Danish Coastal Waters Research Articles

Nitrogen Fixation and Microbial Communities Associated with Decomposing Seagrass Leaves in Temperate Coastal Waters

Seagrass meadows play pivotal roles in coastal biochemical cycles, with nitrogen fixation being a well-established process associated with living seagrass. Here, we tested the hypothesis that nitrogen fixation is also associated with seagrass debris in Danish coastal waters. We conducted a 52-day in situ experiment to investigate nitrogen fixation (proxied by acetylene reduction) and dynamics of the microbial community (16S rRNA gene amplicon sequencing) and the nitrogen fixing community (nifH DNA/RNA amplicon sequencing) associated with decomposing Zostera marina leaves. The leaves harboured distinct microbial communities, including distinct nitrogen fixers, relative to the surrounding seawater and sediment throughout the experiment. Nitrogen fixation rates were measurable on most days, but highest on days 3 (dark, 334.8 nmol N g−1 dw h−1) and 15 (light, 194.6 nmol N g−1 dw h−1). Nitrogen fixation rates were not correlated with the concentration of inorganic nutrients in the surrounding seawater or with carbon:nitrogen ratios in the leaves. The composition of nitrogen fixers shifted from cyanobacterial Sphaerospermopsis to heterotrophic genera like Desulfopila over the decomposition period. On the days with highest fixation, nifH RNA gene transcripts were mainly accounted for by cyanobacteria, in particular by Sphaerospermopsis and an unknown taxon (order Nostocales), alongside Proteobacteria. Our study shows that seagrass debris in temperate coastal waters harbours substantial nitrogen fixation carried out by cyanobacteria and heterotrophic bacteria that are distinct relative to the surrounding seawater and sediments. This suggests that seagrass debris constitutes a selective environment where degradation is affected by the import of nitrogen via nitrogen fixation.

Exploratory study of the Sentinel-3 level 2 product for monitoring chlorophyll-a and assessing ecological status in Danish seas

In situ Chl-a data were used to perform empirical calibration and validation of Sentinel-3 level 2 product in Danish marine waters. Comparing in situ data with both same-day and ±5 days moving averaged Sentiel-3 Chl-a values yielded two similar positive correlations (p > 0.05) with rpearson values of 0.56 and 0.53, respectively. However, as the moving averaged values resulted in significantly more available data than daily matchups (N = 392 vs. N = 1292) at a similar quality of correlation with similar model parameters (slope (1.53 and 1.7) and intercept (−0.28 and −0.33) respectively), which were not significantly different (p > 0.05), the further analyses were focused on ±5 days moving averaged values. A thorough comparison of seasonal and growing season averages (GSA) also showed a very good agreement, except for a few stations characterized by very shallow depth. Overestimation by the Sentinel-3 occurred in shallow coastal areas and was attributed to the interferences from benthic vegetation and high levels of Colored Dissolved Organic matter (CDOM) interfering with the Chl-a signals. Underestimation observed in the inner estuaries with shallow Chl-a rich waters, however, seen as a result of self-shading at high Chl-a concentrations, reducing effective absorption by phytoplankton. Besides the observed minor disagreements, there was no significant difference when the GSA values from in situ and Sentinel-3 were compared for all three water types (p > 0.05, N = 110). Analyzing Chl-a estimates along a depth gradient showed significant (p < 0.001) non-linear trends of declining concentrations from shallow to deeper waters for both in situ (explaining 15.2 % of the variance (N = 109)) and Sentinel-3 data (explaining 36.3 % of the variance (N = 110)), with higher variability in shallow waters. Furthermore, Sentinel-3 enabled full spatial coverage of all 102 monitored water bodies providing GSA data at much higher spatial and temporal resolutions for good ecological status (GES) assessment compared to only 61 through in situ sampling. This underlines the potential of Sentinel-3 for substantially extending the geographical coverage of monitoring and assessment. However, the systematic over- and underestimation of Chl-a in shallow nutrient rich inner estuaries through Sentinel-3 requires further attention to enable routine application of the Sentinel-3 level 2 standard product in the operational Chl-a monitoring in Danish coastal waters. We provide methodological recommendations on how to improve the Sentinel-3 products' representation of in situ Chl-a conditions. Continued frequent in situ sampling remains important for monitoring as these measurements provide essential data for empirical calibration and validation of satellite based estimates to reduce possible systematic bias.

