Molecular diet analysis in mussels and other metazoan filter feeders and an assessment of their utility as natural eDNA samplers.

Molecular gut content analysis is a popular tool to study food web interactions and has recently been suggested as an alternative source for DNA-based biomonitoring. However, the overabundant consumer's DNA often outcompetes that of its diet during PCR. Lineage-specific primers are an efficient means to reduce consumer amplification while retaining broad specificity for dietary taxa. Here, we designed an amplicon sequencing assay to monitor the eukaryotic diet of mussels and other metazoan filter feeders and explore the utility of mussels as natural eDNA samplers to monitor planktonic communities. We designed several lineage-specific rDNA primers with broad taxonomic suitability for eukaryotes. The primers were tested using DNA extracts of different limnic and marine mussel species and the results compared to eDNA water samples collected next to the mussel colonies. In addition, we analysed several 25-year time series samples of mussels from German rivers. Our primer sets efficiently prevent the amplification of mussels and other metazoans. The recovered DNA reflects a broad dietary preference across the eukaryotic tree of life and considerable taxonomic overlap with filtered water samples. We also show the utility of a reversed version of our primers, which prevents amplification of nonmetazoan taxa from complex eukaryote community samples, by enriching fauna associated with the marine brown algae Fucus vesiculosus. Our protocol will enable large-scale dietary analysis in metazoan filter feeders, facilitate aquatic food web analysis and allow surveying of aquacultures for pathogens. Moreover, we show that mussels and other aquatic filter feeders can serve as complementary DNA source for biomonitoring.

Environmental DNA from archived leaves reveals widespread temporal turnover and biotic homogenization in forest arthropod communities.

A major limitation of current reports on insect declines is the lack of standardized, long-term, and taxonomically broad time series. Here, we demonstrate the utility of environmental DNA from archived leaf material to characterize plant-associated arthropod communities. We base our work on several multi-decadal leaf time series from tree canopies in four land use types, which were sampled as part of a long-term environmental monitoring program across Germany. Using these highly standardized and well-preserved samples, we analyze temporal changes in communities of several thousand arthropod species belonging to 23 orders using metabarcoding and quantitative PCR. Our data do not support widespread declines of α-diversity or genetic variation within sites. Instead, we find a gradual community turnover, which results in temporal and spatial biotic homogenization, across all land use types and all arthropod orders. Our results suggest that insect decline is more complex than mere α-diversity loss, but can be driven by ß-diversity decay across space and time.

Insects are a barometer of environmental health. Ecosystems around the world are being subjected to unprecedented man-made stresses, ranging from climate change to pollution and intensive land use. These stresses have been associated with several recent, dramatic declines in insect populations, particularly in areas with heavily industrialised farming practices. Despite this, the links between insect decline, environmental stress, and ecosystem health are still poorly-understood. A decline in one area might look catastrophic, but could simply be part of normal, longer-term variations. Often, we do not know whether insect decline is a local phenomenon or reflects wider environmental trends. Additionally, most studies do not go far back enough in time or cover a wide enough geographical range to make these distinctions. To understand and combat insect decline, we therefore need reliable methods to monitor insect populations over long periods of time. To solve this problem, Krehenwinkel, Weber et al. gathered data on insect communities from a new source: tree leaves. Originally, these samples were collected to study air pollution, but they also happen to contain the DNA of insects that interacted with them before they were collected ­ for example, DNA deposited in chew marks where the insects had nibbled on the leaves. This is called environmental DNA, or eDNA for short. To survey the insect communities that lived in these trees, Krehenwinkel, Weber et al. first extracted eDNA from the leaves and sequenced it. Analysis of the different DNA sequences from the leaf samples revealed not only the number of insect species, but also the abundance (or rarity) of each species within each community. Importantly, the leaves had been collected and stored in stable conditions over several decades, allowing changes in these insect populations to be tracked over time. eDNA analysis revealed subtle changes in the make-up of forest insect communities. In the forests where the leaves were collected, the total number of insect species remained much the same over time. However, many individual species still declined, only to be replaced by newcomer species. These 'colonisers' are also widespread, which will likely lead to an overall pattern of fewer species that are more widely distributed ­ in other words, more homogeneity. The approach of Krehenwinkel, Weber et al. provides a reliable method to study insect populations in detail, over multiple decades, using archived samples from environmental studies. The information gained from this has real-world significance for environmental issues with enormous social impact, ranging from conservation, to agriculture and even public health.

