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Multivariate statistical process monitoring (MSPM) methods provide sensitive indicators of process conditions by harnessing the value of massive process data. Large-scale industrial processes are subject to wide-range time-varying operating conditions such that some variables inevitably exhibit nonstationary behavior, which poses significant challenges for the design of MSPM schemes. In this brief, a novel nonstationary probabilistic slow feature analysis algorithm is developed to comprehensively describe both nonstationary and stationary variations that underlie process measurements during routine operations. For efficient parameter estimation, the expectation–maximization algorithm is employed. By modeling nonstationarity and stationarity as the random walk and stable autoregressive processes, interpretable monitoring statistics are constructed to detect abnormality in nonstationary dynamics, stationary dynamics, and stationary steady conditions. This forms a holistic and pragmatic monitoring framework for industrial processes, which is beneficial for reducing false alarms and providing meaningful operational information for industrial practitioners. The efficacy of the proposed monitoring framework is validated via two case studies.

Asphaltenes comprise the heaviest and least understood fraction of crude petroleum. The asphaltenes are a diverse and complex mixture of organic and organometallic molecules in which most of the molecular constituents are tightly aggregated into more complicated suprastructures. The bulk properties of asphaltenes arise from a broad range of polycyclic aromatics, heteroatoms, and polar functional groups. Despite much analytical effort, the precise molecular architectures of the material remain unresolved. To understand asphaltene characteristics and reactivity, the field has turned to synthetic model compounds that mirror asphaltene structure, aggregation behavior, and thermal chemistry, including the nucleation of coke. Historically, molecular asphaltene modeling was limited to commercial compounds, offering little illumination and few opportunities for hypothesis-driven research. More recently, however, rational molecular design and modern organic synthesis have started to impact this area. This review provides an overview of commercially available model compounds but is principally focused on the design and synthesis of structurally advanced and appropriately functionalized compounds to mimic the physical and chemical behavior of asphaltenes. Efforts to model asphaltene aggregation are briefly discussed, and a prognosis for the field is offered. A referenced tabulation of the synthetic compounds reported to date is provided.

Copper (Cu) appears to be consistently more toxic to anuran species relative to other vertebrate taxa. There are limited Cu toxicity data for salamanders; of the few studies conducted on salamanders, most examined Cu effects on the embryonic, but not the larval, stage. We performed acute toxicity experiments, to quantify LC50s, on Harrison stage 46 larvae (free swimming hatchlings with egg yolk completely absorbed) of three ambystomatid salamander species. Each LC50 experiment used exposure concentrations of 10, 20, 30, 40, 50, and 60 µg/L with 10 replicates per concentration each containing one larva. We found very high toxicity for all species compared to previously published research on the embryo stage. Specifically, the 4-d LC50s for Ambystoma tigrinum and A. opacum were 35.3 and 18.73 µg/L, respectively. The same Cu concentrations caused similar toxicity to A. talpoideum (LC50 = 47.88 µg/L), but exposures required up to 48 d to elicit the same level of mortality. A time-to-event analysis indicated that time to mortality was significantly affected by Cu concentration. Additionally, for A. talpoideum, we observed that elevated levels of Cu decreased growth rate. Comparisons with previously reported Cu toxicity for embryos suggest that, as with fish, Cu may be more toxic to larval salamander stages than for embryos. Further, our data suggest that Cu is an important environmental contaminant that deserves increased scrutiny on the potential for population-level effects where contamination has occurred in wetlands and streams inhabited by salamanders.

