In situ measurement with diffusive gradients in thin films: effect of biofouling in freshwater.

Concerning in situ passive sampler deployment, several technical priorities must be considered. In particular, deployment time must be sufficiently long not only to allow a significant quantity to be accumulated to facilitate analysis but also to ensure that the signal is above the quantification limit and out of the blank influence. Moreover, regarding the diffusive gradient in thin films (DGT) technique, deployment time must also be sufficiently long (at least 5 days) to avoid the interactions of the solutes with the material diffusion layer of the DGT and for the steady state to be reached in the gel. However, biofouling occurs in situ and modifies the surface of the samplers. In this article, we propose a kinetic model which highlights the biofouling effect. This model was able to describe the mitigation of the flux towards the DGT resin observed on Cd, Co, Mn, Ni and Zn during a 22-day deployment in the Seine River. Over a period of 22 days, biofouling had a significant impact on the DGT concentrations measured, which were decreased twofold to threefold when compared to concentrations measured in unaffected DGTs.

Use of low density polyethylene membranes for assessment of genotoxicity of PAHs in the Seine River.

The genotoxicity of river water dissolved contaminants is usually estimated after grab sampling of river water. Water contamination can now be obtained with passive samplers that allow a time-integrated sampling of contaminants. Since it was verified that low density polyethylene membranes (LDPE) accumulate labile hydrophobic compounds, their use was proposed as a passive sampler. This study was designed to test the applicability of passive sampling for combined chemical and genotoxicity measurements. The LDPE extracts were tested with the umu test (TA1535/pSK1002 ± S9) and the Ames assay (TA98, TA100 and YG1041 ± S9). We describe here this new protocol and its application in two field studies on four sites of the Seine River. Field LDPE extracts were negative with the YG1041 and TA100 and weakly positive with the TA98 + S9 and Umu test. Concentrations of labile mutagenic PAHs were higher upstream of Paris than downstream of Paris. Improvement of the method is needed to determine the genotoxicity of low concentrations of labile dissolved organic contaminants.

Assessing the relation between anthropogenic pressure and PAH concentrations in surface water in the Seine River basin using multivariate analysis.

Understanding the relation between polycyclic aromatic hydrocarbons (PAHs) in freshwater and anthropogenic pressure is fundamental to finding a solution to reduce the presence of PAHs in water, and thus their potential impact on aquatic life. In this paper we propose to gain greater insight into the variability, sources and partitioning of PAHs in labile (or freely dissolved=not associated to the organic matter), dissolved and particulate phases in freshwater. This study was conducted using land use data as a marker of anthropogenic pressure and coupling it with chemical measurements. This study was conducted on 30 sites in the Seine River basin, which is subjected to a strong human impact and exhibits a wide range of land uses. Half of the sites were studied twice. Labile PAHs were measured by semi-permeable membrane devices (SPMDs), and dissolved and particulate phases by grab samples. Partial least squares regressions were performed between chemical measurements and data of anthropogenic pressure. The results indicate different sources for the dissolved phase and particles. Dissolved and labile phases were more related to the population density of the watershed, while particles were more related to a local pressure. Season and land use data are necessary information to correctly interpret and compare PAH concentrations from different sites. Furthermore, the whole data set of the 45 field deployments comprising labile, dissolved, total and particulate PAH concentrations as well as the physico-chemical parameters is available in the supplementary information.

Genotoxicity and activation of cellular defenses in transplanted zebra mussels Dreissena polymorpha along the Seine river.

The aim of the present study was to confirm the relevance of studying DNA adduct formation in a field study. In that context, freshwater mussels Dreissena polymorpha, collected in a reference station, were transplanted in different sites with a pollution gradient. After one and two months, mussels were collected and DNA adduct formation was analyzed using the (32)P post labelling technique on both gills and digestive glands. In addition, the expression of genes involved in the detoxification system (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), HSP70, aryl hydrocarbon receptor (AHR), P glycoprotein (PgP), metallothionein (MT)) was assessed by RT-PCR. DNA adducts were observed at amount comparable to data from literature. Increase of DNA adducts after two months of transplantation could be correlated with strong modulation of gene expression implicated in detoxification processes. Indeed, PgP and HSP70 gene expressions were similarly induced in gills and digestive glands while SOD and CAT expressions were down regulated in both tissues. AHR, GST and MT genes were differently regulated depending upon the tissue studied and the level of contamination in the different sites. We demonstrated that mussels transplanted in the different stations with pollution gradient were able to biotransform PAHs, assessed by DNA adduct formation and the high decrease of detoxification genes. Specific DNA adducts pattern obtained after one and two month mussel transplantations demonstrated the relevance of DNA adduct as biomarker of environmental pollution.

