Interrogating pollution sources in a mangrove food web using multiple stable isotopes.

Anthropogenic activities including metal contamination create well-known problems in coastal mangrove ecosystems but understanding and linking specific pollution sources to distinct trophic levels within these environments is challenging. This study evaluated anthropogenic impacts on two contrasting mangrove food webs, by using stable isotopes (δ13C, δ15N, 87Sr/86Sr, 206Pb/207Pb and 208Pb/207Pb) measured in sediments, mangrove trees (Rhizophora mangle, Laguncularia racemosa, Avicennia schaueriana), plankton, shrimps (Macrobranchium sp.), crabs (Aratus sp.), oysters (Crassostrea rhizophorae) and fish (Centropomus parallelus) from both areas. Strontium and Pb isotopes were also analysed in water and atmospheric particulate matter (PM). δ15N indicated that crab, shrimp and oyster are at intermediate levels within the local food web and fish, in this case C. parallelus, was confirmed at the highest trophic level. δ15N also indicates different anthropogenic pressures between both estuaries; Vitória Bay, close to intensive human activities, showed higher δ15N across the food web, apparently influenced by sewage. The ratio87Sr/86Sr showed the primary influence of marine water throughout the entire food web. Pb isotope ratios suggest that PM is primarily influenced by metallurgical activities, with some secondary influence on mangrove plants and crabs sampled in the area adjacent to the smelting works. To our knowledge, this is the first demonstration of the effect of anthropogenic pollution (probable sewage pollution) on the isotopic fingerprint of estuarine-mangrove systems located close to a city compared to less impacted estuarine mangroves. The influence of industrial metallurgical activity detected using Pb isotopic analysis of PM and mangrove plants close to such an impacted area is also notable and illustrates the value of isotopic analysis in tracing the impact and species affected by atmospheric pollution.

Minimising methodological biases to improve the accuracy of partitioning soil respiration using natural abundance 13C.

RATIONALE: Microbial degradation of soil organic matter (heterotrophic respiration) is a key determinant of net ecosystem exchange of carbon, but it is difficult to measure because the CO2 efflux from the soil surface is derived not only from heterotrophic respiration, but also from plant root and rhizosphere respiration (autotrophic). Partitioning total CO2 efflux can be achieved using the different natural abundance stable isotope ratios (δ(13)C) of root and soil CO2. Successful partitioning requires very accurate measurements of total soil efflux δ(13)CO2 and the δ(13)CO2 of the autotrophic and heterotrophic sources, which typically differ by just 2-8‰. METHODS: In Scottish moorland and grass mesocosm studies we systematically tested some of the most commonly used techniques in order to identify and minimise methodological errors. Typical partitioning methods are to sample the total soil-surface CO2 efflux using a chamber, then to sample CO2 from incubated soil-free roots and root-free soil. We investigated the effect of collar depth on chamber measurements of surface efflux δ(13)CO2 and the effect of incubation time on estimates of end-member δ(13)CO2. RESULTS: (1) a 5 cm increase in collar depth affects the measurement of surface efflux δ(13)CO2 by -1.5‰ and there are fundamental inconsistencies between modelled and measured biases; (2) the heterotrophic δ(13)CO2 changes by up to -4‰ within minutes of sampling; we recommend using regression to estimate the in situ δ(13)CO2 values; (3) autotrophic δ(13)CO2 measurements are reliable if root CO2 is sampled within an hour of excavation; (4) correction factors should be used to account for instrument drift of up to 3‰ and concentration-dependent non-linearity of CRDS (cavity ringdown spectroscopy) analysis. CONCLUSIONS: Methodological biases can lead to large inaccuracies in partitioning estimates. The utility of stable isotope partitioning of soil CO2 efflux will be enhanced by consensus on the optimum measurement protocols and by minimising disturbance, particularly during chamber measurements.

Metal-macrofauna interactions determine microbial community structure and function in copper contaminated sediments.