Ecological stoichiometry uses information on a few key biological elements (C, N, and P) to explain complex ecological patterns. Although factors driving variation in these elements are well-established, expanding stoichiometric principles to explore dynamics of the many other essential elements comprising biological tissues (i.e., the ionome) is needed to determine their metabolic relationships and better understand biological control of elemental flows through ecosystems. 2. In this paper, we report observations of ionomic variation in two species of salamander (Ambystoma opacum and A. talpoideum) across ontogenic stages using specimens from biological collections of two wetlands sampled over a 30-year period. This unique data set allowed us to explore the extent of ionomic variation between species, among ontogenic stages, between sites, and through time. 3. We found species- and to a lesser extent site-specific differences in C, N, and P along with 13 other elements forming salamander ionomes but saw no evidence of temporal changes. Salamander ionomic composition was most strongly related to ontogeny with relatively higher concentrations of many elements in adult males (i.e., Ca, P, S, Mg, Zn, and Cu) compared to metamorphic juveniles, which had greater amounts of C, Fe, and Mn. 4. In addition to patterns of individual elements, covariance among elements was used to construct multi-elemental nutrient balances, which revealed differences in salamander elemental composition between species and sites and changes in elemental proportions across ontogenic development. These multi-elemental balances distinguished among species-site-ontogenic stage groups better than using only C, N, and P. 5. Overall, this study highlights the responsiveness of consumer ionomes to life-history and environmental variation while reflecting underlying relationships among elements tied to biological function. As such, ionomic studies can provide important insights into factors shaping consumer elemental composition and for predicting how these changes might affect higher-order ecological processes.

International audience; In the Fall of 2016 a workshop was held which brought together over 50 scientists from the ecological and radio- logical fields to discuss feasibility and challenges of reintegrating ecosystem science into radioecology. There is a growing desire to incorporate attributes of ecosystem science into radiological risk assessment and radioecological research more generally, fueled by recent advances in quantification of emergent ecosystem at- tributes and the desire to accurately reflect impacts of radiological stressors upon ecosystem function. This paper is a synthesis of the discussions and consensus of the workshop participant's responses to three primary questions, which were: 1) How can ecosystem science support radiological risk assessment? 2) What ecosystem level endpoints potentially could be used for radiological risk assessment? and 3) What inference strategies and associated methods would be most appropriate to assess the effects of radionuclides on ecosystem structure and function? The consensus of the participants was that ecosystem science can and should support radiological risk assessment through the incorporation of quantitative metrics that reflect ecosystem functions which are sensi- tive to radiological contaminants. The participants also agreed that many such endpoints exit or are thought to exit and while many are used in ecological risk assessment currently, additional data need to be collected that link the causal mechanisms of radiological exposure to these endpoints. Finally, the participants agreed that ra- diological risk assessments must be designed and informed by rigorous statistical frameworks capable of reveal- ing the causal inference tying radiological exposure to the endpoints selected for measurement.

γ-Lactam derivatives with multiple contiguous stereogenic carbon centers are ubiquitous in physiologically active compounds. The development of straightforward and reliable synthetic routes to such chiral structural motifs in a stereocontrolled manner should thus be of importance. Herein, we report a strategy to construct polycyclic γ-lactam derivatives that contain more than two contiguous stereogenic centers in an enantioselective as well as atom-economic manner. Moreover, we have achieved the first enantioselective synthesis of strigolactam derivative GR-24, a racemic variant of which is a potential seed germination stimulator and plant-growth regulator. A key of the procedure presented here is a nickel(0)/chiral phosphoramidite-catalyzed asymmetric [2+2+1] carbonylative cycloaddition between readily accessible ene-imines and carbon monoxide, which proceeded enantioselectively to furnish up to 90% ee (99% ee after recrystallization). The results of mechanistic studies, including the isolation of a chiral heteronickelacycle, support that the enantioselectivity on the two contiguous carbon atoms of the γ-lactams is determined during the oxidative cyclization on nickel(0).

Despite widespread recognition of the need to transition toward more sustainable production and consumption and numerous initiatives to that end, global resource extraction and corresponding socio-ecological degradation continue to grow. Understanding the causes of this persistent failure is a necessary step towards more effective action. This article contributes to that understanding by synthesizing theory and evidence that links unsustainable production-consumption systems to power and inequality. While sustainable consumption and production research and action mostly focuses on technological or behavioral change, the socio-ecological inequalities driving production-consumption systems built into the organization of our global political economy, remain largely overlooked. In response, we propose a structural political economy orientation that seeks explicitly to reduce these inequalities and advance environmental justice and, thus, create the conditions for sustainable production-consumption systems. We then propose three important arenas of research and action towards sustainable production-consumption systems: justice, governance, and co-production of knowledge and action. These arenas, collectively and individually, can serve as entry points to study and act on the dynamics of (un)sustainable production-consumption systems. This can be done at the micro level, with respect to specific commodity chains or systems of provisioning, or at meso and macro levels with respect to national and global production networks. Our proposed orientation helps distinguish research and practice proposals into those emphasizing management and compensation resulting often in persistence of unsustainability, from those proffering structural changes in unsustainable production-consumption systems. We invite critique and collaboration to develop this research and action agenda further.

