The minimum detectable difference (MDD) is a measure of the difference between the means of a treatment and the control that must exist to detect a statistically significant effect. It is a measure at a defined level of probability and a given variability of the data. It provides an indication for the robustness of statistically derived effect thresholds such as the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) when interpreting treatment-related effects on a population exposed to chemicals in semi-field studies (e.g., micro-/mesocosm studies) or field studies. MDD has been proposed in the guidance on tiered risk assessment for plant protection products in edge of field surface waters (EFSA Journal 11(7):3290, 2013 ), in order to better estimate the robustness of endpoints from such studies for taking regulatory decisions. However, the MDD calculation method as suggested in this framework does not clearly specify the power which is represented by the beta-value (i.e., the level of probability of type II error). This has implications for the interpretation of experimental results, i.e., the derivation of robust effect values and their use in risk assessment of PPPs. In this paper, different methods of MDD calculations are investigated, with an emphasis on their pre-defined levels of type II error-probability. Furthermore, a modification is suggested for an optimal use of the MDD, which ensures a high degree of certainty for decision-makers.

The various uses of copper or copper compounds in industrial, biocidal or pesticidal products lead to inputs of the metal into aquatic environments. To assess effects of copper ions on non-target organisms, the freshwater snail Theodoxus fluviatilis was used as test organism for a three-week laboratory experiment. Snails were exposed to four copper concentrations ranging from 4 to 39 µg Cu2+/L, and besides mortality, several sublethal parameters were evaluated. Concerning survival, an aqueous copper concentration of 6 µg/L was determined as NOEC, and 16 (± 0.1) µg/L as LC50. Negative sublethal effects on reproduction, activity and pathological modifications in the snails were detected in the treatment with an aqueous copper concentration of 15 µg/L. Our results using T. fluviatilis as a mere grazer exclusively feeding on biofilms contribute to findings that field relevant copper concentrations have a significant effect on non-target organisms in aquatic environments.

The replacement of native species by invasive species is one of the most critical threats to the biodiversity of aquatic systems today. However, little is known about potential effects of species invasions on the genetic diversity of indigenous species in cases where the latter coexist with the invader. Here we present an example of the indigenous amphipod Gammarus roeselii, which has been partly replaced by the invasive Dikerogammarus villosus in Lake Constance (C-Europe) and now mostly exists in small, isolated populations. We compared the genetic diversity, population structure, indicators for bottlenecks and migrations rates of G. roeselii before and after the invasion event from samples collected between 1999 and 2013. We expected a genetic impoverishment in the reduced and segregated populations of G. roeselii. However, no genetic measure on G. roeselii differed temporally or spatially over the investigated period, which indicates that D. villosus has not yet had an impact on G. roeselii at the genetic scale. Hence, even though a decline in population size of G. roeselii was found in Lake Constance, our results on the genetic scale contribute to recent findings that the overall impact of D. villosus on native species is not as strong as often discussed.

Dikerogammarus villosus, one of the most successful invaders in European river systems, is commonly regarded as a threat to native biodiversity and a main factor structuring the benthic community of invaded systems. The impact of D. villosus has been intensively studied in small-scale experiments and field observations, but its impact on natural communities on a larger scale remains unclear. Here, we investigated the benthic community structure at ten sites covering a broad range of habitats along the River Rhine (Central Europe) and its tributaries, to determine whether D. villosus is one of the main factors structuring the benthic community. Community composition was analysed using non-metric multidimensional scaling, distance-based redundancy analysis, and correlation analyses. D. villosus was one of nine relevant taxa present that altogether reflected a large part of the variation in the benthic samples, but further analyses indicated that the species might be less important for the community structure than other relevant taxa. Moreover, all nine relevant taxa together can explain only a similar amount of variation in our samples than the five relevant non-faunal environmental factors (water temperature, pH, conductivity, percentage of medium-sized gravel and macrophytes). Overall, our results suggested that rather a combination of non-faunal environmental factors than D. villosus mainly structure the benthic community composition on this larger spatial scale.