Form-Stable Phase Change Materials Based on SEBS and Paraffin: Influence of Molecular Parameters of Styrene-b-(Ethylene-co-Butylene)-b-Styrene on Shape Stability and Retention Behavior.

In this work, the influence of molecular parameters of styrene-b-(ethylene-co-butylene)-b-styrene (SEBS) triblock copolymer as matrix material in form-stable phase change material (FSPCM) on the thermo-mechanical properties and leakage behavior are studied. Various SEBS grades differing in their molecular weight, styrene content, and ethylene/butylene ratio are used as supporting matrix in composites with 90 wt.% paraffin. Thermo-mechanical properties are determined by rheological measurements. The results show phase transitions temperatures from solid to hard gel, hard gel to soft gel, and soft gel to gel fluid. Paraffin leakage in FSPCM is analyzed by mass loss over time in an oven at 60 °C. Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) are applied to determine the thermal energy storage capacity. Finally, the molecular weight and the styrene content are combined to the molecular weight of styrene block which is identified as the authoritative parameter for the thermo-mechanical properties of the SEBS/PCM composite.

Decreasing δ13C and δ15N values in four coastal species at different trophic levels indicate a fundamental food-web shift in the southern North and Baltic Seas between 1988 and 2016.

Marine ecosystems are exposed to increasing human pressures and climatic change worldwide. It has therefore become essential to describe ecosystem statuses with respect to multinational protection schemes, often necessitating long-term monitoring programmes. Changes in the food-web structure, which can be monitored via stable isotope measurements, represent an important descriptor of the status of marine ecosystems. We investigated long-term changes (29 years) in isotopic values (δ13C and δ15N) in four indicative organisms at different trophic levels in the southern North and Baltic Seas: bladderwrack (Fucus vesiculosus), blue mussel (Mytilus ssp.), eelpout (Zoarces viviparus), and herring gull (Larus argentatus). Time series analyses using generalised additive models revealed largely consistent declines in δ13C and δ15N throughout all trophic levels of the coastal food web at all study sites, indicating a clear change in these coastal regions from 1988 to 2016. There were no clear long-term patterns in egg biometrics for herring gulls, except for a consistent increase in eggshell thickness. The declines in stable isotope values were in line with the results of previous long-term studies of single higher-trophic-level species, which suggested that the noted changes were mainly caused by altered foraging patterns of the studied species. The current results demonstrate that declines in δ13C and δ15N have occurred throughout the whole food web, not just in particular species. We discuss the possible reasons for the decrease in stable isotope values, including decreasing eutrophication and an increase in terrestrial carbon sources.

Halogenated flame retardants in tree samples applied as bioindicators for atmospheric pollution.

