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.

Recent findings of halogenated flame retardants (HFR) in the German and Polar environment.

To get an overview about distribution, levels and temporal trends of polybrominated diphenyl ethers (PBDE) and halogenated flame retardants (HFR) of emerging concern, different types of environmental samples archived in the German Environment Specimen Bank as well as fish filet samples from the Arctic (n = 13) and Antarctica (n = 5) were analysed for 43 substances (24 PBDE, 19 HFR) using a multi-column clean-up and GC-API-MS/MS or GC-MS. Sample types were herring gull egg (n = 3), blue mussel (n = 3) and eelpout filet (n = 3) from the German North- and Baltic Sea, bream filet (n = 7), zebra mussel (n = 6) and suspended particulate matter (SPM, n = 7) from German freshwater ecosystems as well as tree leaves (n = 9)/shoots (n = 10), soil (n = 4), earthworm (n = 4) and deer liver (n = 7) as representatives of German terrestrial ecosystems. PBDE and emerging HFR were present in each investigated matrices from Germany and Polar regions showing their widespread distribution. The presence in Arctic and Antarctic fish samples confirms their long-range transport potential. Average concentrations of total emerging HFR were highest in SPM (26 ng g-1 dry weight (dw)), zebra mussel (10 ng g-1 dw) and herring gull egg (2.6 ng g-1 dw). Lowest levels were measured in fish filet samples from Antarctica (0.02 ng g-1 dw). Average total PBDE concentrations were highest in bream filet (154 ng g-1), herring gull egg (61 ng g-1 dw), SPM (21 ng g-1 dw), and zebra mussel 18 (ng g-1) and lowest in deer liver (0.04 ng g-1 dw). The patterns of non-fauna terrestrial samples (leaves, shoots, soil) as well as SPM were dominated by DBDPE and BDE209. Elevated proportions of DPTE and in most cases the absence of DBDPE characterized all fauna samples with the exception of Polar samples. Overall, emerging HFR appeared to be less bioaccumulative than PBDE. Temporal trends were generally decreasing with few exceptions such as DBDPE.

Biota monitoring and the Water Framework Directive-can normalization overcome shortcomings in sampling strategies?

We compare the results of different monitoring programs regarding spatial and temporal trends of priority hazardous substances of the European Water Framework Directive (WFD). Fish monitoring data for hexachlorobenzene (HCB), mercury (Hg), and perfluorooctane sulfonic acid (PFOS) sampled in German freshwaters between the mid-1990s and 2014 were evaluated according to the recommendations of the 2014 adopted WFD guidance document on biota monitoring, i.e., normalization to 5 % lipid content (HCB) or 26 % dry mass (Hg, PFOS) and adjustment to trophic level (TL) 4. Data of the German Environmental Specimen Bank (ESB) (annual pooled samples of bream) were compared to monitoring data of the German federal states (FS), which refer to individual fish of different species. Significant decreasing trends (p < 0.01) were detected for Hg in bream (Abramis brama) sampled by both, the ESB and the FS between 1993 and 2013 but not for FS samples comprising different fish species. Data for HCB and PFOS were more heterogeneous due to a smaller database and gave no consistent results. Obviously, normalization could not compensate differences in sampling strategies. The results suggest that the data treatment procedure proposed in the guidance document has shortcomings and emphasize the importance of highly standardized sampling programs in trend monitoring or whenever results between sites have to be compared.

Current levels and trends of selected EU Water Framework Directive priority substances in freshwater fish from the German environmental specimen bank.

Under the German environmental specimen bank programme bream (Abramis brama) were sampled in six German rivers and analysed for the priority hazardous substances dicofol, hexabromocyclododecane (HBCDD), hexachlorobenzene (HCB), hexachlorobutadiene (HCBD), heptachlor + heptachlor epoxide (HC + HCE), polybrominated diphenylethers (PBDEs), polychlorinated dibenzo-p-dioxins and -furans and dioxin-like polychlorinated biphenyls (PCDD/Fs + dl-PCBs), and perfluorooctane sulfonic acid (PFOS). The aim was to assess compliance with the EU Water Framework Directive environmental quality standards for biota (EQSBiota) for the year 2013, and to analyse temporal trends for those substances that are of special concern. General compliance was observed for dicofol, HBCDD and HCBD whereas PBDEs exceeded the EQSBiota at all sites. For all other substances compliance in 2013 varied between locations. No assessment was possible for HC + HCE at some sites where the analytical sensitivity was not sufficient to cover the EQSBiota. Trend analysis showed decreasing linear trends for HCB and PFOS at most sampling sites between 1995 and 2014 indicating that the emission reduction measures are effective. Mostly decreasing trends or constant levels were also observed for PCDD/Fs and dl-PCBs. In contrast, increasing trends were detected for PBDEs and HBCDD which were especially pronounced at one Saar site located downstream of the industries and conurbation of Saarbrücken and Völklingen. This finding points to new sources of emissions which should be followed in the coming years.

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.

Retrospective study of triclosan and methyl-triclosan residues in fish and suspended particulate matter: results from the German Environmental Specimen Bank.

A retrospective monitoring of triclosan (TCS; period 1994-2003 and 2008) and its potential transformation product methyl-triclosan (MTCS; period 1994-2008) was performed using archived fish samples from German rivers (16 sites, including Elbe and Rhine). At four of these sites suspended particulate matter (SPM) was also investigated covering the period 2005-2007. Samples were analyzed by GC/MS, either directly (MTCS) or after derivatization (TCS). TCS burdens of fish muscle tissue ranged from <0.2-3.4 ng g(-1) ww (wet weight; corresponding to <2-69 ng g(-1) lw, lipid weight) without apparent concentration trends over time. MTCS was detected at considerably higher concentrations in fish ranging from 1.0-33 ng g(-1) ww (47-1010 ng g(-1) lw) and increased until about 2003-2005. Thereafter, concentrations generally were lower, although at some sites single higher values were observed in recent years. In SPM, decreasing MTCS concentrations in the range 1-4 ng g(-1) dry weight were detected while TCS was always below the limit of quantification. Assuming that MTCS concentrations are correlated to TCS consumption, the observed decrease in MTCS levels may be partly a result of the voluntary renunciation of TCS use in detergents for, e.g., laundry or dishwashing declared by a manufacturers' association in 2001. Because of a lack of ecotoxicity studies for MTCS, a QSAR-derived predicted no effect concentration (PNEC) was compared to averaged ambient water concentrations of fish which were calculated from maximum tissue residues by applying an appropriate bioconcentration factor from literature. Since these calculated water concentrations were below the PNEC it is assumed that MTCS alone poses no immediate risk to aquatic organism. The conversion to a PNEC for SPM organisms and comparison with detected SPM levels of MTCS also revealed no risk.

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.