Dissolved nitrogen in soil solution of the Jena Experiment (Main Experiment, year 2004)

This data set contains measurements of dissolved nitrogen (total dissolved nitrogen: TDN, dissolved organic nitrogen: DON, dissolved ammonium: NH4+, and dissolved nitrate: NO3-) in samples of soil water collected from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. In April 2002 glass suction plates with a diameter of 12 cm, 1 cm thickness and a pore size of 1-1.6 µm (UMS GmbH, Munich, Germany) were installed in depths of 10, 20, 30 and 60 cm to collect soil solution. The sampling bottles were continuously evacuated to a negative pressure between 50 and 350 mbar, such that the suction pressure was about 50 mbar above the actual soil water tension. Thus, only the soil leachate was collected. Cumulative soil solution was sampled biweekly and analyzed for nitrate (NO3-) and ammonium (NH4+) concentrations with a continuous flow analyzer (CFA, Skalar, Breda, The Netherlands). Nitrate was analyzed photometrically after reduction to NO2- and reaction with sulfanilamide and naphthylethylenediamine-dihydrochloride to an azo-dye. Our NO3- concentrations contained an unknown contribution of NO2- that is expected to be small. Simultaneously to the NO3- analysis, NH4+ was determined photometrically as 5-aminosalicylate after a modified Berthelot reaction. The detection limits of NO3- and NH4+ were 0.02 and 0.03 mg N L-1, respectively. Total dissolved N in soil solution was analyzed by oxidation with K2S2O8 followed by reduction to NO2- as described above for NO3-. Dissolved organic N (DON) concentrations in soil solution were calculated as the difference between TDN and the sum of mineral N (NO3- + NH4+). In 5% of the samples, TDN was equal to or smaller than mineral N. In these cases, DON was assumed to be zero.

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.

(Table 1) Concentrations of neutral and phenolic organohalogen compounds in adipose tissue, blood, brain and liver of polar bears (Ursus maritimus) from East Greenland

The tissue-specific composition of sum classes of brominated and chlorinated contaminants and metabolic/degradation byproducts was determined in adult male and female polar bears from East Greenland. Significantly (p < 0.05) higher concentrations of SUM-PCBs, various other organochlorines such as SUM-CHL, p,p'-DDE, SUM-CBz, SUM-HCHs, octachlorostyrene (OCS),SUM-mirex, dieldrin, the flame retardants SUM-PBDEs, and total-(R)-hexabromocyclododecane (HBCD), SUM-methylsulfonyl (MeSO2)-PCBs and 3-MeSO2-p,p'-DDE, were found in the adipose and liver tissues relative to whole blood and brain. In contrast, SUM-hydroxyl (OH)-PCB, 4-OH-heptachlorostyrene and SUM-OH-PBDE concentrations were significantly highest (p < 0.05) in whole blood, whereas the highest concentrations of SUM-OH-PBBs were found in the adipose tissue. Based on the total concentrations of all organohalogens in all three tissues and blood, the combined body burden was estimated to be 1.34 ± 0.12 g, where >91% of this amount was accounted for by the adipose tissue alone, followed by the liver, whole blood, and brain. These results show that factors such as protein association and lipid solubility appear to be differentially influencing the toxicokinetics, in terms of tissue composition/localization and burden, of organohalogen classes with respect to chemical structure and properties such as the type of halogenation (e.g., chlorination or bromination), and the presence or absence of additional phenyl group substituents (e.g., MeO and OH groups). The tissue- and blood-specific accumulation (or retention) among organohalogen classes indicates that exposure and any potential contaminant-mediated effects in these polar bears are likely tissue or blood specific.

A sea urchin Na+K+2Cl- cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae

The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na+K+2Cl- cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50 = 6.5 µM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50 = 26.4 µM) and furosemide (IC50 = 315.4 µM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.

