Valve gape behaviour of mussels (Mytilus edulis) exposed to dispersed crude oil as an environmental monitoring endpoint.

Environmental monitoring requires cost-effective and efficient methods for detecting potential effects of pollution, and valve gape behaviour has been used with this purpose for a range of contaminants in freshwater and marine bivalves. The current study investigated the use of a new method for measuring valve behaviour responses in mussels (Mytilus edulis) exposed to dispersed crude oil (DCO). Results confirmed that valve gape is a sensitive parameter; at the high DCO concentration (0.25mgL-1) the mean valve gape was reduced from 49 to 31%, and mussels increased shell movement (measured as distance travelled) or spent more time closed to avoid contact with the oil. At the low DCO concentration (0.015mgL-1) the distance travelled parameter was the most sensitive endpoint. Results also demonstrated that valve gape behaviour is a valid endpoint when monitoring mussels for exposure to DCO.

Effect of dispersed crude oil on the feeding activity, retention efficiency, and filtration rate of differently sized blue mussels (Mytilus edulis).

The use of physiological response endpoints in environmental monitoring represents an opportunity to provide an integrated picture of health status and ecological fitness of individuals, and may provide an indication of potential longer term effects on aquatic organisms in the environment. The feeding behavior response sensitivity of blue mussels (Mytilus edulis) of differing size to dispersed crude oil (DCO) was investigated in a lab exposure experiment. The ability of mussels to recover following a single exposure was also investigated, as well as the response to consecutive exposures, in order to assess the utility of employing the same individuals in chronic environmental monitoring. Feeding physiology was assessed by measuring retention efficiency and filtration rate of individual mussels in a live-algae feeding assay. In addition, the percentage of mussels actively filtering during testing was calculated. The feeding physiology parameters were sensitive and able to discriminate exposed mussels from controls. Further, data indicated that larger mussels appear more suitable in environmental monitoring, as these animals showed both sensitivity and an ability to adapt and recover from exposure while remaining sensitive to subsequent treatments. Smaller mussels were also sensitive to the measured endpoints, even if these animals suffered higher rates of mortality during the exposure. Finally, when exposed to the high concentration of DCO, mussels displayed a tendency to close the valves and terminate filtration.

SELDI-TOF MS analysis of alkylphenol exposed Atlantic cod with phenotypic variation in gonadosomatic index.

Proteomics is a new and promising approach to evaluate potential effects of pollution. In order to investigate if there is a direct link between the protein expression profiles obtained by the SELDI-TOF MS technology and effects observed at the organism level in fish, plasma samples from unexposed and 20 ppb alkylphenol exposed female Atlantic cod (Gadus morhua) with high phenotypic variation in gonadosomatic index (GSI) were analyzed by SELDI-TOF MS. Principle component analysis (PCA) showed that the major proteomic variation present in the dataset (i.e. 23.6%) could be significantly correlated to the individual variation in GSI, which indicates that SELDI-TOF MS data can reflect effects observed at higher levels of organization in fish. Further exploration of the other principal components revealed an additional proteomic pattern specific for the alkylphenol exposed females. Hence, this study supports the usefulness of SELDI-TOF MS as a proteomic tool in ecotoxicological research.

From SELDI-TOF MS to protein identification by on-chip elution.

SELDI-TOF MS has been demonstrated as a powerful tool for biomarker discovery. However, a major disadvantage of SELDI-TOF MS is the lack of direct identification of the discriminatory peaks discovered. We describe a novel experimental identification strategy where peptides/proteins captured to a weak cation exchange ProteinArray surface (CM10) are eluted, and thereafter identified by utilizing a sensitive LC-MS/MS (i.e. LTQ Orbitrap). A mixture of four known proteins was used to test the novel experimental approach described, and all four proteins were successfully identified. Additionally, a biomarker candidate previously discovered in plasma of Atlantic cod (Gadus morhua) by SELDI-TOF MS was identified. Thus, this study indicated that a combination of on-chip elution and a highly sensitive LC-MS/MS system can be an alternative approach to identify biomarker candidates discovered by use of SELDI-TOF MS.

Data processing and classification analysis of proteomic changes: a case study of oil pollution in the mussel, Mytilus edulis.

BACKGROUND: Proteomics may help to detect subtle pollution-related changes, such as responses to mixture pollution at low concentrations, where clear signs of toxicity are absent. The challenges associated with the analysis of large-scale multivariate proteomic datasets have been widely discussed in medical research and biomarker discovery. This concept has been introduced to ecotoxicology only recently, so data processing and classification analysis need to be refined before they can be readily applied in biomarker discovery and monitoring studies. RESULTS: Data sets obtained from a case study of oil pollution in the Blue mussel were investigated for differential protein expression by retentate chromatography-mass spectrometry and decision tree classification. Different tissues and different settings were used to evaluate classifiers towards their discriminatory power. It was found that, due the intrinsic variability of the data sets, reliable classification of unknown samples could only be achieved on a broad statistical basis (n > 60) with the observed expression changes comprising high statistical significance and sufficient amplitude. The application of stringent criteria to guard against overfitting of the models eventually allowed satisfactory classification for only one of the investigated data sets and settings. CONCLUSION: Machine learning techniques provide a promising approach to process and extract informative expression signatures from high-dimensional mass-spectrometry data. Even though characterisation of the proteins forming the expression signatures would be ideal, knowledge of the specific proteins is not mandatory for effective class discrimination. This may constitute a new biomarker approach in ecotoxicology, where working with organisms, which do not have sequenced genomes render protein identification by database searching problematic. However, data processing has to be critically evaluated and statistical constraints have to be considered before supervised classification algorithms are employed.

