Development of an expert system for the integration of biomarker responses in mussels into an animal health index

Biomarkers on sentinel organisms are utilised worldwide in biomonitoring programs. However, the lack of effective interpretational capacity has hampered their uptake for use for assessment of risk in environmental management. The aim of the present study was to develop and test an objective decision-support or expert system capable of integrating biomarker results into a five-level health-status index. The expert system is based on a set of rules derived from available data on responses to natural and contaminant-induced stress of marine mussels. Integration of parameters includes: level of biological organization; biological significance; mutual inter-relationship; and qualitative trends in a stress gradient. The system was tested on a set of biomarker data obtained from the field and subsequently validated with data from previous studies. The results demonstrate that the expert system can effectively quantify the biological effects of different levels of pollution. The system represents a simple tool for risk assessment of the harmful impact of contaminants by providing a clear indication of the degree of stress syndrome induced by pollutants in mussels.     

The use of biomarkers in biomonitoring: a 2-tier approach assessing the level of pollutant-induced stress syndrome in sentinel organisms

The paper outlines a 2-tier approach for wide-scale biomonitoring programmes. To obtain a high level of standardization, we suggest the use of caged organisms (mussels or fish). An "early warning", highly sensitive, low-cost biomarker is employed in tier 1 (i.e. lysosomal membrane stability (LMS) and survival rate, a marker for highly polluted sites). Tier 2 is used only for animals sampled at sites in which LMS changes are evident and there is no mortality, with a complete battery of biomarkers assessing the levels of pollutant-induced stress syndrome. Possible approaches for integrating biomarker data in a synthetic index are discussed, along with our proposal to use a recently developed Expert System. The latter system allows a correct selection of biomarkers at different levels of biological organisation (molecular/cellular/tissue/organism) taking into account trends in pollutant-induced biomarker changes (increasing, decreasing, bell-shape). A selection of biomarkers of stress, genotoxicity and exposure usually employed in biomonitoring programmes is presented, together with a brief overview of new biomolecular approaches.     

Evaluating the performance of sampling plans for phycotoxins in shellfish: Improvement of an existing method

According to the EU Regulation 854/2004, sampling plans must be set up to monitor production areas for the level of okadaic acid (OA) equivalents in live mussel. The level of these toxins, which are produced by harmful algal blooms, must not exceed 160 μg/kg of raw meat (Regulation 853/2004/EC). A sampling plan assessment consists in obtaining an OC (Operating Characteristic) curve showing both consumer and producer risks. The first risk is the risk of opening a shellfish area for harvest while the contamination level is above the threshold; whereas the second risk is the risk of closing a shellfish area having a contamination level under the threshold.For sampling plan validation purposes, a classical mathematical method was improved for the prediction of variance as function of the mean contamination level thanks to prior knowledge of the theoretical distribution fitting the observed OA levels among individual mussels. Indeed, knowing that, thanks to a regression analysis of literature data, for the lognormal distribution the scale parameter was observed to be directly proportional to the location parameter, the regression bias could be lowered. Literature data from Norway and Sweden showed different levels of variability between contamination events and depuration. However, the highest variability level was chosen to propose a best fit sampling plan in order to have a better approach of reality. It consisted of taking two samples of 50 mussels (Mytilus sp.) for this geographic location (Norway and Sweden).     

Multiple biomarkers of pollution effects in caged mussels on the Greek coastline

A suite of biomarkers was measured in caged mussels at areas impacted by different anthropogenic activities along the Greek coastline to assess biological effects of environmental pollution. Mussels were caged at coastal sites in the vicinity of major cities, in areas influenced by major industries, agricultural practices and in islands away from known sources of pollution. Biomarkers indicative of neurotoxicity (acetylcholinesterase, AchE), oxidative stress (catalase, CAT), phase II biotransformation of xenobiotics (glutathione S-transferase, GST), metal exposure (metallothioneins, MTs) and protein synthesis (RNA:DNA ratio) were measured to assess effects of various types of pollutants. AchE activity proved to be the most responsive biomarker with decreased values at sites influenced by agricultural, urban and industrial activities. Decreased CAT and GST activities and increased MTs levels were recorded at a number of anthropogenic-impacted sites. RNA:DNA ratio showed a biphasic response as both high and low values were found at impacted sites. Principal component analysis clearly distinguished sites receiving pollution inputs from non-polluted sites. The combination of the selected biomarkers used in caged mussels resulted useful in the assessment of the effects of environmental pollution.     