Calcareous dinoflagellate cysts analysis of sediment cores from the Equatorial South Atlantic Ocean

An overview is presented of the current state of knowledge on paleo-ecological aspects of calcareous dinoflagellate resting cysts. Apart from literature-based information, a discussion of new results is also provided from Equatorial Atlantic surface plankton samples, surface sediment samples and Late Quaternary sediments from two gravity cores. With the aid of redundancy analysis statistics, variations in the calcareous cyst content of both cores are correlated to variations in total organic carbon (TOC). On a global scale, the calcareous cyst distribution in bottom sediments varies with latitude and inshore-offshore gradients. In the Equatorial Atlantic Ocean, enhanced calcareous cyst production can be observed in regions and time intervals with stratified, oligotrophic conditions in the upper water masses.

Collection of data on particulate and dissolved soil nitrogen in the Jena Experiment (Main Experiment, time series since 2002)

This data set contains four time series of particulate and dissolved soil nitrogen measurements from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. 1. Total nitrogen from solid phase: Stratified soil sampling was performed every two years since before sowing in April 2002 and was repeated in April 2004, 2006 and 2008 to a depth of 30 cm segmented to a depth resolution of 5 cm giving six depth subsamples per core. In 2002 five samples per plot were taken and analyzed independently. Averaged values per depth layer are reported. In later years, three samples per plot were taken, pooled in the field, and measured as a combined sample. Sampling locations were less than 30 cm apart from sampling locations in other years. All soil samples were passed through a sieve with a mesh size of 2 mm in 2002. In later years samples were further sieved to 1 mm. No additional mineral particles were removed by this procedure. Total nitrogen concentration was analyzed on ball-milled subsamples (time 4 min, frequency 30 s-1) by an elemental analyzer at 1150°C (Elementaranalysator vario Max CN; Elementar Analysensysteme GmbH, Hanau, Germany). 2. Total nitrogen from solid phase (high intensity sampling): In block 2 of the Jena Experiment, soil samples were taken to a depth of 1m (segmented to a depth resolution of 5 cm giving 20 depth subsamples per core) with three replicates per block ever 5 years starting before sowing in April 2002. Samples were processed as for the more frequent sampling but were always analyzed independently and never pooled. 3. Mineral nitrogen from KCl extractions: Five soil cores (diameter 0.01 m) were taken at a depth of 0 to 0.15 m (and between 2002 and 2004 also at a depth of 0.15 to 0.3 m) of the mineral soil from each of the experimental plots at various times over the years. In addition also plots of the management experiment, that altered mowing frequency and fertilized subplots (see further details below) were sampled in some later years. Samples of the soil cores per plot (subplots in case of the management experiment) were pooled during each sampling campaign. NO3-N and NH4-N concentrations were determined by extraction of soil samples with 1 M KCl solution and were measured in the soil extract with a Continuous Flow Analyzer (CFA, 2003-2005: Skalar, Breda, Netherlands; 2006-2007: AutoAnalyzer, Seal, Burgess Hill, United Kingdom). 4. Dissolved nitrogen in soil solution: Glass suction plates with a diameter of 12 cm, 1 cm thickness and a pore size of 1-1.6 µm (UMS GmbH, Munich, Germany) were installed in April 2002 in depths of 10, 20, 30 and 60 cm to collect soil solution. The sampling bottles were continuously evacuated to a negative pressure between 50 and 350 mbar, such that the suction pressure was about 50 mbar above the actual soil water tension. Thus, only the soil leachate was collected. Cumulative soil solution was sampled biweekly and analyzed for nitrate (NO3-), ammonium (NH4+) and total dissolved nitrogen concentrations with a continuous flow analyzer (CFA, Skalar, Breda, The Netherlands). Nitrate was analyzed photometrically after reduction to NO2- and reaction with sulfanilamide and naphthylethylenediamine-dihydrochloride to an azo-dye. Our NO3- concentrations contained an unknown contribution of NO2- that is expected to be small. Simultaneously to the NO3- analysis, NH4+ was determined photometrically as 5-aminosalicylate after a modified Berthelot reaction. The detection limits of NO3- and NH4+ were 0.02 and 0.03 mg N L-1, respectively. Total dissolved N in soil solution was analyzed by oxidation with K2S2O8 followed by reduction to NO2- as described above for NO3-. Dissolved organic N (DON) concentrations in soil solution were calculated as the difference between TDN and the sum of mineral N (NO3- + NH4+).

