The practice of historical ecology: What, when, where, how and what for.

In recent decades, there has been a growing number of studies exploring the historical dimensions of the interconnectedness of human societies and the environment. A core approach in this field is historical ecology. We analyzed 544 historical-ecological papers to assess patterns and trends in the field. We found a high degree of interdisciplinarity with a focus on local case studies, of periods of fewer than 500 years, analyzing archival sources through quantitative approaches. The proportion of papers containing management recommendations has increased over time. To make historical ecology globally relevant, more effort should be made to utilize studies across languages, borders and worldviews. We call for high standards regarding the use of social scientific methodologies. Lastly, we argue that fostering longer-term studies and assessing the real-life impact of policy recommendations emerging from historical ecology can help the discipline better contribute solutions to the challenges facing humanity in an uncertain future.

Functional trait trade-offs define plant population stability across different biomes.

Ecological theory posits that temporal stability patterns in plant populations are associated with differences in species' ecological strategies. However, empirical evidence is lacking about which traits, or trade-offs, underlie species stability, especially across different biomes. We compiled a worldwide collection of long-term permanent vegetation records (greater than 7000 plots from 78 datasets) from a large range of habitats which we combined with existing trait databases. We tested whether the observed inter-annual variability in species abundance (coefficient of variation) was related to multiple individual traits. We found that populations with greater leaf dry matter content and seed mass were more stable over time. Despite the variability explained by these traits being low, their effect was consistent across different datasets. Other traits played a significant, albeit weaker, role in species stability, and the inclusion of multi-variate axes or phylogeny did not substantially modify nor improve predictions. These results provide empirical evidence and highlight the relevance of specific ecological trade-offs, i.e. in different resource-use and dispersal strategies, for plant populations stability across multiple biomes. Further research is, however, necessary to integrate and evaluate the role of other specific traits, often not available in databases, and intraspecific trait variability in modulating species stability.

Territory and population attributes affect Florida scrub-jay fecundity in fire-adapted ecosystems.

Fecundity, the number of young produced by a breeding pair during a breeding season, is a primary component in evolutionary and ecological theory and applications. Fecundity can be influenced by many environmental factors and requires long-term study due to the range of variation in ecosystem dynamics. Fecundity data often include a large proportion of zeros when many pairs fail to produce any young during a breeding season due to nest failure or when all young die independently after fledging. We conducted color banding and monthly censuses of Florida scrub-jays (Aphelocoma coerulescens) across 31 years, 15 populations, and 761 territories along central Florida's Atlantic coast. We quantified how fecundity (juveniles/pair-year) was influenced by habitat quality, presence/absence of nonbreeders, population density, breeder experience, and rainfall, with a zero-inflated Bayesian hierarchical model including both a Bernoulli (e.g., brood success) and a Poisson (counts of young) submodel, and random effects for year, population, and territory. The results identified the importance of increasing "strong" quality habitat, which was a mid-successional state related to fire frequency and extent, because strong territories, and the proportion of strong territories in the overall population, influenced fecundity of breeding pairs. Populations subject to supplementary feeding also had greater fecundity. Territory size, population density, breeder experience, and rainfall surprisingly had no or small effects. Different mechanisms appeared to cause annual variation in fecundity, as estimates of random effects were not correlated between the success and count submodels. The increased fecundity for pairs with nonbreeders, compared to pairs without, identified empirical research needed to understand how the proportion of low-quality habitats influences population recovery and sustainability, because dispersal into low-quality habitats can drain nonbreeders from strong territories and decrease overall fecundity. We also describe how long-term study resulted in reversals in our understanding because of complications involving habitat quality, sociobiology, and population density.

Patterns, sources, and consequences of variation in age-specific vital rates: Insights from a long-term study of Weddell seals.