Chemical contamination is often suggested as an important contributing factor to amphibian population declines, but direct links are rarely reported. Population modeling provides a quantitative method to integrate toxicity data with demographic data to understand the long-term effects of contaminants on population persistence. In this study we use laboratory-derived embryo and larval toxicity data for two anuran species to investigate the potential for toxicity to contribute to population declines. We use the southern toad (Anaxyrus terrestris) and the southern leopard frog (Lithobates sphenocephalus) as model species to investigate copper (Cu) toxicity. We use matrix models to project populations through time and quantify extinction risk (the probability of quasi-extinction in 35 yr). Life-history parameters for toads and frogs were obtained from previously published literature or unpublished data from a long-term (>35 yr) data set. In addition to Cu toxicity, we investigate the role of climate change on amphibian populations by including the probability of early pond drying that results in catastrophic reproductive failure (CRF, i.e., complete mortality of all larval individuals). Our models indicate that CRF is an important parameter for both species as both were unable to persist when CRF probability was >50% for toads or 40% for frogs. Copper toxicity alone did not result in significant effects on extinction risk unless toxicity was very high (>50% reduction in survival parameters). For toads, Cu toxicity and high probability of CRF both resulted in high extinction risk but no synergistic (or greater than additive) effects between the two stressors occurred. For leopard frogs, in the absence of CRF survival was high even under Cu toxicity, but with CRF Cu toxicity increased extinction risk. Our analyses highlight the importance of considering multiple stressors as well as species differences in response to those stressors. Our models were consistently most sensitive to juvenile and adult survival, further suggesting the importance of terrestrial stages to population persistence. Future models will incorporate multiple wetlands with different combinations of stressors to understand if our results for a single wetland result in a population sink within the landscape.

Little is known about the propensity of crocodilians to bioaccumulate trace elements as a result of chronic dietary exposure. We exposed 36 juvenile alligators (Alligator mississippiensis) to one of four dietary treatments that varied in the relative frequency of meals containing prey from coal combustion waste (CCW)-contaminated habitats vs. prey from uncontaminated sites, and evaluated tissue residues and growth rates after 12 mo and 25 mo of exposure. Hepatic and renal concentrations of arsenic (As), cadmium (Cd) and selenium (Se) varied significantly among dietary treatment groups in a dose-dependent manner and were higher in kidneys than in livers. Exposure period did not affect Se or As levels but Cd levels were significantly higher after 25 mo than 12 mo of exposure. Kidney As and Se levels were negatively correlated with body size but neither growth rates nor body condition varied significantly among dietary treatment groups. Our study is among the first to experimentally examine bioaccumulation of trace element contaminants in crocodilians as a result of chronic dietary exposure. A combination of field surveys and laboratory experiments will be required to understand the effects of different exposure scenarios on tissue residues, and ultimately link these concentrations with effects on individual health.

Species with complex life cycles are susceptible to environmental stressors across life stages, but the carryover and latent effects between stages remain understudied. For species with biphasic life histories, such as pond-breeding amphibians, delayed effects of aquatic conditions can influence terrestrial juveniles and adults directly or indirectly, usually mediated through fitness correlates such as body size. We collected adult southern toads (Anaxyrus terrestris) from 2 source populations-a natural reference wetland and a metal-contaminated industrial wetland-and exposed their offspring to 2 aquatic stressors (a metal contaminant, copper [Cu], and a dragonfly predator cue) in outdoor mesocosms (n = 24). We then reared metamorphs in terraria for 5 mo to examine delayed effects of early life stage environmental conditions on juvenile performance, growth, and survival. Larval exposure to Cu, as well as having parents from a contaminated wetland, resulted in smaller size at metamorphosis-a response later negated by compensatory growth. Although Cu exposure and parental source did not affect larval survival, we observed latent effects of these stressors on juvenile survival, with elevated Cu conditions and metal-contaminated parents reducing postmetamorphic survival. Parental source and larval Cu exposure affected performance at metamorphosis through carryover effects on body size but, 1 mo later, latent effects of parental source and larval predator exposure directly (i.e., not via body size) influenced performance. The carryover and latent effects of parental source population and aquatic Cu level on postmetamorphic survival and juvenile performance highlight the importance of conducting studies across life stages and generations. Environ Toxicol Chem 2018;37:2660-2669. © 2018 SETAC.

