Ongoing Analytical Procedure Performance Verification Using a Risk-Based Approach to Determine Performance Monitoring Requirements.

The analytical procedure life cycle (APLC) provides a holistic framework to ensure analytical procedure fitness for purpose. USP's general chapter <1220> considers the validation activities that take place across the entire analytical procedure lifecycle and provides a three-stage framework for its implementation. Performing ongoing analytical procedure performance verification (OPPV) (stage 3) ensures that the procedure remains in a state of control across its lifecycle of use post validation (qualification) and involves an ongoing program to collect and analyze data that relate to the performance of the procedure. Knowledge generated during stages 1 (procedure design) and 2 (procedure performance qualification) is used as the basis for the design of the routine monitoring plan to support performance verification (stage 3). The extent of the routine monitoring required should be defined based on risk assessment, considering the complexity of the procedure, its intended purpose, and knowledge about process/procedure variability. The analytical target profile (ATP) can be used to provide or guide the establishment of acceptance criteria used to verify the procedure performance during routine use (e.g., through a system/sample suitability test (SST) or verification criteria applicable to procedure changes or transfers). An ATP however is not essentially required to perform OPPV, and a procedure performance monitoring program can be implemented even if the full APLC framework has not been applied. In these situations, verification criteria can be derived from existing validation or system suitability criteria. Elements of the life cycle approach can also be applied retrospectively if deemed useful.

Contrasts in riverscape patterns of intraspecific genetic variation in a diverse Neotropical fish community of high conservation value.

Spatial patterns of genetic variation compared across species provide information about the predictability of genetic diversity in natural populations, and areas requiring conservation measures. Due to their remarkable fish diversity, rivers in Neotropical regions are ideal systems to confront theory with observations and would benefit greatly from such approaches given their increasing vulnerability to anthropogenic pressures. We used SNP data from 18 fish species with contrasting life-history traits, co-sampled across 12 sites in the Maroni- a major river system from the Guiana Shield -, to compare patterns of intraspecific genetic variation and identify their underlying drivers. Analyses of covariance revealed a decrease in genetic diversity as distance from the river outlet increased for 5 of the 18 species, illustrating a pattern commonly observed in riverscapes for species with low-to-medium dispersal abilities. However, the mean within-site genetic diversity was lowest in the two easternmost tributaries of the Upper Maroni and around an urbanized location downstream, indicating the need to address the potential influence of local pressures in these areas, such as gold mining or fishing. Finally, the relative influence of isolation by stream distance, isolation by discontinuous river flow, and isolation by spatial heterogeneity in effective size on pairwise genetic differentiation varied across species. Species with similar dispersal and reproductive guilds did not necessarily display shared patterns of population structure. Increasing the knowledge of specific life history traits and ecological requirements of fish species in these remote areas should help further understand factors that influence their current patterns of genetic variation.

Automatic detection, identification and counting of anguilliform fish using in situ acoustic camera data: Development of a cross-camera morphological analysis approach.

Acoustic cameras are increasingly used in monitoring studies of diadromous fish populations, even though analyzing them is time-consuming. In complex in situ contexts, anguilliform fish may be especially difficult to identify automatically using acoustic camera data because the undulation of their body frequently results in fragmented targets. Our study aimed to develop a method based on a succession of computer vision techniques, in order to automatically detect, identify and count anguilliform fish using data from multiple models of acoustic cameras. Indeed, several models of cameras, owning specific technical characteristics, are used to monitor fish populations, causing major differences in the recorded data shapes and resolutions. The method was applied to two large datasets recorded at two distinct monitoring sites with populations of European eels with different length distributions. The method yielded promising results for large eels, with more than 75% of eels automatically identified successfully using datasets from ARIS and BlueView cameras. However, only 42% of eels shorter than 60 cm were detected, with the best model performances observed for detection ranges of 4-9 m. Although improvements are required to compensate for fish-length limitations, our cross-camera method is promising for automatically detecting and counting large eels in long-term monitoring studies in complex environments.

European flounder foraging movements in an estuarine nursery seascape inferred from otolith microchemistry and stable isotopes.

Despite the importance of estuarine nurseries in the regulation of many fish stocks, temporal and spatial movements and habitat use patterns of juvenile fish remain poorly understood. Overall, combining several movement metrics allowed us to characterize dispersal patterns of juvenile flounder, Platichthys flesus, along an estuarine seascape. Specifically, we investigated otolith microchemistry signatures (Sr:Ca and Ba:Ca ratios) and stable isotope ratios (δ13C and δ15N) in muscles of these juveniles, during three consecutive years to assess inter-annual fluctuations in their home range and isotopic niches. The morphological condition and lipid content of individuals were lower in years of high as compared to low dispersal along the estuarine gradient. We discuss these results in relation to the ecosystem productivity and intra- and inter-specific competition level, which in turn affects movements and foraging behaviors of juvenile flounders.