Invasive species can cause indirect effects on native biota by modifying parasite-host interactions and disease occurrence in native species. This study investigated the role of the invasive Pacific oyster ( Crassostrea gigas ) in potential spillover (co-introduced parasites infect native hosts) and spillback (native or established parasites infect invasive hosts and re-infect native hosts) scenarios of recently introduced ( Mytilicola orientalis ) and previously established ( Mytilicola intestinalis ) marine parasitic copepods in two regions in northern Europe, the Dutch Delta and the Wadden Sea. By examining 3416 individuals of 11 potential host species from sympatric host populations, we found that the recently introduced parasite M. orientalis does not only infect its principal host, the invasive Pacific oyster (prevalence at infected sites 2–43 %, mean intensity 4.1 ± 0.6 SE), but also native blue mussels ( Mytilus edulis; 3–63 %, 2.1 ± 0.2), common cockles ( Cerastoderma edule ; 2–13 %, 1.2 ± 0.3) and Baltic tellins ( Macoma balthica ; 6–7 %, 1.0 ± 0), confirming a spillover effect. Spillback effects were not observed as the previously established M. intestinalis was exclusively found in blue mussels (prevalence at infected locations 3–72 %, mean intensity 2.4 ± 0.3 SE). The high frequency of M. orientalis spillover, in particular to native mussels, suggests that Pacific oysters may cause strong parasite-mediated indirect impacts on native bivalve populations.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (δ15N and δ13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Thermal requirements of larval weatherfish Misgurnus fossilis were investigated in terms of growth, survival and aerobic performance. Growth and survival of M. fossilis larvae acclimated to five temperatures (11, 15, 19, 23 and 27° C) were measured over 25 days. In the upper temperature treatments (19, 23 and 27° C), survival of larvae was stable throughout the entire rearing period (>75%), whereas 11 and 15° C resulted in severe declines in survival (to <10%). Growth of larvae (expressed as dry mass and total length) was highest at 19 and 23° C, but significantly decreased at 27° C. Routine metabolic rate of 3 days post-hatch larvae was estimated as oxygen consumption rate (ṀO2 ) during acute exposure (30 min to 1 h) to seven temperatures (11, 15, 19, 23, 27, 31 and 35° C). Larval oxygen uptake increased with each consecutive temperature step from 11 to 27° C, until a plateau was reached at temperatures >27° C. All larvae of the 35° C regime, however, died within the ṀO2 measurement period. M. fossilis larvae show greater than expected tolerance of high temperatures. On the other hand, low temperatures that are within the range of likely habitat conditions are critical because they might lead to high mortality rates when larvae are exposed over periods >10 days. These findings help to improve rearing conditions and to identify suitable waters for stocking and thus support the management of re-introduction activities for endangered M. fossilis.

Communities and food web structures of aquatic ecosystems can be strongly affected by the establishment of alien macroinvertebrate species. In many European waters, the invasion of the Ponto-Caspian amphipod Dikerogammarus villosus has led to displacement of other macroinvertebrates. Predation by D. villosus is often assumed to be the key driver of the displacement based on results of laboratory studies, but this has not been verified in the field. Here, we report our investigation of the relevance of D. villosus predation in the River Rhine system using both stable isotope analyses of δ13C and δ15N, and molecular analyses of D. villosus gut contents with group-specific primers aiming at macroinvertebrate prey taxa. Stable isotope analyses of D. villosus from ten sites showed mean δ15N values comparable to those of primary consumers. Overall, only approximately 1% of all tested primer/gut content combinations revealed DNA of the respective taxa. Both indicate minor importance of predation by D. villosus as a driver of the observed macroinvertebrate displacement. Conceivably, competitive strength due to opportunistic feeding, indicated by different niche widths between and a strong intraspecific variation of δ13C values of D. villosus within sites of our study, is much more important for its invasion success.