Distribution and speed of recreational boats in Danish waters based on coastal observations and satellite images: Predicting where boats may affect harbour porpoises

Danish coastal waters are a hotspot for recreational boating and harbour porpoises (Phocoena phocoena), but as there is little data on the boats’ speeds and distributions, it is difficult to determine where porpoises are likely to be affected by boat noise. To gain knowledge on the potential impacts of small boats we 1) used coastal observations to assess how boat speed is related to boat size, type, distance to marinas, and wave height, and 2) used satellite images to assess the spatial distribution of small boats in Kattegat, the Sound, the Belt Sea and the Western Baltic areas (see map in Sveegaard et al., 2015). This made it possible to determine how the probability of observing boats was related to distance to the coast and number of people living within a radius of 5 km from a given area. We found that boat speeds varied among marinas and that speed was positively correlated with distance to the marina. The relationship between boat speed and size depended on boat type, with motorboats being the fastest. The satellite image analysis showed that motorboats were the dominant type in coastal waters, constituting 61.4% of the observed boats. The probability of observing boats on a satellite image covering 0.25 km2 dropped from 0.13 by the shore to 0.04 at a distance of 5 km from shore. The probability of observing boats was >10% in 6.6% of the study area, and the probability of observing boats and porpoises in the same area was >10% in 3.0% of the study area. Coastal waters are important habitats for many aquatic species, e.g., fish, invertebrates and porpoises, that use sound for communication or navigation. Our study uses a combination of observation and satellite data to identity areas where boats overlap with harbour porpoise presence and thus where the animals may be affected by boat noise. The study also illustrates how combining these two methods can yield important information on the potential impacts of boats on wildlife.

Taxonomic re-evaluation of Ericiolus and Mercedesia (Prymnesiophyceae) and description of three new species

ABSTRACT The genera Ericiolus and Mercedesia are distinctive extant coccolithophores that are characterized by monothecate, monomorphic coccospheres with one type of triradiate star-shaped nannoliths. The two genera were described from the Danish coastal waters, the surface waters of the Arctic, and the Southern Oceans. During a study of samples from the low photic zone of the Mediterranean and Sargasso Seas, and from the subtropical gyres of the South-eastern Pacific and the South Atlantic Oceans, 44 collapsed coccospheres with triradiate star-like nannoliths were observed via scanning electron microscopy. Observations on the morphologies and biometric assessments of these specimens revealed that three distinct sets of nannoliths can be distinguished and that these were morphologically differentiated from all currently known species of Ericiolus and Mercedesia. The new forms and the previously described species of Ericiolus and Mercedesia were, however, similar, as they all demonstrated a distinctive set of collectively shared morphological characters and almost identical size ranges. On the basis of this, and instead of describing a third genus for the same group of nannoliths, we preferred to taxonomically synonymize Mercedesia with Ericiolus and revise the definition of Ericiolus. Therefore, we describe three new species, Ericiolus bendifii sp. nov., Ericiolus sheldoniae sp. nov. and Ericiolus mattioliae sp. nov., and an incompletely defined taxon, as Ericiolus cf. bendifii, and establish the new combinations E. aspiphorus comb. nov., E. multistellatus comb. nov. and E. pusillus comb. nov.