Copper is essential for healthy cellular functioning, but this heavy metal quickly becomes toxic when supply exceeds demand. Marine sediments receive widespread and increasing levels of copper contamination from antifouling paints owing to the 2008 global ban of organotin-based products. The toxicity of copper will increase in the coming years as seawater pH decreases and temperature increases. We used a factorial mesocosm experiment to investigate how increasing sediment copper concentrations and the presence of a cosmopolitan bioturbating amphipod, Corophium volutator, affected a range of ecosystem functions in a soft sediment microbial community. The effects of copper on benthic nutrient release, bacterial biomass, microbial community structure and the isotopic composition of individual microbial membrane [phospholipid] fatty acids (PLFAs) all differed in the presence of C. volutator. Our data consistently demonstrate that copper contamination of global waterways will have pervasive effects on the metabolic functioning of benthic communities that cannot be predicted from copper concentrations alone; impacts will depend upon the resident macrofauna and their capacity for bioturbation. This finding poses a major challenge for those attempting to manage the impacts of copper contamination on ecosystem services, e.g. carbon and nutrient cycling, across different habitats. Our work also highlights the paucity of information on the processes that result in isotopic fractionation in natural marine microbial communities. We conclude that the assimilative capacity of benthic microbes will become progressively impaired as copper concentrations increase. These effects will, to an extent, be mitigated by the presence of bioturbating animals and possibly other processes that increase the influx of oxygenated seawater into the sediments. Our findings support the move towards an ecosystem approach for environmental management.

Can gas chromatography combustion isotope ratio mass spectrometry be used to quantify organic compound abundance?

Quantifying the concentrations of organics such as phospholipid fatty acids (PLFAs) and n-alkanes and measuring their corresponding (13)C/(12)C isotope ratios often involves two separate analyses; (1) quantification by gas chromatography flame ionisation detection (GC-FID) or gas chromatography/mass spectrometry (GC/MS), and (2) (13) C-isotope abundance analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS). This requirement for two separate analyses has obvious disadvantages in terms of cost and time. However, there is a history of using the data output of isotope ratio mass spectrometers to quantify various components; including the N and C concentrations of solid materials and CO(2) concentrations in gaseous samples. Here we explore the possibility of quantifying n-alkanes extracted from sheeps' faeces and fatty acid methyl esters (FAMEs) derivatised from PLFAs extracted from grassland soil, using GC-C-IRMS. The results were compared with those from GC-FID analysis of the same extracts. For GC-C-IRMS the combined area of the masses for all the ions (m/z 44, 45 and 46) was collected, referred to as 'area all', while for the GC-FID analysis the peak area data were collected. Following normalisation to a common value for added internal standards, the GC-C-IRMS 'area all' values and the GC-FID peak area data were directly compared. Strong linear relationships were found for both n-alkanes and FAMEs. For the n-alkanes the relationships were 1:1 while, for the FAMEs, GC-C-IRMS overestimated the areas relative to the GC-FID results. However, with suitable reference material 1:1 relationships were established. The output of a GC-C-IRMS system can form the basis for the quantification of certain organics including FAMEs and n-alkanes.

Challenges in measuring the δ13C of the soil surface CO2 efflux.

The δ13C of the soil surface efflux of carbon dioxide (δ13CRS) has emerged as a powerful tool enabling investigation of a wide range of soil processes from characterising entire ecosystem respiration to detailed compound-specific isotope studies. δ13CRS can be used to trace assimilated carbon transfer below ground and to partition the overall surface efflux into heterotrophic and autotrophic components. Despite this wide range of applications no consensus currently exists on the most appropriate method of sampling this surface efflux of CO2 in order to measure δ13CRS. Here we consider and compare the methods which have been used, and examine the pitfalls. We also consider a number of analysis options, isotope ratio mass spectrometry (IRMS), tuneable diode laser spectroscopy (TDLS) and cavity ring-down laser spectroscopy (CRDS). δ13CRS is typically measured using chamber systems, which fall into three types: closed, open and dynamic. All are imperfect. Closed chambers often rely on Keeling plots to estimate δ13CRS, which may not be appropriate without free turbulent air mixing. Open chambers have the advantage of being able to maintain steady-state conditions but analytical errors may become limiting with low efflux rates. Dynamic chambers like open chambers are complex, and controlling pressure fluctuations caused by air movement is a key concern. Both open and dynamic chambers in conjunction with field portable TDLS and CRDS analysis systems have opened up the possibility of measuring δ13CRS in real time permitting new research opportunities and are on balance the most suited to this type of measurement.