The estuarine maximum turbidity zone (MTZ) can be assumed to be a stressful environment featuring special conditions of great biological importance with an excess of organic matter, high-deposition rates, large variations in salinity, and dredging activities. Under such harsh conditions, populations may remain below the carrying capacity and competition is assumed to be of little importance, as predicted by the stress-gradient hypothesis. Therefore, we hypothesized that invertebrates of similar feeding types may utilize the same resources. To test our hypothesis, we chose the three most abundant taxa classified in literature as deposit feeders (Bathyporeia pilosa, Boccardiella ligerica, Marenzelleria sp.) and two taxa classified as predominately predacious (Palaemon longirostris, Crangon crangon) and determined their isotopic niches based on a stable isotope analysis for the MTZ of the Elbe Estuary (Germany). We expected the isotopic niches of similar feeding types to show a clear overlap if our hypothesis was true. Our results showed that the isotopic niches of no two taxa overlapped within each feeding group, indicating different resource use and the absence of competition. The sediment analysis revealed that two of the deposit feeders inhabited significantly different mean grain sizes. The lack of overlap of isotopic niches within each feeding group may be due to differences in habitat and feeding behavior in the case of the deposit feeders and due to different migration behavior in the case of the predominately predacious species. However, competition may have occurred in the past, resulting in a divergence of feeding niches during evolution.

Summary River ecosystems are threatened by multiple stressors, including habitat degradation, pollution and invasive species. However, freshwater ecologists have largely disregarded the contribution of toxicants to stress in rivers, whereas ecotoxicologists have primarily examined toxicant effects in artificial systems. As a result, there is a paucity of information on the co‐occurrence of organic toxicants with other stressors and on the relative importance of toxicants for overall ecological risk in rivers. We used monitoring data for German rivers to analyse the individual and joint occurrence of four stressors: habitat degradation, invasive species, nutrient pollution and organic toxicants. All stressors were examined for ecological risks in terms of whether they exceeded low‐ and high‐risk thresholds derived from published studies and regulatory thresholds. Nutrients and habitat degradation exceeded low and high risk thresholds at c. 85% of the sites and invasive species and organic toxicants at c. 50% of the sites. At least one stressor exceeded thresholds at all sites for which data on all four stressors were available. Toxicity showed weak positive correlations with nutrients and habitat degradation (0.2 < Spearman's ρ < 0.34, 0.009 < P < 0.08). The risks of ecological effects arising from habitat degradation and invasive species were higher in lowland rivers, particularly for invasive species. Our assessment shows that organic toxicants contribute notably to risks of ecological effects in rivers, to a similar extent as invasive species, although habitat degradation and nutrients are the dominant stressors. Exposure to multiple stressors is the typical situation prevailing in rivers. Consequently, mitigation measures focusing on individual stressors may not be effective at reducing ecological risks. This suggests that integrating concepts and data from freshwater ecology and ecotoxicology is essential to meet the challenge of managing multiple stressors in river ecosystems.

While recent research has provided evidence that the emergence of merolimnic insects (species with an aquatic larval stage) provides a considerable energy subsidy to riparian food webs, it has also shown that merolimnic insects may serve as a vector for contaminants. Therefore, riparian food webs may be at risk from either an aquatic-terrestrial transfer of contaminants or from the contaminant-driven reductions of emerging merolimnic insects. The objective of the present study was to develop an integrated stream mesocosms test design capable of identifying these inter-ecosystem boundary effects and to provide a comprehensive approach as a basis for ecotoxicological testing. We chose the widely distributed web-building spider Tetragnatha extensa as a representative species for riparian predators. Trophic aspects of riparian food webs were investigated by stable isotope analysis of carbon (δ13C) and nitrogen (δ15N). Utilization of stable isotope ratios provided detailed information on the riparian food web structure and the dietary composition of T. extensa. Merolimnic invertebrates (mainly Cloeon spp. and Chironomidae) were found to contribute up to 71 % of T. extensa’s diet, demonstrating their importance in riparian food webs in ecotoxicological mesocosm testing. This study provides a conceptual and methodological basis for assessing aquatic insect emergence-related pollutant transfer or effect translation from aquatic to adjacent terrestrial systems.