Predicting eider predation potentials on mussels in Danish coastal areas—implications for mussel farming site-selection

Selecting optimal locations for mussel farming is vital for the optimization of production yield and for the minimization of environmental impact. Although predation by sea ducks may induce large stock losses and hence severe economic loss for mussel farmers, predation potential is rarely included in site-selection tools. In this paper we present a GIS-based spatial model predicting the potential of eider predation on blue mussel farms in Danish coastal waters. The model incorporates national survey data on eiders, as well as knowledge of eider behavior and habitat preferences, and was calibrated with predated/non-predated observations of eiders from 9 experimental mussel farms or test lines in Danish coastal waters. Except for 1 case study area, our model successfully confirmed a higher predation potential at test sites where predation had been observed. Our resulting predation potential map revealed potentials ranging from very low in inner parts of narrow estuaries to very high in more open coastal areas. Integration of the predation map into an existing site-selection tool showed that areas optimal for mussel growth were also associated with the highest modelled predation potential. Nonetheless, it was possible to identify areas having a very low potential of predation and only a 10% lower mussel production potential. These results underpin the potential for reducing production loss and increasing income by including predation potential in site-selection tools. In addition, the eider predation model can be used to identify and subsequently protect key foraging areas to support eider conservation.

A network analysis of connected biophysical pathways to advice eelgrass (Zostera marina) restoration

The North Sea and the Baltic Sea, including Danish coastal waters, have experienced a drastic decline in eelgrass Zostera marina coverage during the past century. Around 1900, eelgrass meadows covered about 6700 km2 of Danish coastal waters while the current potential distribution area is only about one third of this. In some areas, the potential distribution area is far from realized, and restoration efforts are needed to assist recovery. Such efforts are challenging, and resource-demanding and careful site selection is, therefore, important. In the present study, we aim to identify the connectivity of eelgrass populations as a basis for guiding site selection for restoration. We developed a coupled biophysical model to study eelgrass dispersal in the Kattegat. Partly submerged particles simulated the dispersal of reproductive eelgrass shoots containing seeds during the flowering season July–September. We then used network analysis to identify the potential connectivity between populations. We evaluated connectivity based on In-strength, Betweenness and Eigenvector centrality metrics and identified key areas in the Kattegat such as the central part of Aalborg Bay, to be considered to restore the network of Z. marina patches. The study proves the potentials of combining hydrodynamic models and network analysis to support marine conservation and planning, and highlights the importance of collaboration between ecologists, oceanographers, and practitioners in this endeavour.

Land-use and agriculture in Denmark around year 1900 and the quest for EU Water Framework Directive reference conditions in coastal waters

The EU Water Framework Directive (WFD) aims to protect the ecological status of coastal waters. To establish acceptable boundaries between good and moderate ecological status, the WFD calls for reference conditions practically undisturbed by human impact. For Denmark, the nitrogen (N) concentrations present around year 1900 have been suggested to represent reference conditions. As the N load of coastal waters relates closely to runoff from land, any reduction in load links to agricultural activity. We challenge the current use of historical N balances to establish WFD reference conditions and initiate an alternative approach based on parish-level land-use statistics collected 1896/1900 and N concentrations in root zone percolates from experiments with year 1900-relevant management. This approach may be more widely applicable for landscapes with detailed historic information on agricultural activity. Using this approach, we find an average N concentration in root zone percolates that is close to that of current agriculture. Thus, considering Danish coastal waters to be practically unaffected by human activity around year 1900 remains futile as 75% of the land area was subject to agricultural activity with a substantial potential for N loss to the environment. It appears unlikely that the ecological state of coastal waters around year 1900 may serve as WFD reference condition.

Revisiting the toxin profile of Alexandrium pseudogonyaulax; Formation of a desmethyl congener of goniodomin A

During a survey of the production of goniodomin A (GDA) by Alexandrium pseudogonyaulax in Danish coastal waters, Krock et al. (2018) obtained mass spectral evidence for the presence of a truncated congener, herein termed GD754, having a molecular weight 14 Da lower than GDA and assigned it as goniodomin B (GDB). An erroneous structure of GDB involving deletion of a methylene group between rings B and D had previously been reported by Espiña et al. (2016) but without experimental details. HPLC properties reported by Krock for GD754 point to it being a homolog of GDA. Comparison of mass spectral fragmentation data reported for GD754 with fragmentation data for GDA, show it to be a truncated form of GDA with the deletion involving a CH2 group from ring F or one of the two methyl substituents on ring F, not elsewhere on the molecule. On biosynthetic grounds, the GD754 congener is proposed to be 34-desmethyl-GDA. Further experimental work will be required to confirm this hypothesis.

