Effects of fish farm loading on sea grass Posidonia oceanica at Vrgada Island (Central Adriatic): a nitrogen stable isotope study

Analyses of nitrogen stable isotopes in the marine sea grass Posidonia oceanica were used to investigate the influence of fish farming on the coastal ecosystem of Vrgada Island in the Murter Sea, Central Adriatic. The results show a statistically significant ¹⁵N enrichment (up to 4.7‰ at p?<?0.005) in P. oceanica leaf and shoot tissues from fish cage sites with respect to the unaffected offshore reference site of Lumbarda Reef Flat (Kornati Islands). Heavy nitrogen enrichment was also detected in other benthic organisms analysed during this study and is attributed to the absorption and assimilation of ¹⁵N-enriched fish farm derived nitrogen waste.     

Fish host-cestode parasite stable isotope enrichment patterns in marine, estuarine and freshwater fishes from Northern Canada

Cestode parasites from freshwater (threespine stickleback, Gasterosteus aculeatus), estuarine (brook charr, Salvelinus fontinalis) and marine (Greenland cod, Gadus ogac) fish from northern Quebec, Canada, were used to investigate the hypotheses that cestode parasites are (13)C and (15)N enriched relative to host food sources, but (15)N depleted with respect to host muscle tissue as a result of differential enrichment during the assimilation of common nutrient sources. Cestode parasites and fish were generally similarly enriched in (13)C with respect to common food sources and, in the case of Greenland cod, cestode parasites were (13)C enriched relative to host tissue. Cestode parasites were also generally (15)N enriched with respect to mean host dietary signatures, but depleted with respect to host muscle tissue. In the case of Greenland cod cestode parasites, no significant (15)N enrichment relative to host dietary signature was observed. Cestode parasites appear generally to experience smaller (15)N enrichment than hosts as a result of trophic transfer of common dietary sources. Differential (15)N enrichment patterns in parasites and fish may be attributed to differences in parasite and host metabolism, particularly the anaerobic and aerobic natures of their respective metabolisms. Results imply that isotope enrichment paradigms developed for the study of aquatic foodwebs cannot be routinely applied to quantitatively assess the role of parasites in aquatic foodwebs and that reference to host muscle tissue measures will not allow accurate characterization of parasite foodweb position. Appropriate reference to assimilated food sources is required to accurately estimate parasite isotopic enrichment patterns and to determine parasite trophic position relative to the host.     

Enrichment of the nitrogen atomic component with the <Superscript>15</Superscript>N isotope in a post-discharge zone

Isotope atomic composition in the post-discharge zone of a pulsed discharge in a nitrogen flow was studied by electron paramagnetic resonance. It is shown that, while the atomic concentration in the post-discharge zone decreases, the relative content of the ¹⁵N isotope increases more than 30 times compared to its natural abundance. Such a high isotope enrichment exceeds more than fourfold the corresponding maximal value that is predicted for the nitrogen atoms by the existing theoretical model. Analysis of the experimental results shows that the isotope-selective dissociation of nitrogen molecules proceeds in the post-discharge zone after the most part of atoms created in the discharge zone had recombined at the tube surface. A mechanism that explains that a nitrogen isotope enrichment as high as that is quite possible is proposed.     

Aufnahme von 15NO2 und Einbau des 15NO2-Stickstoffs in verschiedene Stickstofffraktionen bei Sonnenblumen (Uptake of 15NO2 and Metabolic Transfer of the 15NO2 Nitrogen to Various Nitrogen Fractions of Sunflowers)

Abstract

Sunflowers were exposed to ¹⁵NO₂ at a range between 4,9 and 42,0 ppb. The ¹⁵NO₂ uptake was quantified and the fate of the ¹⁵N in different parts and pools of nitrogen was investigated. The very high δ¹⁵N-values of the free amino acid pool can't give an answer to the question whether the NO₂-nitrogen is incorporated by a different pathway, compared to the nitrogen which derives from root uptake. Or does a compartmentation of the nitrate pool in the plant cell cause a dilution of the ¹⁵N enrichment in the mineral nitrogen pool during sample preparation?     