Mass spectrometric profiling - a diagnostic tool in fish?

The development of rapid and sensitive diagnostic tools to assess the effect of stressors on organisms is a principal objective of environmental proteomics. This study is focused on evaluating the potential of using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) to assess stress in Atlantic salmon (Salmo salar). Plasma and mucus samples were taken from fish that had previously been maintained in a range of high density conditions, together with control fish maintained under low density conditions. Samples were collected during the post-density stress period for protein profile analysis. The mass spectra were analysed to evaluate reproducibility and to search for condition specific changes in protein expression. Multivariate analysis of the peak relative intensity data indicated a segregation of the data into three entities in accordance with the density level fish had been subjected to during the density stress period. This segregation was seen in both plasma and mucus data.

Comparison of protein expression in plasma from nonylphenol and bisphenol A-exposed Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) by use of SELDI-TOF.

The overall objective of this study was to compare the expression of plasma proteins in juvenile cod and turbot after a 3 week exposure to two different chemicals known to be estrogenic: 4-nonylphenol (NP, 29 microg/L) and bisphenol A (BPA, 59 microg/L). ProteinChip) array technology in combination with surfaced enhanced laser desorption ionisation-time of flight (SELDI-TOF) was used to investigate general responses in plasma proteins. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) was used to analyse two specific biomarkers of estrogenic exposure, vitellogenin (Vtg) and zona radiata protein (Zrp) in plasma. Both methods revealed clear species specific responses. In cod, 67% of significantly altered proteins showed the same response (up or down regulated) in NP and BPA exposed animals (males and females combined). The rest were either specific to NP (10%), BPA (19%) or they showed opposite responses to the two chemicals (4%). In contrast, only 20% of significantly altered proteins were common for NP and BPA exposed turbot: 60% were altered only in NP and 17% only in BPA. Furthermore, in BPA exposed cod, 77% of the responses were common for male and females, whereas turbot showed only 21% similarity for the two genders. However, NP exposed male and female turbot showed 88% similarity in responses. As gender was not determined in NP exposed cod, gender specific responses could not be determined. ELISA results supported that cod responded clearly to both chemicals as a large increase was observed in Vtg and Zrp levels. Turbot responded strongly to NP, but seemed only slightly affected by BPA. Overall, the results indicated that cod are more sensitive or respond with less specificity to estrogenic chemicals than turbot. The relatively large degree of common responses in NP and BPA exposed cod may indicate that in cod BPA have similar mode of action as NP. Generally, the results show the potential of SELDI-TOF as a tool for comparing multiple responses, and for identifying exposure as well as gender specific responses.

Investigation of micronuclei and other nuclear abnormalities in peripheral blood and kidney of marine fish treated with crude oil.

The induction of micronuclei and other nuclear abnormalities (nuclear buds, bi-nucleated and fragmented-apoptotic cells) was analyzed in the erythrocytes of peripheral blood and cephalic kidney of turbot (Scophthalmus maximus) and Atlantic cod (Gadus morua), treated with crude oil (Statfjord B, Norway) and with nonylphenol. Significant increase in MN was observed in turbot kidney and blood after exposure to 30 ppb of nonylphenol, 0.5 ppm of oil, and after co-exposure to 0.5 ppm of oil spiked with additional mixture of alkylphenols and PAHs (P varied between 0.0054 and <0.0001). The induction of micronuclei was observed only in cod kidney after exposure to spiked oil (P=0.0317). Significant inter-specific differences after the exposure to 0.5 ppm of oil (P=0.0385) and after treatment with spiked oil (P=0.0067) were observed. In turbot cephalic kidney, the elevated levels of bi-nucleated cells were observed in all treatment groups (P values varied in a range from 0.05 to 0.0025) while the increase in cells with nuclear buds was noted after the exposure to 0.5 ppm of oil (P=0.05). The fragmented-apoptotic cells appeared after the exposure to nonylphenol (P=0.0039) and to spiked oil (P<0.0001). In turbot blood, only the significant induction in nuclear buds was detected. Statistically significant inter-tissue differences were found only in the induction of fragmented-apoptotic cells after the exposure to nonylphenol and to spiked oil.

The BEEP Stavanger Workshop: Mesocosm exposures.