Occurrence and distribution of Amphidomataceae (Dinophyceae) in Danish coastal waters of the North Sea, the Limfjord and the Kattegat/Belt area

Some species of the dinophytes Azadinium and Amphidoma (Amphidomataceae) produce azaspiracids (AZA), lipophilic polyether compounds responsible for Azaspiracid Shellfish Poisoning (AZP) in humans after consumption of contaminated seafood. Toxigenic Amphidomataceae are known to occur in the North Atlantic and the North Sea area, but little is known about their importance in Danish coastal waters. In 2016, 44 Stations were sampled on a survey along the Danish coastline, covering the German Bight, Limfjord, the Kattegat area, Great Belt and Kiel Bight. Samples were analysed by live microscopy, liquid chromatography-tandem mass spectrometry (LC–MS/MS) and quantitative polymerase-chain-reaction (qPCR) on the presence of Amphidomataceae and AZA. Amphidomataceae were widely distributed in the area, but were below detection limit on most of the inner Limfjord stations. Cell abundances of the three toxigenic species, determined with species-specific qPCR assays on Azadinium spinosum, Az. poporum and Amphidoma languida, were generally low and restricted to the North Sea and the northern Kattegat, which was in agreement with the distribution of the generally low AZA abundances in plankton samples. Among the toxigenic species, Amphidoma languida was dominant with highest cell densities up to 3 × 103 cells L−1 on North Sea stations and at the western entrance of the Limfjord. Azaspiracids detected in plankton samples include low levels of AZA-1 at one station of the North Sea, and higher levels of AZA-38 and -39 (up to 1.5 ng L−1) in the North Sea and the Limfjord entrance. Furthermore, one new AZA (named AZA-63) was discovered in plankton of two North Sea stations. Morphological, molecular, and toxinological characterisation of 26 newly established strains from the area confirmed the presence of four amphidomatacean species (Az. obesum, Az. dalianense, Az. poporum and Am. languida). The single new strain of Az. poporum turned out as a member of Ribotype A2, which was previously only known from the Mediterranean. Consistent with some of these Mediterranean A2 strains, but different to the previously established AZA-37 producing Az. poporum Ribotype A1 strains from Denmark, the new strain did not contain any AZA. Azaspiracids were also absent in all Az. obesum and Az. dalianense strains, but AZA-38 and -39 were found in all Am. languida strains with total AZA cell quotas ranging from 0.08 up to 94 fg cell−1. In conclusion, AZA-producing microalgae and their respective toxins were low in abundance but widely present in the area, and thus might be considered in local monitoring programs to preserve seafood safety in Danish coastal waters.

Habitat Model of Eelgrass in Danish Coastal Waters: Development, Validation and Management Perspectives

During the last century, eutrophication significantly reduced the depth distribution and density of the habitat forming eelgrass meadows (Zostera marina) in Danish coastal waters. Despite large reductions in nutrient loadings and improved water quality, Danish eelgrass meadows are currently not as widely distributed as expected from improvements in water clarity alone. This point to the importance of other environmental conditions such as sediment quality, wave exposure, oxygen conditions and water temperature that may limit eelgrass growth and contribute to constraining current distributions. Recently, detailed local models have been set up to evaluate the importance of such regulating factors in selected Danish coastal areas, but nationwide maps of eelgrass distribution and large-scale evaluations of regulating factors are still lacking. To provide such nationwide information, we applied a spatial habitat GIS modeling approach, which combines information on six key eelgrass habitat requirements (light availability, water temperature, salinity, frequency of low oxygen concentration, wave exposure and sediment type) for which we were able to obtain national coverage. The modeled potential current distribution area of Danish eelgrass meadows was 2204 km2 compared to historical estimates of around 7000 km2, indicating a great potential for further distribution. While validating the modeled eelgrass distribution area in three areas (83-111 km2) that hold large eelgrass meadows, we found an agreement of 67% with in situ monitoring data and 77% for eelgrass areas as identified from summer orthophotos. The GIS model predicted higher coverage especially in shallow waters and near the depth limits. Areas of disagreement between GIS-modelled and observed coverage generally exhibited higher exposure level, mean summer temperature and salinity compared to areas of agreement. A sensitivity analysis showed that the modeled area distribution of eelgrass was highly sensitive to light conditions, with 18 to 38% increase in coverage following an increase in light availability of 20%. Modelled coverage of eelgrass was also sensitive to wave exposure and temperature conditions while less sensitive to changes in oxygen and salinity conditions. Large regional differences in habitat conditions suggest spatial variation in the factors currently limiting the recovery of eelgrass and, hence, variations in actions required for sustainable management.

