Significant Turning Point: Common Buzzard (Buteo buteo) Exposure to Second-Generation Anticoagulant Rodenticides in the United Kingdom.

Second-generation anticoagulant rodenticides (SGARs) are widely used to control rodent populations, resulting in the serious secondary exposure of predators to these contaminants. In the United Kingdom (UK), professional use and purchase of SGARs were revised in the 2010s. Certain highly toxic SGARs have been authorized since then to be used outdoors around buildings as resistance-breaking chemicals under risk mitigation procedures. However, it is still uncertain whether and how these regulatory changes have influenced the secondary exposure of birds of prey to SGARs. Based on biomonitoring of the UK Common Buzzard (Buteo buteo) collected from 2001 to 2019, we assessed the temporal trend of exposure to SGARs and statistically determined potential turning points. The magnitude of difenacoum decreased over time with a seasonal fluctuation, while the magnitude and prevalence of more toxic brodifacoum, authorized to be used outdoors around buildings after the regulatory changes, increased. The summer of 2016 was statistically identified as a turning point for exposure to brodifacoum and summed SGARs that increased after this point. This time point coincided with the aforementioned regulatory changes. Our findings suggest a possible shift in SGAR use to brodifacoum from difenacoum over the decades, which may pose higher risks of impacts on wildlife.

Modelling the effects of the pyrethroid insecticide cypermethrin on the life cycle of the soil dwelling annelid Enchytraeus crypticus, an original experimental design to calibrate a DEB-TKTD model.

The Dynamic Energy Budget theory (DEB) enables ecotoxicologists to model the effects of chemical stressors on organism life cycles through the coupling of toxicokinetic-toxicodynamic (TK-TD) models. While good progress has been made in the application of DEB-TKTD models for aquatic organisms, applications for soil fauna are scarce, due to the lack of dedicated experimental designs suitable for collecting the required time series effect data. Enchytraeids (Annelida: Clitellata) are model organisms in soil ecology and ecotoxicology. They are recognised as indicators of biological activity in soil, and chemical stress in terrestrial ecosystems. Despite this, the application of DEB-TKTD models to investigate the impact of chemicals has not yet been tested on this family. Here we assessed the impact of the pyrethroid insecticide cypermethrin on the life cycle of Enchytraeus crypticus. We developed an original experimental design to collect the data required for the calibration of a DEB-TKTD model for this species. E. crypticus presented a slow initial growth phase that has been successfully simulated with the addition of a size-dependent food limitation for juveniles in the DEB model. The DEB-TKTD model simulations successfully agreed with the data for all endpoints and treatments over time. The highlighted physiological mode of action (pMoA) for cypermethrin was an increase of the growth energy cost. The threshold for effects on survival was estimated at 73.14 mg kg- 1, and the threshold for effects on energy budget (i.e., sublethal effects) at 19.21 mg kg- 1. This study demonstrates that DEB-TKTD models can be successfully applied to E. crypticus as a representative soil species, and may improve the ecological risk assessment for terrestrial ecosystems, and our mechanistic understanding of chemical effects on non-target species.

Constitutive changes in pigment concentrations: implications for estimating isoprene emissions using the photochemical reflectance index.

The photochemical reflectance index (PRI), through its relationship with light use efficiency (LUE) and xanthophyll cycle activity, has recently been shown to hold potential for tracking isoprene emissions from vegetation. However, both PRI and isoprene emissions can also be influenced by changes in carotenoid pigment concentrations. Xanthophyll cycle activity and changes in carotenoid concentrations operate over different timescales, but the importance of constitutive changes in pigment concentrations for accurately estimating isoprene emissions using PRI is unknown. To clarify the physiological mechanisms behind the PRI-isoprene relationship, the light environment of potted Salix viminalis (osier willow) trees was modified to induce acclimation in photosynthetic rates, phytopigments, isoprene emissions and PRI. Acclimation resulted in differences in pigment concentrations, isoprene emissions and PRI. Constitutive changes in carotenoid concentration were significantly correlated with both isoprene emissions and PRI, suggesting that the relationship between PRI and isoprene emissions is significantly influenced by constitutive pigment changes. Consequently knowledge regarding how isoprene emissions are affected by both longer term changes in total carotenoid concentrations and shorter term dynamic adjustments of LUE is required to facilitate interpretation of PRI for monitoring isoprene emissions.

