Valutazione del rischio ecologico e sviluppo di strategie innovative per il monitoraggio della qualita’ dei corpi idrici: applicazione di un nuovo approccio integrato in un bacino pilota.

Freshwater is extremely precious; but even more precious than freshwater is clean freshwater. From the time that 2/3 of our planet is covered in water, we have contaminated our globe with chemicals that have been used by industrial activities over the last century in a unprecedented way causing harm to humans and wildlife. We have to adopt a new scientific mindset in order to face this problem so to protect this important resource. The Water Framework Directive (European Parliament and the Council, 2000) is a milestone legislative document that transformed the way that water quality monitoring is undertaken across all Member States by introducing the Ecological and Chemical Status. A “good or higher” Ecological Status is expected to be achieved for all waterbodies in Europe by 2015. Yet, most of the European waterbodies, which are determined to be at risk, or of moderate to bad quality, further information will be required so that adequate remediation strategies can be implemented. To date, water quality evaluation is based on five biological components (phytoplankton, macrophytes and benthic algae, macroinvertebrates and fishes) and various hydromorphological and physicochemical elements. The evaluation of the chemical status is principally based on 33 priority substances and on 12 xenobiotics, considered as dangerous for the environment. This approach takes into account only a part of the numerous xenobiotics that can be present in surface waters and could not evidence all the possible causes of ecotoxicological stress that can act in a water section. The mixtures of toxic chemicals may constitute an ecological risk not predictable on the basis of the single component concentration. To improve water quality, sources of contamination and causes of ecological alterations need to be identified. On the other hand, the analysis of the community structure, which is the result of multiple processes, including hydrological constrains and physico-chemical stress, give back only a “photograph” of the actual status of a site without revealing causes and sources of the perturbation. A multidisciplinary approach, able to integrate the information obtained by different methods, such as community structure analysis and eco-genotoxicological studies, could help overcome some of the difficulties in properly identifying the different causes of stress in risk assessment. In synthesis, the river ecological status is the result of a combination of multiple pressures that, for management purposes and quality improvement, have to be disentangled from each other. To reduce actual uncertainty in risk assessment, methods that establish quantitative links between levels of contamination and community alterations are needed. The analysis of macrobenthic invertebrate community structure has been widely used to identify sites subjected to perturbation. Trait-based descriptors of community structure constitute a useful method in ecological risk assessment. The diagnostic capacity of freshwater biomonitoring could be improved by chronic sublethal toxicity testing of water and sediment samples. Requiring an exposure time that covers most of the species’ life cycle, chronic toxicity tests are able to reveal negative effects on life-history traits at contaminant concentrations well below the acute toxicity level. Furthermore, the responses of high-level endpoints (growth, fecundity, mortality) can be integrated in order to evaluate the impact on population’s dynamics, a highly relevant endpoint from the ecological point of view. To gain more accurate information about potential causes and consequences of environmental contamination, the evaluation of adverse effects at physiological, biochemical and genetic level is also needed. The use of different biomarkers and toxicity tests can give information about the sub-lethal and toxic load of environmental compartments. Biomarkers give essential information about the exposure to toxicants, such as endocrine disruptor compounds and genotoxic substances whose negative effects cannot be evidenced by using only high-level toxicological endpoints. The increasing presence of genotoxic pollutants in the environment has caused concern regarding the potential harmful effects of xenobiotics on human health, and interest on the development of new and more sensitive methods for the assessment of mutagenic and cancerogenic risk. Within the WFD, biomarkers and bioassays are regarded as important tools to gain lines of evidence for cause-effect relationship in ecological quality assessment. Despite the scientific community clearly addresses the advantages and necessity of an ecotoxicological approach within the ecological quality assessment, a recent review reports that, more than one decade after the publication of the WFD, only few studies have attempted to integrate ecological water status assessment and biological methods (namely biomarkers or bioassays). None of the fifteen reviewed studies included both biomarkers and bioassays. The integrated approach developed in this PhD Thesis comprises a set of laboratory bioassays (Daphnia magna acute and chronic toxicity tests, Comet Assay and FPG-Comet) newly-developed, modified tacking a cue from standardized existing protocols or applied for freshwater quality testing (ecotoxicological, genotoxicological and toxicogenomic assays), coupled with field investigations on macrobenthic community structures (SPEAR and EBI indexes). Together with the development of new bioassays with Daphnia magna, the feasibility of eco-genotoxicological testing of freshwater and sediment quality with Heterocypris incongruens was evaluated (Comet Assay and a protocol for chronic toxicity). However, the Comet Assay, although standardized, was not applied to freshwater samples due to the lack of sensitivity of this species observed after 24h of exposure to relatively high (and not environmentally relevant) concentrations of reference genotoxicants. Furthermore, this species demonstrated to be unsuitable also for chronic toxicity testing due to the difficult evaluation of fecundity as sub-lethal endpoint of exposure and complications due to its biology and behaviour. The study was applied to a pilot hydrographic sub-Basin, by selecting section subjected to different levels of anthropogenic pressure: this allowed us to establish the reference conditions, to select the most significant endpoints and to evaluate the coherence of the responses of the different lines of evidence (alteration of community structure, eco-genotoxicological responses, alteration of gene expression profiles) and, finally, the diagnostic capacity of the monitoring strategy. Significant correlations were found between the genotoxicological parameter Tail Intensity % (TI%) and macrobenthic community descriptors SPEAR (p<0.001) and EBI (p<0.05), between the genotoxicological parameter describing DNA oxidative stress (ΔTI%) and mean levels of nitrates (p<0.01) and between reproductive impairment (Failed Development % from D. magna chronic bioassays) and TI% (p<0.001) as well as EBI (p<0.001). While correlation among parameters demonstrates a general coherence in the response to increasing impacts, the concomitant ability of each single endpoint to be responsive to specific sources of stress is at the basis of the diagnostic capacity of the integrated approach as demonstrated by stations presenting a mismatch among the different lines of evidence. The chosen set of bioassays, as well as the selected endpoints, are not providing redundant indications on the water quality status but, on the contrary, are contributing with complementary pieces of information about the several stressors that insist simultaneously on a waterbody section providing this monitoring strategy with a solid diagnostic capacity. Our approach should provide opportunities for the integration of biological effects into monitoring programmes for surface water, especially in investigative monitoring. Moreover, it should provide a more realistic assessment of impact and exposure of aquatic organisms to contaminants. Finally this approach should provide an evaluation of drivers of change in biodiversity and its causalities on ecosystem function/services provision, that is the direct and indirect contributions to human well-being.