After stocking with larvae from the Gironde-Garonne-Dordogne population, in 2013, three young-of-the-year Allis shad Alosa alosa, probably originating from natural reproduction, were documented for the first time in a period of nearly 100years in the River Rhine. In 2014, a further increase was observed when 57 juveniles and eight adults were caught; seven of these eight adults were derived from stocking, indicating the success of stocking measures within the framework of the EU-LIFE project.

The invasive amphipod Dikerogammarus villosus has become increasingly dominant in German river ecosystems since the River Rhine invasion in the mid-1990s. Because it is preying on other invertebrate taxa, its appearance is often assumed to be associated with a drastic decrease in species diversity and changes in natural benthic communities. Despite this, the trophic function of D. villosus and its predation potential are rarely studied in natural river communities. Here, we assess the trophic function of D. villosus in two invaded systems, the Elbe River and the River Rhine, using stable carbon and nitrogen isotope analyses. In the two studied river food webs, D. villosus had a generally low trophic position (TP), indicating a likely function as primary consumer or at most as an omnivore. The significantly higher TP in the River Rhine (TP = 2.6) than in the Elbe River (TP = 1.9) suggested a partial use of animal prey in the River Rhine. This was supported by the results of the isotope mixing model SIAR which predicted in addition to a high importance of herbivory in both rivers a likely feeding on other invasive amphipod species in the Rhine community. We conclude that D. villosus has a variable but mostly low predacious behaviour, probably depending on the benthic community structure. To allow more realistic trophic analyses for D. villosus in the field, we determined the trophic enrichment factors (TEFs) for nitrogen and carbon isotopes of two diet types in the tissue of D. villosus in an eight-week laboratory experiment. The carbon isotope enrichment ( 1 3 C) from the chironomid diet was negative in the consumer (-1.75 ‰), whereas the carbon from leaf litter was enriched positively in the tissue (3.27 ‰). The TEFs for nitrogen ( 1 5 N) were more similar for plant and animal diet (leaf litter: 3.56 ‰, chironomids: 2.29 ‰). Because of the high specificity of the enrichment factors with respect to species and food source, we recommend their use in further trophic analyses.

Land use related habitat degradation in freshwater ecosystems has considerably increased over the past decades, resulting in effects on the aquatic and the riparian communities. Previous studies, mainly in undisturbed systems, have shown that aquatic emergent insects contribute substantially to the diet of riparian predators. To evaluate the effect of land use on aquatic prey subsidies of riparian spiders, we performed a longitudinal study from June to August 2012 along a first order stream (Rhineland-Palatinate, Germany) covering three land use types: forest, meadow and vineyard. We determined the contribution of aquatic and terrestrial resources to the diet of web-weaving (Tetragnathidae spp.) and ground-dwelling (Pardosa sp.) riparian spiders using stable isotope analyses of aquatic emergent insects and terrestrial arthropods. The contribution of aquatic and terrestrial sources differed between Tetragnathidae spp. and Pardosa sp. as well as among land use types. Tetragnathidae spp. consumed 80–100% of aquatic insects in the meadows and 45–65% in the forest and vineyards. Pardosa sp. consumed 5–15% of aquatic insects in the forest, whereas the proportions of aquatic and terrestrial sources were approximately 50% in the meadow and vineyard. Thus, aquatic emergent insects are an important subsidy to riparian spiders and land use is likely to affect the proportion of aquatic sources in the spider diet.