Evaluation of cadmium,lead and metallothionein contents in the tissues of mussels (Mytilus galloprovincialis) from the Campania coast (Italy): Levels and seasonal trends

The biological effect of seasonality on cadmium, lead and metallothionein contents was assessed in mussels Mytilus galloprovincialis from natural banks located along the coastline of the Gulf of Naples (Campania, Italy). Heavy metals and metallothionein concentrations were measured in digestive and reproductive glands. The results showed a clear correlation between metallothionein content and the reproductive gland status determined during the seasons; on the contrary, no correlation was found between metallothionein and metal contents. Data allow us to hypothesize that metallothionein functions go beyond metal detoxification, thus opening new scenarios for these proteins in invertebrates. The effect of seasons on metals concentration in mussel tissues showed similar seasonal patterns between the sites, regardless of their anthropogenic impacts. Cadmium content was not strictly related to seasonal periods, whereas lead content was significantly lower in summer. The results also indicate that the metal contents in mussels from the Gulf of Naples do not represent a risk to human health, even in the period of their maximum accumulation, and that the relaying of mussels before marketing could improve the animal stress conditions, but having a slight effect on metal excretion.     

Adaptation of the cholinergic system of <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (Bivalvia,Mytilidae) to the impact of natural and anthropogenic conditions

The effects of environmental conditions on cholinesterase activity and kinetic parameters of substrate hydrolysis in the hemolymph of the mussel Crenomytilus grayanus were studied. Under seasonal upwellings, the cholinergic system efficiency is provided for by a wide range of efficient concentrations of the substrate, i.e., under such conditions the mussels at the molecular level have a quantitative adaptation strategy of the enzyme. In mussels from the stationary upwelling zone (at a steady low temperature of water) for efficiency of the cholinergic system, the quantitative strategy of enzyme adaptation is realized. In mussels from a highly contaminated site, irreversible damages to the cholinergic process were observed. The affinity of the substrate to the enzyme is highly informative and an appropriate biomarker for the load level and the adaptation capacity of the organism. The affinity of the substrate to the enzyme is recommended as a new biomarker.     

Consequences of keepingMytilus in the laboratory as assessed by different cellular condition indices

Mytilus galloprovincialis Lmk. were maintained in the laboratory for three months in a semicontinuous water flow system. Animals were fed a commercial filter-feeder food and sampled after 0, 21, 35, 49, 77, and 91 days. In order to establish whether laboratory conditions and the food used were deleterious to mussels, their health status was assessed by quantifying different histological parameters of the digestive gland tissue. It was concluded that mussels kept for more than 35 days under the described laboratory conditions showed signs of stress presumably caused by the reproductive state of the mussels investigated. The food used and the nutrition-related health status of the animals were adequate, as shown by transmission electron microscopical studies after the 91-day maintenance period. A stress response was also evoked by a 10-day starvation period, which was reflected by an increased proportion of type I and type IV digestive tubules, and a reduced “Mean Epithelial Thickness” (MET). Finally, the results demonstrate the sensitivity of quantitative histological diagnosis in comparison to subjective tubule grading procedures in the assessment of the degree of stress experienced by mussels.     