Phosphorus retention and remobilization along hydrological pathways in karst terrain.

Karst landscapes are often perceived as highly vulnerable to agricultural phosphorus (P) loss, via solution-enlarged conduits that bypass P retention processes. Although attenuation of P concentrations has been widely reported within karst drainage, the extent to which this results from hydrological dilution, rather than P retention, is poorly understood. This is of strategic importance for understanding the resilience of karst landscapes to P inputs, given increasing pressures for intensified agricultural production. Here hydrochemical tracers were used to account for dilution of P, and to quantify net P retention, along transport pathways between agricultural fields and emergent springs, for the karst of the Ozark Plateau, midcontinent USA. Up to ∼ 70% of the annual total P flux and ∼ 90% of the annual soluble reactive P flux was retained, with preferential retention of the most bioavailable (soluble reactive) P fractions. Our results suggest that, in some cases, karst drainage may provide a greater P sink than previously considered. However, the subsequent remobilization and release of the retained P may become a long-term source of slowly released "legacy" P to surface waters.

Long-term trends of second generation anticoagulant rodenticides (SGARs) show widespread contamination of a bird-eating predator, the Eurasian Sparrowhawk (Accipiter nisus) in Britain.

Second generation anticoagulant rodenticides (SGARs) are widely used to control rodents around the world. However, contamination by SGARs is detectable in many non-target species, particularly carnivorous mammals or birds-of-prey that hunt or scavenge on poisoned rodents. The SGAR trophic transfer pathway via rodents and their predators/scavengers appears widespread, but little is known of other pathways of SGAR contamination in non-target wildlife. This is despite the detection of SGARs in predators that do not eat rodents, such as specialist bird-eating hawks. We used a Bayesian modelling framework to examine the extent and spatio-temporal trends of SGAR contamination in the livers of 259 Eurasian Sparrowhawks, a specialist bird-eating raptor, in regions of Britain during 1995-2015. SGARs, predominantly difenacoum, were detected in 81% of birds, with highest concentrations in males and adults. SGAR concentrations in birds were lowest in Scotland and higher or increasing in other regions of Britain, which had a greater arable or urban land cover where SGARs may be widely deployed for rodent control. However, there was no overall trend for Britain, and 97% of SGAR residues in Eurasian Sparrowhawks were below 100 ng/g (wet weight), which is a potential threshold for lethal effects. The results have potential implications for the population decline of Eurasian Sparrowhawks in Britain. Fundamentally, the results indicate an extensive and persistent contamination of the avian trophic transfer pathway on a national scale, where bird-eating raptors and, by extension, their prey appear to be widely exposed to SGARs. Consequently, these findings have implications for wildlife contamination worldwide, wherever these common rodenticides are deployed, as widespread exposure of non-target species can apparently occur via multiple trophic transfer pathways involving birds as well as rodents.

Citizen science monitoring reveals links between honeybee health, pesticide exposure and seasonal availability of floral resources.

We use a national citizen science monitoring scheme to quantify how agricultural intensification affects honeybee diet breadth (number of plant species). To do this we used DNA metabarcoding to identify the plants present in 527 honey samples collected in 2019 across Great Britain. The species richness of forage plants was negatively correlated with arable cropping area, although this was only found early in the year when the abundance of flowering plants was more limited. Within intensively farmed areas, honeybee diets were dominated by Brassica crops (including oilseed rape). We demonstrate how the structure and complexity of honeybee foraging relationships with plants is negatively affected by the area of arable crops surrounding hives. Using information collected from the beekeepers on the incidence of an economically damaging bee disease (Deformed Wing Virus) we found that the occurrence of this disease increased where bees foraged in agricultural land where there was a high use of foliar insecticides. Understanding impacts of land use on resource availability is fundamental to assessing long-term viability of pollinator populations. These findings highlight the importance of supporting temporally timed resources as mitigation strategies to support wider pollinator population viability.

Generation of priority research questions to inform conservation policy and management at a national level.