Quantifying the relationship between demographic patterns and genetic diversity are important in predicting temporal population genetic changes. To make predictions there must be an understanding of the relationship between census size, the effective number of breeders (Nb) and effective population size (Ne). We evaluate the temporal genetic variation between three populations of Ambystoma salamanders, and compare genetic and demographic estimates of Nb and Ne. We sampled two wetlands, RB and GB for A. opacum and sampled A. talpoideum at RB, over a 20-year period. Ambystoma opacum colonized the RB wetland in 1980 and the population has steadily expanded as A. talpoideum has declined towards local extinction; the GB population of A. opacum has remained relatively large and stable over this same time period. Genetic variation at 10 microsatellites remained stable at each population over the sampled time frame, and did not reflect changes in population size. Genetic estimates of Nb also did not reflect demographic trends, and were lower than demographic estimates of Nb. Genetic methods of determining Ne gave similar estimates to demographic methods. Our findings indicate that sample sizes and number of markers typically used in genetic studies do not provide enough precision to monitor population size changes in amphibians, which likely violate many of the assumptions of Nb and Ne estimation models. These findings should be considered when using Ne in conservation and management assessments of amphibian populations.

Many metals are acutely toxic to aquatic organisms at high concentrations and for some metals, such as copper (Cu), even low-level chronic contamination may be cause for conservation concern. Amphibian susceptibility to Cu has been examined in only a few species, and susceptibility is highly variable. The lethal and sublethal effects were examined of chronic aqueous Cu exposure on embryonic and larval eastern narrowmouth toads, Gastrophryne carolinensis. Copper levels as low as 10 μg Cu/L reduced embryonic and larval survival. Embryonic survivorship varied within- and between-source populations, with embryos derived from uncontaminated-wetland parents having greater survival at lower Cu levels than embryos from parents from a metal-contaminated constructed wetland. At 30 μg/L, embryos from the contaminated site had greater survival. Overall survival from oviposition to metamorphosis was 68.9% at 0 μg/L and 5.4% at 10 μg/L. Similarly, embryos exposed to ≥50 μg/L demonstrated developmental delays in transition from embryo to free-swimming larva. These results demonstrate a negative population-specific response to environmentally relevant levels of Cu. Environ Toxicol Chem 2015;34:575–582. © 2014 SETAC

In this paper, we report a highly efficient, scalable approach to the total synthesis of conformationally unrestricted, electronically isolated arrays of alkyl-tethered polycyclic aromatic chromophores. This new class of modular molecules consists of polycyclic aromatic "islands" comprising significant structural fragments present in unrefined heavy petroleum, tethered together by short saturated alkyl chains, as represented in the "archipelago model" of asphaltene structure. The most highly branched archipelago compounds reported here share an architecture with first-generation dendrimeric constructs, making the convergent, chromatography-free synthesis described herein particularly attractive for further extensions in scope and applications to materials chemistry. The syntheses are efficient, selective, and readily adaptable to a multigram scale, requiring only inexpensive, "earth-abundant" transition-metal catalysts for cross-coupling reactions and extraction and fractional crystallization for purification. This approach avoids typical limitations in cost, scale, and operational practicality. All of the archipelago compounds and synthetic intermediates have been fully characterized spectroscopically and analytically. The solid-state structure of one archipelago model compound has been determined by X-ray crystallography.