Assignment of congested NMR spectra: carbonyl backbone enrichment via the Entner-Doudoroff pathway

In NMR spectra of complex proteins, sparse isotope enrichment can be important, in that the removal of many (13)C-(13)C homonuclear J-couplings can narrow the lines and thereby facilitate the process of spectral assignment and structure elucidation. We present a simple scheme for selective yet extensive isotopic enrichment applicable for production of proteins in organisms utilizing the Entner-Doudoroff (ED) metabolic pathway. An enrichment scheme so derived is demonstrated in the context of a magic-angle spinning solid-state NMR (MAS SSNMR) study of Pf1 bacteriophage, the host of which is Pseudomonas aeruginosa, strain K (PAK), an organism that uses the ED pathway for glucose catabolism. The intact and infectious Pf1 phage in this study was produced by infected PAK cells grown on a minimal medium containing 1-(13)C d-glucose ((13)C in position 1) as the sole carbon source, as well as (15)NH(4)Cl as the only nitrogen source. The 37MDa Pf1 phage consists of about 93% major coat protein, 1% minor coat proteins, and 6% single-stranded, circular DNA. As a consequence of this composition and the enrichment scheme, the resonances in the MAS SSNMR spectra of the Pf1 sample were almost exclusively due to carbonyl carbons in the major coat protein. Moreover, 3D heteronuclear NCOCX correlation experiments also show that the amino acids leucine, serine, glycine, and tyrosine were not isotopically enriched in their carbonyl positions (although most other amino acids were), which is as expected based upon considerations of the ED metabolic pathway. 3D NCOCX NMR data and 2D (15)N-(15)N data provided strong verification of many previous assignments of (15)N amide and (13)C carbonyl shifts in this highly congested spectrum; both the semi-selective enrichment patterns and the narrowed linewidths allowed for greater certainty in the assignments as compared with use of uniformly enriched samples alone.     

Stable isotopes in animal ecology: the effect of ration size on the trophic shift of C and N isotopes between feed and carcass

The feeding ecology of living or extinct animal species is now frequently studied by analysis of stable isotope ratios in small quantities of carcass or remains such as bones or teeth. Although there are numerous papers on these applications in natural systems, the theoretical and experimental basis of this method is weak. In order to evaluate the effect of different feeding levels on the carbon and nitrogen trophic shift, an experiment was carried out in which fish (Nile tilapia, Oreochromis niloticus, initial weight 40.8 g) were fed for 4 weeks at three levels from slightly above maintenance to almost satiation. For each treatment, three fish were reared individually. The isotopic ratios of carbon and nitrogen in feeds and fish carcasses were determined and in the case of carbon this was done separately for lipids and lipid-free matter. The trophic shift was calculated at each feeding level from the delta13C and delta15N ratios of feed and fish. There was a significant trend towards higher values for the trophic shift at higher feeding rates in all fractions analysed. Although further research is required, it can be concluded that the effect of feeding level cannot be ignored when the diet of an animal has to be calculated from the ratios of isotopes in its body.     

Elemental Analyzer-Quadrupole MS Coupling—A Low Cost Equipment for the Simultaneous Determination of Carbon/13C and Nitrogen/15N in Isotopically Enriched Soil and Plant Samples

Abstract

Since the end of the 80s elemental analyzer-isotope ratio mass spectrometer connections have been used for the fast, automatic and highly precise determination of carbon and nitrogen content as well as their isotopic composition in one run. But for artificially enriched stable isotopes as tracer in biological processes and since these processes have a high biological variability anyway (e.g. soil processes) the use of these highly precise but also sophisticated and expensive instruments is not required. In this case the use of a quadrupole mass spectrometer connected with an elemental analyzer can offer a low cost alternative. As shown, such coupling is suitable for automatic simultaneous routine analysis of total nitrogen and carbon and their isotopic enrichment (¹⁵N, ¹³C) in plant material and soils. The relative standard deviation for ¹⁵N and ¹³C determination is 2% To meet this precision a careful sample homogenization by grinding is very important. The duration of one measurement is 6–8 min. depending on whether nitrogen alone or both nitrogen and carbon are determined. This enables a high sample throughput.     

Plant delta(15)N associated with arbuscular mycorrhization, drought and nitrogen deficiency

It has long been evident that plant (15)N chiefly reflects the processes which fractionate (15)N/(14)N rather than the (15)N of plant N source(s). It has emerged recently that one of the most important fractionating processes contributing to the whole plant (15)N is the presence/absence, type or species of mycorrhiza, especially when interacting with nutrient deficiency. Ecto- and ericoid mycorrhizas are frequently associated with (15)N-depleted foliar (15)N, commonly as low as -12 per thousand. As shown by the present study, plants having no mycorrhiza, or those infected with various species of arbuscular mycorrhiza (AM)-forming fungi, interact with varying concentrations of soil nitrogen [N] and moisture to enrich plant (15)N by as much as 3.5 per thousand. Hence the lack of a mycorrhiza, or variation in the species of AM-forming fungal associations, can account for about 25% of the usually reported variations of foliar (15)N found in field situations and do so by (15)N enrichment rather than depletion. Copyright 1999 John Wiley & Sons, Ltd.     