Integrating knowledge from across the natural and social sciences is necessary to effectively address societal tradeoffs between human use of biological diversity and its preservation. Collaborative processes can change the ways decision makers think about scientific evidence, enhance levels of mutual trust and credibility, and advance the conservation policy discourse. Canada has responsibility for a large fraction of some major ecosystems, such as boreal forests, Arctic tundra, wetlands, and temperate and Arctic oceans. Stressors to biological diversity within these ecosystems arise from activities of the country's resource-based economy, as well as external drivers of environmental change. Effective management is complicated by incongruence between ecological and political boundaries and conflicting perspectives on social and economic goals. Many knowledge gaps about stressors and their management might be reduced through targeted, timely research. We identify 40 questions that, if addressed or answered, would advance research that has a high probability of supporting development of effective policies and management strategies for species, ecosystems, and ecological processes in Canada. A total of 396 candidate questions drawn from natural and social science disciplines were contributed by individuals with diverse organizational affiliations. These were collaboratively winnowed to 40 by our team of collaborators. The questions emphasize understanding ecosystems, the effects and mitigation of climate change, coordinating governance and management efforts across multiple jurisdictions, and examining relations between conservation policy and the social and economic well-being of Aboriginal peoples. The questions we identified provide potential links between evidence from the conservation sciences and formulation of policies for conservation and resource management. Our collaborative process of communication and engagement between scientists and decision makers for generating and prioritizing research questions at a national level could be a model for similar efforts beyond Canada.

Aluminium in UK rivers: a need for integrated research related to kinetic factors, colloidal transport, carbon and habitat.

Dissolved aluminium concentrations ([Al]) in the <0.45 µm filtered fraction are described for 54 UK river sites covering rural, acidic/acid sensitive, agricultural and urban typologies, and wide pH range (4 to 11). High [Al] occurred under acidic conditions and for acid runoff neutralised by bicarbonate rich groundwater. Thermodynamic analysis indicates Al hydroxide/hydroxy-silicate oversaturation at circumneutral pH across the rivers, but undersaturation at lower/higher pH. The oversaturation reflects in part the presence of Al bearing colloids as indicated by (1) [Al] being correlated with components associated with both lithogenic (Fe, Ti and lanthanides) colloids and organic carbon, (2) baseflow studies using cross-flow ultrafiltration and (3) comparison of our data with Acid Waters Monitoring Network (AWMN) information on labile and non-labile Al. Tree harvesting and emission reductions of SO(x) in acidic and acid sensitive catchments in mid-Wales led to acidification reversal, lower [Al] and changing [H(+)] - [Al] relationships. The [Al] decline was confined to acidic conditions while [Al] increased during the later part of the monitoring period with a peak around 2002 for moorland and forested systems. Colloidal production across the flow range was indicated late in the record by comparison of our data with information collected by the AWMN for a site in mid-Wales. This production seems interlinked with organic carbon and with dissolved CO(2) changes. In order for further understanding of Al hydrogeochemistry in river systems there is a need to integrate research that moves from equilibrium to kinetic and colloidal consideration including the critical issues of organic and inorganic controls within the context of bioavailability and aquatic stress. The colloidal Al may well be of low environmental concern to fish and other factors such as habitat may well be critical.

Chemicals with increasingly complex modes of action result in greater variation in sensitivity between earthworm species.

The scale of variation in species sensitivity to toxicants has been theoretically linked to mode of action. Specifically, it has been proposed there will be greater variations for chemicals with a putative specific biological target than for toxicants with a non-specific narcotic mechanism. Here we test the hypothesis that mode of action is related to variation in sensitivity in a specifically designed experiment for species from a single ecologically important terrestrial taxa, namely earthworms. Earthworm toxicity tests were conducted with five species for four chemicals, providing a series of increasingly complex modes of action: a putative narcotic polycyclic aromatic hydrocarbon (fluoranthene), and three insecticides (chlorpyrifos, cypermethrin, imidacloprid) with known neuronal receptor targets. Across all the chemicals, the standard epigeic test species Eisenia fetida and Lumbricus rubellus, were generally among the two least sensitive, while the endogenic Aporrectodea caliginosa and Megascolecidae Amynthas gracilis were generally more sensitive (never being among the two least sensitive species). This indicates a potential for bias in the earthworm ecotoxicology literature, which is dominated by studies in epigeic Lumbricidae, but contains few endogeic or Megascolecidae data. Results confirmed the lowest range of variation in sensitivities for effects on reproduction was for fluoranthene (2.5 fold). All insecticides showed greater variation for species sensitivity (cypermethrin: 7.5 fold, chlorpyrifos: 10.3 fold, imidacloprid: 31.5 fold) consistent with the specific mechanisms of the pesticides. Difference in toxicodynamics, based on mode of action specificity and receptor complexity was reflected in the magnitude of sensitivity variation. However, measurements of tissue concentrations also indicated the potential importance of toxicokinetics in explaining species sensitivity variations for chlorpyrifos and cypermethrin.