Organoclastic sulfate reduction in the sulfate-methane transition of marine sediments

Abstract Sulfate and methane diffuse vertically along opposed concentration gradients into the sulfate-methane transition in subsurface marine sediments where anaerobic oxidation of methane with sulfate takes place. The stoichiometry of this process is 1:1, yet the calculated sulfate flux often exceeds the calculated methane flux. Our aim was to determine whether organoclastic sulfate reduction, fueled by the oxidation of buried organic matter, explains this excess sulfate flux into the sulfate-methane transition. We analyzed data for sulfate, methane and sulfate reduction from eight sites in Danish coastal waters. Fluxes of sulfate and methane were determined by diffusion-reaction modeling whereas sulfate reduction rates were determined by 35S radiotracer experiments at high depth resolution. The sulfate reduction data showed that the organic carbon mineralization rates followed a log-log linear depth trend throughout the sulfate zone. Extrapolation of this power law trend down into the sulfate-methane transition showed organic carbon oxidation corresponded to 14–59% of the total sulfate flux into the zone and in average explained 82% of the excess sulfate consumption above the 1:1 stoichiometry. A part of the excess sulfate was consumed directly by organoclastic sulfate reduction while a part drove a cryptic methane cycle by which the organic matter was degraded to methane and the methane concurrently oxidized to CO2 with sulfate. We extrapolated the same power law trend down through the methanogenic zone and found that this provided a good estimate of total methanogenesis in the sediment column when compared to the upward flux of methane.

Development of a LC-MS/MS method for the quantification of goniodomins A and B and its application to Alexandrium pseudogonyaulax strains and plankton field samples of Danish coastal waters

An external standard of goniodomin A (GDA) was prepared from a strain of Alexandrium pseudogonyaulax originating from New Zealand and its chemical structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. Using the GDA standard, an ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method in selected reaction monitoring (SRM) mode was developed for separation and quantification of GDA. This method was successfully applied to planktonic field samples collected during an oceanographic expedition conducted with R/V Uthörn along the Danish west coast, Limfjord and Kattegat in June 2016. In addition, this method was used to characterize goniodomin (GD) profiles of 17 A. pseudogonyaulax strains from the coastal North Sea and from Limfjord. Highest GDA levels were found in Limfjord (up to 590 ng NT−1 m−1), but GDA was also detected in the North Sea appearing at the latitude of Sylt Island northwards and in Kattegat from the eastern mouth of Limfjord down to the Kiel Bight, but at lower abundances than within Limfjord. This is the first reported detection of GDA in planktonic field samples. Chemical analysis of 17 strains of A. pseudogonyaulax revealed that all strains were producers of GDA (5–35 pg cell−1) as well as in most cases minor amounts (0.01–0.07 pg cell−1, expressed as GDA equivalents) of goniodomin B (GDB).