By incorporating the free-swimming nematode Turbatrix aceti into early feeding regimes of the European whitefish Coregonus maraena, the suitability of this nematode species was investigated as an alternative to Artemia nauplii. During a 14-day feeding trial in a total of 25 aquaria each 1.7 L (each treatment n = 5, 255 larvae/tank) T. aceti was used either as the sole live food or in combination with Artemia nauplii or microdiet to determine the effect of T. aceti on growth performance and survival rate of C. maraena. By analysing the fatty acid composition of T. aceti prior to and after enrichment with INVE spresso® it was investigated whether the amount of n3-polyunsaturated fatty acids (n3-PUFA) in T. aceti could be further enhanced. Supplementation of Artemia nauplii with T. aceti increased growth significantly within the first 5 days of rearing in comparison to the non-supplemented food treatments (14.39 ± 0.15 mm compared to 13.44 ± 0.18 mm; mean ± SE). However, growth and survival of juvenile C. maraena on nematode-supplemented Artemia nauplii did not differ significantly from non-supplemented Artemia nauplii at the end of the 14-day rearing period (15.22 ± 0.15 mm compared to 14.86 ± 0.24 mm). All feeding treatments containing Artemia nauplii showed significantly higher growth and lower mortality at the end of the experiment in comparison to diets containing only the microdiet or T. aceti or a combination thereof. The overall low performance of T. aceti alone can most likely be explained by an insufficient capacity of C. maraena to digest this nematode species efficiently. Enrichment with INVE spresso® successfully increased the proportion of DHA in the T. aceti tissue. The results reveal that T. aceti cannot be considered a full alternative to Artemia nauplii, at least not in the rearing of C. maraena, but might be a useful vector of essential fatty acids within the early rearing period of this and potentially other fish species when provided as live food along with Artemia nauplii.

Up to now our knowledge of water mite diet has been fragmentary. It is derived from observations in the field and laboratory or from a few selective laboratory experiments on food choice. In the present study, we were able to detect chironomid DNA in water mite bodies for the first time using molecular methods. Prey DNA was detected in virtually all Hygrobates fluviatilis (Hygrobatidae) that were fed on chironomid larvae after a starvation period of up to approximately 1 week. From the shortest interval (1 h after feeding) to the longest period after feeding (50 h) the relative amount of detected prey DNA was significantly reduced. In addition, there was a relationship between the relative amount of prey DNA and the assumed amount of the ingested prey (classified in categories of the dead prey which reflect the increasing ingestion of the mites and the decreasing body content of the prey individuals). The results of our study indicate that similar molecular analyses will be a powerful tool for diet investigations of mites from the field on various taxonomic resolutions of prey taxa. Moreover, the results of food selection experiments from the laboratory could be compared to evidence of predation by individuals from the field. For many mite taxa, especially ones which turned out to be difficult to breed in the laboratory (e.g. by unknown diet), the new methods might enable us to gain the first ever data on diet and thus may help us to consider the role of water mites in food webs more adequately in the future.

Prior species distribution models identified temperature as one of the most important environmental variables defining the present and future distribution of anadromous allis shad (Alosa alosa). The current study analysed effects of temperature on the recruitment-potential of allis shad by investigating growth and survival at 16, 20, 24 and 28 °C during a ten day rearing trial and by measuring mass-specific respiration. Highest growth in length was at 28 °C; growth at 16 °C was minimal. At the end of the rearing-period, no significant differences in survival between tested rearing-temperatures were found. Exposure to temperatures of 13 to 30 °C and subsequent measurement of mass-specific respiration revealed tolerance of temperatures up to 30 °C and a lower temperature limitation close to 16 °C. After acclimatization of larvae to temperatures ranging from 16−28 °C for 10 days, Q10-values of mass-specific respiration indicated a high adaptive capacity to increasing temperatures, but also the ability to adapt to temperatures as low as 16 °C. Our results indicate that the predicted temperature sensitivity of A. alosa cannot be explained by a direct physiological relationship. The obtained results can help to improve predictive modelling and the conservation of allis shad throughout its current distribution range.

The endangered freshwater snail Theodoxus fluviatilis is a widely distributed European member of the gastropod family Neritidae. This taxon was abundantly found in the River Rhine until the end of the 20th century, and was considered to be extinct there since the late 1990s. Since 2006, a new, but morphological different form of T. fluviatilis has been recorded in the Upper Rhine region. Our aim was to identify the source of the recent populations by analysing individuals from five sites throughout the current known distribution along the River Rhine. Therefore, we sequenced the mitochondrial cytochrome c oxidase subunit I (COI) gene, and compared the data with those from individuals collected in the early 1990s and 40 already known haplotypes from a pan-European study. Our results show that all studied recent Rhine individuals harbour only one COI haplotype that corresponds to an already known haplotype described from the River Danube and the Ukraine region near the Black Sea. This suggests that a re-colonisation of the River Rhine by T. fluviatilis from the River Danube is the most likely scenario of the re-establishment of the species. This route of invasion is known for other freshwater taxa that originate from the Ponto-Caspian region. Even though the current Rhine populations belong to T. fluviatilis their invasion may have consequences for the native Central European populations. Therefore, we recommend considering the current Rhine population as ‘cryptic invader’.