Stable isotope enrichment techniques can aid in understanding dispersal of animals. Pond-breeding amphibians often have spatially disjunct populations that depend on immigration for persistence, yet obtaining direct estimates of dispersal rates among wetlands is challenging. We enriched aquatic mesocosms with 15N to “mark” amphibian larvae and determine the feasibility of using enrichment techniques to study dispersal in pond-breeding amphibians. Because newly metamorphosed amphibians in mark-release-recapture studies may not be recaptured until adulthood, we estimated persistence of the 15N enrichment signature up to 3 years post-metamorphosis. We reared larval marbled salamanders (Ambystoma opacum) in artificial mesocosms dosed with 15NH4Cl, and maintained metamorphs on unlabeled prey for 7 months to estimate the biological half life (BHL) of 15N in tissue. Metamorphs in spiked treatments attained δ15N levels >1000 times higher than reference animals (5 ± 1‰), and levels remained ~225 times higher than controls after 7 months. The average 15N BHL was 2.49 ± 0.24 months, indicating that the elevated signature should be discernible for a minimum of 20–28 months after metamorphosis. Our results suggest that 15N enrichment is feasible for field studies of amphibian dispersal, as metamorphs will retain isotope-enriched tissues that persist until at least the second year of breeding.

The creation of wetlands, such as urban and industrial ponds, has increased in recent decades, and these wetlands often become enriched in pollutants over time. One metal contaminant trapped in created wetlands is copper (Cu2+). Copper concentrations in sediments and overlying water may affect amphibian species that breed in created wetlands. The authors analyzed the Cu concentration in dried sediments from a contaminated wetland and the levels of aqueous Cu released after flooding the sediments with different volumes of water, mimicking low, medium, and high pond-filling events. Eggs and larvae of Ambystoma opacum Gravenhorst, a salamander that lays eggs on the sediments in dry pond beds that hatch on pond-filling, were exposed to a range of Cu concentrations that bracketed potential aqueous Cu levels in created wetlands. Embryo survival varied among clutches, but increased Cu levels did not affect embryo survival. At Cu concentrations of 500 µg/L or greater, however, embryos hatched earlier, and the aquatic larvae died shortly after hatching. Because Cu concentrations in sediments increase over time in created wetlands, even relatively tolerant species such as A. opacum may be affected by Cu levels in the posthatching environment. Environ Toxicol Chem 2014;33:1631–1637. © 2014 SETAC

Terrestrial habitats surrounding isolated wetlands are a critical resource for many pond-breeding amphibian species, yet few studies have examined the terrestrial distribution of post-metamorphic juveniles and adults. We used an encircling drift fence at a breeding pond in conjunction with partial fences at 90, 172, and 332 m from the wetland to estimate the terrestrial distribution of adult marbled salamanders (Ambystoma opacum; four breeding seasons) and mole salamanders (A. talpoideum; two seasons), as well as the dispersion of newly metamorphosed A. opacum (one summer). For newly metamorphosed A. opacum, 79% emigrated ,90 m from the wetland, and 8% moved beyond 172 m; movement distance was unrelated to body size. Distribution of adult A. opacum varied among years, with an average of 28% (range 23-31%) occurring beyond 172 m in all years. Averaged across two years, 51% of adult A. talpoideum occurred beyond 172 m. Lognormal models provided a good fit to both the juvenile and adult ambystomatid distributions, and parameters differed between age classes, sexes, species, and years within species. For adult A. opacum a buffer radius of 300 m or 340 m, depending on the year, is estimated to include 95% of adults; for A. talpoideum the estimate is 464 m or 501 m. A reanalysis of distribution data for seven ambystomatid species shows that a previous estimate of a 164-m radius to protect 95% of a population underestimates the needed buffer radius by 185 m. Because our study wetland requires a nearly 500 m wide radius to protect 95% of its ambystomatid adults, preservation of similar communities may require much more surrounding terrestrial habitat than previously thought.

Environmental contaminants are implicated in the global decline of amphibian populations. Copper (Cu) is a widespread contaminant that can be toxic at concentrations just above the normal physiological range. In the present study we examined the effects of chronic Cu aqueous exposure on embryos and larvae of southern toads, Anaxyrus (Bufo) terrestris. Measurable levels of Cu were found in larvae, with tissue concentrations up to 27.5 μg Cu/g dry mass. Aqueous concentrations of Cu as low as 10 μg/L significantly reduced survival to the free-swimming stage and no larvae reached metamorphosis at concentrations above 15 μg/L. Clutches from populations with prior Cu exposure had the lowest survivorship. Among several populations there was significant variation in survivorship at different levels of Cu. More data are needed to understand the underlying causes of within- and among-population resilience to anthropogenic stressors.