Coniferous shoots and deciduous tree leaf samples from 10 sites in Germany were taken in 2015 or 2016 within the German Environmental Specimen Bank sampling program and analysed for 24 polybrominated biphenyl ethers (PBDEs) and 19 additional halogenated flame retardants (HFRs). At one site, additional historic samples dating back till 2003 were also investigated. Samples were Soxhlet-extracted, cleaned-up by a non-destructive multi-step procedure involving gel permeation chromatography, and detected by GC-API-MS/MS as well as GC-MS. Besides PBDEs as classical HFRs, emerging HFRs such as Dechlorane Plus, DPTE, DBDPE, or ATE were region-wide observed demonstrating their widespread occurrence in the atmosphere. Highest concentrations in recent samples were found for DBDPE (<230-2760 pg g-1 dry weight (dw)) followed by DPTE (91-1540 pg g-1 dw), BDE209 (<156-461 pg g-1 dw), and BDE47 (<27-505 pg g-1 dw) or DP (31-122 pg g-1 dw). The overall uniform and widespread distribution as well as similar HFR levels and composition profiles observed in recent conifer shoots and corresponding deciduous tree leaves from the same area indicate a prolonged medium to long-range transport as sources. Furthermore, it is demonstrated that both tree types are generally suitable bioindicators for atmospheric pollution with HFRs, although accumulation may vary depending on HFR properties and accumulation period. The historic samples showed decreasing PBDE levels whereas no clear trend could be observed for other investigated HFRs at this site.

Transcriptional changes measured in rice roots after exposure to arsenite-contaminated sediments.

Transcriptional analyses are discussed to provide a deeper understanding of the molecular mechanisms underlying toxic effects. Thus, they can complement classic ecotoxicological test methods and potentially allow the identification of biomarkers associated to the exposure of chemical stressors and or adverse biological effects. This feasibility study intended to identify a set of potential gene expression biomarkers for arsenite-exposure in rice roots that could complement the informative value of an existing sediment-contact test with rice. A sediment-contact test with Oryza sativa with the parameters inhibition of root and shoot elongation as phenotypic endpoints was used as basis. Rice plants were exposed to arsenite-spiked sediments. Transcriptomic changes in response to arsenite were observed by means of cDNA-microarray analysis regarding the whole-transcriptome at two sublethal arsenite concentrations. In order to identify candidate biomarker genes, differentially expressed genes were identified. Arsenite-induced differentially expressed genes were significantly associated with gene ontology (GO)-terms that indicated a general stress response. Of the differentially expressed genes, five genes were selected and their expression was measured at seven arsenite concentrations by means of qPCR in order to obtain their expression profiles. Three candidate biomarker genes showed a dose-dependent upregulation, while two showed no clear dose-dependent expression. The expression of all candidate biomarkers was also assessed in rice plants grown on two arsenic-contaminated natural sediments, but only one biomarker gene showed the expected upregulation.

Assuring quality in microplastic monitoring: About the value of clean-air devices as essentials for verified data.

Avoiding aerial microfibre contamination of environmental samples is essential for reliable analyses when it comes to the detection of ubiquitous microplastics. Almost all laboratories have contamination problems which are largely unavoidable without investments in clean-air devices. Therefore, our study supplies an approach to assess background microfibre contamination of samples in the laboratory under particle-free air conditions. We tested aerial contamination of samples indoor, in a mobile laboratory, within a laboratory fume hood and on a clean bench with particles filtration during the examining process of a fish. The used clean bench reduced aerial microfibre contamination in our laboratory by 96.5%. This highlights the value of suitable clean-air devices for valid microplastic pollution data. Our results indicate, that pollution levels by microfibres have been overestimated and actual pollution levels may be many times lower. Accordingly, such clean-air devices are recommended for microplastic laboratory applications in future research work to significantly lower error rates.

Hexabromocyclododecane diastereomers in fish and suspended particulate matter from selected European waters-trend monitoring and environmental quality standard compliance.

The brominated flame retardant hexabromocyclododecane (HBCD) was monitored in fish and sediment (from one lake) or suspended particulate matter (SPM; from five rivers) at European freshwater sites to study the effects of reduction measures implemented by HBCD producers and users in recent years. Bream (Abramis brama) were sampled annually between 2007 and 2013 in the rivers Götaälv/SE, Rhône/FR, Western Scheldt/NL, Mersey/UK, and Tees/UK and in Lake Belau/DE. Sediment/SPM was taken every second year between 2008 and 2014. HBCD was analyzed by LC/MS/MS allowing the determination of the alpha-, beta-, and gamma-diastereomers. For most sites, a decrease in ∑HBCD was observed in fish (e.g., in the Rhône and Western Scheldt by about 80 and 60%, respectively, with significantly decreasing trends, p < 0.01). In the Rhône, HBCD also decreased in SPM. At the sampling site in the Tees which was impacted by a former HBCD point source, fish HBCD levels decreased only after a major flood event in 2013. While fish data indicate a decline in environmental HBCD concentrations at most sites with diffuse emissions, SPM data were less conclusive. The European environmental quality standard for HBCD in fish of 167 µg kg-1 wet weight was met by all fish samples in 2013.