Ecological studies on high-rate biological filters with special reference to microbial biosynthesis and nitrification

Pilot scale studies of high rate filtration were initiated to assess its potential as either a primary 'roughing' filter to alleviate the seasonal overloading of low rate filters on Hereford sewage treatment works - caused by wastes from cider production - or as a two stage high rate process to provide complete sewage treatment. Four mineral and four plastic primary filter media and two plastic secondary filter media were studied. The hydraulic loading applied to the primary plastic media (11.2 m3 /m3 .d) was twice that applied to the mineral media. The plastic media removed an average around 66 percent and the mineral media around 73 percent of the BOD applied when the 90 percentile BOD concentration was 563 mg/1. At a hydraulic loading of 4 m3 /m3 .d the secondary filters removed most of the POD from partially settled primary filter effluents, with one secondary effluent satisfying a 25 mg/1 BOD and 30 mg/1 SS standard. No significant degree of nitrification was achieved. Fungi dominated the biological film of the primary filters, with invertebrate grazers having little influence on film levels. Ponding did not arise, and modular media supported lower film levels than random-fill types. Secondary filter film levels were low, being dominated by bacteria. The biological loading applied to the filters was related to sludge dewaterability, with the most readily conditionable sludges produced by filters supporting heavy film. Sludges produced by random-fill media could be dewatered as readily as those produced by low rate filters treating the same sewage. Laboratory scale studies showed a relationship between log effluent BOD and nitrification achieved by biological filters. This relationship and the relationship between BOD load applied and removed observed in all filter media could he used to optimise operating conditions required in biological filters to achieve given effluent BOD and ammoniacal nitrogen standards.