Physiological and proteomic responses in Mytilus edulis exposed to PCBs and PAHs extracted from Baltic Sea sediments

Stress responses in blue mussels (Mytilus edulis. L.) exposed to rganic pollutants were measured using several physiological measures and as changes in protein expression. Blue mussels from the Baltic Sea were exposed for 6 days in a flow-through system to two fractions of extracted Baltic sediments (containing primarily PAHs or PCBs) from one industrially impacted site and one off-shore site. Exposure to Aroclor1248 (a commercial PCB mixture) was included as a reference treatment. Physiological response was measured as changes in respiration, excretion, clearance rates and scope for growth. Of the physiological responses, only clearance rate and scope for growth in the Aroclor and impacted site PCB treatments differed significantly (p<0.05) from control organisms, perhaps due to a large variation among individuals. Seven proteins were observed, presumed to be from stress protein families (hsp60, hsp70 and hsp90) on one-dimensional electrophoresis gels. All protein levels, except three proteins, 62, 73 and 90 kDa, in response to PCB exposure from the industrial site, were significantly higher (p<0.05) in treated than in control organisms, suggesting the use of stress-inducible proteins as diagnostics in risk assessment. A wider sample of proteins was observed using two-dimensional gel electrophoresis. The presence or absence of protein spots compared to control organisms was used as an indication of stress. Between 23 and 76 proteins or spots were present and 15 to 23 absent compared to controls, and the results supported the physiological and one-dimensional gel results, suggesting that the mussels were indeed suffering from stress. The methods used here represent stress monitoring at two different levels of biological organization; the cellular- and the level of individual organisms. In this experiment the protein response showed less variation among individuals compared to the physiological parameters. The protein response, however, still suffers from the lack of interpretation into commonly used monitoring terms, which emphasizes the need for more knowledge of whether the response is a momentary reflection of exposure or an early warning of higher order effects.     

Application of two SH-based methods for metallothionein determination in mussels and intercalibration of the spectrophotometric method: laboratory and field studies in the Mediterranean Sea

Metallothionein (MT) induction is widely used as a biomarker of exposure to metals in mussels. The aims of the present work were first to compare the suitability of spectrophotometry and differential pulse polarography (DPP) for MT detection in mussels exposed to 200 ppb cadmium for 9 days in a laboratory experiment and in mussels sampled in different seasons from expected pollution gradients along the Mediterranean Sea; second, to intercalibrate the widely used spectrophotometric method using mussels from Saronikos Gulf. In the intercalibration of the spectrophotometric method, similar results (p>0.05) were obtained by two different research teams indicating a good reproducibility of the technique. However, polarographic and spectrophotometric methods gave significantly (p<0.05) different results in laboratory and field studies. In the laboratory experiment, MT values detected with DPP were nine times higher than with spectrophotometry. The results obtained by the two methods were significantly correlated. Both methods could discriminate between control and exposed mussels. In field studies, MT values obtained by DPP were 34–38-fold higher than with spectrophotometry, and MT concentrations measured by both methods were not correlated. This discrepancy could be due to several factors, including the low levels of bioavailable metals in the studied areas and the possibility that the different methods can measure MT isoforms differentially. Further work is needed to decipher the functions of MT isoforms in mussels. This information is relevant for the application of MT as a biomarker in biomonitoring programmes.     

Effect of air exposure on lysosomal tissues of Mytilus edulis L. from natural intertidal wild beds and submerged culture ropes

Blue mussels collected from suspended culture ropes and from three natural intertidal wild beds from different areas of the German Bight were tested for their ability to cope with hypoxic conditions. During the experiment mussels were exposed to air from 0 to 72 h. Mussels from all sampling sites displayed high tolerance to aerial exposure with moderate levels of mortality after 12 to 48 h of exposure. Lysosomal membrane stability (LMS), a biomarker of general stress, changed notably between minimum values after 12 h and maximum values after 24 h of aerial exposure in intertidal mussels. In contrast, labilization times of mussels from the hanging culture increased continuously up to 48 h of exposure. Intertidal mussels from the island of Heligoland exhibited significantly decreased membrane stability after 72 h of air exposure, correlating to higher mortality rates. Intertidal mussels, although adapted to daily aerial exposure in their natural environment, showed a similar pattern of mortality and lower LMS values during the experiment than mussels from the suspended culture site. The increase of LMS values of mussels under hypoxic conditions at the beginning of the experiment at all sites was tested for the influence of macro-autophagic processes using immune labelling techniques. With this approach it could be demonstrated that high LMS values significantly correlate with low autophagic activity. However, hypoxic conditions do not enhance autophagic processes during the early periods of aerial exposure. Only at the end of the experiment, high values for autophagy were measured in mussels from an intertidal site accompanied with high mortalities. The results indicate that autophagic processes are not involved in the early adaptive processes that enable the mussel to cope with periods of aerial exposure.     