In the southeastern U.S., changes in temperature and precipitation over the last three decades have been the most dramatic in winter and spring seasons. Continuation of these trends could negatively impact pond-breeding amphibians, especially those that rely on winter and spring rains to fill seasonal wetlands, trigger breeding, and ensure reproductive success. From 2009 to 2012, we monitored Spring and Fall presence of aquatic stages (larval and paedomorphic, gilled adult) of a winter-breeding amphibian (the mole salamander, Ambystoma talpoideum) and used multi-season models to estimate occupancy, local colonization and extinction. Seasonal estimates of occupancy, corrected for imperfect detection, declined from 22.3 % of ponds in Spring 2009 to 9.9 % in Fall 2012. Our best supported model suggested that changes in occupancy were driven by increased rates of extinction that corresponded with drought-related drying of ponds. Based on uncertainty in climate change projections for the Southeast, we present a conceptual model of predicted changes in wetland hydroperiods across a landscape with projected decreases and increases in future precipitation. Such precipitation changes could alter wetland hydroperiods, facilitate extinctions of species adapted to short, intermediate or long hydroperiod environments and, ultimately, modify the composition of amphibian communities within freshwater wetland ecosystems.

Bioaccumulation of contaminants and subsequent maternal transfer to offspring are important factors that affect the reproductive success of wildlife. However, maternal transfer of contaminants has rarely been investigated in amphibians. We examined maternal transfer of trace elements in southern toads ( Bufo[Anaxyrus] terrestris) residing in two locations: (1) an active coal combustion waste (CCW) disposal basin and adjacent 40-ha floodplain contaminated with CCW over 35 years ago and (2) an uncontaminated reference site. Our study is among the few to document tissue concentration-dependent maternal transfer of contaminants and associated adverse effects in amphibians. We found that females collected from the CCW-contaminated area had elevated concentrations of Ni, Se, and Sr; these females also transferred elevated levels of Cu, Pb, Se, and Sr to their eggs compared to females from the reference site. Overall reproductive success, estimated as a function of clutch size and offspring viability, was reduced by 27% in clutches collected from parents from the contaminated site compared to the reference site. Offspring viability negatively correlated with female and/or egg concentrations of Se and Ni. Reproductive success negatively correlated with Se and Cu concentrations in females, and Se concentrations in eggs. Our study highlights how exposure to CCW can negatively affect amphibian reproduction.

An all-PDMS on-line microdialysis-microchip electrophoresis with on-chip derivatization and electrophoretic separation for near real-time monitoring of primary amine-containing analytes is described. Each part of the chip was optimized separately, and the effect of each of the components on temporal resolution, lag time, and separation efficiency of the device was determined. Aspartate and glutamate were employed as test analytes. Derivatization was accomplished with naphthalene-2,3,-dicarboxyaldehyde/cyanide (NDA/CN−), and the separation was performed using a 15-cm serpentine channel. The analytes were detected using LIF detection.

Understanding the demography of species over recent history (e.g.

Natural and anthropogenic stressors typically do not occur in isolation; therefore, understanding ecological risk of contaminant exposure should account for potential interactions of multiple stressors. Realistically, common contaminants can also occur chronically in the environment. Because parental exposure to stressors may cause transgenerational effects on offspring, affecting their ability to cope with the same or novel environmental stressors, the exposure histories of generations preceding that being tested should be considered. To examine multiple stressor and parental exposure effects we employed a 2 × 2 × 2 factorial design in outdoor 1000-L mesocosms (n = 24). Larval southern toads (Anaxyrus terrestris), bred from parents collected from reference and metal-contaminated sites, were exposed to two levels of both an anthropogenic (copper—0, 30 µg/L Cu) and natural (predator cue − present/absent) stressor and reared to metamorphosis. Toads from the metal-contaminated parental source population were smaller at metamorphosis and had delayed development; i.e., a prolonged larval period. Similarly, larval Cu exposure also reduced size at metamorphosis and prolonged the larval period. We, additionally, observed a significant interaction between larval Cu and predator-cue exposure on larval period, wherein delayed emergence was only present in the 30-µg/L Cu treatments in the absence of predator cues. The presence of parental effects as well as an interaction between aquatic stressors on commonly measured endpoints highlight the importance of conducting multistressor studies across generations to obtain data that are more relevant to field conditions in order to determine population-level effects of contaminant exposure.