Ecological studies on river pollution control

This collection of papers records a series of studies, carried out over a period of some 50 years, on two aspects of river pollution control - the prevention of pollution by sewage biological filtration and the monitoring of river pollution by biological surveillance. The earlier studies were carried out to develop methods of controlling flies which bred in the filters and caused serious nuisance and possible public health hazard, when they dispersed to surrounding villages. Although the application of insecticides proved effective as an alleviate measure, because it resulted in only a temporary disturbance of the ecological balance, it was considered ecologically unsound as a long-term solution. Subsequent investigations showed that the fly populations in filters were largely determined by the amount of food available to the grazing larval stage in the form of filter film. It was also established that the winter deterioration in filter performance was due to the excessive accumulation of film. Subsequent investigations were therefore carried out to determine the factors responsible for the accumulation of film in different types of filter. Methods of filtration which were considered to control film accumulation by increasing the flushing action of the sewage, were found to control fungal film by creating nutrient limiting conditions. In some filters increasing the hydraulic flushing reduced the grazing fauna population in the surface layers and resulted in an increase in film. The results of these investigations were successfully applied in modifying filters and in the design of a Double Filtration process. These studies on biological filters lead to the conclusion that they should be designed and operated as ecological systems and not merely as hydraulic ones. Studies on the effects of sewage effluents on Birmingham streams confirmed the findings of earlier workers justifying their claim for using biological methods for detecting and assessing river pollution. Further ecological studies showed the sensitivity of benthic riffle communities to organic pollution. Using experimental channels and laboratory studies the different environmental conditions associated with organic pollution were investigated. The degree and duration of the oxygen depletion during the dark hours were found to be a critical factor. The relative tolerance of different taxa to other pollutants, such as ammonia, differed. Although colonisation samplers proved of value in sampling difficult sites, the invertebrate data generated were not suitable for processing as any of the commonly used biotic indexes. Several of the papers, which were written by request for presentation at conferences etc., presented the biological viewpoint on river pollution and water quality issues at the time and advocated the use of biological methods. The information and experiences gained in these investigations was used as the "domain expert" in the development of artificial intelligence systems for use in the biological surveillance of river water quality.

Zoocoenological state of microhabitats and its seasonal dynamics in an aquatic macroinvertebrate assembly (Hydrobiological case studies on lake Balaton, No. 1.)

In the years 2002, 2003 and 2004 we collected samples of macroinvertebrates on a total of 36 occasions in Badacsony bay, in areas of open water (in the years 2003 and 2004 reed-grassy) as well as populated by reed (Phragmites australis) and cattail (Typha angustifolia). Samples were taken using a stiff hand net. The sampling site includes three microhabitats differentiated only by the aquatic plants inhabiting these areas. Our data was gathered from processing 208 individual samples. The quantity of macroinvertebrates is represented by biovolume value based on volume estimates. We can identify taxa in abundant numbers found in all water types and ooze; as well as groups associated with individual microhabitats with various aquatic plants. We can observe a notable difference between the years in the volume of invertebrate macrofauna caused by the drop of water level, and the multiplication of submerged macrophytes. There are smaller differences between the samples taken in reeds and cattail stands. In the second half of 2003 – which was a year of drought – the Najas marina appeared in open waters and allowed to support larger quantities of macroinvertebrates. In 2004 with higher water levels, the Potamogeton perfoliatus occurring in the same area has had an even more significant effect. This type of reed-grass may support the most macroinvertebrates during the summer. From the aspect of diversity relations we may suspect different characteristics. The reeds sampling site proved to be the richest, while the cattail microhabitat is close behind, open water (with submerged macrophytes) is the least diverse microhabitat.

Ecology of the Ráckeve-Sorokság Danube – a review

Present paper is a review on the Ráckeve-Soroksár Danube in ecological standpoint. The goal of this study is to collect and evaluate all of available publications in that conception, concerning this Danube arm. Phytoplankton, zooplankton, macroinvertebrates, vertebrates, macrophytes and also water chemistry, water management, geographical description are presented. The review comprises the main studies beginning with the earliest faunistic publications up to the recent ecological, multidisciplinary investigations. Spatial and temporal patterns likewise water quality are considered as important. Additionally checklist of aquatic invertebrate and vertebrate fauna are given based on data from literature.