Application of prediction intervals to the interpretation of the robustness study of a UHPLC method for the separation of cannabinoids.

Design of Experiments (DoE) is a well-established tool used for analytical methods robustness studies, because of its ability to assess the effect of a great number of factors in a minimal number of experiments. However, when assessing the robustness of an analytical method the analysis of the individual effect of each factor is not sufficient on its own. Some factors may not influence the robustness of the method, but their effect combined with the effects of other factors may have a significant contribution on the robustness of the method, which is not given by conventional analysis of DoE results. The aim of this work is to propose, in addition to the analysis of the individual effects of the factors, to estimate the joint effect of the factors by means of the matrix experimental results prediction interval. This prediction interval is the interval in which, with a given probability, should fall the next results, therefore it is an interesting tool to estimate the variation limits of the method results during routine use. We also propose the use of two other prediction intervals which can help to analyze the DoE results and give a conclusion on the method robustness. The first one is based on the DoE experimental error information, and it gives an estimation of the experimental error component impact on the factors joint effect. The second one is based on the factors non-significance limits, and it provides the information regarding the factors impact on the responses in the case where the conditions are, by definition, robust. We applied these proposals to the robustness study of a UHPLC method for the separation of phytocannabinoids and we could demonstrate that, in addition to the calculated effects values and robustness information, the use of the prediction intervals information provided additional information that allowed a better interpretation of the method performance parameters.

Assessing spatial deposition of aquatic subsidies by insects emerging from agricultural streams.

The role of winged aquatic insects that emerge from streams and subsidize terrestrial ecosystems has been demonstrated for natural forest landscapes, but almost no information is available for intensive agricultural landscapes. This study is the first to estimate aquatic subsidies provided by flying insects that emerge from streams and land on cropland. We investigated three major groups of aquatic insects - Trichoptera, Ephemeroptera and Chironomidae (Diptera) - that emerged from 12 third-order temperate, agricultural streams. We simultaneously monitored their emergence using floating traps and their terrestrial dispersal using passive interception traps. We estimated that the annual aquatic emerging dry mass (DM) of these groups varied from 1.4-7.5 g m-2 yr-1, depending on the stream. We used a Bayesian approach to estimate parameters of the terrestrial dispersal function of each group. We combined emerging DM and the dispersal parameters to estimate how terrestrial deposition of aquatic insect DM varied with increasing distance from streams. The results highlighted that emerging DM and dispersal to land could be higher in intensive agricultural landscapes than that previously described in natural settings. We estimated that 12.5 kg ha-1 yr-1 of winged aquatic insect DM fell to the ground 0-10 m from stream edges, composed mainly of Ephemeroptera and Trichoptera. We also estimated that 2.2 kg DM ha-1 yr-1 fell 10-50 m from the stream, especially small-bodied species of Chironomidae, throughout the year, except for the coldest weeks of winter. By influencing aquatic insect communities that emerge from streams, intensive agricultural practices change the magnitude and spatial extent of aquatic subsidy deposition on land. Implications for terrestrial food webs and ecosystem services provided to agriculture are discussed.

Molecular detection of the swim bladder parasite Anguillicola crassus (Nematoda) in fecal samples of the endangered European eel Anguilla anguilla.

The European eel Anguilla anguilla is listed as critically endangered by the IUCN. Among many threats, the introduced parasitic nematode Anguillicola crassus is suspected to alter the eels' swim bladder and jeopardize their reproductive oceanic migration. To date, gaining knowledge about the distribution and prevalence of A. crassus requires individual sacrifice (over 50,000 eels were sacrificed for epidemiology studies since 2010). This paper describes a non-lethal molecular protocol for identifying prevalence of A. crassus in A. anguilla, based on searching for A. crassus DNA in the feces of eels. Tests using three DNA microsatellite markers specific to the nematode showed that molecular detection provided similar results to visual examination of the swim bladder in up to 80% of the cases, and allowed for comparison of prevalence among sites. Easy to implement, this non-lethal protocol for detecting A. crassus could be valuable for management plans of this endangered species.

Application of prediction intervals to the interpretation of analytical methods robustness studies.