Variations in the reproductive and survival abilities of individuals within a population are ubiquitous in nature, key to individual fitness, and affect population dynamics, which leads to strong interest in understanding causes and consequences of vital-rate variation. For long-lived species, long-term studies of large samples of known-age individuals are ideal for evaluating vital-rate variation. A population of Weddell seals in Erebus Bay, Antarctica, has been studied each Austral spring since the 1960s. Since 1982, all newborns have been tagged each year and multiple capture-mark-recapture (CMR) surveys have been conducted annually. Over the past 20 years, a series of analyses have built on results of earlier research by taking advantage of steady improvements in the project's long-term CMR data and available analytical methods. Here, I summarize progress made on four major topics related to variation in age-specific vital rates for females: early-life survival and age at first reproduction, costs of reproduction, demographic buffering, and demographic senescence. Multistate modelling found that age at first reproduction varies widely (4-14 years of age) and identified contrasting influences of maternal age on survival and recruitment rates of offspring. Subsequent analyses of data for females after recruitment revealed costs of reproduction to both survival and future reproduction and provided strong evidence of demographic buffering. Recent results indicated that important levels of among-individual variation exist in vital rates and revealed contrasting patterns for senescence in reproduction and survival. Sources of variation in vital rates include age, reproductive state, year, and individual. The combination of luck and individual quality results in strong variation in individual fitness outcomes: ~80% of females born in the population produce no offspring, and the remaining 20% vary strongly in lifetime reproductive output (range: 1-23 pups). Further research is needed to identify the specific environmental conditions that lead to annual variation in vital rates and to better understand the origins of individual heterogeneity. Work is also needed to better quantify the relative roles of luck, maternal effects, and environmental conditions on variation in vital rates and to learn the importance of such variation to demographic performance of offspring and on overall population dynamics.

Zoo-Led Initiatives and Their Role in Lemur Conservation In Situ.

We examined wider society's ability to achieve biodiversity conservation and management targets using lemurs as a case study. We evaluated the impact on lemur conservation in situ by conservation initiatives led by European zoos in Madagascar exploring the European Association of Zoos and Aquaria's Conservation Database projects in terms of performed actions and achieved goals as well as communication to the public. We found that zoo-led conservation initiatives may have a positive impact on the ground and tend to achieve most conservation goals related to wild lemurs. However, we suggest that such conservation programmes should underline that they target further lemur species beyond the flagship charismatic species and that enhanced communication efforts would be beneficial to further raise public awareness.

Eight things you should never do in a monitoring program: an Australian perspective.

Monitoring is critical to gauge the effect of environmental management interventions as well as to measure the effects of human disturbances such as climate change. Recognition of the critical need for monitoring means that, at irregular intervals, recommendations are made for new government-instigated programs or to revamp existing ones. Using insights from past well-intentioned (but sadly also often failed) attempts to establish and maintain government-instigated monitoring programs in Australia, we outline eight things that should never be done in environmental monitoring programs (if they aim to be useful). These are the following: (1) Never commence a new environmental management initiative without also committing to a monitoring program. (2) Never start a monitoring program without clear questions. (3) Never implement a monitoring program without first doing a proper experimental design. (4) Never ignore the importance of matching the purpose and objectives of a monitoring program to the design of that program. (5) Never change the way you monitor something without ensuring new methods can be calibrated with the old ones. (6) Never try to monitor everything. (7) Never collect data without planning to curate and report on it. (8) If possible, avoid starting a monitoring program without the necessary resources secured. To balance our "nevers", we provide a checklist of actions that will increase the chances a monitoring program will actually measure the effectiveness of environmental management. Scientists and resource management practitioners need to be part of a stronger narrative for, and key participants in, well-designed, implemented, and maintained government-led monitoring programs. We argue that monitoring programs should be mandated in threatened species conservation programs and all new environmental management initiatives.

Safety Assessment of 1-Hydroxyethyl 4,5-Diamino Pyrazole Sulfate as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of 1-Hydroxyethyl 4,5-Diamino Pyrazole Sulfate, which functions as an oxidative hair dye ingredient. The Panel reviewed relevant data provided in this safety assessment, and concluded that 1-Hydroxyethyl 4,5-Diamino Pyrazole Sulfate is safe in the present practices of use and concentration in oxidative hair dye formulations.

Exploring the Definition of "Similar Toxicities": Case Studies Illustrating Industry and Regulatory Interpretation of ICH S6(R1) for Long-Term Toxicity Studies in One or Two Species.

