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A riqueza do património genético vitícola existente em Portugal leva a considerá-lo como um dos países da Europa com maior densidade de castas autóctones, embora cerca de 85 % dessas castas só ocupem aproximadamente 10 % da área total de vinha. Apesar da preservação destas castas muito pouco cultivadas, que se designa de castas minoritárias, estar praticamente garantida em coleções ampelográficas, torna-se necessário a sua valorização e difusão pelo sector vitivinícola. Este trabalho constitui uma primeira abordagem para a caracterização físico-química e sensorial de vinhos produzidos, em duas vindimas, a partir de quatro castas brancas e de quatro castas tintas. No caso dos vinhos das castas tintas, a caracterização físico-química incluiu também o estudo do perfil de antocianinas, bem como a determinação dos teores de antocianas totais e de polifenóis totais. Os resultados obtidos mostraram uma diferenciação dos vinhos das duas vindimas, verificando-se também, ao nível organolético, apreciações globais superiores para os vinhos brancos das castas ‘Galego Dourado’ e ‘Códega do Larinho’ e para os vinhos tintos das castas ‘Malvasia Preta’ e ‘Cornifesto’. Verificou-se, ainda, que os vinhos que apresentaram teores mais elevados de antocianas foram os das castas ‘Cornifesto’ e ‘Malvasia Preta’, sendo os vinhos das castas ‘Tinta Negra’ e ‘Malvasia Preta’ os que evidenciaram ser mais ricos em polifenóis totais. Um resultado importante de notar é que os teores de polifenóis totais dos vinhos da mesma casta mostraram ser idênticos ou muito similares, nas duas vindimas. Contudo, considera-se que sejam necessários mais estudos para uma caracterização mais robusta dos vinhos destas castas.

The lack of graft success is a problem that causes economic loss to nursery growers and wine growers, especially because it is often expressed when the vineyard is already established. Grafting is a complex phenomenon, involving anatomical, physiological, biochemical and molecular interactions between the scion and the rootstock. For this reason, the lack of graft success is a complex problem to study. However, it is believed that through a joint analysis of the different mechanisms of graft success, one can advance more quickly in the knowledge of this phenomenon. Despite the scarce information available on the role of phenolic compounds in Vitis grafts, an analytical method was developed and validated for phenolic compounds quantification in vine graft tissues reviling some chemical markers for the cv. ‘Syrah’ with 110R and SO4 rootstocks. This work aims to determine, among phenolic acids and two flavanols, the chemical markers of graft compatibility between scions from two clones of cv. ‘Touriga Nacional’ (TN 21 and TN 112) and the rootstock 110R. For this purpose, extracts of grafting tissues were prepared at three different locations (above, below and at the grafting union), and were analyzed in three different stages of the grafting cycle. From all sections analyzed, the graft union tissue is the one to discriminate better the two combinations. Gallic acid, catechin and sinapic acid were found in higher amount at graft union in the less compatible combination later in graft development. Furthermore, the chlorogenic acid had a higher content in the more compatible combination at the early stage when compared to the less compatible graft.

This work focused on the characterization of the volatile compounds and sensory profile of white wines produced from three minority grapevine varieties of Portugal namely ‘Malvasia’ (Colares), ‘Verdelho’ and ‘Galego Dourado’. The characterization took place using sensory and gas chromatography analysis. Furthermore, the data obtained were analysed through the use of multivariate analysis, which made it possible to evaluate the similarities and dissimilarities between the varieties. The results obtained show a differentiation of the wines produced from each grapevine variety but above all a differentiation of the two vintages was verified. The results obtained, both from a sensory and a chemical point of view, show an interesting oenological potential of these varieties, but still require further studies, in order to evaluate the influence of climatic effects on the profile of volatile compounds and also on the sensory profile Este trabalho centrou-se na caracterização sensorial e da composição volátil de vinhos brancos produzidos a partir de três castas minoritárias, designadamente ‘Malvasia’ (Colares), ‘Verdelho’ e ‘Galego Dourado’. A caracterização ocorreu por meio de análise sensorial e por cromatografia gás líquido de alta resolução, e os resultados obtidos foram analisados através de análise multivariada, que permitiu avaliar as semelhanças e as diferenças entre as castas. Os resultados obtidos mostram uma diferenciação dos vinhos produzidos a partir de cada casta, mas acima de tudo uma diferenciação das duas colheitas. Os resultados obtidos, tanto do ponto de vista sensorial como na composição química, mostram um potencial enológico interessante destas castas, embora sejam necessários mais estudos para avaliar a influência dos efeitos climáticos no perfil de compostos voláteis e também no perfil sensorial info:eu-repo/semantics/publishedVersion