Quantifying diet-borne metal uptake in Gammarus pulex using stable isotope tracers.

Gammarids are aquatic amphipods widely used for water quality monitoring. To investigate the copper and cadmium diet-borne metal uptake in Gammarus pulex, we adapted the pulse-chase stable isotopes-based approach to determine the food ingestion rate (IR), the gut retention time (GRT) and the metal assimilation efficiencies (AE). G. pulex were fed with (65)Cu-, (106)Cd-, and (53)Cr-labeled alder leaves for 7.5h and then with unlabeled leaves for 5d. The metal stable isotope contents in the gammarids, leaves, filtered water and periodically collected feces were determined. Chromium was poorly assimilated by the gammarids; thus, Cr was used as an unassimilated tracer. The first tracer defecation occurred before the first feces harvest, indicating a gut passage time of less than 9h. A 24-h GRT and a 0.69gg(-1)d(-1) IR were estimated. The Cd AE value was estimated as 5-47%, depending on the assimilation determination method applied. The Cu AE value could not be evaluated regardless of the determination method used, most likely because of the rapid Cu regulation in gammarids in addition to analytical uncertainties when determining the Cu content in leaves. Application of the Cd AE value in the framework of the biodynamic bioaccumulation model shows that the diet-borne uptake of Cd significantly contributes (66-95%) to the metal bioaccumulation in G. pulex fed with alder leaves.

DNA adduct formation and induction of detoxification mechanisms in Dreissena polymorpha exposed to nitro-PAHs.

Derived polycyclic aromatic hydrocarbons (PAHs) such as nitro-PAHs are present in the environment and are known to be much more toxic than PAHs compounds. However, very few studies have analysed their effects on the aquatic environment and none have investigated the freshwater environment. In the present study, we determined whether 1-nitropyrene (1-NP), a model of nitro-PAHs, can induce DNA adducts in gills and digestive glands of the freshwater mussel Dreissena polymorpha. Two concentrations of 1-NP (50 and 500 µM) were tested. In addition, in order to understand the metabolic pathways involved in 1-NP genotoxicity, mRNA expression of genes implicated in biotransformation mechanisms was assessed by quantitative reverse transcription-PCR. Results showed the presence of DNA adducts in both gills and digestive glands, with highest levels obtained after 5 days of exposure to 500 µM. Metallothionein mRNA levels were enhanced in digestive glands exposed to 50 µM. Surprisingly, at the higher concentration (500 µM), aryl hydrocarbon receptor and HSP70 genes were only up-regulated in digestive glands while PgP mRNA levels were increased in both tissues. Results suggested a cytotoxic and genotoxic effect of 1-NP. Mussels seemed to be able to partially detoxify this compound, in view of the low amount of DNA adducts observed after 5 days exposure to 50 µM. For the first time, 1-NP biotransformation and detoxification systems have been characterised in D. polymorpha.

Bioaccumulation of polybrominated diphenyl ethers (PBDEs) in Gammarus pulex: relative importance of different exposure routes and multipathway modeling.

Characterizing the exposure routes of an organism and its ability to regulate accumulated contaminants is a crucial step toward developing a biomonitor. To date, very little data are available on the bioaccumulation kinetics of PBDEs in freshwater biota. This study aims at investigating the potential use of a litter-degrader widely distributed in European freshwaters, Gammarus pulex, as an indicator of exposure to PBDEs. In aquatic microcosms, gammarids were exposed to a mixture of brominated congeners (BDE-28, 47, 66, 85, 99, 100, 153, 154 and 183) to assess their ability to bioconcentrate PBDEs. Results show that all tested congeners are highly internalized by G. pulex and uptake rates of PBDEs are closely related to their partition coefficients (Kow). The determination of the elimination rate of BDE-47, the congener most readily accumulated by gammarids, indicated that metabolism and excretion of this congener are low in G. pulex, which argues in favor of its use as a quantitative biomonitor. Finally, bioaccumulation experiments were performed using contaminated leaves to determine the relative importance of dietary uptake in the contamination of gammarids. Even though water is the preeminent exposure route, a significant uptake of BDE-47 through food was observed (27%). We propose a biodynamic model that takes into account both exposure routes to describe BDE-47 bioaccumulation. This study supports the use of this ubiquitous amphipod as an early warning monitor of the bioavailable contamination of freshwaters by PBDEs.