From seeds to plasma: Confirmed exposure of multiple farmland bird species to clothianidin during sowing of winter cereals.

Neonicotinoids are the largest group of systemic insecticides worldwide and are most commonly applied as agricultural seed treatments. However, little is known about the extent to which farmland birds are exposed to these compounds during standard agricultural practices. This study uses winter cereal, treated with the neonicotinoid clothianidin, as a test system to examine patterns of exposure in farmland birds during a typical sowing period. The availability of neonicotinoid-treated seed was recorded post-sowing at 39 fields (25 farms), and camera traps were used to monitor seed consumption by wild birds in situ. The concentration of clothianidin in treated seeds and crop seedlings was measured via liquid chromatography-tandem mass spectrometry, and avian blood samples were collected from 11 species of farmland bird from a further six capture sites to quantify the prevalence and level of clothianidin exposure associated with seed treatments. Neonicotinoid-treated seeds were found on the soil surface at all but one of the fields surveyed at an average density of 2.8 seeds/m2. The concentration of clothianidin in seeds varied around the target application rate, whilst crop seedlings contained on average 5.9% of the clothianidin measured in seeds. Exposure was confirmed in 32% of bird species observed in treated fields and 50% of individual birds post-sowing; the median concentration recorded in positive samples was 12 ng/mL. Results here provide clear evidence that a variety of farmland birds are subject to neonicotinoid exposure following normal agricultural sowing of neonicotinoid-treated cereal seed. Furthermore, the widespread availability of seeds at the soil surface was identified as a primary source of exposure. Overall, these data are likely to have global implications for bird species and current agricultural policies where neonicotinoids are in use, and may be pertinent to any future risk assessments for systemic insecticide seed treatments.

Model systems to elucidate minimum requirements for protected areas networks.

In conservation biology there have been varying answers to the question of "How much land to protect?" Simulation models using decision-support software such as Marxan show that the answer is sensitive to target type and amount, and issues of scale. We used a novel model system for landscape ecology to test empirically whether the minimum conservation requirements to represent all species at least once are consistent across replicate landscapes, and if not, whether these minimum conservation requirements are linked to biodiversity patterns. Our model system of replicated microcosms could be scaled to larger systems once patterns and mechanisms are better understood. We found that the minimum representation requirements for lichen species along the microlandscapes of tree trunks were remarkably consistent (4-6 planning units) across 24 balsam fir trees in a single stand, as well as for 21 more widely dispersed fir and yellow birch trees. Variation in minimum number of planning units required correlated positively with gamma diversity. Our results demonstrate that model landscapes are useful to determine whether minimum representation requirements are consistent across different landscapes, as well as what factors (life history, diversity patterns, dispersal strategies) affect variation in these conservation requirements. This system holds promise for further investigation into factors that should be considered when developing conservation designs, thus yielding scientifically-defensible requirements that can be applied more broadly.

Detecting fluoxetine and norfluoxetine in wild bird tissues and feathers.