Transparent mediation-based access to multiple yeast data sources using an ontology driven interface

BACKGROUND: Saccharomyces cerevisiae is recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Information solicited by scientists on its biological entities (Proteins, Genes, RNAs...) is scattered within several data sources like SGD, Yeastract, CYGD-MIPS, BioGrid, PhosphoGrid, etc. Because of the heterogeneity of these sources, querying them separately and then manually combining the returned results is a complex and time-consuming task for biologists most of whom are not bioinformatics expert. It also reduces and limits the use that can be made on the available data. RESULTS: To provide transparent and simultaneous access to yeast sources, we have developed YeastMed: an XML and mediator-based system. In this paper, we present our approach in developing this system which takes advantage of SB-KOM to perform the query transformation needed and a set of Data Services to reach the integrated data sources. The system is composed of a set of modules that depend heavily on XML and Semantic Web technologies. User queries are expressed in terms of a domain ontology through a simple form-based web interface. CONCLUSIONS: YeastMed is the first mediation-based system specific for integrating yeast data sources. It was conceived mainly to help biologists to find simultaneously relevant data from multiple data sources. It has a biologist-friendly interface easy to use. The system is available at http://www.khaos.uma.es/yeastmed/.     

Application of two SH-based methods for metallothionein determination in mussels and intercalibration of the spectrophotometric method: laboratory and field studies in the Mediterranean Sea.

Metallothionein (MT) induction is widely used as a biomarker of exposure to metals in mussels. The aims of the present work were first to compare the suitability of spectrophotometry and differential pulse polarography (DPP) for MT detection in mussels exposed to 200 ppb cadmium for 9 days in a laboratory experiment and in mussels sampled in different seasons from expected pollution gradients along the Mediterranean Sea; second, to intercalibrate the widely used spectrophotometric method using mussels from Saronikos Gulf. In the intercalibration of the spectrophotometric method, similar results (p>0.05) were obtained by two different research teams indicating a good reproducibility of the technique. However, polarographic and spectrophotometric methods gave significantly (p<0.05) different results in laboratory and field studies. In the laboratory experiment, MT values detected with DPP were nine times higher than with spectrophotometry. The results obtained by the two methods were significantly correlated. Both methods could discriminate between control and exposed mussels. In field studies, MT values obtained by DPP were 34-38-fold higher than with spectrophotometry, and MT concentrations measured by both methods were not correlated. This discrepancy could be due to several factors, including the low levels of bioavailable metals in the studied areas and the possibility that the different methods can measure MT isoforms differentially. Further work is needed to decipher the functions of MT isoforms in mussels. This information is relevant for the application of MT as a biomarker in biomonitoring programmes.     

Formalized reproduction of an expert‐based phytosociological classification: A case study of subalpine tall‐forb vegetation