Within the BEEP project (Biological Effects of Environmental Pollution in Marine Ecosystems) the Work Package 1 was addressed to the development of new and more sensitive biomarkers of exposure in several sentinel organisms. Within this framework, common mesocosm exposures of organic pollutants relevant for marine ecosystems were conducted in the facilities of Akvamiljø a/s (Stavanger, Norway). In the first experiment, Atlantic cod (Gadus morhua), turbot (Scophthalmus maximus) and shore crab (Carcinus maenas) were exposed to nonylphenol, North Sea crude oil and a combination of crude oil and alkylated phenols. Mussels (Mytilus edulis) were exposed to North Sea crude oil and a combination of crude oil, alkylated phenols and PAHs. In the second experiment, Atlantic cod, turbot, mussel and spider crab (Hyas araneus) were exposed to the plasticizers bisphenol A and diallyl phatalate and the brominated flame retardant BDE-47. The main purpose of the present study was to provide the 30 participating Institutes with samples which had been exposed to defined contaminant concentrations in a controlled laboratory exposure for 3 weeks. This paper describes the mesocosm experimental design, the transplantation and treatment of the organisms, and the contaminant exposures.

Proteome modifications of blue mussel (Mytilus edulis L.) gills as an effect of water pollution.

The discharge of chemicals such as oil associated or not with derived products constitutes a real threat for the environment. We report here the differential expression of the blue mussel (Mytilus edulis) gill proteins corresponding to two contaminated environmental conditions: crude oil and offshore produced water. In order to evaluate and understand contaminants, effects and adaptive response of these organisms, we identified proteins using MS. The latter can be grouped into three main classes: proteins involved in the cellular structure, in metabolism, and in defence proteins.

Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry approach to biomarker discovery in blue mussels (Mytilus edulis) exposed to polyaromatic hydrocarbons and heavy metals under field conditions.

Proteomics provide potential in the discovery of new sensitive biomarkers for environmental pollution. To evaluate this potential, we have utilized ProteinChip technology to analyze the proteomic profile of blue mussels (Mytilus edulis) from polluted marine habitats surrounding the island of Karmøy, Norway. Two different types of contamination, heavy metals and polyaromatic hydrocarbons (PAHs), were compared to a clean reference site. Differentially expressed proteins/peptides were found, which showed a specific induction or a general suppression associated with the field site of origin. By combining sets of protein markers in a tree-building algorithm, we were able to correctly classify samples from these sites with an accuracy of 90%.

Analysis of micronuclei in blue mussels and fish from the Baltic and North Seas.

Micronuclei (MN) were analyzed in erythrocytes of flounder (Platichthys flesus) and wrasse (Symphodus melops) and in gill cells of blue mussels (Mytilus edulis). The organisms were collected from three study stations in the Baltic Sea and from seven stations in the North Sea (Karmsund area, Norway) 4 times. The statistically significant differences obtained were related to the season, sex of the fish, and sampling locality. Higher MN frequencies were found in fish and mussels collected from the most polluted study stations in the North Sea. The same tendency could be described in the Baltic Sea; however, it was masked by the recent oil spill from the Butinge oil terminal. Our results showing higher MN frequencies in presumably what were the most polluted study locations suggest that MN tests in fish and mussels may be used for the detection of genotoxic effects in a marine environment. The endpoint is well characterized and can be easily recognized, and the technique is convenient to use in field samplings following standard procedures and protocols.

Rapid assessment of polycyclic aromatic hydrocarbon (PAH) exposure in decapod crustaceans by fluorimetric analysis of urine and haemolymph.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and potentially harmful contaminants of the coastal and marine environment. Studies of their bioavailability, disposition and metabolism in marine organisms are therefore important for environmental monitoring purposes. Detecting PAH compounds in the biological fluids of marine organisms provides a measure of their environmental exposure to PAHs. In the present study, the shore crab Carcinus maenas was exposed to waterborne pyrene for 48h. Urine and haemolymph samples were analysed by direct fluorimetry utilising both fixed wavelength (FF) and synchronous scanning fluorescence (SFS) techniques. Samples from exposed crabs exhibited fluorescence due to 1-OH pyrene equivalents, whilst samples from control crabs did not. Levels of equivalents were exposure dependent. Urine was shown to be a more suitable medium for the analysis of PAH equivalents. In a separate experiment, depuration of pyrene equivalents in urine was monitored over time. Urinary levels reached a maximum 2-4 days after initial exposure and decreased steadily thereafter. No unchanged parent pyrene was detected in samples from exposed crabs. While fluorimetric techniques could discriminate between 1-OH pyrene equivalents and parent pyrene, identification of specific metabolites was only possible with HPLC/F analysis. This revealed crabs had bio-transformed pyrene into 3 major conjugates of 1-OH pyrene, which were excreted in the urine. While such biotransformation of PAH is well documented in fish and several crustaceans, this is the first study to use direct fluorimetry to detect PAH equivalents in exposed crustacean urine. Fluorimetric results correlated well with those obtained by HPLC/F and ELISA techniques. The technique has great potential as a rapid, inexpensive and non-destructive technique for field biomonitoring of PAH exposure in crustaceans.