The domesticated grapevine spread along the Mediterranean basin from the primary Near East domestication area, where the greatest genetic diversity is found in its ancestor, the wild vine populations. Portuguese wild populations are on the southwestern fringe of the distribution of the Vitis vinifera L. ssp. sylvestris (C.C. Gmel.) Hegi in Europe. During the last Glacial Period they became isolated from the previous continuum that had been the territory of wild vine populations. Archaeological remains of domesticated vinifera grapevines in Portugal date back from 795 Before Common Era (BCE) in the lower Tagus river basin. In this work, 258 Portuguese vinifera varieties and sylvestris plants were characterized using 261 single nucleotide polymorphism (SNP) markers. The study of the genetic diversity of this local germplasm, its population structure and kinship, all framed in their historical and geographical backgrounds, revealed a complex network of first-degree relationships, where only Iberian varieties are involved. Some Iberian genotypes, like Alfrocheiro (Bruñal, in Spain), Sarigo (Cayetana Blanca), Mourisco Branco (Hebén), Amaral (Caiño Bravo), and Marufo (Moravia Dulce) are ancestors of a considerable fraction of all the autochthonous analyzed varieties. A part of the diversity developed was mostly local in some cases as shown by the closeness of several varieties (Vinhos Verdes) to the wild cluster in different analyses. Besides, several evidences of introgression of domesticated germplasm into wild vines was found, substantiating the high risk of genetic contamination of the sylvestris subspecies. All these findings together to the known matching between the wild maternal lineage of the Iberian Peninsula and an important number of Portuguese grapevine varieties (chlorotype A), point out that some of these varieties derive, directly or indirectly, from originally local wild populations, supporting the possible occurrence of secondary events of local domestication, or, at least, of an introgression process of wild into cultivated grapevines.

Background Grafting is an intensive commercial practice required to protect the European grapevine against the Phylloxera pest. Rootstocks resistant to this pest are hybrids of American vine species with different levels of compatibility with European Vitis vinifera varieties. Aiming to understand what drives grafting compatibility in grapevine, a transcriptomic approach was used to search for master regulators of graft success. Two scion/rootstock combinations, with different levels of compatibility, were compared in a nursery-grafting context at two stages, at 21 and 80 days after grafting. Results In the most compatible combination, an earlier and higher expression of genes signaling the metabolic and hormonal pathways as well as a reduced expression of genes of the phenolic metabolism and of the oxidative stress response was observed. At 80 days after grafting a higher expression of transcription factors regulating vascular maintenance, differentiation and proliferation was obtained in the most compatible combination. Moreover, lower expression levels of microRNAs potentially targeting important transcription factors related to plant development was observed in the more compatible combination when compared to the less compatible one. Conclusion In this context, a set of regulators was selected as potential expression markers for early prediction of a compatible grafting.