Abstract. Delimitation of vegetation units in phytosociology is traditionally based on expert knowledge. Applications of expert‐based classifications are often inconsistent because criteria for assigning relevés to vegetation units are seldom given explicitly. Still, there is, e.g. in nature conservation, an increasing need for a consistent application of vegetation classification using computer expert systems for unit identification. We propose a procedure for formalized reproduction of an expert‐based vegetation classification, which is applicable to large phytosociological data sets. This procedure combines Bruelheide's Cocktail method with a similarity‐based assignment of relevés to constancy columns of a vegetation table. As a test of this method we attempt to reproduce the expert‐based phytosociological classification of subalpine tall‐forb vegetation of the Czech Republic which has been made by combination of expert judgement and stepwise numerical classification of 718 relevés by TWINSPAN. Applying the Cocktail method to a geographically stratified data set of 21794 relevés of all Czech vegetation types, we defined groups of species with the statistical tendency of joint occurrences in vegetation. Combinations of 12 of these species groups by logical operators AND, OR and AND NOT yielded formal definitions of 14 of 16 associations which had been accepted in the expert‐based classification. Application of these formal definitions to the original data set of 718 relevés resulted in an assignment of 376 relevés to the associations. This assignment agreed well with the original expert‐based classification. Relevés that remained un‐assigned because they had not met the requirements of any of the formal definitions, were subsequently assigned to the associations by calculating similarity to relevé groups that had already been assigned to the associations. A new index, based on frequency and fidelity, was proposed for calculating similarity. The agreement with the expert‐based classification achieved by the formal definitions was still improved after applying the similarity‐based assignment. Results indicate that the expert‐based classification can be successfully formalized and converted into a computer expert system.     

The Passaic River Creel/Angler Survey: Expert Panel Review,Findings, and Recommendations

A Creel/Angler Survey (CAS) was conducted to provide site-specific information on recreational fishing in the lower six miles of the Passaic River (Study Area). Information collected during the CAS will be used to develop site-specific exposure factors, including fish consumption rates, for use in the human health risk assessment required by an Administrative Order on Consent as part of the Remedial Investigation/Feasibility Study for the Study Area. An expert panel was convened to provide an independent opinion regarding the need for, design of, and implementation of the CAS. The expert panel was charged with evaluating whether the conduct of a CAS is necessary to support an accurate risk assessment for the Study Area and whether the proposed CAS is sufficient to characterize local fish consumption behavior for risk assessment purposes. The expert panel agreed that a CAS is necessary and concluded that the proposed CAS, with specific modifications to the study design and data analysis, would provide the information necessary to estimate site-specific fish consumption rates. Revision of the CAS to accommodate the expert panel recommendations enhanced the quality of the data collected and ensured that the data will support the assessment of human health risks from consumption of fish from the Study Area.     

Macrozoobenthos communities and biocoenotic assessment of ecological status in relation to degree of human impact in small streams in southwest Germany

One of the major objectives of the VALIMAR project is to determine the ecological significance of various fish biomarker studies as indicators of chronic pollution in small streams in southwest Germany. Results of these fish biomarker investigations were compared to information from complementary studies on the meiobenthos, macrobenthos, and fish community studies to assess the ecological significance of these biomarker investigations. The main objective of this study was to provide biological assessments of the biomarker sites on the basis of the macrozoobenthos communities. Since no validated framework for the assessment of the biological integrity existed in Germany, two multimetric approaches were adapted to the whole stream system by investigating benthos and fish communities of 46 sites of varying degrees of human disturbance. Assessment of the communities was conducted in accordance with the European Community Water Framework Directive. Species distribution of benthos depended upon stream type and pollution status of streams. Biological attributes and bioindices of benthos communities, however, did not correlate with typological parameters like stream size or dominant substrate but correlated better with pollution parameters like conductivity or chloride concentration. Using a set of 18 measures, such as portion of sessile individuals, Rheoindex, oxygen availability index, and portion of pool dwellers, the benthos communities were characterized and evaluated. The composition of the fish communities was mainly determined by stream type, pollution and migration barriers. The influence of chemical parameters could be assessed by developing a“fish chemistry index”, which calculatesthe similarity of the present fish community with the potential natural community, but excludes those species strongly effected by deficits in stream channel morphology. Both fish chemistry index and benthic indices strongly correlated with pollution index parameters, clearly distinguishing between the more polluted Körsch sites and the less pollutedKrähenbach and Aich sites. Most of the single bioindices as well as overall assessment by multimetric indices indicated a gradient of decreasing quality from the reference stream Krähenbach to theslightly polluted Aich and Körsch upstream site (KE, upstream of all sewage treatment plants) and finally to the most polluted Körsch site directlybelow the most upstream sewage treatment plant (KD). According to the Water Framework Directive, the classification of ecological status of the benthos communities ranges from “high” (best of 5 classes) forthe reference stream to “bad” (5th class) for KD. Assessment of the fish community tends to score somewhat worse than the benthos due to deficits in morphological quality of the stream reaches. The benthos assessment and the newlydeveloped “fish chemistry index” correlated well with chemical water quality and hence with biomarkers, whereas ecological status of fish and overall ecological status was also influenced by river morphology. In conclusion all tested assessment methods on biocoenotic level are reliable indicators for the degree of human disturbance on small streams, whereas biomarkers are more suited for risk assessment and the investigation ofcause-effect-relationships.     