The contamination of the environment with human pharmaceuticals is widespread and demand for such products is mounting globally. Wild vertebrates may be at particular risk from any effects from pharmaceuticals, because of the evolutionary conservation of drug targets. However, exposure of wildlife to pharmaceuticals is poorly characterised, partly due to challenges associated with detecting rapidly metabolised compounds. As part of a wider study on the behavioural effects of fluoxetine (Prozac) on Eurasian starlings (Sturnus vulgaris), we investigated which avian samples are best suited for detecting exposure to fluoxetine in free-living birds. We analysed plasma, various tissues and tail feathers (grown both in the wild and in captivity during the dosing period) from fluoxetine-treated birds (dosed daily with 0.035 mg kg-1 bodyweight for 28 weeks), and liver tissue and tail feathers from sham-dosed birds. We detected fluoxetine in only two of twelve plasma samples from dosed birds. In dosed birds, median concentrations of free fluoxetine/norfluoxetine in tissues (two hour post-final dose) were: 111.2/67.6 ng g-1 in liver, 29.6/5.7 ng g-1 in kidney, 14.2/4.0 ng g-1 in lung, 15.1/1.6 ng g-1 in brain. We estimated that fluoxetine would remain detectable in liver and kidney approximately 4.5 times longer (90 h) than in brain (20h). In dosed birds, fluoxetine was detected in feathers regrown during the dosing period (median concentration = 11.4 ng g-1) at concentrations significantly higher than in regrown feathers from control birds. Fluoxetine residues were detected in wild-grown feathers (grown before the birds were brought into captivity) at concentrations up to 27.0 ng g-1, providing some evidence of likely exposure in the wild. Our results show liver and kidney can be used for detecting fluoxetine in avian carcasses and provide a first indication that feathers may be useful for assessing exposure to fluoxetine, and possibly other pharmaceuticals.

Cross-scale effects of spruce budworm outbreaks on boreal warblers in eastern Canada.

Insect outbreaks are major natural disturbance events that affect communities of forest birds, either directly by affecting the food supply or indirectly by changing the vegetation composition of forest canopies. An examination of correlations between measures of bird and insect abundance across different spatial scales and over varying time lag effects may provide insight into underlying mechanisms. We developed a hierarchical Bayesian model to assess correlations between counts of eight warbler species from the Breeding Bird Survey in eastern Canada, 1966 to 2009, with the presence of spruce budworm (Choristoneura fumiferana Clem.) at immediate local scales and time-lagged regional scales, as measured by extent of defoliation on host tree species. Budworm-associated species Cape May warbler (Setophaga tigrina), bay-breasted warbler (Setophaga castanea), and Tennessee warbler (Oreothlypis peregrina) responded strongly and positively to both local and regional effects. In contrast, non-budworm-associated species, Blackburnian warbler (Setophaga fusca), magnolia warbler (Setophaga magnolia), Canada warbler (Cardellina canadensis), black-throated blue warbler (Setophaga caerulescens), and black-throated green warbler (Setophaga virens), only responded to regional effects in a manner that varied across eastern Canada. The complex responses by forest birds to insect outbreaks involve both increased numerical responses to food supply and to longer term responses to changes in forest structure and composition. These effects can vary across spatial scales and be captured in hierarchical population models, which can serve to disentangle common trends from data when examining drivers of population dynamics like forest management or climate change.

Long-term increase in secondary exposure to anticoagulant rodenticides in European polecats Mustela putorius in Great Britain.

As a result of legal protection and population recovery, European polecats (Mustela putorius) in Great Britain are expanding into areas associated with greater usage of second-generation anticoagulant rodenticides (SGARs). We analysed polecat livers collected from road casualties from 2013 to 2016 for residues of five SGARs. We related variation in residues to polecat traits and potential exposure pathways, by analysing stable isotopes of carbon (δ13C) and nitrogen (δ15N) in their whiskers. 54 of 68 (79%) polecats had detectable residues of at least one SGAR. Bromadiolone (71%) was the most frequently detected compound, followed by difenacoum (53%) and brodifacoum (35%). Applying historical limits of detection to allow comparison between these new data and previous assessments, we show that in the 25 years from 1992 to 2016 inclusive, the rate of detection of SGARs in polecats in Britain has increased by a factor of 1.7. The probability of SGAR detection was positively related to increasing values of δ15N, suggesting that polecats feeding at a higher trophic level were more likely to be exposed. Total concentrations of SGARs in polecats with detectable residues were higher in polecats collected in arable compared to pastoral habitats, and in the west compared to the east of Britain. The number of compounds detected and total concentrations of SGARs increased with polecat age. There was no evidence of regional or seasonal variation in the probability of detecting SGARs, suggesting that the current risk of exposure to SGARs does not vary seasonally and has increased (from that in the 1990s) throughout the polecat's range. We recommend quantification of current practices in rodenticide usage, particularly in the light of recent regulatory changes, to enable assessment and mitigation of the risks of secondary exposure to rodenticides in non-target wildlife.