ICH S6 (R1) states that safety evaluation of biotherapeutics should normally include 2 relevant species when available (i.e., a rodent and non-rodent species in which the test material is pharmacologically active), at least for short-term toxicology studies (generally supporting Phase I trials). For subsequent long-term toxicology studies (e.g., chronic studies up to 6 months dosing duration), there are options to reduce to only one species when justified, including when the mechanism of action of the biologic is well-understood or the toxicity findings in the short-term studies are "similar" in both the rodent and non-rodent species. Across the industry, around 25 to 33% of biologics assess multiple species within short-term toxicity studies but it is often unclear how different companies and regulators are applying the ICH S6 (R1) principles of "similar toxicity profiles" to progress with either 1 or 2 species in the long-term studies, in particular whether the absence of toxicities is considered within this definition. Sponsors may potentially continue to use 2 species to avoid regulatory risk and potential delays in development timelines, representing missed opportunities for reducing animal use, particularly of non-human primates, during drug development.This article summarizes presentations from a symposium at the 41st Annual meeting of the American College of Toxicology (ACT) in November 2020, in which industry case studies and regulatory perspectives addressed considerations and decisions for using 1 or 2 species for long-term toxicity studies, highlighting any common themes or experience that could be applicable for use in future decision-making.

Potential Effects of Sucralose and Saccharin on Gut Microbiota: A Review.

Artificial sweeteners are additives widely used in our diet. Although there is no consensus, current evidence indicates that sucralose and saccharin could influence the gut microbiota. The aim of this study was to analyze the existing scientific evidence on the effects of saccharin and sucralose consumption on gut microbiota in humans. Different databases were used with the following search terms: sweeteners, non-caloric-sweeteners, sucralose, splenda, saccharin, sugartwin, sweet'n low, microbiota, gut microbiota, humans, animal model, mice, rats, and/or in vitro studies. In vitro and animal model studies indicate a dose-dependent relationship between the intake of both sweeteners and gut microbiota affecting both diversity and composition. In humans, long-term study suggests the existence of a positive correlation between sweetener consumption and some bacterial groups; however, most short-term interventions with saccharin and sucralose, in amounts below the ADI, found no significant effect on those groups, but there seems to be a different basal microbiota-dependent response of metabolic markers. Although studies in vitro and in animal models seem to relate saccharin and sucralose consumption to changes in the gut microbiota, more long-term studies are needed in humans considering the basal microbiota of participants and their dietary and lifestyle habits in all population groups. Toxicological and basal gut microbiota effects must be included as relevant factors to evaluate food safety and nutritional consequences of non-calorie sweeteners. In humans, doses, duration of interventions, and number of subjects included in the studies are key factors to interpret the results.

Long-term monitoring reveals widespread and severe declines of understory birds in a protected Neotropical forest.

Long-term studies on the population dynamics of tropical resident birds are few, and it remains poorly understood how their populations have fared in recent decades. Here, we analyzed a 44-y population study of a Neotropical understory bird assemblage from a protected forest reserve in central Panama to determine if and how populations have changed from 1977 to 2020. Using the number of birds captured in mist nets as an index of local abundance, we estimated trends over time for a diverse suite of 57 resident species that comprised a broad range of ecological and behavioral traits. Estimated abundances of 40 (∼70%) species declined over the sampling period, whereas only 2 increased. Furthermore, declines were severe: 35 of the 40 declining species exhibited large proportional losses in estimated abundance, amounting to ≥50% of their initial estimated abundances. Declines were largely independent of ecology (i.e., body mass, foraging guild, or initial abundance) or phylogenetic affiliation. These widespread, severe declines are particularly alarming, given that they occurred in a relatively large (∼22,000-ha) forested area in the absence of local fragmentation or recent land-use change. Our findings provide robust evidence of tropical bird declines in intact forests and bolster a large body of literature from temperate regions suggesting that bird populations may be declining at a global scale. Identifying the ecological mechanisms underlying these declines should be an urgent conservation priority.

Cover and density of southwestern ponderosa pine understory plants in permanent chart quadrats (2002-2020).