Finessing atlas data for species distribution models

Aim The spatial resolution of species atlases and therefore resulting model predictions are often too coarse for local applications. Collecting distribution data at a finer resolution for large numbers of species requires a comprehensive sampling effort, making it impractical and expensive. This study outlines the incorporation of existing knowledge into a conventional approach to predict the distribution of Bonelli’s eagle (Aquila fasciata) at a resolution 100 times finer than available atlas data. Location Malaga province, Andalusia, southern Spain. Methods A Bayesian expert system was proposed to utilize the knowledge from distribution models to yield the probability of a species being recorded at a finer resolution (1 × 1 km) than the original atlas data (10 × 10 km). The recorded probability was then used as a weight vector to generate a sampling scheme from the species atlas to enhance the accuracy of the modelling procedure. The maximum entropy for species distribution modelling (MaxEnt) was used as the species distribution model. A comparison was made between the results of the MaxEnt using the enhanced and, the random sampling scheme, based on four groups of environmental variables: topographic, climatic, biological and anthropogenic. Results The models with the sampling scheme enhanced by an expert system had a higher discriminative capacity than the baseline models. The downscaled (i.e. finer scale) species distribution maps using a hybrid MaxEnt/expert system approach were more specific to the nest locations and were more contrasted than those of the baseline model. Main conclusions The proposed method is a feasible substitute for comprehensive field work. The approach developed in this study is applicable for predicting the distribution of Bonelli’s eagle at a local scale from a national‐level occurrence data set; however, the usefulness of this approach may be limited to well‐known species.     

Integrative Biology of Idas iwaotakii (Habe, 1958), a ‘Model Species’ Associated with Sunken Organic Substrates

The giant bathymodioline mussels from vents have been studied as models to understand the adaptation of organisms to deep-sea chemosynthetic environments. These mussels are closely related to minute mussels associated to organic remains decaying on the deep-sea floor. Whereas biological data accumulate for the giant mussels, the small mussels remain poorly studied. Despite this lack of data for species living on organic remains it has been hypothesized that during evolution, contrary to their relatives from vents or seeps, they did not acquire highly specialized biological features. We aim at testing this hypothesis by providing new biological data for species associated with organic falls. Within Bathymodiolinae a close phylogenetic relationship was revealed between the Bathymodiolus sensu stricto lineage (i.e. “thermophilus” lineage) which includes exclusively vent and seep species, and a diversified lineage of small mussels, attributed to the genus Idas, that includes mostly species from organic falls. We selected Idas iwaotakii (Habe, 1958) from this latter lineage to analyse population structure and to document biological features. Mitochondrial and nuclear markers reveal a north-south genetic structure at an oceanic scale in the Western Pacific but no structure was revealed at a regional scale or as correlated with the kind of substrate or depth. The morphology of larval shells suggests substantial dispersal abilities. Nutritional features were assessed by examining bacterial diversity coupled by a microscopic analysis of the digestive tract. Molecular data demonstrated the presence of sulphur-oxidizing bacteria resembling those identified in other Bathymodiolinae. In contrast with most Bathymodiolus s.s. species the digestive tract of I. iwaotakii is not reduced. Combining data from literature with the present data shows that most of the important biological features are shared between Bathymodiolus s.s. species and its sister-lineage. However Bathymodiolus s.s. species are ecologically more restricted and also display a lower species richness than Idas species.     