Application of 15N NMR Spectroscopy to Studies of the Intermolecular Interaction of Biomolecules

Abstract

Nitrogen nuclei are frequently located at the interaction sites of biomolecules; for example, amide nitrogens in the peptide are the key to maintaining the peptide backbone conformation by hydrogen bonding. Histidine, tryptophan, and arginine side chains contain nitrogen atoms which are often located at the active sites of enzymes. Heterocyclic compounds like purine and pyrimidine bases are substances essential to information transfer by base pair formation of nucleic acids. Also some other co-factors and dyes, such as flavins, porphyrins and chlorophylls, are nitrogen-containing substances which regulate energy tranduction in biological systems. Lecithin and phosphatidylethanolamine are the principal components of the phospholipids from biomembranes. To detect interaction sites and to study the interaction mechanism of these biomolecules, the use of nitrogen NMR seems promising. Although more than 99% of naturally occurring nitrogen element has the ¹⁴N nuclei, a disadvantage of the use of ¹⁴N magnetic resonance has been recognized. It has a spin quantum number I = 1 and the associated quadrupole moment provides a very broad resonance signal of about 100～1000 Hz. Thus, for detection of small changes of the chemical environment, use of ¹⁴N magnetic resonance is not adequate. The natural abundance of ¹⁵N nuclei with a spin quantum number I = 1/2 (which give a sharp resonance signal) is only 0.3% (Table 1). But recent developments in the instrumentation of NMR spectroscopy have made it possible to observe the resonance of the nuclei with low natural abundance. Fourier transform (FT) NMR can save thousands of times the accumulation time to improve the signal to noise ratio of ¹⁵N spectra [1-3]. Also superconducting magnets with wide bores have made possible the use of thick sample tubes of 25 mmΦ and observation of the ¹⁵N resonance of substances of low solubility [4]. In spite of such instrumental development, the observation of the 15N resonance is still not easy because of its low sensitivity; about of proton magnetic resonance. In the application of ¹⁵N NMR in biological systems, we often encounter quite low solubility of biomacromolecules and also sometimes need to measure the concentration dependency of ¹⁴N chemical shifts. For such experiments, enrichment of ¹⁵N nuclei in the molecules is required. Chemical syntheses starting from the simple ¹⁵N containing compounds as an ¹⁵N source and also biological syntheses by bacterial fermentation using the ¹⁵N source in culture media are employed for ¹⁵N enrichment. Enrichment at specific positions of biomolecules is useful for spectral assignments and also for analyses of the pathways of biosyntheses [5].     

Development of non-lethal sampling of carbon and nitrogen stable isotope ratios in salmonids: effects of lipid and inorganic components of fins

The preferred tissue for analyses of fish stable isotope ratios for most researchers is muscle, the sampling of which typically requires the specimen to be sacrificed. The use of non-destructive methods in fish isotopic research has been increasing recently, but as yet is not a standard procedure. Previous studies have reported varying levels of success regarding the utility of non-lethally obtained stable isotope materials, e.g. fins, but none have accounted for the potential compounding effects of inorganic components of fin rays or lipids. Comparisons of carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope ratios of muscle with adipose and caudal fin of two salmonids, Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.), revealed that caudal fin can be used as a non-destructive surrogate for muscle in stable isotope analysis, but that adipose fin, where available, is a better proxy. The use of a published model to inexpensively counteract the confounding effect of lipids, which are depleted in ¹³C, greatly improved the relationship between fish muscle and fins. However, efforts to account for the inorganic components of fin rays were counterproductive and required twice the biomass of fins clipped from each fish. As this experiment was conducted on wild fish, controlled laboratory studies are required to confirm these field observations.     

Soil delta15N patterns in old-growth forests of southern Chile as integrator for N-cycling

Old-growth forests of southern Chile represent an important reserve of temperate (rain) forests in the world. Wetter and colder forest ecosystems appear to be more efficient in conserving and recycling N such that mostly non-plant available N species are lost, which could be indicated by more depleted delta15N values of the soil and plants. Hydrological N loss from the old-growth forests in southern Chile occurs mainly via dissolved organic nitrogen and not via dissolved inorganic N. Forest disturbances (e.g. fire, clear-cutting or enhanced N deposition) cause (abrupt) changes in ecosystem N-cycling processes. In this study, we hypothesized that delta15N signatures of soil profiles under old-growth forests could be used as an integrator for ecosystem N-cycling, and changes of these delta15N profiles could be valuable to assess ecosystem resilience towards disturbances. Six old-growth forests were selected in the phytogeographical region of the Valdivian rain forest in southern Chile. One of the sites has been partly burned in February 2002. First, we observed that ecosystems with higher mean annual precipitation and lower mean annual temperature were relatively more depleted in 15N. Secondly, we found that a forest fire caused a 100-fold increase of the nitrate export and induced an enrichment of the soil delta15N signal in the upper 20 cm.     