Quantification of effects of season and nitrogen supply on tree below-ground carbon transfer to ectomycorrhizal fungi and other soil organisms in a boreal pine forest.

SUMMARY: *The flux of carbon from tree photosynthesis through roots to ectomycorrhizal (ECM) fungi and other soil organisms is assumed to vary with season and with edaphic factors such as nitrogen availability, but these effects have not been quantified directly in the field. *To address this deficiency, we conducted high temporal-resolution tracing of (13)C from canopy photosynthesis to different groups of soil organisms in a young boreal Pinus sylvestris forest. *There was a 500% higher below-ground allocation of plant C in the late (August) season compared with the early season (June). Labelled C was primarily found in fungal fatty acid biomarkers (and rarely in bacterial biomarkers), and in Collembola, but not in Acari and Enchytraeidae. The production of sporocarps of ECM fungi was totally dependent on allocation of recent photosynthate in the late season. There was no short-term (2 wk) effect of additions of N to the soil, but after 1 yr, there was a 60% reduction of below-ground C allocation to soil biota. *Thus, organisms in forest soils, and their roles in ecosystem functions, appear highly sensitive to plant physiological responses to two major aspects of global change: changes in seasonal weather patterns and N eutrophication.

Through the eye of the needle: a review of isotope approaches to quantify microbial processes mediating soil carbon balance.

For soils in carbon balance, losses of soil carbon from biological activity are balanced by organic inputs from vegetation. Perturbations, such as climate or land use change, have the potential to disrupt this balance and alter soil-atmosphere carbon exchanges. As the quantification of soil organic matter stocks is an insensitive means of detecting changes, certainly over short timescales, there is a need to apply methods that facilitate a quantitative understanding of the biological processes underlying soil carbon balance. We outline the processes by which plant carbon enters the soil and critically evaluate isotopic methods to quantify them. Then, we consider the balancing CO(2) flux from soil and detail the importance of partitioning the sources of this flux into those from recent plant assimilate and those from native soil organic matter. Finally, we consider the interactions between the inputs of carbon to soil and the losses from soil mediated by biological activity. We emphasize the key functional role of the microbiota in the concurrent processing of carbon from recent plant inputs and native soil organic matter. We conclude that quantitative isotope labelling and partitioning methods, coupled to those for the quantification of microbial community substrate use, offer the potential to resolve the functioning of the microbial control point of soil carbon balance in unprecedented detail.

Measuring the 13C content of soil-respired CO2 using a novel open chamber system.

Carbon dioxide respired by soils comes from both autotrophic and heterotrophic respiration. 13C has proved useful in differentiating between these two sources, but requires the collection and analysis of CO2 efflux from the soil. We have developed a novel, open chamber system which allows for the accurate and precise quantification of the delta13C of soil-respired CO2. The chamber was tested using online analyses, by configuring a GasBench II and continuous flow isotope ratio mass spectrometer, to measure the delta13C of the chamber air every 120 s. CO2 of known delta13C value was passed through a column of sand and, using the chamber, the CO2 concentration stabilized rapidly, but 60 min was required before the delta13C value was stable and identical to the cylinder gas (-33.3 per thousand). Changing the chamber CO2 concentration between 200 and 900 micromol.mol(-1) did not affect the measured delta13C of the efflux. Measuring the delta13C of the CO2 efflux from soil cores in the laboratory gave a spread of +/-2 per thousand, attributed to heterogeneity in the soil organic matter and roots. Lateral air movement through dry sand led to a change in the delta13C of the surface efflux of up to 8 per thousand. The chamber was used to measure small transient changes (+/-2 per thousand) in the delta13C of soil-respired CO2 from a peaty podzol after gradual heating from 12 to 35 degrees C over 12 h. Finally, soil-respired CO2 was partitioned in a labelling study and the contribution of autotrophic and heterotrophic respiration to the total efflux determined. Potential applications for the chamber in the study of soil respiration are discussed.

Collection and storage of CO2 for 13C analysis: An application to separate soil CO2 efflux into root- and soil-derived components.