Filtration via Conventional Glass Fiber Filters in 15N2 Tracer Assays Fails to Capture All Nitrogen-Fixing Prokaryotes

Biological dinitrogen fixation (BNF) represents a major input of reduced nitrogen (N) to the oceans. Accurate direct measurements of BNF rates are crucial for reliably determining the biogeochemical significance of diazotrophy at local and global scales. Traditionally, borosilicate glass fiber filters (GF/F, Whatman) with a nominal pore size of 0.7 µm are used to collect suspended particles by filtration after incubations with added 15N2 tracer. We carried out BNF experiments in the Baltic Sea, Danish coastal waters, and the Pacific Ocean comparing the retentive characteristics of precombusted GF/F filters with newer Advantec glass fiber filters which have a smaller nominal pore size of 0.3 µm. Where BNF was detected, rates were nearly always higher, and sometimes even exclusively detectable, when using Advantec filters. In the majority of samples across tested habitats, significantly more cells were lost to GF/F filtrate (average = 51 %, range = 10 – 70 % of cells) than to Advantec filtrate (average = 40 %, range = 10 – 54 %). Using Illumina sequencing of nitrogenase (nifH) gene amplicons, we show that diazotroph communities can markedly differ between bulk water and filtrates from GF/F and Advantec filtrations, suggesting that different diazotrophs can pass through the filter types. In order to reduce the potential underestimations of BNF due to filtration loss of diazotrophs, we recommend using Advantec filters or alternatively silver membranes with 0.2 µm pore size, especially in waters expected to be inhabited by relatively small, unicellular diazotrophs.

Modelling stressors on the eelgrass recovery process in two Danish estuaries

Eelgrass (Zostera marina L.) depth limit is used as an environmental indicator in Danish coastal waters in the Water Framework Directive (WFD) to evaluate coastal waters and their ecological condition. Even after decades of reduced nutrient loadings the reestablishment of eelgrass has not yet succeeded. The mechanisms hindering/delaying eelgrass recovery were recently identified: 1) lack of sediment anchoring capacity, 2) resuspension created by drifting ephemeral macroalgae, 3) seedling uprooting created by current and wave forces, 4) ballistic stress from attached macroalgae and 5) burial of seeds and seedlings by lugworms. These processes were quantified and introduced to an ecological MIKE 3D model. The developed model was calibrated and validated on two Danish estuaries, Odense Fjord and Roskilde Fjord. Analyses of the simulations were performed on area distribution maps. The parameterized stressors impact has been investigated over a three-year period.The results indicate accumulated effects from multiple stressors weakening the capability of eelgrass to recolonize. Combining all stressors in the model decreased the total area covered by eelgrass 83.72% in Odense Fjord and 80.30% in Roskilde Fjord compared to simulation without stressors. Eelgrass peak biomass declined in both fjords from 33.4 to 4.55 tonCkm−2 in Odense Fjord and from 24.42 to 5.58tonCkm−2 in Roskilde Fjord. Combining lugworm burial of seeds and seedlings with resuspension from macroalgae and wave forcing had the second strongest negative impact on eelgrass growth, area reduction of 78.31% and 73.14% in Odense and Roskilde Fjord was seen. Ballistic stress from attached macroalgae also reduced growth drastically. Light conditions, sediment organic content along with shear stress at the sediment surface impact the ability of eelgrass to cope with above mentioned stressors. The spatial resolution of the model setup made it possible to generate maps where eelgrass is exposed to lowest stress, revealing areas for potential eelgrass recovery. The developed eelgrass model is now used as a national tool to predict areas where eelgrass restoration effort may be initiated.

Successful reduction of diffuse nitrogen emissions at catchment scale: example from the pilot River Odense, Denmark.