15N tracer application to evaluate nitrogen dynamics of food webs in two subtropical small-scale aquaculture ponds under different managements

Small, semi-intensively managed aquaculture ponds contribute significantly to the food security of small-scale farmers around the world. However, little is known about nutrient flows within natural food webs in such ponds in which fish production depends on the productivity of natural food resources. ¹⁵N was applied as ammonium at 1.1 and 0.4 % of total nitrogen in a traditionally managed flow-through pond and a semi-intensively managed stagnant pond belonging to small-scale farmers in Northern Vietnam and traced through the natural food resources over 7 days. Small-sized plankton (1–60 μ m) was the dominant pelagic biomass in both ponds with higher biomass in the stagnant pond. This plankton assimilated major portions of the applied tracer and showed a high sedimentation and turnover rate. High re-activation of settled nutrients into the pelagic food web was observed. The tracer was removed more quickly from the flow-through pond than from the stagnant pond. A steady nutrient supply could increase fish production.     

挥发性盐基氮的光谱分析方法

报道了一种挥发性盐基氮(TVB-N)测定方法。通过奈斯勒试剂处理的分子筛吸收挥发性盐基氮,形成NH₂Hg₂IO固相显色体,采用漫反射光谱装置直接测定。探讨了测量原理、条件及影响因素,表明该方法具有操作方便,灵敏度高,试剂用量少等优点。方法的线性范围为1～8 μg·mL-1,检测限达0.1 g·mL-1。用于鱼、肉食品新鲜度的监测,发现鱼肉随放置时间的延长,挥发性盐基氮迅速增加,表明鱼肉的腐败变质是一个加速过程,并且鱼的变质速率相对猪肉更快一些。     

A methodological approach to improve estimates of nutrient gains by partially myco-heterotrophic plants†

The stable isotopes ¹⁵N and ¹³C can be used to investigate the nutritional mode of terrestrial orchids and pyroloids (Monotropoideae, Ericaceae). Some of these plants are putatively autotrophic but meet their nitrogen and carbon demands by gaining organic compounds (e.g. amino acids) from mycorrhizal fungi. This so-called partially myco-heterotrophic nutrition is reflected by their isotope signature. The application of a two-source linear mixing model on δ values of such plants allows calculating the percentage of N and C derived from their associated mycorrhizal fungi. Here we present an approach to improve estimates of the plants’ degree of myco-heterotrophy. Due to the presented conversion of δ values into enrichment factors (?), results obtain a better resolution and data from various studies become normalised which facilitates combined representations and meta-analyses.     

氢化物的敢相富集及其在超痕量分析中的应用

本文从富集原理及其在光谱超痕量分析的应用方面分别对这几种氢化物发生后的气相富集技术进行了较详细地综述。这些气相富集方法包括液氮冷却捕集技术、气球收集技术、石墨炉原位捕集技术、吸收液吸收技术及固体吸附技术等。并对这些氢化物气相富集技术的潜力，及与各种光谱分析法联用的发展前景也进行了讨论。     

Feeding level and diet quality influence trophic shift of C and N isotopes in Nile tilapia (Oreochromis niloticus (L.))

Many scientists use naturally occurring stable isotopes to reconstruct the diets of animals. However, isotopic ratios may be affected not only by the composition of the diet but also by the amount of food consumed. Thus, an experiment using tilapia (Oreochromis niloticus) was carried out to test the influence of feeding level on delta13C and delta15N of fish given a semi-synthetic wheat gluten/wheat starch based diet. In addition, the effect of diet quality was tested by comparing tilapia given this feed with tilapia fed a fish meal/wheat meal based diet. Forty-four tilapia were reared individually. After a prefeeding phase, fish were randomly assigned to five groups, four on the semi-synthetic diet at different feeding levels and one group on the fish meal/wheat meal based diet fed at the equivalent of the highest level of the semi-synthetic diet. The experiment lasted eight weeks. Proximate composition, gross energy content and delta13C and delta15N values were determined in feed and fish, for delta13C separately in the lipids and the lipid-free matter. Delta13C in the lipids and the lipid-free matter and delta15N of tilapia fed the semi-synthetic diet decreased significantly with increasing feeding rate. The absolute values of the trophic shift in fish fed the semi-synthetic wheat based diet were significantly higher than in fish fed the fish meal/wheat meal based diet. The different delta13C and delta15N values in tilapia fed the same diet at different feeding levels and the influence of feed quality on the trophic shift add to the uncertainty involved in the use of stable isotopes in ecological research.     