Soil surface CO2 efflux is comprised of CO2 from (i) root respiration and rhizosphere microbes and (ii) heterotrophic respiration from the breakdown of soil organic matter (SOM). This efflux may be partitioned between these sources using delta13C measurements. To achieve this, continuous flow isotope ratio mass spectrometry can be used and, in conjunction with 10 mL septum-capped vials, large numbers of samples may be analysed using a Finnigan MAT Delta(plus)XP interfaced to a Gas Bench II. Here we describe a number of advances to facilitate such work, including: (i) a technique for monitoring mass spectrometer performance, (ii) improvements to sample storage, and (iii) a gas-handling system for incubating and sampling the CO2 derived from roots and soils. Mass spectrometer performance was monitored using an automated refillable vial. Compressed air analysed with this system had mean delta13C of -9.61 +/- 0.16 per thousand (+/- 1sigma, n = 28) collected over four runs. Heating the butyl rubber septa used to seal the vials at 105 degrees C for 12 h improved the sample storage. After air transportation over 12 days, the isotope composition of the CO2 at ambient concentrations was unchanged (before: -35.2 +/- 0.10 per thousand, n = 4; after: -35.3 +/- 0.10 per thousand, n = 15); without heat treatment of the septa the CO2 became slightly enriched (-35.0 +/- 0.14 per thousand, n = 15). The linearity of the Gas Bench II was found to decline above 8000 micromol CO2 mol(-1). To stay within a linear range and to allow the incubation of soil and root material we describe a gas-handling system based around a peristaltic pump. Finally, we demonstrate these methods by growing a C-4 grass (Guinea grass, Panicum maximum Jacq.) in a C-3 soil. Root respiration was found to contribute between 5 and 22% to the soil surface CO2 efflux. These methodologies will facilitate experiments aimed at measuring the isotopic composition of soil-derived CO2 across a range of ecological applications.

Contribution of current carbon assimilation in supplying root exudates of Lolium perenne measured using steady-state C labelling.

Coupling growth of Lolium perenne L. in sterile solution culture with steady-state (13)CO(2) labelling allowed quantification of the contribution of C, assimilated either before or after a specific time point, both to plant compartments and root exudates. Plants were grown for 27 days in atmospheres containing CO(2) with delta(13)C signatures of either -13.5 or -36.1 per thousand. Air supplies to plants were then reciprocally switched to the opposing signature (day 0), plants were destructively harvested and root exudates collected over the next 8 days. Following the switch, C assimilated after day 0 and transported to the roots initially only appeared in root tips, later appearing in both tip and non-tip material. The delta(13)C signature of the root exudate changed exponentially with time. Assimilation pre- and post-day 0 contributed equally to exudate C at 4.5 days. Beyond day 8, assimilation pre-day 0 still contributed 41.7% of exudate C. Over all 8 days, a linear relationship existed between the delta(13)C signatures of root tips and exudate, suggesting that all newly assimilated C in the exudate was from root tips. Results imply pulse-labelling approaches to study root exudates are discriminative in the sources of exudates labelled and in the sites from which exudation occurs.

Determination of Cd and Zn by isotope dilution-thermal ionisation mass spectrometry using a sequential analysis procedure.

Isotope dilution-thermal ionisation mass spectrometry (ID-TIMS) was used to examine the certified Cd and Zn content of 4 Certified Reference Materials (CRMs); 2 soils: GBW07401 and GBW07405, 1 plant CRM060 and an animal tissue SRM1566a. The CRMs were chosen to be of contrasting origin and Cd:Zn content. Three digestion procedures were compared: (i) an open tube aqua regia procedure (ii) microwave digestion using Teflon bombs and (iii) hydrofluoric acid (HF) digestion using PTFE bombs. The Cd and Zn levels obtained using ID-TIMS all fell within the published certified range for the CRMs. This was the case regardless of the digestion procedure used, although HF digestion tended to yield marginally higher levels than the other procedures and in one instance, Cd in GBW07401, was significantly different (P<0.05) from the certified range. A filament loading procedure was developed, to allow sequential analysis of Cd and Zn on the same single filament during thermal ionisation mass spectrometry analysis. The sequential analysis technique was evaluated to ensure that Zn did not fractionate during Cd analysis and there was no inter-element interference. No marked difference in the precision and accuracy of the isotope ratio measurements were obtained from sequential element analyses on the same filament when compared to individual element analyses for a range of standard solutions or for sample digests. The most efficient procedure in terms of costs and productivity for future work of this kind would be a combination of microwave digestion and sequential analysis of Cd and Zn on the same filament.