Terrestrial inputs into freshwater ecosystems are a classical field of environmental science. Resource fluxes (subsidy) from aquatic to terrestrial systems have been less studied, although they are of high ecological relevance particularly for the receiving ecosystem. These fluxes may, however, be impacted by anthropogenically driven alterations modifying structure and functioning of aquatic ecosystems. In this context, we reviewed the peer-reviewed literature for studies addressing the subsidy of terrestrial by aquatic ecosystems with special emphasis on the role that anthropogenic alterations play in this water–land coupling. Our analysis revealed a continuously increasing interest in the coupling of aquatic to terrestrial ecosystems between 1990 and 2014 (total: 661 studies), while the research domains focusing on abiotic (502 studies) and biotic (159 studies) processes are strongly separated. Approximately 35% (abiotic) and 25% (biotic) of the studies focused on the propagation of anthropogenic alterations from the aquatic to the terrestrial system. Among these studies, hydromorphological and hydrological alterations were predominantly assessed, whereas water pollution and invasive species were less frequently investigated. Less than 5% of these studies considered indirect effects in the terrestrial system e.g. via food web responses, as a result of anthropogenic alterations in aquatic ecosystems. Nonetheless, these very few publications indicate far-reaching consequences in the receiving terrestrial ecosystem. For example, bottom-up mediated responses via soil quality can cascade over plant communities up to the level of herbivorous arthropods, while top-down mediated responses via predatory spiders can cascade down to herbivorous arthropods and even plants. Overall, the current state of knowledge calls for an integrated assessment on how these interactions within terrestrial ecosystems are affected by propagation of aquatic ecosystem alterations. To fill these gaps, we propose a scientific framework, which considers abiotic and biotic aspects based on an interdisciplinary approach.

RATIONALE:Dietary sterol deficiencies can have severe life history consequences for consumers. Compound-specific stable isotope analysis (CSIA) was applied to the exploration of the sterol metabolic constraints and bioconversion capacities of the amphipod Gammarus roeselii. Evaluating structural sterol requirements has great potential to improve our understanding of the ecological relevance of sterols as limiting nutrients.METHODS:Juvenile G. roeselii were reared on food mixtures consisting of different ratios of the two algae Scenedesmus obliquus (cultivated with (13)C-labeled NaHCO3) and Nannochloropsis limnetica (unlabeled), which have been shown previously to differ in food quality. We measured the sterol content and composition using a gas chromatograph equipped with a flame ionization detector and the δ(13)C values of sterols using compound-specific isotope ratio mass spectrometry to examine potential sterol-mediated nutritional constraints of G. roeselii.RESULTS:In the food mixtures, δ(13)C values of cholesterol, synthesized by N. limnetica, were -25‰ and those of the Δ(7)-phytosterols, chondrillasterol and fungisterol, synthesized by S. obliquus, were 7 and 18‰, respectively. Although the cholesterol concentrations in G. roeselii decreased with increasing proportion of dietary S. obliquus, the δ(13)C values remained constant at -25‰. Lathosterol, which appeared in G. roeselii at high dietary proportions of S. obliquus, had a δ(13)C value of 35‰.CONCLUSIONS:We provide evidence that the the Δ(7)-phytosterols present in S. obliquus cannot be metabolized to cholesterol in G. roeselii, resulting in the accumulation of lathosterol in the animals and potentially in sterol-limited growth. These findings emphasize the advantage of CSIA in revealing the physiological mechanisms associated with nutritional constraints.