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Terrestrial dissolved organic matter (DOM) in forested watersheds is a known precursor of disinfection byproducts (DBPs) in drinking water. Although the characteristics of terrestrial DOM may change with increasing nitrogen (N) deposition in forests, how these changes alter formation potential and toxicity of DBPs remains unexplored. We analyzed the speciation and toxicity of DBPs from chlorination of DOM derived from soils (O, A, and B horizons) in an experimental temperate forest with 22 years of N addition. With long-term N addition, the DOM reactivity toward the formation of trihalomethanes (from 27.7-51.8 to 22.8-31.1 µg/mg-dissolved organic carbon (DOC)) and chloral hydrate (from 1.25-1.63 to 1.14-1.36 µg/mg-DOC) decreased, but that toward the formation of haloketones increased (from 0.23-0.26 to 0.26-0.33 µg/mg-DOC). The DOM reactivity toward the formation of haloacetonitriles was increased in the deeper soil but reduced in the surface soil. The DBP formation potential of DOM draining from a certain area of forest soils (in µg-DBP/m2-soil) was estimated to be reduced by 20.3% for trihalomethanes and increased by 37.5% for haloketones and have minor changes for haloacetonitriles and chloral hydrate (both <7%). Furthermore, the DBPs from chlorination of the soil-derived DOM showed lowered microtoxicity with N addition possibly due to reduced brominated DBP formation. Overall, this study highlights that N deposition may not increase drinking water toxicity through altering terrestrial DOM characteristics.

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Ecological research networks functioning across climatic and edaphic gradients are critical for improving predictive understanding of biogeochemical cycles at local through global scales. One international network, the Detrital Input and Removal Treatment (DIRT) Project, was established to assess how rates and sources of plant litter inputs influence accumulations or losses of organic matter in forest soils. DIRT employs chronic additions and exclusions of aboveground litter inputs and exclusion of root ingrowth to permanent plots at eight forested and two shrub/grass sites to investigate how soil organic matter (SOM) dynamics are influenced by plant detrital inputs across ecosystem and soil types. Across the DIRT network described here, SOM pools responded only slightly, or not at all, to chronic doubling of aboveground litter inputs. Explanations for the slow or even negative response of SOM to litter additions include increased decomposition of new inputs and priming of old SOM. Evidence of priming includes increased soil respiration in litter addition plots, decreased dissolved organic carbon (DOC) output from increased microbial activity, and biochemical markers in soil indicating enhanced SOM degradation. SOM pools decreased in response to chronic exclusion of aboveground litter, which had a greater effect on soil C than did excluding roots, providing evidence that root-derived C is not more critical than aboveground litter C to soil C sequestration. Partitioning of belowground contributions to total soil respiration were predictable based on site-level soil C and N as estimates of site fertility; contributions to soil respiration from root respiration were negatively related to soil fertility and inversely, contributions from decomposing aboveground litter in soil were positively related to site fertility. The commonality of approaches and manipulations across the DIRT network has provided greater insights into soil C cycling than could have been revealed at a single site.

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Understanding soil organic matter (OM) biogeochemistry at the molecular-level is essential for assessing potential impacts from management practices and climate change on shifts in soil carbon storage. Biomarker analyses and nuclear magnetic resonance (NMR) spectroscopy were used in an ongoing detrital input and removal treatment experiment in a temperate deciduous forest in Pennsylvania, USA, to examine how above- and below-ground plant inputs control soil OM quantity and quality at the molecular-level. From plant material to surface soils, the free acyclic lipids and cutin, suberin, and lignin biomarkers were preferentially retained over free sugars and free cyclic lipids. After 20years of above-ground litter addition (Double Litter) or exclusion (No Litter) treatments, soil OM composition was relatively more degraded, as revealed by solid-state 13C NMR spectroscopy. Under Doubled Litter inputs, soil carbon and phospholipid fatty acid (PLFA) concentrations were unchanged, suggesting that the current OM degradation status is a reflection of microbial-mediated degradation that occurred prior to the 20-year sampling campaign. Soil OM degradation was higher in the No Litter treatments, likely due to the decline in fresh, above-ground litter inputs over time. Furthermore, root and root and litter exclusion treatments (No Roots and No Inputs, respectively) both significantly reduced free sugars and PLFAs and increased preservation of suberin-derived compounds. PLFA stress ratios and the low N-acetyl resonances from diffusion edited 1H NMR also indicate substrate limitations and reduced microbial biomass with these treatments. Overall, we highlight that storage of soil carbon and its biochemical composition do not linearly increase with plant inputs because the microbial processing of soil OM is also likely altered in the studied forest.

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Stress within tree roots may influence whole-tree responses to nutrient deficiencies or toxic ion accumulation, but the mechanisms that govern root responses to the belowground chemical environment are poorly quantified. Currently, root production is modeled using rates of forest production and stoichiometry, but this approach alone may be insufficient to forecast variability in forest responses when physical and chemical stressors alter root lifespan, rooting depth or mycorrhizal colonization directly. Here, we review key research priorities for improving predictions of tree responses to changes in the belowground biogeochemical environment resulting from nitrogen deposition, including: limits of the optimum allocation paradigm, root physiological stress and lifespan, contingency effects that determine threshold responses across broad gradients, coupled water-biogeochemical interactions on roots, mycorrhizal dynamics that mediate root resilience and model frameworks to better simulate root feedbacks to aboveground function. We conclude that models incorporating physiological feedbacks, dynamic responses to coupled stressors, mycorrhizal interactions, and which challenge widely-accepted notions of optimum allocation, can elucidate potential thresholds of tree responses to biogeochemical stressors. Emphasis on comparative studies across species and environmental gradients, and which incorporates insights at the cellular and ecosystem level, is critical for forecasting whole-tree responses to altered biogeochemical landscapes.

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The Comet assay (single cell gel electrophoresis assay) measures DNA strand breaks in individual cells. In the assay cells are embedded in agarose, lysed, and electrophoresed under low voltage, allowing migration of damaged DNA. The DNA is stained and subsequently viewed with an epifluorescent microscope. If DNA damage has occurred the electrophoresed DNA fragments appear as a diffuse tail behind the nucleus known as a "comet". Many computer-aided analysis systems are currently in use to quantify the amount of DNA damage that is represented by a comet image. Here, we present a novel method of analysis known as "tail profile". This method of analysis provides several advantages over currently employed methods, which rely primarily on the "tail moment" method of analysis. We compared the amount of DNA damage reported from both the tail profile and tail moment methods of analysis and observed a 26% (P<0.0001) increase in damage detected by tail profile across the 10-25 microm range of tail length, where the majority of the relevant comet data is concentrated. We further report that this increase in sensitivity is not only limited to assessing DNA damage, but also to gathering data from DNA repair assays. Furthermore, we demonstrate increased functionality and extended data analysis capabilities with the use of a compressed collection of images called a "comet chip" and through a visual representation of data called a "profile plot". Use of the custom macros enabled us to detect an unexpected characteristic of the electrophoretic profile, giving us novel insight into the nature of comet analysis. In addition to the increased analytical sensitivity proffered by this system, the tail profile macros are upgradeable and platform independent.

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