess biological effects of pollutants and other stressors. In the present study, Mytilus galloprovincialis has been employed as a sentinel organism to assess biological pollution effects in the Mar Piccolo of Taranto (Southern Italy), a coastal lagoon divided into two small inlets, connected to the open sea through one natural and one artificial narrow openings. Mussels were collected in June 2013 at three sites located within each of the two inlets of the Mar Piccolo. Biological effects were investigated through a suite of biomarkers suitable to reflect effects and/or exposure to contaminants at biochemical and cellular levels. Biochemical biomarkers included glutathione-S-transferase (GST) and acetylcholinesterase (AChE) enzyme activities; as histochemical biomarkers, lysosomal membrane stability, lipofuscin and neutral lipid accumulation, and lysosomal structural changes were considered. As a whole, results highlighted differences among the three study sites, particularly for GST, AChE, and lipofuscins, which are consistent with the variations of the chemical pollutants in sediments. The applied biomarkers showed that a stress syndrome likely to be ascribed to environmental pollutants is occurring in mussels living in the Mar Piccolo of Taranto, in particular, the ones inhabiting the first inlet.     

Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles

Engineered nano-sized Cu oxide particles are extensively used in diverse applications. Because aquatic environments are the ultimate “sink” for all contaminants, it is expected that nanoparticles (NP) will follow the same fate. In this study, two marine invertebrates Scrobicularia plana and Hediste diversicolor were chosen as ecotoxicological models. The aim was to evaluate behavioural (burrowing kinetics, feeding rate) and biochemical (biomarkers) responses of S. plana and H. diversicolor exposed in the laboratory to Cu (10 μg L⁻¹) added in natural seawater either in the form of engineered nanoparticles (NPs) of CuO or as dissolved Cu in 2% HNO₃. Exposure was characterized by considering (i) the physico-chemical fate of NP (ii) the fraction of labile Cu in experimental media and (iii) Cu bioaccumulation. Results showed high aggregation of CuO NPs in seawater and no additional bioavailable Cu concentrations. Behavioural impairments were observed in S. plana exposed to CuO NPs or soluble Cu whereas in H. diversicolor, only the exposure to soluble Cu led to a burrowing decrease. No obvious neurotoxicity effects were revealed since in both species, no changes in cholinesterasic activity occurred in response to both forms of Cu exposure. Biomarkers of oxidative-stress catalase and glutathione-S-transferase were enhanced in both species whereas superoxide dismutase was increased only in S. plana exposed to CuO NPs. Metallothionein-like protein was increased in bivalves exposed to both forms of Cu. Since, no detectable release of soluble Cu from CuO NPs occurred during the time of experiment, ecotoxicity effects seem to be related to CuO NPs themselves.     

Development of enzyme-linked immunosorbent assay (ELISA) for glutathione S-transferase (GST-S) protein in the intertidal copepod Tigriopus japonicus and its application for environmental monitoring

To utilize the GST-S protein as a useful biomarker for environmental contamination, we developed a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) in the intertidal copepod Tigriopus japonicus. Two polyclonal antibodies, TJ-GST-S1 and TJ-GST-S2, were raised against two TJ-GST-S synthetic peptides. Also a recombinant TJ-GST-S protein was purified as a standard for ELISA development. Each polyclonal antibody was tested by Western blot analysis and indirect ELISA. Of two polyclonal antibodies, TJ-GST-S2 ELISA was further employed due to its wide range of detection and the limit of specificity compared to those of TJ-GST-S1 ELISA system. After exposure to 4 metals (Ag, As, Cd, and Cu) to T. japonicus, the amount of TJ-GST-S protein was significantly elevated in a concentration-dependent manner. Also, TJ-GST-S protein was upregulated at relative high concentrations of B[α]P, PCB, and TBT. In this paper, we suggest that T. japonicas ELISA for TJ-GST-S2 is useful as a potential indicator system for marine contaminants.     

The differentially-expressed proteome in Zn/Cd hyperaccumulator Arabis paniculata Franch. in response to Zn and Cd

The Zn/Cd hyperaccumulator Arabis paniculata is able to tolerate high level of Zn and Cd. To clarify the molecular basis of Zn and Cd tolerance, proteomic approaches were applied to identify proteins involved in Zn and Cd stress response in A. paniculata. Plants were exposed to both low and high Zn or Cd levels for 10 d. Proteins of leaves in each treatment were separated by 2-DE (two-dimensional electrophoresis). Nineteen differentially-expressed proteins upon Zn treatments and 18 proteins upon Cd treatments were observed. Seventeen out of 19 of Zn-responsive proteins and 16 out of 18 of Cd-responsive proteins were identified using MALDI-TOF/TOF-MS (matrix-assisted laser desorption/ionization time of flight mass spectrometry). The most of identified proteins were known to function in energy metabolism, xenobiotic/antioxidant defense, cellular metabolism, protein metabolism, suggesting the responses of A. paniculata to Zn and Cd share similar pathway to certain extend. However, the different metal defense was also revealed between Zn and Cd treatment in A. paniculata. These results indicated that A. paniculata against to Zn stress mainly by enhancement of energy metabolism including auxin biosynthesis and protein metabolism to maintain plant growth and correct misfolded proteins. In the case of Cd, plants adopted antioxidative/xenobiotic defense and cellular metabolism to keep cellular redox homeostasis and metal-transportation under Cd stress.     