Towards the suitable monitoring of ingestion of microplastics by marine biota: A review.

Monitoring plastic ingestion in marine biota is a difficult task, especially regarding ubiquitous microplastics (particles of <5 mm). Due to their microscopic size, evidence for microplastic ingestion is often limited to laboratory studies. The following review provides a comparison and assessment of different microplastic ingestion monitoring procedures. Emphasis is given to the most important steps of current monitoring practice: (1) selecting suitable indicator species, (2) sampling and sample processing, (3) analytical procedures and (4) the prevention of secondary contamination of the sample. Moreover, an overview on ingestion records of microplastics by different marine feeding guilds is presented, including filter, suspension and deposit feeders as well as predators and scavengers. Lastly, monitoring processes are addressed critically in terms of their suitability for achieving the aims of an appropriate monitoring programme. Recommendations for future research priorities are presented with a focus on the necessity of standardised and comparable monitoring procedures in microplastic detection.

No microplastics in benthic eelpout (Zoarces viviparus): An urgent need for spectroscopic analyses in microplastic detection.

Monitoring the ingestion of microplastics is challenging and suitable detection techniques are insufficiently used. Thus, misidentifying natural for synthetic microfibres cannot be avoided. As part of a framework to monitor the ingestion of microplastics in eelpout, this short report addresses the accurate identification of microfibres. We show that, following visual inspections, putatively synthetic microfibres are indeed of natural origin, as ascertained by spectrometric analyses. Consequently, we call for an inclusion of spectroscopic techniques in standardized microplastic monitoring schemes.

Historical Invasion Records Can Be Misleading: Genetic Evidence for Multiple Introductions of Invasive Raccoons (Procyon lotor) in Germany.

Biological invasions provide excellent study systems to understand evolutionary, genetic and ecological processes during range expansions. There is strong evidence for positive effects of high propagule pressure and the associated higher genetic diversity on invasion success, but some species have become invasive despite small founder numbers. The raccoon (Procyon lotor) is often considered as a typical example for such a successful invasion resulting from a small number of founders. The species' largest non-native population in Germany is commonly assumed to stem from a small number of founders and two separate founding events in the 1930s and 1940s. In the present study we analyzed 407 raccoons at 20 microsatellite loci sampled from the invasive range in Western Europe to test if these assumptions are correct. Contrary to the expectations, different genetic clustering methods detected evidence for at least four independent introduction events that gave rise to genetically differentiated subpopulations. Further smaller clusters were either artifacts or resulted from founder events at the range margin and recent release of captive individuals. We also found genetic evidence for on-going introductions of individuals. Furthermore a novel randomization process was used to determine the potential range of founder population size that would suffice to capture all the alleles present in a cluster. Our results falsify the assumption that this species has become widespread and abundant despite being genetically depauperate and show that historical records of species introductions may be misleading.

Development of a sediment-contact test with rice for the assessment of sediment-bound pollutants.