Vitamin D plays a vital role not only in bone health but also in pathophysiology of many other body functions. In recent years, there has been significant increase in testing of 25-hydroxyvitamin D (25-OH vitamin D), a marker of vitamin D deficiency. The most commonly used methods for the measurement of 25-OH vitamin D are immunoassays and liquid chromatography tandem mass spectrometry (LC-MS-MS). Since immunoassays suffer from inaccuracies and interferences, LC-MS-MS is a preferred method. In LC-MS-MS methods, 25-OH vitamin D is extracted from serum or plasma by solid-phase or liquid-phase extraction. Because these extraction methods are time consuming, we developed an easy method that uses simple protein precipitation followed by injection of the supernatant to LC-MS-MS. Several mass-to-charge (m/z) ratio transitions, including commonly used transitions based on water loss, were evaluated and several tube types were tested. The optimal transitions for 25-OH vitamin D2 and D3 were 395.5 > 269.5 and 383.4 > 257.3, respectively. The reportable range of the method was 1–100 ng/mL, and repeatability (within-run) and within-laboratory imprecision were

Exposure to environmental contaminants contributes to the global decline of amphibian populations. The impacts of organic contaminants on amphibians are well documented. However, substantially less is known concerning the potential effects of metals on amphibian populations. Copper (Cu) is an essential element, but it can be toxic at concentrations only slightly higher than the normal physiological range. The present study examines the effects of chronic Cu exposure on embryos and larvae of southern leopard frogs, Lithobates (Rana) sphenocephalus. Groups of eggs from multiple clutches were collected from two wetlands and exposed to a range of Cu concentrations (0-150 mg/L) until they reached the free-swimming stage, and then individual larvae were reared to metamorphosis. Higher Cu concentrations significantly reduced embryo survival to the free-swimming stage but did not further reduce survival to metamorphosis. Larval period was affected by Cu treatment, but the clutch from which larvae originated (i.e., parentage) explained a higher proportion of the variation. Embryo survival to hatching varied significantly among clutches, ranging from 42.9 to 79.2%. Measurable levels of Cu were found in larvae with body burdens up to 595 mg Cu/g dry mass in the 100 mg/L treatment, and larval Cu body burdens were higher than in metamorphs. The present study also demonstrated that higher initial egg density ameliorated embryo mortality at higher Cu levels and should be accounted for in future studies. Environ. Toxicol. Chem. 2012;31:1587-1594. # 2012 SETAC Keywords—Lithobates (Rana) sphenocephalus Copper Amphibian Ecotoxicology Metal toxicity

We conducted a mesocosm study to assess the individual and interactive effects of previous maternal exposure and larval exposure to trace element-laden sediments on southern toads (Bufo terrestris). Previous maternal exposure to coal combustion wastes (CCW) reduced larval survival to metamorphosis up to 57% compared to larvae of unexposed females. Larvae reared on CCW accumulated significant concentrations of trace elements resulting in extended larval periods, reduced growth rates, and reduced mass at metamorphosis. However, the effects were dependent on age of sediments, suggesting the effects of contaminants from CCW may be partially ameliorated over time through the reduced bioavailability of trace elements in aged CCW. Most importantly, maternal exposure to contaminants coupled with larval exposure to fresh CCW interacted to reduce survival to metamorphosis by 85% compared to reference conditions. Our study yields further evidence that disposal of CCW in aquatic basins potentially creates ecological traps for some amphibian populations.