Since the emergence of the Analytical Quality by Design concept and the announcement of the future ICH Q14 "Analytical Procedure Development" guideline [1], a significant revival of the interest of the laboratories for the robustness study of their methods can be seen. Design of Experiments is a well-established tool used for this task, because of its ability to study the individual effect of a great number of factors in a minimal number of experiments. However, when assessing the robustness of an analytical method the analysis of the individual effect of each factor is not sufficient on its own. Indeed, the role of this study is also to verify, regardless of the very significance of the factors, that the chosen variations of the operating parameters of the method do not modify the experimental variance measured under the operating conditions for which the parameters are strictly defined. We therefore propose, in addition to the analysis of the individuals effects of the factors, an original tool which makes it possible to verify that the studied factors, when they vary together, do not lead to a significant variation in the measured responses. This tool is based on the prediction interval of the matrix experimental results, for each of the studied responses. This concept of prediction interval is already used in the context of analytical methods accuracy study. In a robustness design of experiments, it can represent the interval in which should be found, with a given probability, the response value if complementary experiments are conducted. It therefore represents the variation induced simultaneously by the different factors and can be easily compared to a maximum acceptable deviation of the considered response. We will detail this proposal and illustrate it with an example from a robustness study carried out on a liquid chromatography assay method.

The analysis of cannabinoids in cannabis samples by supercritical fluid chromatography and ultra-high-performance liquid chromatography: A comparison study.

The aim of this work was to develop a supercritical fluid chromatographic method to study the applicability of this emerging technique to cannabinoid analysis and showcase its advantages. During method development, the authors focused on nine phyto-cannabinoids to assess the selectivity needed to potentially perform the quantitation of each cannabinoid. After method development, robustness studies were carried out on this method to gain more information about its qualitative behavior (in terms of critical resolutions) when varying some crucial parameters (concentration of additive, column temperature, starting gradient conditions and column batch). Once the robustness was evaluated and the parameters most affecting the selected responses were individuated, the SFC method was applied for a simulated routine use to generate quantitative results concerning the concentrations of the main cannabinoids in real cannabis samples. The samples were also analyzed by means of an ultra-high-performance liquid chromatographic method currently used in the laboratory for the same objective. Finally, the results obtained with both analytical methods were compared to evaluate their accordance. The Bland-Altman method was applied as a statistical strategy to evaluate the degree of accordance between the results generated and display the data in a difference plot. The ultra-high performance supercritical fluid chromatography quantitative results were in accordance with the ultra-high performance liquid chromatography results, demonstrating the applicability of this technique for cannabinoid analysis.

Seasonal and spatial variations of stream insect emergence in an intensive agricultural landscape.

A growing amount of literature exists on reciprocal fluxes of matter and energy between ecosystems. Aquatic subsidies of winged aquatic insects can affect terrestrial ecosystems significantly, but this issue is rarely addressed in agroecosystems. By altering the production of benthic macroinvertebrates, agricultural practices could increase or decrease the strength of aquatic subsidies and subsequently the provision of several ecosystem services to agriculture. Effects of seasons and environmental variables on aquatic insect emergence were investigated in third-order agricultural streams in northwestern France. Most emerging dry mass (DM) of caught insects belonged to Trichoptera (56%), Chironomidae (25%) and Ephemeroptera (19%). We estimated that annual emerging dry mass of aquatic insects ranged between 1445 and 7374 mg/m2/y depending on the stream. Seasonal variations were taxon-specific, with Ephemeroptera emerging only in spring, Trichoptera emerging in spring and early summer, and Chironomidae emerging throughout the year. The percentage of watershed area covered by agriculture, ammonium concentration and hypoxia positively influenced emerging DM of Chironomidae but negatively influenced Ephemeroptera. Emerging DM of Trichoptera and Ephemeroptera increased significantly as water conductivity and temperature increased. Channel openness increased the emerging DM of all taxonomic groups, but Chironomidae were more abundant in narrow, incised streams. Assuming that the biomass of aquatic invertebrates ultimately disperse toward terrestrial habitats, nutrient accumulations on land near streams were estimated to reach 0.5-2.3 kg C ha-1 y-1, 0.1-0.5 kg N ha-1 y-1 and 0.005-0.03 kg P ha-1 y-1, depending on the stream. This suggests a significant flux of aquatic nutrients to agroecosystems and the need for future studies of its potential influence on the ecosystem services provided to agriculture.

Stability of a Screw Dislocation in a ⟨011⟩ Copper Nanowire.

The stability of a screw dislocation in a free ⟨011⟩ copper nanowire is investigated using atomistic calculations. This study reveals a strong anisotropy of the Eshelby potential well (EPW) that traps the dislocation. Moreover the depth of the EPW is found to vanish when the radius of the nanowire decreases. It is demonstrated that this behavior is due to the dissociated state of the dislocation.

High-performance thin-layer chromatography analysis of steviol glycosides in Stevia formulations and sugar-free food products, and benchmarking with (ultra) high-performance liquid chromatography.