Despite the key role of higher plants in aquatic ecosystems as functional and structural elements, sediment-contact tests with macrophytes are still scarce. Moreover, due to large differences in exposure routes for pollutants as well as in life cycles between the diverse taxa of macrophytes, sensitivities to pollutants vary between taxa. Therefore, the development of new test systems with aquatic macrophytes, in general, is favorable. This study proposes a protocol for a sediment-contact test with Oryza sativa and addresses the main question whether the rice plant is a suitable test organism for sediment toxicity testing with higher plants. As a first evaluation step, the variability and sensitivity of the test was investigated using spiked artificial sediments. Thus, according to the protocol, rice was exposed to arsenic-, cadmium-, chromium-, and nickel-spiked sediments. Additionally, it was investigated which classical endpoints for plant bioassays, such as root and shoot elongation, are suitable for this bioassay. As a second evaluation step, the test system was used for assessment of natural sediments. Thereupon, a sensitivity profile of the presented test protocol was analyzed in comparison to other plant-based test systems. Inhibition of root and shoot elongation turned out to be the most sensitive endpoints for single-substance testing in spiked artificial sediments. However, regarding testing of natural sediments, rice shoots responded more sensitive than rice roots. In conclusion, the rice plant clearly showed pollutant-induced effects on growth in sediments, and thus, it is likely a promising test organism to complement sediment-contact tests with higher plants.

Biometric parameters of the bream (Abramis brama) as indicators for long-term changes in fish health and environmental quality--data from the German ESB.

Piscifaunal health depends upon the state and quality of the aquatic environment. Variations in physical condition of fish may therefore be attributed to changes in environmental quality. Based on time series of up to 20 years of biometric data of bream from multiple sampling sites of the German environmental specimen bank (ESB), this study assessed whether changes in biometric parameters are able to indicate long-term alterations in fish health and environmental quality. Evaluated biometric parameters of fish health comprised length and weight of individuals of a defined age class, the condition factor, lipid content and hepatosomatic index (HSI). Although there are negative trends of the HSI, the overall development of health parameters can be interpreted as positive. This seems to suggest that health parameters conclusively mirror the long-term improvement of water quality in the selected rivers. However, the applicability of the condition factor as well as lipid content as indicators for fish health remained subject to restrictions. Altogether, the results from the ESB confirmed the high value of biometric parameters for monitoring of long-term changes in state and quality of aquatic ecosystems.

Standardization of egg collection from aquatic birds for biomonitoring--a critical review.

Collecting bird eggs is an established method of biomonitoring for specific pollution hazards. One of the most critical problems with this method is the extreme biological variability in bird eggs, but standardizing the collection and preservation of eggs can reduce these problems. Furthermore, standard practices are required so that the results can be compared among studies because mistakes cannot be corrected by laboratory analysis. Therefore, a standard procedure for collecting and preserving bird eggs may be necessary. The objective of this review is to investigate the current standard of quality assurance in the field by analyzing 86 peer-reviewed papers describing egg collection and use for aquatic birds. We show that little attention has been paid to standardizing how eggs are collected and stored in the field. Important information is often absent, including crucial aspects of sample collection and preservation, such as the freshness of the eggs, the position of the eggs in the laying sequence, the selection criteria, random sampling, and the duration and temperature of transport. Potential standards are suggested and discussed as a foundation for the development of quality assurance standards in the field.

Monitoring of hexabromocyclododecane diastereomers in fish from European freshwaters and estuaries.

BACKGROUND AND AIMS: Hexabromocyclododecane (HBCD) is a brominated flame retardant used mainly in polystyrene foam as well as in textile applications. In recent years, measures were taken to reduce HBCD emissions during its production and use. To evaluate the efficacy of these measures, a monitoring project was initiated with fish as bioaccumulation indicators. METHODS: Since 2007 bream was sampled annually at several European freshwater sites applying standardized methods. Muscle samples were analyzed by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method which allows the quantification of α-, ß-, and γ-diastereomers of HBCD. DISCUSSION: In most samples, α-HBCD was predominant. The lowest HBCD levels (11 µg/kg lipid weight (lw), sum of three HBCD diastereomers) were detected in bream sampled in 2009 from Lake Belau which is situated in a rural area of Northern Germany. During the 4-year study period, statistical significant decreases of HBCD concentrations were detected in bream from the rivers Rhone (France; -85%, level 2010, 205 µg/kg lw) and Western Scheldt (the Netherlands; -60%, level 2010, 36 µg/kg lw). High HBCD concentrations (9,480-14,500 µg/kg lw) without a clear time trend were observed in bream from the river Tees (UK). These high levels are in line with published data for fish from contaminated regions. CONCLUSIONS: Overall, the results indicate that environmental burdens of HBCD are decreasing at those locations where point sources and more diffuse emissions are expected.