Accumulation and fate of mercury in an Everglades aquatic food web

This project examined the pathways of mercury (Hg) bioaccumulation and its relation to trophic position and hydroperiod in the Everglades. I described fish-diet differences across habitats and seasons by analyzing stomach contents of 4,000 fishes of 32 native and introduced species. Major foods included periphyton, detritus/algal conglomerate, small invertebrates, aquatic insects, decapods, and fishes. Florida gar, largemouth bass, pike killifish, and bowfin were at the top of the piscine food web. Using prey volumes, I quantitatively classified the fishes into trophic groups of herbivores, omnivores, and carnivores. Stable-isotope analysis of fishes and invertebrates gave an independent and similar assessment of trophic placement. Trophic patterns were similar to those from tropical communities. I tested for correlations of trophic position and total mercury. Over 4,000 fish, 620 invertebrate, and 46 plant samples were analyzed for mercury with an atomic-fluorescence spectrometer. Mercury varied within and among taxa. Invertebrates ranged from 25–200 ng g −1 ww. Small-bodied fishes varied from 78–>400 ng g −1 ww. Large predatory fishes were highest, reaching a maximum of 1,515 ng−1 ww. Hg concentrations in both fishes and invertebrates were positively correlated with trophic position. I examined the effects of season and hydroperiod on mercury in wild and caged mosquitofish at three pairs of marshes. Nine monthly collections of wild mosquitofish were analyzed. Hydroperiod-within-site significantly affected concentrations but it interacted with sampling period. To control for wild-fish dispersal, and to measure in situ uptake and growth, I placed captive-reared, neonate mosquitofish with mercury levels from 7–14 ng g−1 ww into field cages in the six study marshes in six trials. Uptake rates ranged from 0.25–3.61 ng g−1 ww d −1. As with the wild fish, hydroperiod-within-site was a significant main effect that also interacted with sampling period. Survival exceeded 80%. Growth varied with season and hydroperiod, with greatest growth in short-hydroperiod marshes. The results suggest that dietary bioaccumulation determined mercury levels in Everglades aquatic animals, and that, although hydroperiod affected mercury uptake, its effect varied with season. ^

Immunophylogenetic aspects of a gorgonian coral

One goal of comparative immunology is to derive inferences about evolutionary pathways in the development of immune-defense systems. Almost 700 million years ago, a major divergence occurred in the phylogeny of animals, spitting all descendants into either the protostome or deuterostome (includes vertebrates) lineages. Genes have evolved independently along these lineages for that amount of time. Cnidarians originated before that divergence event, and can hold clues as to which immune response genes are homologous to both lineages. This work uses the gorgonian coral, Swiftia exserta, for two major reasons: (1) because of their phylogenetic position, corals are an important animal model in studies concerning the phylogeny of immune-response genes, and (2) nothing is known about the genes controlling immunocompetence in corals. The work described here has important implications in both innate and adaptive immunity. ^ The vertebrate complement system is a major component of innate immunity. C3 is a critical component of the three pathways of complement. Because of its opsonic properties, a C3-like protein is expected to have evolved early. However, currently available data suggests that complement-like components are unique to the deuterostome lineage. This work describes the cloning and characterization of a C3-like gene from S. exserta. The deduced polypeptide sequence reveals conservation of multiple, functionally critical, sites while sharing physiochemical and structural properties with the complement components C3/C4/C5. ^ Antigen processing, via intracellular enzymatic proteasomes, is a major requirement of vertebrate adaptive immunity. These organelles have a catalytic core, through which pass intracellular proteins for degradation into peptides presentable to the immune system. LMP 7 is one component of the paralogous “immuno-proteasome”. LMP 7 is a paralog of the ubiquitous LMP X, but is restricted to vertebrates. While LMP 7 is absent in the coral, this work describes a coral LMP X gene. Phylogenetic analyses, along with hydropathy profiling of a critical portion of the invertebrate and vertebrate paralogous genes, suggests that some invertebrates have two diverging LMP X genes. In some cases, one LMP X protein shares characteristics with vertebrate LMP 7. This work presents new evidence for how the LMP X and 7 genes evolved. ^

Antibacterial proteins and peptides in nurse shark ( Ginglymostoma cirratum) peripheral blood leukocytes

In many vertebrate and invertebrate species mediators of innate immunity include antimicrobial peptides (AMPs) such as peptide fragments of histones and other proteins with previously ascribed different functions. Shark AMPs have not been described and this research examines the antibacterial activity of nurse shark (Ginglymostoma cirratum) peripheral blood leukocyte lysates. Screening of lysates prepared by homogenizing unstimulated peripheral blood leukocytes identified muramidase (lysozyme-like) and non-muramidase antibacterial activity. Lysates were tested for lysozyme using the lysoplate assays, and antibacterial (AB) activity was assayed for by a microdilution growth assay that was developed using Planococcus citreus as the target bacterium. Fractionation of crude lysates by ion exchange and affinity chromatography was followed by a combination of SDS-PAGE with LC/MS-MS and/or N-terminal sequence analysis of low molecular weight protein bands (<20 kDa). This yielded several peptides with amino acid sequence similarity to lysozyme, ubiquitin, hemoglobin, human histones H2A, H2B and H4 and to antibacterial histone fragments of the catfish and the Asian toad. Not all peptide sequences corresponded to peptides potentially antibacterial. The correlation of a specific protein band in active lysate fractions was accomplished by employing the acid-urea gel overlay assays in which AB activity was seen as zones of growth inhibition on a lawn of P. citreus at a position corresponding to that of the putative AB protein band. This study is the first to describe putative AMPs in the shark and their potential role in innate immunity.^