Can Hediste diversicolor (Nereidae, Polychaete) be considered a good candidate in evaluating PAH contamination? A multimarker approach

The aim of this study was to develop a preliminary characterization of the biological responses of Hediste diversicolor to polycyclic aromatic hydrocarbons (PAHs) under controlled laboratory conditions. In order to test the effects of PAH exposure, a battery of biomarkers was applied to the polychaetes by exposing them to sublethal concentrations of benzo[a]pyrene (0.1 and 0.5 mg L⁻¹) for 10 d under laboratory conditions. The battery of biomarkers tested included oxidative stress biomarkers (glutathione content, enzymatic activities of catalase, glutathione S-transferases, glutathione reductase, glutathione peroxidases), total oxyradical scavenging capacity (TOSC) toward peroxyl and hydroxyl radicals and activity of acyl CoA oxidase (AOX) as a marker of peroxisome proliferation measured in the entire body; lipofuscin and neutral lipid accumulations and levels of Ca²⁺-ATPase activity analyzed in the intestinal epithelium; lysosomal membrane stability and genotoxic effects measured as DNA strand breaks and frequency of micronuclei in coelomocytes. Chemical analyses were also carried out to verify the polychaete’s benzo[a]pyrene (B[a]P) bioaccumulation levels after the exposure period. The results obtained indicate that B[a]P caused significant changes in most of the parameters measured in H. diversicolor. Biological responses to the organic compound were particularly significant for the biomarkers measured in the intestinal epithelium and in coelomocytes, emphasizing that these tissues were more affected during our experimental conditions. Considering the key trophic role of this benthic species in estuarine and coastal ecosystems, this study confirmed that H. diversicolor is an appropriate bioindicator of organic contamination.     

Influence of mercury and selenium chemistries on the progression of cardiomyopathy in pygmy sperm whales, Kogia breviceps

More than half of pygmy sperm whales (Kogia breviceps) that strand exhibit signs of cardiomyopathy (CMP). Many factors may contribute to the development of idiopathic CMP in K. breviceps, including genetics, infectious agents, contaminants, biotoxins, and dietary intake (e.g. selenium, mercury, and pro-oxidants). This study assessed trace elements in K. breviceps at various stages of CMP progression using fresh frozen liver and heart samples collected from individuals that stranded along US Atlantic and Gulf coasts between 1993 and 2007. Standard addition calibration and collision cell inductively coupled plasma mass spectrometry (ICP-MS) were employed for total Se analysis and pyrolysis atomic absorption (AA) was utilized for total Hg analysis to examine if the Se/Hg detoxification pathway inhibits the bioavailability of Se. Double spike speciated isotope dilution gas chromatography ICP-MS was utilized to measure methyl Hg and inorganic Hg. Immunoblot detection and colorimetric assays were used to assess protein oxidation status. Data collected on trace elements, selenoproteins, and oxidative status were evaluated in the context of animal life history and other complementary histological information to gain insight into the biochemical pathways contributing to the development of CMP in K. breviceps. Cardiomyopathy was only observed in adult pygmy sperm whales, predominantly in male animals. Both Hg:Se molar ratios and overall protein oxidation were greater in males than females and increased with progression of CMP.     

Accumulation features and temporal trends of PCDDs, PCDFs and PCBs in Baikal seals (Pusa sibirica)

This study investigated the accumulation features and temporal trends of PCDD/Fs, dioxin-like PCBs (DL-PCBs) and non-dioxin-like PCBs (NDL-PCBs) in the blubber of Baikal seals collected in 1992 and 2005. DL-PCBs (480-3600 ng/g) and NDL-PCBs (980-35,000 ng/g) were dominant contaminants. Concentrations of PCDDs and PCBs in males were significantly higher than in females. In males, age-dependent accumulation was observed for PCDDs, mono-ortho PCBs and NDL-PCBs. PCDFs and non-ortho PCBs showed no such trends, implying that exposure of seals to these contaminants has been decreasing in recent years. No decreasing temporal trend was observed for PCDDs, mono-ortho PCBs and NDL-PCBs, suggesting that Baikal seals are still exposed to PCDDs and PCBs. TEQs of PCDDs and mono-ortho PCBs in seals collected in 2005 accounted for 62-77% of total TEQs. The TEQ levels in 40% of the specimens exceeded the threshold level for immunosuppression observed in harbor seals (209 pg/g).     

Effects of dioxin exposure in Eisenia andrei: integration of biomarker data by an Expert System to rank the development of pollutant-induced stress syndrome in earthworms

A battery of biomarkers has recently been developed in the earthworm Eisenia andrei. In this study, different biomarkers (i.e. Ca²⁺-ATPase activity, lysosomal membrane stability-LMS, lysosomal lipofuscin and neutral lipid content) were utilized to evaluate the alterations in the physiological status of animals, induced by exposure for 3 d to different sublethal concentrations of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) (1.5 × 10⁻³, 1.5 × 10⁻², 1.5 × 10⁻¹ ng mL⁻¹) utilizing the paper contact toxicity test. Lysosome/cytoplasm volume ratio and DNA damage were also evaluated as a biomarker at the tissue level and as a biomarker of genotoxicity, respectively. Moreover, the NR retention time assay conditions were optimized for the determination of in vivo LMS in earthworm coelomocytes. The results demonstrate that LMS and Ca²⁺-ATPase activity were early warning biomarkers able to detect the effects of minimal amounts of TCDD and that biomarkers evaluated at the tissue level are important for following the evolution of the stress syndrome in earthworms. To evaluate the health status of the animals, an Earthworm Expert System (EES) for biomarker data integration and interpretation was developed. The EES proved to be a suitable tool able to rank, objectively, the different levels of the stress syndrome in E. andrei induced by the different concentrations of TCDD.