Water-free synthesis of polyurethane foams using highly reactive diisocyanates derived from 5-hydroxymethylfurfural.

This paper reports on the synthesis of a new highly reactive diisocyanate monomer based on hydroxymethylfurfural. It further describes its catalyst-free conversion to linear-chain thermoplastic polyurethanes as well as to cross-linked polyurethane foams. In addition, a novel strategy for the synthesis of polyurethane foams without the necessity of using water is developed. Nitrogen is utilized herein as blowing agent which is formed during Curtius rearrangement of a new furan based carboxylic azide into its corresponding diisocyanate.

Silacyclobutane-based diblock copolymers with vinylferrocene, ferrocenylmethyl methacrylate, and [1]dimethylsilaferrocenophane.

Well-defined diblock copolymers have been prepared in which three different ferrocene-based monomers are combined with 1,1-dimethylsilacyclobutane (DMSB) and 1-methylsilacyclobutane, respectively, as their carbosilane counterparts. Optimized procedures are reported for the living anionic chain growth following sequential monomer addition protocols, ensuring narrow polydispersities and high blocking efficiencies. The DMSB-containing copolymers show phase segregation in the bulk state, leading to micromorphologies composed of crystalline DMSB phases and amorphous polymetallocene phases.

Defined Poly[styrene-block-(ferrocenylmethyl methacrylate)] Diblock Copolymers via Living Anionic Polymerization.

Ferrocenylmethyl methacrylate (FMMA) is one of the very few metallocene-based monomers that are promising candidates for truly living anionic polymerization. Nevertheless, FMMA homopolymers with a narrow polydispersity, or block copolymerization studies that result in satisfying blocking efficiencies, are unknown so far. Here we describe a procedure that leads to highly regular FMMA-based polymers for the first time, characterized by polydispersity indices (PDI) of less that 1.05 and very high blocking efficiencies (>95%) in sequential copolymerization with styrene. Some of the obtained poly[styrene-block-(ferrocenylmethyl methacrylate)]s show unusual microphase morphologies, presumably the consequence of high T(g) s causing 'frustrated' non-equilibrium states.

Is there a linkage between bioaccumulation and the effects of alkylphenols on male breams (Abramis brama)?

There was some evidence from a previous study that estrogenic disruptors, like alkylphenols, could effect fish in the small River Saar of Southwestern Germany. Concentrations of 4NP and 4NP1EO found in breams (Abramis brama) in the Saar River were much higher than those found in other sampling sites of the German Environmental Specimen Bank, including those from sampling sites in the Rivers Elbe, Rhine, Mulde, and Saale and in Lake Belau. We studied the relationship between accumulation and effect using vitellogenin (vtg) and a hepatosomatic index (HSI) of estrogenic effects and by measuring concentrations of AP and APE accumulated in breams caught at six sampling sites in the River Saar and one in the River Mosel. To link these results with those of the previous study we standardized our sampling efforts to obtain comparable data. Elevated vtg levels were found in the breams at all sampling sites near to or downstream of sewage plant discharges, whereas low vtg levels corresponded to sampling sites not influenced by municipal waste water. While HSI values did not correspond to the location of sampling sites, there was a weak but statistically significant correlation to vtg concentrations. Concentrations of four AP and APE were much more lower, as in the previous study, and were neither linked with sewage treatment plant discharges nor correlated with vtg levels. In conclusion, a linkage between accumulation and the effects of AP and APE could not be established, but the relationship between elevated vtg concentrations and municipal waste water, which contains other important endocrine disruptors, was clear.