This data set consists of 101 permanent 1 m × 1 m (1-m2 ) quadrats located within southwestern ponderosa pine ecosystems near Flagstaff, Arizona, USA. Individual plants in these quadrats were identified and mapped annually for 19 years (2002-2020). The original chart quadrats were established between 1912 and 1927 to determine the effects of domestic livestock grazing on herbaceous plants and pine seedlings. Today these data provide opportunities to examine the effects of climate and land-use change on plant demography, population dynamics, and community processes. We provide the following data and data formats: (1) digitized maps of all plant locations in shapefile and geodatabase format, (2) shapefiles showing annual locations of each individual plant species, (3) annual maps of each quadrat in TIFF and PDF format, (4) annual basal area of each species per quadrat for species mapped as polygons, (5) tabular representation of polygon areas and centroid locations for plant species mapped as polygons, (6) tabular representation of point locations for plant species mapped as points, (7) plot-scale 20 m × 20 m overstory tree canopy cover, tree basal area, parent material, and elevation, (8) quadrat-scale information (e.g., quadrat site and number, coordinates in UTM Zone 12 and latitude/longitude, and descriptive comments for each quadrat), (9) plant species list, (10) summary of plant species observed in each quadrat, (11) summary of quadrats mapped by site and year, and (12) data formatted for use in Integral Projection Models (IPM) and plant population analyses. There are no copyright restrictions; please cite this paper and the associated data set when data are used in publications.

Increasing nutrient inputs over the last 500 years in an Italian low-impacted seagrass meadow.

Posidonia oceanica is a seagrass endemic to the Mediterranean and it has been widely used as a bioindicator. We studied the layers of a 500-year-old matte using a multiproxy approach (δ13C, δ15N, 14C and C and N concentrations in seagrass debris) in order to evaluate the potential of P. oceanica as a long-term environmental indicator of N pollution and CO2 emissions. From 1581 to 1800, accumulation rate was ca. 0.35 cm year-1, while in the last 100 years it has amounted to ca. 0.51 cm year-1. We observed increasing δ15N values with height in the vertical matte profile, indicating an increase in anthropogenic organic N inputs over time. In contrast, no clear trend in the δ13C values was observed. This study reconstructs the long-term impact of human activities on a seagrass meadow located off the Italian coast, yielding long-term background information that can help managers to implement efficient plans.

Diet of Eastern Imperial Eagle (Aquilaheliaca) in Bulgaria: composition, distribution and variation.

The Eastern Imperial Eagle (EIE) is a top predator exploiting different prey in different parts of its distribution. In this study, we summarise data collected over a long period of time (for 25 consecutive years), identifying key prey species in the different regions, as well as clarifying seasonal preferences in the eagle's diet. Most studies on the EIE food composition covering different parts of the species distribution range analyse the breeding season, while data about the winter diet are scarce. To the best of our knowledge, this is the first study detailing the differences in EIE's dietary preferences between the breeding and the winter periods. We identified 4891 specimens belonging to 196 different taxa, which represents the most comprehensive study considering the diet diversity of this threatened species. Mammals represented the largest proportion of the diet, followed by birds and reptiles. Northern White-breasted Hedgehog was the most common prey, accounting for 25.7% of the total prey caught and 26.75% of the biomass. The European Souslik was the second most important prey with 14.35% participation in the eagle's diet, but with a 3.75% contribution to the biomass. As we predicted, prey composition and main prey species varied spatially and seasonally. Modelling differences in the EIE diet, we found that the "territory effect" had the strongest impact on the dietary variations. Diet diversity differed significantly between regions (F = 12.6, df = 4, p = 0.01). During the breeding season, eagles fed mainly on Hedgehogs (29.88%), Sousliks (16.85%) and Storks (7.74%), while the winter diet was predominantly small rodents (44.17%) and songbirds (13.96%). We found that top predators, such as EIE, have successfully adapted to a novel food source, which is abundant in the area. The detected flexibility in the diet of the species and its ability to switch to alternative prey, if available, when the primary prey decreased, should be considered when planning species conservation efforts. Investigating the temporal change of the main prey in the eagle's diet is also crucial for further species conservation measures.

Inviting ecologists to delve deeper into traditional ecological knowledge.

Ecologists and conservationists increasingly acknowledge that traditional ecological knowledge (TEK) is vital for a better understanding and conservation of biodiversity; for example, for a more complex socioecological understanding of long-term processes, ecosystem resilience, the impacts of traditional management practices, and the worldviews underpinning these practices. To gain a deeper understanding of the ecological dimensions of TEK, ecologists and conservation biologists should conduct participatory long-term collaborative research on TEK. To conduct TEK research properly, however, ecologists need to familiarize themselves more deeply with the methodologies of social sciences, further develop their links with social scientists, and adopt new approaches, such as strengthening respect towards other knowledge systems and being inclusive in research and open to new types of validation.