Metallothionein mRNA induction is correlated with the decrease of DNA strand breaks in cadmium exposed zebra mussels.

We have previously shown that cadmium (Cd) and benzo[a]pyrene (BaP) induced early DNA damages in zebra mussels, and that the level of DNA strand breaks (SB) returned to a basal level after 3 days of exposure to Cd. The aim of the present study was to go further in the mechanisms of Cd and BaP detoxification. For that purpose, expression of genes encoding for metallothionein (MT), aryl hydrocarbon receptor (AHR), P-gp, catalase, glutathione S-transferase and heat shock protein 70 (HSP70) proteins have been measured using RT-qPCR. Data reported here show that Cd is a strong inducer of MT and HSP70 genes, and that BaP is a strong inducer of P-gp and AHR genes. Exposure to Cd and BaP resulted in moderate changes in antioxidant enzymes mRNA. Since the increase of MT mRNA occurred when the DNA SB level returned to its basal level, we can suggest that MT is implicated in cadmium detoxification.

Linking community tolerance and structure with low metallic contamination: a field study on 13 biofilms sampled across the Seine river basin.

It is difficult to assess the biological consequences of diffuse water contamination by micropollutants which are present in rivers at low, even sublethal levels. River biofilms, which respond quickly to changes of environmental parameters, are good candidates to acquire knowledge on the response of aquatic organisms to diffuse chemical contamination in the field. The study was designed as an attempt to link biofilm metal tolerance and metallic contamination in a field survey covering 13 different sampling sites in the Seine river basin (north of France) with low contamination levels. Cd and Zn tolerance of heterotrophic communities was assessed using a short-term toxicity test based on ß-glucosidase activity. Metal tolerance levels varied between sites but there was no obvious correlation between tolerance and corresponding water contamination levels for Cd and Zn. Indeed, metallic contamination at the sampling sites remained subtle when compared to water quality standards (only two sampling sites had either Zn or both Cu and Zn concentrations exceeding the Environmental Quality Standards set by the EU Water Framework Directive). Yet, multivariate analysis of the data using Partial Least Squares Regression revealed that both metallic and environmental parameters were important variables explaining the variability of metal tolerance levels. Automated Ribosomal Intergenic Spacer Analysis (ARISA) was also performed on both bacterial and eukaryotic biofilm communities from the 13 sampling sites. Multivariate analysis of ARISA fingerprints revealed that biofilms with similar tolerance levels have similar ARISA profiles. Those results confirm that river biofilms are potential indicators of low, diffuse contamination levels of aquatic systems.

Essential metal contents in indigenous gammarids related to exposure levels at the river basin scale: metal-dependent models of bioaccumulation and geochemical correlations.

Biomonitoring, assumed to be an integrative measurement of the chemical exposure of aquatic organisms, is not straightforward for essential metals because they can be actively regulated by animals. Although increasing bioaccumulation with exposure levels is a crucial endpoint for the development of biomonitors, it is rarely verified in real environments, where the metal concentrations are rather low and vary little. This study was designed at the scale of a river basin to assess the ability of Gammarus pulex indigenous populations to accumulate Cu, Zn and Mn in realistic exposure conditions. During two annual campaigns, water and gammarids were collected at various sites contrasted in terms of physicochemistry and contamination. The results show significant relationships between metal concentrations in animals and in freshwaters established by conceptual models of bioaccumulation, but with patterns specific to each metal (base level, internal regulation and maximal accumulation). In particular, a saturation process of Cu accumulation occurs at environmental exposure levels, unlike Mn and Zn. Statistical analyses performed from field data show that Cu and Zn bioaccumulations may be influenced by a complex combination of geochemical variables, unlike Mn. We conclude that G. pulex is a useful candidate to monitor metal bioavailability in freshwaters due to its responsiveness to low exposures of surrounding environments. Nevertheless, a reliable quantification of bioavailability of essential metals requires characterizing some geochemical effects on metal bioaccumulation.