Land-based total nitrogen (N) loadings to Danish coastal waters have been markedly reduced since 2000. This has been achieved by general measures reducing discharges from all point sources and N leaching from farmed land supplemented with more local and targeted mitigation measures such as restoration of wetlands to increase the catchment-specific N retention. In the catchment of River Odense, restoration of wetlands has been extensive. Thus, in the major gauged catchment (485 km(2)) eleven wetlands (860 ha) have been restored since 2000. A comparison of data on N concentrations and loss from a gauging station in the River Odense with data from a control catchment (772 km(2)), in which a significantly less intensive wetland restoration programme has been undertaken, showed an excess downward trend in N, amounting to 124 t N yr(-1), which can be ascribed to the intensive wetland restoration programme carried out in the River Odense catchment. In total, the N load in the River Odense has been reduced by 377 t N yr(-1) (39%) since 2000. The observed downward trend is supported by monitoring data from two wetlands restored in 2001 and 2004 in the River Odense catchment.

Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species

Harmful algal blooms (HAB) pose serious economic and health risks worldwide. Current methods of identification require high levels of taxonomic skill and can be highly time-consuming thus limiting sample throughput. So, new rapid and reliable methods for detection and enumeration of HAB species are required. Here we describe a high-throughput, multiplex-qPCR (M-qPCR) method using hydrolysis probe technology for the simultaneous detection of four HAB species commonly found in many coastal areas worldwide: Alexandrium tamarense, Karenia mikimotoi, Karlodinium veneficum and Prymnesium parvum. Primers and probes were species-specific and highly efficient when tested in simplex. Species were then added in succession and the assay conditions adjusted until all four species could be quantitatively evaluated simultaneously. Enumeration accuracy of the M-qPCR assay as a monitoring tool was evaluated using spiked natural environmental samples from Danish coastal waters. Comparison of estimates of cell abundances obtained by the M-qPCR technique with those obtained by light microscopy (Sedgwick Rafter technique) showed no statistically significant difference across a range of concentrations. We were also able to identify and enumerate target cells that would be below the detection limit of light microscopy making this a suitable method for early bloom detection or for low biomass species. With the development of molecular probes for a greater number of algal species M-qPCR will be of great benefit to phytoplankton monitoring programmes and the aquaculture industry worldwide.

Recovery of Danish Coastal Ecosystems After Reductions in Nutrient Loading: A Holistic Ecosystem Approach

In the 1980s, Danish coastal waters suffered from eutrophication and several nutrient management plans have been implemented during the years to improve ecological status. This study aims at giving a holistic ecosystem perspective on 25 years of mitigation measures. We report trends of nutrient inputs and the responses to these in various chemical and biological components. Nutrient inputs from land were reduced by ~50 % for nitrogen (N) and 56 % for phosphorus (P) since 1990. These reductions resulted in significant and parallel declines in nutrient concentrations, and initiated a shift in the dominance of primary producers towards reduced phytoplankton biomass (chlorophyll a concentration) and increased cover of macroalgae in deeper waters. In the last 5 years, eelgrass meadows have also expanded towards deeper waters, in response to improving water clarity. An expected improvement of bottom water oxygen conditions has not been observed, presumably because more frequent stratification and higher water temperatures have counteracted the expected positive effects of reduced nutrient inputs. The biomass of the benthic macrofauna decreased as expected, but it was composed of a drastic decline of filter feeders paralleled by a more moderate increase of deposit feeders. This shift was most likely induced by increasing stratification. The reduced benthic filtration along with the limited eelgrass cover probably kept relatively more particles in suspension, which can explain why improvements in the Secchi depths were modest. Overall, several ecosystem components demonstrated clear signs of improvement, suggesting that at least partial recovery is attainable. On this basis, we propose a conceptual scheme for recovery of shallow coastal ecosystems following marked reductions in nutrient inputs.