Dissolved nitrogen in soil solution of the Jena Experiment (Main Experiment, year 2002)

This data set contains measurements of dissolved nitrogen (total dissolved nitrogen: TDN, dissolved organic nitrogen: DON, dissolved ammonium: NH4+, and dissolved nitrate: NO3-) in samples of soil water collected from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. In April 2002 glass suction plates with a diameter of 12 cm, 1 cm thickness and a pore size of 1-1.6 µm (UMS GmbH, Munich, Germany) were installed in depths of 10, 20, 30 and 60 cm to collect soil solution. The sampling bottles were continuously evacuated to a negative pressure between 50 and 350 mbar, such that the suction pressure was about 50 mbar above the actual soil water tension. Thus, only the soil leachate was collected. Cumulative soil solution was sampled biweekly and analyzed for nitrate (NO3-) and ammonium (NH4+) concentrations with a continuous flow analyzer (CFA, Skalar, Breda, The Netherlands). Nitrate was analyzed photometrically after reduction to NO2- and reaction with sulfanilamide and naphthylethylenediamine-dihydrochloride to an azo-dye. Our NO3- concentrations contained an unknown contribution of NO2- that is expected to be small. Simultaneously to the NO3- analysis, NH4+ was determined photometrically as 5-aminosalicylate after a modified Berthelot reaction. The detection limits of NO3- and NH4+ were 0.02 and 0.03 mg N L-1, respectively. Total dissolved N in soil solution was analyzed by oxidation with K2S2O8 followed by reduction to NO2- as described above for NO3-. Dissolved organic N (DON) concentrations in soil solution were calculated as the difference between TDN and the sum of mineral N (NO3- + NH4+). In 5% of the samples, TDN was equal to or smaller than mineral N. In these cases, DON was assumed to be zero.

Dissolved nitrogen in soil solution of the Jena Experiment (Main Experiment, year 2003)

This data set contains measurements of dissolved nitrogen (total dissolved nitrogen: TDN, dissolved organic nitrogen: DON, dissolved ammonium: NH4+, and dissolved nitrate: NO3-) in samples of soil water collected from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. In April 2002 glass suction plates with a diameter of 12 cm, 1 cm thickness and a pore size of 1-1.6 µm (UMS GmbH, Munich, Germany) were installed in depths of 10, 20, 30 and 60 cm to collect soil solution. The sampling bottles were continuously evacuated to a negative pressure between 50 and 350 mbar, such that the suction pressure was about 50 mbar above the actual soil water tension. Thus, only the soil leachate was collected. Cumulative soil solution was sampled biweekly and analyzed for nitrate (NO3-) and ammonium (NH4+) concentrations with a continuous flow analyzer (CFA, Skalar, Breda, The Netherlands). Nitrate was analyzed photometrically after reduction to NO2- and reaction with sulfanilamide and naphthylethylenediamine-dihydrochloride to an azo-dye. Our NO3- concentrations contained an unknown contribution of NO2- that is expected to be small. Simultaneously to the NO3- analysis, NH4+ was determined photometrically as 5-aminosalicylate after a modified Berthelot reaction. The detection limits of NO3- and NH4+ were 0.02 and 0.03 mg N L-1, respectively. Total dissolved N in soil solution was analyzed by oxidation with K2S2O8 followed by reduction to NO2- as described above for NO3-. Dissolved organic N (DON) concentrations in soil solution were calculated as the difference between TDN and the sum of mineral N (NO3- + NH4+). In 5% of the samples, TDN was equal to or smaller than mineral N. In these cases, DON was assumed to be zero.