Cu~(2 )、Zn~(2 )诱导稀有鮈鲫应激蛋白质的研究

以稀有鮈鲫为材料,研究了应激蛋白质作为生物学指标的敏感性。结果表明,在无可观察效应浓度下,经5d亚慢性胁迫暴露,以Cu2+为胁迫因子,稀有鮈鲫被诱导出约54KDa的应激蛋白质;以Zn2+为胁迫因子,稀有鮈鲫被诱导出约94KDa,67KDa和40KDa的应激蛋白质。应激蛋白质有可能成为一种生物学指标运用于生态风险性早期预警。     

Appropriate experimental design for transplanting mussels (Mytilus sp.) in analyses of environmental stress: an example in Sydney Harbour (Australia)

Some locations in Sydney Harbour (Sydney, Australia) contain large amounts of contaminants (heavy metals and hydrocarbons), sometimes in concentrations thought to affect biological systems. In order to estimate effects of sediment-bound contaminants on the physiology of organisms living above the sediment, the rates of clearance and respiration and the efficiency of absorption of mussels, Mytilus sp., living in a contaminated location were measured, the scope for growth (SfG) was calculated and compared to that of mussels living in uncontaminated locations. Two different models were proposed to explain expected differences. The first was that the contaminants at the impacted location reduced the SfG of local mussels; the second was that at the contaminated location only those mussels survived that had a small SfG (genetic differences between populations might be a reason for differential survival). To test which model applied, mussels were transplanted between the contaminated and uncontaminated locations. Moving and disturbing mussels (handling, tagging and caging) required the inclusion of two control treatments. These treatments were essential for a proper evaluation of the results but have generally not been included in similar studies. Effects of moving were estimated by translocating mussels from the uncontaminated and from the contaminated location to similar locations. To estimate effects of disturbance, mussels in the experimental locations were given the same treatment as the experimental mussels, but were returned to the place of origin. It was predicted that translocating and disturbing mussels would have no effect on the SfG, which would be similar to that of mussels at the place of origin. As expected, SfG was smaller (because rate of clearance was reduced) in undisturbed mussels at the contaminated location compared with those of mussels at the uncontaminated location. Because there were significant effects of disturbance on the SfG of mussels at the contaminated location, it was concluded that this difference was not caused by differences between the amounts of contaminants in the two locations, but was caused by other confounding factors (physical disturbance by crabs and fouling organisms). That the interpretation of the results would have been different if proper controls were not included is discussed in this paper and the importance of proper experimental controls is stressed.     

Evaluating the Predictive Value of Biomarkers with Stratified Case‐Cohort Design

Summary Identification of novel biomarkers for risk assessment is important for both effective disease prevention and optimal treatment recommendation. Discovery relies on the precious yet limited resource of stored biological samples from large prospective cohort studies. Case‐cohort sampling design provides a cost‐effective tool in the context of biomarker evaluation, especially when the clinical condition of interest is rare. Existing statistical methods focus on making efficient inference on relative hazard parameters from the Cox regression model. Drawing on recent theoretical development on the weighted likelihood for semiparametric models under two‐phase studies ( Breslow and Wellner, 2007 ), we propose statistical methods to evaluate accuracy and predictiveness of a risk prediction biomarker, with censored time‐to‐event outcome under stratified case‐cohort sampling. We consider nonparametric methods and a semiparametric method. We derive large sample properties of proposed estimators and evaluate their finite sample performance using numerical studies. We illustrate new procedures using data from Framingham Offspring Study to evaluate the accuracy of a recently developed risk score incorporating biomarker information for predicting cardiovascular disease.