DGT measurement in low flow conditions: diffusive boundary layer and lability considerations.

Recent papers have alerted the scientific community that a diffusive boundary layer (DBL) forming in front of diffusive gradients in thin film (DGT) devices when they are immersed in water might have a significant impact on the results and have suggested a method to assess the DBL. This paper aims at evaluating to what extent the DBL impacts the results of metal measurement in water by DGT and providing new information on the dissociation kinetics of metal complexes in wastewater by using DBL calculation. A careful study of the influence of the water velocity on the measurement with DGTs equipped with restricted gels is presented. Deployments took place in the laboratory with a range of stirring speeds (0-400 rpm) and in a canal receiving treated wastewater with increasing controlled water velocity (0.07-3 cm s(-1)). Even under extreme low flow conditions, the error made in using the equation that does not take into account that the DBL was lower than the analytical error. Nevertheless, the DBL is the seat of dissociation of complexes and increases the lability window beyond the steric constraints of the hydrogel. The capacity of restricted gels to only sample inorganic species under these conditions is questioned. This study also is an opportunity to provide information on metal-ligand interactions in wastewater by creating the kinetic signature of the wastewater. Unlike previous studies which used different types of water, Pb was the more limited metal and interacted strongly with the ligands.

Seasonal and PAH impact on DNA strand-break levels in gills of transplanted zebra mussels.

Genotoxicity endpoints are useful tools to biomonitor the physicochemical and biological quality of aquatic ecosystems. A caging study on the freshwater bivalve Dreissena polymorpha was planned to run over four seasons in the Seine River basin in order to assess whether DNA damage measured in transplanted mussels to polluted area vary according to seasonal changes. Three sites were chosen along the Seine River, one upstream from Paris and two downstream, corresponding to a chemical gradient of water contamination. The DNA strand break (comet assay) and chromosomal damage (micronucleus test) were measured in caged mussels at each site and in winter, spring and summer, along with PAH water contamination, PAH bioaccumulation, the mussel condition index (CI), the gonado-somatic index (GSI) and the filtration rate (FR). The level of DNA strand break measured in winter was low and increased in spring, concomitantly with FR and GSI. Over the same period, micronucleus (MN) frequency and PAH bioaccumulation decreased significantly in caged mussels, with both parameters positively correlated to each other. DNA strand-break levels and MN frequencies showed inter-site variations corresponding to the chemical contamination gradient. These two genotoxicity endpoints usefully complement each other in field studies. These results show that the MN test and comet assay, when applied to gill cells of caged zebra mussels, are sensitive tools for freshwater genotoxicity monitoring.

Genotoxicity assessment and detoxification induction in Dreissena polymorpha exposed to benzo[a]pyrene.

The use of DNA adduct analysis has previously focused on the use of marine organisms for biomonitoring, whereas similar investigations in freshwater organisms are sparse. In that context, we have investigated the relevance of DNA adducts as biomarkers of genotoxicity in the freshwater mussels Dreissena polymorpha. The objective of the present study is to determine the stability of DNA adducts induced by benzo[a]pyrene (B[a]P) in zebra mussels. Mussels were exposed to dissolved B[a]P (10-100 µg/l) for 4 days. Afterwards, mussels were kept in clean water for 28 days and DNA adduct levels were subsequently measured in two different organs, the digestive glands and the gills, using the (32)P-postlabelling technique. In parallel, the expression of genes involved in the detoxification system was assessed by qPCR (catalase, superoxide dismutase, glutathione S transferase, HSP70, aryl hydrocarbon receptor, P glycoprotein). We observed a higher level of DNA adducts in the digestive glands compared to the gills. Moreover, in gills, the level of DNA adduct was dependent on the B[a]P concentration. The levels of adducts tended to decrease in both organs after 28 days in clean water. In addition, an early induction of HSP70, PgP, AHR and SOD mRNA levels was noticed in the gills compared to the digestive glands. CAT and GST gene expression increased from 12h exposure in both organs. A higher gene expression level of those genes was observed in the gills, except for AHR and CAT genes. Data converge towards the fact that DNA adducts hence represent a very promising biomarker of B[a]P contamination and potentially of exposure to polycyclic aromatic hydrocarbons. In addition, for the first time in this study, B[a]P detoxification system was characterised in D. polymorpha.