Connecting the data landscape of long-term ecological studies: The SPI-Birds data hub.

The integration and synthesis of the data in different areas of science is drastically slowed and hindered by a lack of standards and networking programmes. Long-term studies of individually marked animals are not an exception. These studies are especially important as instrumental for understanding evolutionary and ecological processes in the wild. Furthermore, their number and global distribution provides a unique opportunity to assess the generality of patterns and to address broad-scale global issues (e.g. climate change). To solve data integration issues and enable a new scale of ecological and evolutionary research based on long-term studies of birds, we have created the SPI-Birds Network and Database (www.spibirds.org)-a large-scale initiative that connects data from, and researchers working on, studies of wild populations of individually recognizable (usually ringed) birds. Within year and a half since the establishment, SPI-Birds has recruited over 120 members, and currently hosts data on almost 1.5 million individual birds collected in 80 populations over 2,000 cumulative years, and counting. SPI-Birds acts as a data hub and a catalogue of studied populations. It prevents data loss, secures easy data finding, use and integration and thus facilitates collaboration and synthesis. We provide community-derived data and meta-data standards and improve data integrity guided by the principles of Findable, Accessible, Interoperable and Reusable (FAIR), and aligned with the existing metadata languages (e.g. ecological meta-data language). The encouraging community involvement stems from SPI-Bird's decentralized approach: research groups retain full control over data use and their way of data management, while SPI-Birds creates tailored pipelines to convert each unique data format into a standard format. We outline the lessons learned, so that other communities (e.g. those working on other taxa) can adapt our successful model. Creating community-specific hubs (such as ours, COMADRE for animal demography, etc.) will aid much-needed large-scale ecological data integration.

Following the infection dynamics of the tropical trematode Oligogonotylus mayae in its intermediate and definitive hosts for 13 years.

We present a time series of 13 years (2003-2016) of continuous monthly data on the prevalence and mean abundance of the trematode Oligogonotylus mayae for all the hosts involved in its life cycle. We aimed to determine whether annual (or longer than annual) environmental fluctuations affect these infection parameters of O. mayae in its intermediate snail host Pyrgophorus coronatus, and its second and definitive fish host Mayaheros urophthalmus from the Celestun tropical coastal lagoon, Yucatan, Mexico. Fourier time series analysis was used to identify infection peaks over time, and cross-correlation among environmental forcings and infection parameters. Our results suggest that the transmission of O. mayae in all its hosts was influenced by the annual patterns of temperature, salinity and rainfall. However, there was a biannual accumulation of metacercarial stages of O. mayae in M. urophthalmus, apparently associated with the temporal range of the El Niño-Southern Oscillation (five years) and the recovery of the trematode population after a devasting hurricane. Taking O. mayae as an example of what could be happening to other trematodes, it is becoming clear that environmental forcings acting at long-term temporal scales affect the population dynamics of these parasites.

Rethinking toxicity testing: Influence of aging on the outcome of long-term toxicity testing and possible remediation.

Traditionally, toxicity testing is conducted at fixed dose rates (i.e., mg/kg/day) without considering life-changing events, e.g., stress, sickness, aging- and/or pregnancy-related changes in physical, physiological and biochemical parameters. In humans, life-changing events may cause systemic dose non-proportionality requiring modulation of drug dosage; similar changes occur in animals altering systemic dose during chronic/carcinogenic testing leading to "late-occurring" effects in some studies. For example, propylene monomethyl ether, an industrial chemical, initially induced sedation in rats and mice with recovery upon induction of hepatic CYPs after ~1 week. Sedation reappeared in rats but not in mice after ~12 months of exposure due to decreased CYP activity in rats, elderly mice were able to maintain slightly higher CYP activity avoiding recurrence of sedation. The systemic dose of two pharmaceuticals (doxazosin and brimonidine tartrate) increased up to 6-fold in ≥12-month old rats with no toxicity. In a rat reproductive toxicity study, systemic dose of 2,4-D, an herbicide, rapidly increased due to increased consumption of 2,4-D-fortified diet during pregnancy, lactation and neonatal growth, requiring adjustment to maintain the targeted systemic dose. Ideally, toxicological studies should be based on systemic dose with the option of modulating external dose rates to maintain the targeted systemic dose. Systemic dose can easily be monitored in selected core study animals at desired intervals considering recent developments in sampling and analysis at a fraction of the overall cost of a study.