Nutrient enrichment, grazer identity, and their effects on epiphytic algal assemblages: field experiments in subtropical turtlegrass Thalassia testudinum meadows

We tested the relative importance of top-down and bottom-up effects by experimentally evaluating the combined and separate effects of nutrient availability and grazer species composition on epiphyte communities and seagrass condition in Florida Bay. Although we succeeded in substantially enriching our experimental cylinders, as indicated by elevated nitrogen concentrations in epiphytes and seagrass leaves, we did not observe any major increases in epiphyte biomass or major loss of Thalassia testudinum by algal overgrowth. Additionally, we did not detect any strong grazer effects and found very few significant nutrient-grazer interactions. While this might suggest that there was no important differential response to nutrients by individual grazer species or by various combinations of grazers, our results were complicated by the lack of significant differences between control and grazer treatments, and as such, these results are best explained by the presence of unwanted amphipod grazers (mean = 471 ind. m–2) in the control cylinders. Our estimates of grazing rates and epiphyte productivities indicate that amphipods in the control cylinders could have lowered epiphyte biomass to the same level that the experimental grazers did, thus effectively transforming the control treatments into grazer treatments. If so, our experiments suggest that the effects of invertebrate grazing (and those of amphipods alone) were stronger than the effects of nutrient enrichment on epiphytic algae, and that it does not require a large density

No Free Lunch: Displaced Marsh Consumers Regulate a Prey Subsidy to an Estuarine Consumer

Pulse subsidies account for a substantial proportion of resource availability in many systems, having persistent and cascading effects on consumer population dynamics, and energy flow within and across ecosystem boundaries. Although the importance of pulsed resource subsidies is well-established, the mechanisms that regulate resource fluxes across ecosystem boundaries are not well understood. The aim of our study was to determine the extent that marsh consumers regulated a marsh prey subsidy to estuarine consumers in the oligohaline reaches of an Everglades estuary. We characterized a marsh pulsed subsidy of cyprinodontoid, invertebrate and sunfish prey that move into the upper estuary from adjacent drying marshes. In response to the prey pulse, we examined the numerical, fitness and dietary responses of three focal consumers in the upper estuary; two marsh species (largemouth bass and bowfin) that accompanied the subsidy as a result of marsh drying, and one estuarine consumer (snook). At the onset of marsh drying and the prey subsidy, estuarine consumers switched diets to consume the larger marsh prey (sunfishes), while bass and bowfin maintained similar diets (cyprinodontoids and invertebrates respectively) than pre and post subsidy. From the consumption of this subsidy, bass (marsh species) and snook (estuarine species) exhibited fitness gains while bowfin did not. Although both marsh and estuarine consumers benefitted from the subsidy, we found evidence that freshwater consumers shunted some of the subsidy away from snook. Of the prey sampled in consumer stomachs, 41% of marsh prey biomass was eaten by marsh consumers, while 59% was consumed by the estuarine consumer. We conclude that the amount of the marsh prey available to estuarine consumers may be greater in the absence of marsh consumers, thus the magnitude of the prey subsidy could depend on the dynamics of the marsh consumers from donor communities.

PROTEOMIC ANALYSIS OF WOLBACHIA SYMBIOSIS WITHIN THE DROSOPHILA OVARY

Wolbachia pipientis are bacterial endosymbionts carried by millions of invertebrate species, including ~40% of insect species and some filarial nematodes. In insects, basic Wolbachia research has potential applications in controlling vector borne disease. Conversely, Wolbachia of filarial nematodes are causative agents of neglected tropical diseases such as lymphatic filariasis and African river blindness. However, remarkably little is known about how Wolbachia interact with their hosts at the molecular level. Understanding this is important to inform the basis for symbiosis and help prevent human disease. I used a high-throughput proteomics approach to study how Drosophila host cells are modified by Wolbachia infection. This analysis identified 23 Drosophila proteins that significantly changed in amount as a result of Wolbachia infection. A subset of differentially abundant host proteins were consistent with Wolbachia-associated phenotypes reported previously. This study also provides the first ever discovery-based evidence for a Wolbachia-associated change in maternal germline histone loads, which has possible implications in Rescue of a common Wolbachia-induced reproductive manipulation known as Cytoplasmic Incompatibility.