Background concentrations and loadings of nitrogen in Danish surface waters

Quantitative knowledge of background nitrogen (N) concentrations and loadings is essential for as it determines the minimally disturbed conditions for N in surface waters as a near reference condition. To determine background N concentrations, a total of 39 smaller Danish streams draining relatively undisturbed catchments in different regions were selected and screened for N concentrations and discharge during a 1-year period (2004–2005). Only 19 of the streams fulfilled the threshold set for least disturbed conditions (LDC) in their catchments (proportion of agricultural land <10%). Within the five sampled dominant landscape types in Denmark, the concentrations of ammonium N and total organic N in the LDC streams were found to be nearly constant: 0.05 ± 0.01 mg N L−1 and 0.53 ± 0.07 mg N L−1, respectively. In contrast, the concentration of nitrate N differed significantly (P < 0.05) between the five landscape types, with variations between 0.06 and 0.83 mg N L−1. An analysis of all the 39 streams sampled demonstrated significantly different relationships between the proportion of land use and the flow-weighted annual concentration of nitrate N for two dominant soil types with a Y-axis intercept equal to 0.12 mg N L−1 for streams draining coarse textural soils and 0.76 mg N L−1 for streams draining finer textural soils. A significant (P < 0.05) downward trend (18–41%) emerged for flow-weighted annual total N concentrations in four of the five streams that were sampled during the period 1990–2011. A 5 × 5 km grid map of Denmark showing nitrate N and total N background concentrations was produced and used for estimating background N losses to Danish surface waters. Based on the grid map, the average annual background loss of total N was calculated to be 13,000 tonnes N or 19% of the total N loading to Danish coastal waters during the period 2004–2005.

Reduced Cover of Drifting Macroalgae Following Nutrient Reduction in Danish Coastal Waters

Based on a large dataset from the national Danish monitoring programme, we analysed the temporal variability of drifting algae cover in shallow (1–2 m) water during a period of reduced nutrient loadings. Algal cover was analysed both in absolute terms and relative to eelgrass, Zostera marina, cover to test the hypotheses that (1) the cover of drifting algae and the relative dominance of algae versus eelgrass in shallow waters have declined in parallel to reductions in nutrient levels during the last decades, and (2) spatio-temporal differences in algal cover can be related to differences in nutrient conditions and environmental characteristics. The cover of drifting algae was positively related to total nitrogen concentration and Secchi depth but negatively related to exposure, salinity and mean summer temperature. The cover of drifting macroalgae showed significant declines over the past two decades, paralleling the reduction in nutrient concentrations. The present cover of drifting algae is low (<10 %) and probably pose little threat to the general distribution of eelgrass in shallow Danish waters though local accumulations may still be harmful. However, the ratio between drifting algae cover and eelgrass cover showed no significant trend, reflecting that eelgrass cover had not increased despite the reduced levels of nutrients and drifting algae. This ratio also showed no consistent relationship to water quality probably because different regulation mechanisms govern drifting algae and eelgrass, and feedback mechanisms may delay eelgrass recovery. Reduced drift algal cover may be an early sign of improved ecological status, while further improvements in terms of recovery of eelgrass meadows have longer perspectives.

A new highly sensitive method to assess respiration rates and kinetics of natural planktonic communities by use of the switchable trace oxygen sensor and reduced oxygen concentrations.

Oxygen respiration rates in pelagic environments are often difficult to quantify as the resolutions of our methods for O2 concentration determination are marginal for observing significant decreases during bottle incubations of less than 24 hours. Here we present the assessment of a new highly sensitive method, that combine Switchable Trace Oxygen (STOX) sensors and all-glass bottle incubations, where the O2 concentration was artificially lowered. The detection limit of respiration rate by this method is inversely proportional to the O2 concentration, down to <2 nmol L−1 h−1 for water with an initial O2 concentration of 500 nmol L−1. The method was tested in Danish coastal waters and in oceanic hypoxic waters. It proved to give precise measurements also with low oxygen consumption rates (∼7 nmol L−1 h−1), and to significantly decrease the time required for incubations (≤14 hours) compared to traditional methods. This method provides continuous real time measurements, allowing for a number of diverse possibilities, such as modeling the rate of oxygen decrease to obtain kinetic parameters. Our data revealed apparent half-saturation concentrations (Km values) one order of magnitude lower than previously reported for marine bacteria, varying between 66 and 234 nmol L−1 O2. Km values vary between different microbial planktonic communities, but our data show that it is possible to measure reliable respiration rates at concentrations ∼0.5–1 µmol L−1 O2 that are comparable to the ones measured at full air saturation.