Contaminants often occur as mixtures in the environment, but investigations into toxicity usually employ a single chemical. Metal contaminant mixtures from anthropogenic activities such as mining and coal combustion energy are widespread, yet relatively little research has been performed on effects of these mixtures on amphibians. Considering that amphibians tend to be highly sensitive to copper (Cu) and that metal contaminants often occur as mixtures in the environment, it is important to understand the interactive effects that may result from multiple metals. Interactive effects of Cu and zinc (Zn) on amphibians have been reported as antagonistic and, conversely, synergistic. The goal of our study was to investigate the role of Zn in Cu toxicity to amphibians throughout the embryonic developmental period. We also considered maternal effects and population differences by collecting multiple egg masses from contaminated and reference areas for use in four experiments across three species. We performed acute toxicity experiments with Cu concentrations that cause toxicity (10–200 μg/L) in the absence of other contaminants combined with sublethal concentrations of Zn (100 and 1000 μg/L). Our results suggest very few effects of Zn on Cu toxicity at these concentrations of Zn. As has been previously reported, we found that maternal effects and population history had significant influence on Cu toxicity. The explanation for a lack of interaction between Cu and Zn in this experiment is unknown but may be due to the use of sublethal Zn concentrations when previous experiments have used Zn concentrations associated with acute toxicity. Understanding the inconsistency of amphibian Cu/Zn mixture toxicity studies is an important research direction in order to create generalities that can be used to understand risk of contaminant mixtures in the environment.

A multicomponent cyclocondensation reaction between 2-aminoanthracene, aromatic aldehydes, and 5-α-cholestan-3-one has been used to synthesize model asphaltene compounds. The active catalyst for this reaction has been identified as hydriodic acid, which is formed in situ from the reaction of iodine with water, while iodine is not a catalyst under anhydrous conditions. The products, which contain a tetrahydro[4]helicene moiety, are optically active, and the stereochemical characteristics have been examined by VT-NMR and VT-CD spectroscopies, as well as X-ray crystallography.

Intermediate filaments (IFs) of nestin and vimentin are expressed in immature astrocytes. In this study, we examined the re-expression of these early glial traits in rat reactive astrocytes in affected neuronal perikarya in supraoptic (SON) and paraventricular (PVN) nuclei induced by hypophysectomy. Double-labeling immunofluorescence confocal laser microscopy demonstrated that by 7 days post-lesion, both nestin and vimentin were present intensely in hypertrophied GFAP-IR reactive astrocytes in the area of hypophysectomized magnocellular neurons in SON and PVN, while nestin and vimentin are absent in the normal or sham-operated animals. As the gliotic reaction progressed, the morphology of nestin or vimentin-positive reactive astrocytes in SON but not PVN changed from stellate form at 7 days to thin and elongated shape, morphologically compatible with radial glia during development, at 14 days post-lesion. By 28 days post-lesion, while vimentin-IR persisted in reactive astrocytes in SON and PVN, nestin-IR could hardly be detected. The spatiotemporal pattern of nestin-IR and/or vimentin-IR in reactive astrocytes suggests astrocytes attempt to revert to a more primitive glia form indicated by changes in morphology and phenotype following hypophysectomy, which may contribute to neuronal trophism and plasticity in the lesioned HNS favoring neuronal maintenance and fiber outgrowth.

In organisms that have complex life cycles, factors in the larval environment may affect both larval and adult traits. For amphibians, the postmetamorphic transition from the aquatic environment to terrestrial habitat may be a period of high juvenile mortality. We hypothesized that lipid stores at metamorphosis may affect an animal's success during this critical transition period. We examined variation in total lipid levels among years and sites in recently metamorphosed individuals of two pond-breeding salamander species, the marbled salamander (Ambystoma opacum) and the mole salamander (A. talpoideum), with limited data for one anuran species (southern leopard frog, Rana sphenocephala). Lipid levels were allometrically related to body size and ranged from 1.9 to 23.8% of body dry mass. The two salamander species differed in lipid allocation patterns, with A. opacum apportioning a higher percentage of total lipid reserves into fat bodies than A. talpoideum. Species differences in lipid allocation patterns may primarily reflect that large metamorphs will mature as one-year olds, and, regardless of species, will alter lipid compartmentalization accordingly. We used mark-recapture data obtained at drift fences encircling breeding ponds for 13 A. opacum cohorts to estimate the proportion of postmetamorphic individuals that survived to breed (age 1-4) and the mean age at first reproduction. Regression models indicated that size-corrected lipid level at metamorphosis (i.e., lipid residuals), and to a lesser extent rainfall following metamorphosis, was positively related to adult survival. Snout-vent length at metamorphosis was negatively related to age at first reproduction. We suggest that lipid stores at metamorphosis are vital to juvenile survival in the months following the transition from aquatic to terrestrial habitat, and that a trade-off shaped by postmetamorphic selection in the terrestrial habitat exists between allocation to energy stores versus structural growth in the larval environment.