Macroinvertebrate abundance in montane tropical streams of varying land-use, Ecuador

Despite the importance of tropical montane cloud forest streams, studies investigating aquatic communities in these regions are rare and knowledge on the driving factors of community structure is missing. The objectives of this study therefore were to understand how land-use influences habitat structure and macroinvertebrate communities in cloud forest streams of southern Ecuador. We evaluated these relationships in headwater streams with variable land cover, using multivariate statistics to identify relationships between key habitat variables and assemblage structure, and to resolve differences in composition among sites. Results show that shading intensity, substrate type and pH were the environmental parameters most closely related to variation in community composition observed among sites. In addition, macroinvertebrate density and partly diversity was lower in forested sites, possibly because the pH in forested streams lowered to almost 5 during spates. Standard bioindicator metrics were unable to detect the changes in assemblage structure between disturbed and forested streams. In general, our results indicate that tropical montane headwater streams are complex and heterogeneous ecosystems with low invertebrate densities. We also found that some amount of disturbance, i.e. patchy deforestation, can lead at least initially to an increase in macroinvertebrate taxa richness of these streams.

(Table 1) Individual codes, haplotype codes, GenBank accession numbers for DNA sequences and sample locality of the analysed Betamorpha fusiformis specimens from the ANDEEP III expedition

Based on our current knowledge about population genetics, phylogeography and speciation, we begin to understand that the deep sea harbours more species than suggested in the past. Deep-sea soft-sediment environment in particular hosts a diverse and highly endemic invertebrate fauna. Very little is known about evolutionary processes that generate this remarkable species richness, the genetic variability and spatial distribution of deep-sea animals. In this study, phylogeographic patterns and the genetic variability among eight populations of the abundant and widespread deep-sea isopod morphospecies Betamorpha fusiformis [Barnard, K.H., 1920. Contributions to the crustacean fauna of South Africa. 6. Further additions to the list of marine isopods. Annals of the South African Museum 17, 319-438] were examined. A fragment of the mitochondrial 16S rRNA gene of 50 specimens and the complete nuclear 18S rRNA gene of 7 specimens were sequenced. The molecular data reveal high levels of genetic variability of both genes between populations, giving evidence for distinct monophyletic groups of haplotypes with average p-distances ranging from 0.0470 to 0.1440 (d-distances: 0.0592-0.2850) of the 16S rDNA, and 18S rDNA p-distances ranging between 0.0032 and 0.0174 (d-distances: 0.0033-0.0195). Intermediate values are absent. Our results show that widely distributed benthic deep-sea organisms of a homogeneous phenotype can be differentiated into genetically highly divergent populations. Sympatry of some genotypes indicates the existence of cryptic speciation. Flocks of closely related but genetically distinct species probably exist in other widespread benthic deep-sea asellotes and other Peracarida. Based on existing data we hypothesize that many widespread morphospecies are complexes of cryptic biological species (patchwork hypothesis).

Potential impact of invasive anuran species in western Newfoundland

Successful dispersal and establishment of invasive anurans (frogs and toads) may be influenced by competitive exclusion and/or niche differentiation with competing species. I investigated the dispersal of anurans in western Newfoundland using anuran calling surveys and pond-edge visual encounter surveys. The Mink Frog, Lithobates septentrionalis, had dispersed ~50 km northeast from the original (2001) discovery location and ~34 km southwest; displaying spatial separation from Green Frogs, Lithobates clamitans, at landscape and local scales. Visual encounter surveys did not reveal any correlation between adult Mink Frogs and odonate competitors. Additionally, I assessed the impact of varying tadpole densities on removal of epilithic periphyton by providing epilithon covered substrates for American Toad, Anaxyrus americanus, tadpoles raised in laboratory or field enclosures. Higher tadpole densities resulted in smaller tadpoles that removed more periphyton from substrates. As anuran population ranges expand, there may be effects on ecological resources for vertebrate and invertebrate competitors.