Neonicotinoid residues in UK honey despite European Union moratorium.

Due to concerns over negative impacts on insect pollinators, the European Union has implemented a moratorium on the use of three neonicotinoid pesticide seed dressings for mass-flowering crops. We assessed the effectiveness of this policy in reducing the exposure risk to honeybees by collecting 130 samples of honey from bee keepers across the UK before (2014: N = 21) and after the moratorium was in effect (2015: N = 109). Neonicotinoids were present in about half of the honey samples taken before the moratorium, and they were present in over a fifth of honey samples following the moratorium. Clothianidin was the most frequently detected neonicotinoid. Neonicotinoid concentrations declined from May to September in the year following the ban. However, the majority of post-moratorium neonicotinoid residues were from honey harvested early in the year, coinciding with oilseed rape flowering. Neonicotinoid concentrations were correlated with the area of oilseed rape surrounding the hive location. These results suggest mass flowering crops may contain neonicotinoid residues where they have been grown on soils contaminated by previously seed treated crops. This may include winter seed treatments applied to cereals that are currently exempt from EU restrictions. Although concentrations of neonicotinoids were low (<2.0 ng g-1), and posed no risk to human health, they may represent a continued risk to honeybees through long-term chronic exposure.

Insights into the fate of a 13C labelled phenol pulse for stable isotope probing (SIP) experiments.

Stable isotope probing (SIP) using DNA or RNA as a biomarker has proven to be a useful method for attributing substrate utilisation to specific microbial taxa. In this study we followed the transfer of a (13)C(6)-phenol pulse in an activated sludge micro-reactor to examine the resulting distribution of labelled carbon in the context of SIP. Most of the added phenol was metabolically converted within the first 100 min after (13)C(6)-phenol addition, with 49% incorporated into microbial biomass and 6% respired as CO(2). Less than 1% of the total (13)C labelled carbon supplied was incorporated into microbial RNA and DNA, with RNA labelling 6.5 times faster than DNA. The remainder of the added (13)C was adsorbed and/or complexed to suspended solids within the sludge. The (13)C content of nucleic acids increased beyond the initial consumption of the (13)C-phenol pulse. This study confirms that RNA labels more efficiently than DNA and reveals that only a small proportion of a pulse is incorporated into nucleic acids. Evidence of continued (13)C incorporation into nucleic acids suggests that cross-feeding of the SIP substrate was rapid. This highlights both the benefits of using a biomarker that is rapidly labelled and the importance of sampling within appropriate timescales to avoid or capture the effects of cross-feeding, depending on the goal of the study.

Impact of water table depth on forest soil methane turnover in laboratory soil cores deduced from natural abundance and tracer 13C stable isotope experiments.

We investigated turnover of methane (CH4) in soils from a poorly drained UK forest. In situ, this forest exhibited a negligible soil-atmosphere CH4 flux, whereas adjacent grassland plots were sources of CH4. We hypothesised that the forest plots exhibited reduced anaerobic CH4 production through water-table draw down. Consequently, we exposed soil cores from under oak to high and low water-table conditions in the laboratory. Methane fluxes increased significantly in the high water-table (1925+/-1702 mug CH4 m(-2) h(-1)) compared to the low one (-3.5+/-6.8 microg CH4 m(-2) h(-1)). Natural abundance delta13C values of CH4 showed a strong depletion in high water-table cores (-56.7+/-2.9 per thousand) compared to methane in ambient air (-46.0 per thousand) indicative of methanogenic processes. The delta13C values of CH4 from low water-table cores (delta13C-46.8+/-0.2 per thousand) was similar to ambient air and suggested little alteration of headspace CH4 by the soil microbial community. In order to assess the CH4 oxidizing activity of the two treatments conclusively, a 13CH4 spike was added to the cores and 13CO2 production was measured as the by-product of CH4 oxidation. 13CH4 oxidation rates were 57.5 (+/-12.7) and 0.5 (+/-0.1) microg CH4 m(-2) h(-1) for high and low water-tables, respectively. These data show that the lower water-table hydrology treatment impacted methanogenic processes without stimulating methanotrophy.