Modifying connectivity to promote state change reversal: the importance of geomorphic context and plant-soil feedbacks.

Alternative states maintained by feedbacks are notoriously difficult, if not impossible, to reverse. Although positive interactions that modify soil conditions may have the greatest potential to alter self-reinforcing feedbacks, the conditions leading to these state change reversals have not been resolved. In a 9-yr study, we modified horizontal connectivity of resources by wind or water on different geomorphic surfaces in an attempt to alter plant-soil feedbacks and shift woody-plant-dominated states back toward perennial grass dominance. Modifying connectivity resulted in an increase in litter cover regardless of the vector of transport (wind, water) followed by an increase in perennial grass cover 2 yr later. Modifying connectivity was most effective on sandy soils where wind is the dominant vector, and least effective on gravelly soils on stable surfaces with low sediment movement by water. We found that grass cover was related to precipitation in the first 5 yr of our study, and plant-soil feedbacks developed following 6 yr of modified connectivity to overwhelm effects of precipitation on sandy, wind-blown soils. These feedbacks persisted through time under variable annual rainfall. On alluvial soils, either plant-soil feedbacks developed after 7 yr that were not persistent (active soils) or did not develop (stable soils). This novel approach has application to drylands globally where desertified lands have suffered losses in ecosystem services, and to other ecosystems where connectivity-mediated feedbacks modified at fine scales can be expected to impact plant recovery and state change reversals at larger scales, in particular for wind-impacted sites.

Natural population variability may be masking the more-individuals hypothesis.

Species richness and productivity are correlated at global and regional scales, but the mechanisms linking them are inconclusive. The most commonly invoked mechanism, the more-individuals hypothesis (MIH), hypothesizes that increased productivity leads to increased food resource availability, which leads to an increased number of individuals supporting more species. Empirical evidence for the MIH remains mixed despite a substantial literature. Here we used simulations to determine whether interannual population variability could be masking a "true" MIH relationship. In each simulation, fixed linear relationships between productivity, richness, and 50-yr average abundance mimicked the MIH mechanism. Abundance was allowed to vary annually and sampled for 1-40 yr. Linear regressions of richness on sampled abundance assessed the probability of detecting the fixed MIH relationship. Medium to high population variability with short-term sampling (1-3 yr) led to poor detection of the fixed MIH relationship. Notably, this level of sampling and population variability describes nearly all MIH studies to date. Long-term sampling (5+ yr) led to improved detection of the fixed relationship; thus it is necessary to detect support for the MIH reliably. Such sampling duration is nonexistent in the MIH literature. Robust future studies of the MIH necessitate consideration of interannual population variability.

Untargeted and Targeted Fingerprinting of Extra Virgin Olive Oil Volatiles by Comprehensive Two-Dimensional Gas Chromatography with Mass Spectrometry: Challenges in Long-Term Studies.

Comprehensive two-dimensional gas chromatography coupled with mass spectrometric detection (GC × GC-MS) offers an information-rich basis for effective chemical fingerprinting of food. However, GC × GC-MS yields 2D-peak patterns (i.e., sample 2D fingerprints) whose consistency may be affected by variables related to either the analytical platform or to the experimental parameters adopted for the analysis. This study focuses on the complex volatile fraction of extra-virgin olive oil and addresses 2D-peak patterns variations, including MS signal fluctuations, as they may occur in long-term studies where pedo-climatic, harvest year, or shelf life changes are studied. The 2D-pattern misalignments are forced by changing chromatographic settings and MS acquisition. All procedural steps, preceding pattern recognition by template matching, are analyzed and a rational workflow defined to accurately realign patterns and analytes metadata. Signal-to-noise ratio (SNR) detection threshold, reference spectra extraction, and similarity match factor threshold are critical to avoid false-negative matches. Distance thresholds and polynomial transform parameters are key for effective template matching. In targeted analysis (supervised workflow) with optimized parameters, method accuracy reaches 92.5% (i.e., % of true-positive matches) while for combined untargeted and targeted ( UT) fingerprinting (unsupervised workflow), accuracy reaches 97.9%. Response normalization also is examined, evidencing good performance of multiple internal standard normalization that effectively compensates for discriminations occurring during injection of highly volatile compounds. The resulting workflow is simple, effective, and time efficient.