A high-performance TLC (HPTLC) method was newly developed and validated for analysis of 7 steviol glycosides in 6 different types of food and Stevia formulations. After a minimized one-step sample preparation, 21 samples were developed in parallel, allowing an effective food screening. Depending on the sample application volume, the method was suited to analyze food sample concentrations in the mg/kg range. LOQs of stevioside in natural yoghurt matrix spiked at 0.02, 0.13 and 0.2% were determined by the calibration curve method to be 12ng/band (peak height). ANOVA was successfully passed to prove data homogeneity in the working range (30-600ng/band). The accuracy (recovery tolerance limit, 92-120%), repeatability (3.1-5.4%) and intermediate precision (4.0-8.4%) were determined for stevioside in milk-based matrix including sample preparation and recovery rates at 3 different concentration levels. For the first time, the recording of HPTLC-ESI-MS spectra via the TLC-MS Interface was demonstrated for rebaudioside A. HPTLC contents for rebaudioside A were compared with results of two (U)HPLC methods. The running costs and analysis time of the three different methods were discussed in detail with regard to screening of food products.

Stable isotope analyses on archived fish scales reveal the long-term effect of nitrogen loads on carbon cycling in rivers.

Stable isotope analysis of organic matter in sediment records has long been used to track historical changes in productivity and carbon cycling in marine and lacustrine ecosystems. While flow dynamics preclude stratigraphic measurements of riverine sediments, such retrospective analysis is important for understanding biogeochemical cycling in running waters. Unique collections of riverine fish scales were used to analyse δ(15) N and δ(13) C variations in the food web of two European rivers that experience different degrees of anthropogenic pressure. Over the past four decades, dissolved inorganic N loading remained low and constant in the Teno River (70°N, Finland); in contrast, N loading increased fourfold in the Scorff River (47°N, France) over the same period. Archived scales of Atlantic salmon parr, a riverine life-stage that feeds on aquatic invertebrates, revealed high δ(15) N values in the Scorff River reflecting anthropogenic N inputs to that riverine environment. A strong correlation between dissolved inorganic N loads and δ(13) C values in fish scales was observed in the Scorff River, whereas no trend was found in the Teno River. This result suggests that anthropogenic N-nutrients enhanced atmospheric C uptake by primary producers and its transfer to fish. Our results illustrate for the first time that, as for lakes and marine ecosystems, historical changes in anthropogenic N loading can affect C cycling in riverine food webs, and confirm the long-term interactions between N and C biogeochemical cycles in running waters.

Natal departure timing from spatially varying environments is dependent of individual ontogenetic status.

Natal departure timing represents one of the first crucial decisions for juveniles born in spatially varying environments that ultimately disappear, but our knowledge on its determinants is limited. The present study aimed at understanding the determinants of juvenile natal departure by releasing individually tagged juvenile pike (Esox lucius L.) with variable body size and trophic position in a temporary flooded grassland. Specifically, we investigated whether natal departure depends on individual competitive status ('competition hypothesis'), physiological tolerance to environmental conditions ('physiological hypothesis') or individual trophic position and the spatial heterogeneity of trophic resources ('trophic hypothesis'). The results indicated that departure timing was negatively correlated with body size at release, showing that the dominance status among competing individuals was not the main trigger of juvenile departure. A positive correlation between departure timing and individual body size at departure was observed, suggesting that inter-individual variability in physiological tolerance did not explain departure patterns. While individual growth performances were similar irrespective of the timing of natal departure, stable isotope analyses revealed that juveniles with higher trophic position departed significantly earlier than individuals with lower trophic position. Therefore, the trade-off driving the use of spatially varying environments was most likely dependent upon the benefits associated with energetic returns than the costs associated with inter-individual competition or physiological stress. This result highlighted how ontogeny, and particularly ontogenetic niche shift, can play a central role in juvenile's decision to depart from natal habitats in a predatory species.

Experimental designs dedicated to the evaluation of a membrane extraction method: membrane-assisted solvent extraction for compounds having different polarities by means of gas chromatography-mass detection.

Membrane-assisted solvent extraction was applied for the determination of different classes of compounds in water, having K(o/w) (octanol-water partition coefficient) values between 10(1) (aniline) and 10(8) (methyl stearate), by means of experimental designs. Four solvents were investigated--propan-2-ol, ethyl acetate, diisopropyl ether and cyclohexane--as well as extraction time, temperature, salt impact, pH and methanol addition. The best choice was diisopropyl ether, 50 degrees C, 30 min and an addition of 3 g of sodium chloride at pH 2 for polar compounds. The relative standard deviation (n = 3) was found in the range from 5 to 17%. Recoveries ranged between 34 and 100%. Membrane-assisted solvent extraction was successfully applied to a fast screening method dedicated to an unknown wastewater sample.