ESCA studies of nitrogen-containing stainless steels

ESCA examination of films formed on nitrogen-containing stainless steels after immersion in 0.1 M NaCl revealed that nitrogen was enriched in the outer 0.5 nm of the film at a concentration a few times higher than in the substrate. The N(1s) binding energy varied from 399.6–399.7 eV at the outermost surface of the film to 398.3–398.8 eV in the film at a depth of about 0.5 nm. This indicates a change in the chemical state of nitrogen upon exposure of the nitrogen-containing stainless steels to the solution, and furthermore, that the surface nitrogen enrichment could be of significance in the improved pitting resistance.     

Nitrogen stable isotopes reveal age-dependent dietary shift in the Japanese scallop Mizuhopecten yessoensis

Ontogenetic niche shifts in diet are a consequence of changes in body size or resource partitioning between age classes. To better resolve the feeding patterns of the Japanese scallop Mizuhopecten yessoensis, we examined the relative importance of age and size in the diet of this species using stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) from 2006 to 2009. Contribution of food sources was quantified using an isotope mixing model by comparing the muscle tissue isotope ratios to those of suspended particulate organic matter (SPOM) and their zooplankton prey (e.g. micro- and meso-zooplankton). Unlike the δ¹³C values, which remained constant with age and size, muscle δ¹⁵N values were more positively correlated with age accounting for 69?% of variations than size with only 46?%. Increasing ¹⁵N values with age suggested that shifts in diet from SPOM to micro- and meso-zooplankton occurred during ontogeny in M. yessoensis. Results of the isotope mixing model indicated that SPOM contribution to scallop’s diet decreased from 68 to 8?% while those of zooplankton increased from 15 to 50?% with increasing age. This study concludes that age-related dietary shift explains the enrichment of ¹⁵N, as a result of predation on zooplankton by M. yessoensis.     

Diets of nesting laughing gulls (Larus atricilla) at the Virginia Coast Reserve: observations from stable isotope analysis

Food web studies often ignore details of temporal, spatial, and intrapopulation dietary variation in top-level consumers. In this study, intrapopulation dietary variation of a dominant carnivore, the Laughing Gull (Larus atricilla), was examined using carbon, nitrogen, and sulfur isotope analysis of gull tissues as well as their prey (fish, invertebrates, and insects) from the Virginia Coast Reserve estuarine system. As earlier traditional diet studies found evidence of individual dietary specialization within gull populations, this study used stable isotope analysis to assess specialization in a coastal Laughing Gull population. Specifically, blood, muscle, and feather isotope values indicated significant intrapopulation dietary specialization. Some gulls relied more heavily on estuarine prey (mean blood delta13C = -17.5, delta15N = 12.6, and delta34S = 9.3), whereas others appeared to consume more foods of marine origin (mean blood delta13C = -19.4, delta15N = 14.8, and delta34S = 10.4). It is important to account for such dietary variability when assessing trophic linkages in dynamic estuarine systems.     

Soil δ15N patterns in old-growth forests of southern Chile as integrator for N-cycling

Old-growth forests of southern Chile represent an important reserve of temperate (rain) forests in the world. Wetter and colder forest ecosystems appear to be more efficient in conserving and recycling N such that mostly non-plant available N species are lost, which could be indicated by more depleted δ¹⁵N values of the soil and plants. Hydrological N loss from the old-growth forests in southern Chile occurs mainly via dissolved organic nitrogen and not via dissolved inorganic N. Forest disturbances (e.g. fire, clear-cutting or enhanced N deposition) cause (abrupt) changes in ecosystem N-cycling processes. In this study, we hypothesized that δ¹⁵N signatures of soil profiles under old-growth forests could be used as an integrator for ecosystem N-cycling, and changes of these δ¹⁵N profiles could be valuable to assess ecosystem resilience towards disturbances. Six old-growth forests were selected in the phytogeographical region of the Valdivian rain forest in southern Chile. One of the sites has been partly burned in February 2002. First, we observed that ecosystems with higher mean annual precipitation and lower mean annual temperature were relatively more depleted in ¹⁵N. Secondly, we found that a forest fire caused a 100-fold increase of the nitrate export and induced an enrichment of the soil δ¹⁵N signal in the upper 20 cm.