Pollution-induced community tolerance of freshwater biofilms: measuring heterotrophic tolerance to Pb using an enzymatic toxicity test.

This study aims at investigating the impacts of Pb on freshwater biofilms with a pollution-induced community tolerance (PICT) approach using a recently developed short-term toxicity test based on ß-glucosidase activity to measure biofilms' tolerance to Pb. We first investigated more closely the influence of the total suspended solids (TSS) concentrations of biofilm suspensions used for short-term toxicity tests performed to assess Pb tolerance. The Pb EC(50) values of four dilutions of the same biofilm suspension increased with their TSS concentrations. TSS-normalization allowed to obtain a unique measure of Pb tolerance, thus confirming that TSS-normalization of EC(50) values is a good means to estimate biofilm tolerance to Pb. The experiment was repeated with three different biofilm samples collected at different sites and dates. Second, biofilms were exposed to Pb (0, 1, 10 and 100 µg/L) for 3 weeks in microcosms to assess the impacts of Pb exposure on the communities. An increase in Pb tolerance was observed for the biofilm exposed to 100 µg/L. Automated Ribosomal Intergenic Spacer Analysis revealed modifications of bacterial and eukaryotic community structure with Pb exposure. Moreover, exposure to 100 µg/L Pb also led to an increase in Zn tolerance but not Cu tolerance. This study shows that tolerance acquisition to Pb can be detected after exposure to environmental concentrations of Pb using a PICT methodology and normalized EC(50) values as measures of Pb tolerance.

Modelling copper bioaccumulation in Gammarus pulex and alterations of digestive metabolism.

Bioaccumulation enables to integrate the ability of aquatic organisms to regulate metals and effects of water chemistry on metal bioavailability. Linking this process to biological responses offers thus promising lines of enquiry for protecting aquatic ecosystems. This study aims at characterizing the mechanisms involved in waterborne Cu bioaccumulation and assessing metal impact on digestive metabolism in an ecosystem engineer widely distributed in Europe, Gammarus pulex. The organism was exposed to several Cu concentrations (from 0.5 to 100 µg/L) in aquatic microcosms to establish kinetic parameters for the construction and comparison of two bioaccumulation models, i.e. the biodynamic and saturation models. Cu uptake was recorded in waters exhibiting various concentrations of Na, Mg and Ca at environmental levels to assess the influence of cationic composition on bioaccumulation. Then, the effect of increasing Cu in exposure media on the digestive metabolism of G. pulex was investigated by measuring enzymatic activities (ß-glucosidase, N-acetyl-ß-glucosaminidase, ß-galactosidase). We showed that the saturation model is more suitable than the biodynamic model to describe Cu bioaccumulation in gammarids due to a maximal capacity of animals to accumulate the metal. Cationic composition of water affected insignificantly Cu uptake. All activities of tested enzymes decreased with increasing Cu in exposure media but with different degrees. High correlations were established between the inhibition of enzymatic activities and amounts of Cu bioaccumulated by gammarids. These biological responses could thus provide early-warming of Cu impact on aquatic biota.

Impact of biofouling on diffusive gradient in thin film measurements in water.

The technique of diffusive gradient in thin film (DGT) is commonly used to assess metal contamination in natural waters. In this paper, we assess the effect of biofouling on DGT measured labile concentrations in water and investigate whether an additional nuclepore polycarbonate membrane on the surface of DGT devices can limit biofilm growth. Simultaneous field deployments of DGT equipped with and without the additional membrane in a canal receiving wastewater were compared. The effect of the biofilm was also assessed in controlled laboratory experiments, completed by the experimental determination of several metals diffusion coefficients in the hydrogel and membrane systems. The biofilms effect was problematic only from the 10th day of accumulation. Accumulation of some elements is highly biased by the presence of a thick biofilm (Zn, Ni, Cd). The polycarbonate membrane improved the quantification of Cd and Ni but adversely affects the quantification of Cr and Co. A kinetic model is proposed to explain the biofilm role on the DGT measurement. Depending on the metals of interest, it is possible to limit bias due to biofilms by using an additional polycarbonate membrane.

Diffuse urban pollution increases metal tolerance of natural heterotrophic biofilms.