Differential responses of grass and a dwarf shrub to long-term changes in soil microbial biomass C, N and P following factorial addition of NPK fertilizer, fungicide and labile carbon to a heath.

Microbial immobilization may decrease the inorganic nutrient concentrations of the soil to the extent of affecting plant nutrient uptake and growth. We have hypothesized that graminoids with opportunistic nutrient-acquisition strategies are strongly influenced by nutrient limitation imposed by microbes, whereas growth forms such as dwarf shrubs are less affected by the mobilization-immobilization cycles in microbes. By adding NPK fertilizer, labile C (sugar) and fungicide (benomyl) over a 5 yr period in a fully factorial design, we aimed to manipulate the sink-source potential for nutrients in a non-acidic heath tundra soil. After 2 yr, N and P accumulated in the microbial biomass after fertilization with no change in microbial C, which suggests that nutrients did not limit microbial biomass growth. After 5 yr, microbial C was enhanced by 60% in plots with addition of labile C, which points to C-limitation of the microbial biomass. Microbial biomass N and P tended to increase following addition of labile C, by 10 and 25%, respectively. This caused decreased availability of NH4 + and P, showing close microbial control of nutrient availability. The most common graminoid, Festuca ovina, responded to fertilizer addition with a strong increase, and to labile C addition with a strong decrease in cover, providing the first direct field evidence that nutrient limitation imposed by immobilizing microbes can affect the growth of tundra plants. Also in support of our hypothesis, following addition of labile C the concentrations of N and K in leaves and that of N in roots of F. ovina decreased, whilst the demand of roots for P increased. In contrast, the most common dwarf shrub, Vaccinium uliginosum, was only slightly sensitive to changes in resource availability, showing no cover change after 4 yr addition of labile C and fertilizer, and little change in leaf nutrient concentrations. We suggest that the differential responses of the two growth forms are due to differences in storage and nutrient uptake pathways, with the dwarf shrub having large nutrient storage capacity and access to organic forms of N through its mycorrhizal association. While the fungicide had no effect on ericoid mycorrhizal colonization of roots or symbiotic function inferred from plant 15 N natural abundance, it decreased microbial biomass C and N after 2 yr. Throughout the fifth season, the availability of soil NO3 - and inorganic P was decreased with no change in microbial biomass C, N or P, suggesting a negative impact of benomyl on N and P mineralization.

Vascular plant 15N natural abundance in heath and forest tundra ecosystems is closely correlated with presence and type of mycorrhizal fungi in roots.

In this study we show that the natural abundance of the nitrogen isotope 15, δ15N, of plants in heath tundra and at the tundra-forest ecocline is closely correlated with the presence and type of mycorrhizal association in the plant roots. A total of 56 vascular plant species, 7 moss species, 2 lichens and 6 species of fungi from four heath and forest tundra sites in Greenland, Siberia and Sweden were analysed for δ15N and N concentration. Roots of vascular plants were examined for mycorrhizal colonization, and the soil organic matter was analysed for δ15N, N concentration and soil inorganic, dissolved organic and microbial N. No arbuscular mycorrhizal (AM) colonizations were found although potential host plants were present in all sites. The dominant species were either ectomycorrhizal (ECM) or ericoid mycorrhizal (ERI). The δ15N of ECM or ERI plants was 3.5-7.7‰ lower than that of non-mycorrhizal (NON) species in three of the four sites. This corresponds to the results in our earlier study of mycorrhiza and plant δ15N which was limited to one heath and one fellfield in N Sweden. Hence, our data suggest that the δ15N pattern: NON/AM plants > ECM plants ≥ ERI plants is a general phenomenon in ecosystems with nutrient-deficient organogenic soils. In the fourth site, a␣birch forest with a lush herb/shrub understorey, the differences between functional groups were considerably smaller, and only the ERI species differed (by 1.1‰) from the NON species. Plants of all functional groups from this site had nearly twice the leaf N concentration as that found in the same species at the other three sites. It is likely that low inorganic N availability is a prerequisite for strong δ15N separation among functional groups. Both ECM roots and fruitbodies were 15N enriched compared to leaves which suggests that the difference in δ15N between plants with different kinds of mycorrhiza could be due to isotopic fractionation at the␣fungal-plant interface. However, differences in δ15N between soil N forms absorbed by the plants could also contribute to the wide differences in plant δ15N found in most heath and forest tundra ecosystems. We hypothesize that during microbial immobilization of soil ammonium the microbial N pool could become 15N-depleted and the remaining, plant-available soil ammonium 15N-enriched. The latter could be a main source of N for NON/AM plants which usually have high δ15N. In contrast, amino acids and other soil organic N compounds presumably are 15N-depleted, similar to plant litter, and ECM and ERI plants with high uptake of these N forms hence have low leaf δ15N. Further indications come from the δ15N of mosses and lichens which was similar to that of ECM plants. Tundra cryptogams (and ECM and ERI plants) have previously been shown to have higher uptake of amino acid than ammonium N; their low δ15N might therefore reflect the δ15N of free amino acids in the soil. The concentration of dissolved organic N was 3-16 times higher than that of inorganic N in the sites. Organic nitrogen could be an important N source for ECM and, in particular, ERI plants in heath and forest tundra ecosystems with low release rate of inorganic N from the soil organic matter.

Shoot biomass, δ13C, nitrogen and chlorophyll responses of two arctic dwarf shrubs to in situ shading, nutrient application and warming simulating climatic change.

As climatic change might induce ecophysiological changes in plants which affect their long-term performance, we investigated responses in above-ground biomass, δ13C, nitrogen and chlorophyll of two evergreen arctic dwarf shrubs, Cassiope tetragona and Empetrum hermaphroditum, to 5 (biomass, N) or 6 years of shading, nutrient application and air/soil warming at a dwarf shrub dominated tree-line heath (450 m a.s.l) and a high altitude fellfield (1100 m a.s.l.) in Swedish Lapland. Warming enhanced the green biomass (equivalent to the last 3-4 years of leaf production) and the ratio of green to brown biomass of C. tetragona at the fellfield, and diluted the shoot N concentration. Fertilizer application led to higher shoot N concentration and larger green-to-brown biomass ratio at both sites, and fertilizer application and warming generally had an additive effect on the green biomass. We conclude that both warming and increased soil nutrient availability stimulated the growth of C. tetragona at the fellfield whereas at the heath there was a clear increase in production only if enhanced temperature was combined with nutrient application. Across treatments C. tetragona at the fellfield had 0.6‰ higher δ13C and 1.4 mg g-1 more leaf N, and the soil organic matter δ13C was 1.0‰ higher at the fellfield than at the heath. However, an increase in shoot N concentration with altitude does not necessarily lead to higher δ13C as no differences in δ13C were observed when leaf N of the two dwarf shrubs was increased by fertilizer application c. tetragona in non-warmed plots had higher δ13C values than those from warmed plots at the same altitude, which provides the first in situ experimental validation of the theory that temperature partly is responsible for altitudinal trends in plant carbon isotope discrimination. Increased biomass and chlorophyll concentration of C. tetragona in warmed plots points to increased assimilation, at least at the fellfield. As the δ13C-based and, therefore, time-integrated estimate of the ratio of CO2 concentration in the leaf intercellular spaces to that in the atmosphere (C i/C a) also increased, warming probably enhanced the stomatal conductance relatively more than the C assimilation, which may be harmful if climatic change leads to reduced soil moisture content and increased plant competition for water. At both sites C. tetragona and E. hermaphroditum responded to shade by increasing the concentration of shoot N and photosynthetic pigments whereas biomass production (and therefore also net photosynthesis) did not decline. Shade was accompanied by a 0.6-1.3‰ (E. hermaphroditum) or 1.2-2.2‰ (C. tetragona) decrease in δ13C. This could be due to enhanced stomatal conductance with shading, and perhaps to shade reducing the ericoid mycorrhizal uptake of soil organic C, a factor which has been overlooked as an influence on plant δ13C.