This study is a first attempt to investigate the impact of urban contamination on metal tolerance of heterotrophic river biofilms using a short-term test based on ß-glucosidase activity. Tolerance levels to Cu, Cd, Zn, Ni and Pb were evaluated for biofilms collected at three sites along an urban gradient in the Seine river (France). Metallic pollution increased along the river, but concentrations remained low compared to environmental quality standards. Biofilm metal tolerance increased downstream from the urban area. Multivariate analysis confirmed the correlation between tolerance and contamination and between multi-metallic and physico-chemical gradients. Therefore, tolerance levels have to be interpreted in relation to the whole chemical and physical characteristics and not solely metal exposure. We conclude that community tolerance is a sensitive biological response to urban pressure and that mixtures of contaminants at levels lower than quality standards might have a significant impact on periphytic communities.

Adaptation of copper community tolerance levels after biofilm transplantation in an urban river.

The Water Framework Directive requires the development of biological tools which can act as early-warning indicators of a sudden increase (accidental pollution) or decrease (recovery due to prevention) of the chemical status of aquatic systems. River biofilms, which respond quickly to modifications of environmental parameters and also play a key part in the functioning of aquatic ecosystems, are therefore good candidates to monitor an increase or a decrease of water pollution. In the present study, we investigated the biological response of biofilms transplanted either upstream (recovery) or downstream (deterioration of exposure levels) the urban area of Paris (France). Both modifications of Cu community tolerance levels and of global bacterial and eukaryotic community structure using automated ribosomal intergenic spacer analysis (ARISA) fingerprints were examined 15 and 30 days after the transplantation. Cu tolerance levels of the heterotrophic component of biofilms were assessed using a short-term toxicity test based on ß-glucosidase (heterotrophic) activity. Cu tolerance increased for biofilms transplanted upstream to downstream Paris (5-fold increase on day 30) and conversely decreased for biofilms transplanted downstream to upstream (8-fold decrease on day 30). ARISA fingerprints revealed that bacterial and eukaryotic community structures of transplanted biofilms were closer to the structures of biofilms from the transplantation sites (or sites with similar contamination levels) than to biofilms from their sites of origin. Statistical analysis of the data confirmed that the key factor explaining biofilm Cu tolerance levels is the sampling site and not the site of origin. It also showed that Cu tolerance levels are related to the global urban contamination (both metals and nutrients). The study shows that biofilms adapt fast to modifications of their surroundings. In particular, community tolerance varies quickly and reflects the new exposure levels only 15 days after transplantation. Those results support the use of biofilms as reliable early-warning indicators of diffuse urban contamination.

Towards a renewed research agenda in ecotoxicology.

New concerns about biodiversity, ecosystem services and human health triggered several new regulations increasing the need for sound ecotoxicological risk assessment. The PEER network aims to share its view on the research issues that this challenges. PEER scientists call for an improved biologically relevant exposure assessment. They promote comprehensive effect assessment at several biological levels. Biological traits should be used for Environmental risk assessment (ERA) as promising tools to better understand relationships between structure and functioning of ecosystems. The use of modern high throughput methods could also enhance the amount of data for a better risk assessment. Improved models coping with multiple stressors or biological levels are necessary to answer for a more scientifically based risk assessment. Those methods must be embedded within life cycle analysis or economical models for efficient regulations. Joint research programmes involving humanities with ecological sciences should be developed for a sound risk management.

Bioavailability of particulate metal to zebra mussels: biodynamic modelling shows that assimilation efficiencies are site-specific.

This study investigates the ability of the biodynamic model to predict the trophic bioaccumulation of cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni) and zinc (Zn) in a freshwater bivalve. Zebra mussels were transplanted to three sites along the Seine River (France) and collected monthly for 11 months. Measurements of the metal body burdens in mussels were compared with the predictions from the biodynamic model. The exchangeable fraction of metal particles did not account for the bioavailability of particulate metals, since it did not capture the differences between sites. The assimilation efficiency (AE) parameter is necessary to take into account biotic factors influencing particulate metal bioavailability. The biodynamic model, applied with AEs from the literature, overestimated the measured concentrations in zebra mussels, the extent of overestimation being site-specific. Therefore, an original methodology was proposed for in situ AE measurements for each site and metal.