The assessment of wine authenticity is of utmost importance in the current context of a growing market globalization. The strontium isotopic ratio 87Sr/86Sr is a well-established tool for dating and tracing the origin of rocks and minerals with special interest for wine traceability. A study was developed to examine the variation of 87Sr/86Sr in wines from Douro Valley taking into account the effects of vineyard location and grape variety. The 87Sr/86Sr of soils and wines from six vineyards were determined by using an ICP-MS based analytical procedure. A total of twenty-two monovarietal wines, obtained at micro vinification scale, from relevant white and red grapevine varieties for Douro region, were analysed. The range of 87Sr/86Sr values observed in soils and wines was of 0.708–0.725 and 0.711–0.717, respectively. The present study updates the scarce knowledge available on strontium isotopic ratios in soils and wines from Douro Valley, and its results will enlarge global databank on wine composition and support comparison with other world regions.

The role of phenolic compounds in graft compatibility has been widely studied in various fruit species. However, little information is available for Vitis spp. This work aims at investigating the differences in phenolic acid and flavanol content in various scion/rootstock systems and understanding if they can be used as chemical markers of graft compatibility, using combinations of the Syrah cultivar (clones FR 470 and FR 383) with two rootstocks (110R and SO4). For this purpose, extracts of grafting tissues collected above graft union, at the graft union and below graft union from each combination at crucial stages of the grafting cycle (callusing, rooting and end of cycle) were prepared and analysed by HPLC. The results obtained revealed that the contents in gallic, ferulic and sinapic acids are closely related to the level of compatibility of the studied grafting combinations. The less compatible combinations (Syrah383/110R and Syrah383/SO4) exhibit higher content of gallic acid and lower contents of ferulic and sinapic acids than the more compatible ones (Syrah470/110R and Syrah470/SO4). In addition, these phenolic acids accumulated in the graft union and in the adjacent sections as a response to grafting. A significant difference in their levels was detected between the above and below graft union sections, especially at the rooting stage. Therefore, they can be seen as chemical markers to be used in an early detection of graft compatibility, preferably performed at the rooting stage and targeting the sections above and below the graft union.

Background and Aims Grapevine cultivation has a long tradition in Portugal. Presently, 343 cultivars are legally accepted for wine production. Two hundred and eighty eight accessions from the Portuguese National Ampelographic Collection (including 27 wild grapevines) were genotyped using single-nucleotide polymorphisms (SNP) and compared with the Instituto de Ciencias de la Vid y del Vino-SNP database to reveal identities, synonymies and homonymies. Methods and Results A set of 48 SNPs was used to profile the 288 accessions and 263 different genotypes were detected. A comparison with the Instituto de Ciencias de la Vid y del Vino-SNP database unveiled 14 new synonymies among Portuguese, Spanish and other international cultivars and confirmed known synonyms and homonyms. Most of the genotyped cultivars are not cultivated in Spain. Clustering using hierarchical and non-hierarchical methods did not reflect the Portuguese geographical viticulture regions. Conclusions New synonyms and homonyms were identified by SNP profiling of the Portuguese grapevine germplasm. Most of the genotyped cultivars are circumscribed to Portugal. Some cultivars are important in the Iberian context. Significance of the Study This study confirmed the uniqueness of the Portuguese grapevine genetic pool and disclosed many new synonyms within the cultivars authorised for wine production in Portugal. This study confirmed the suitability of the 48 SNP set for grapevine profiling. Results will help to manage the Portuguese germplasm and to adjust the Portuguese legal framework on the denomination of the cultivars authorised for wine production.

Congruence among analyses of plant genomic data partitions (nuclear, chloroplast and mitochondrial) is a strong indicator of accuracy in plant molecular phylogenetics. Recent analyses of both nuclear and chloroplast genome data of land plants (embryophytes) have, controversially, been shown to support monophyly of both bryophytes (mosses, liverworts, and hornworts) and tracheophytes (lycopods, ferns, and seed plants), with mosses and liverworts forming the clade Setaphyta. However, relationships inferred from mitochondria are incongruent with these results, and typically indicate paraphyly of bryophytes with liverworts alone resolved as the earliest-branching land plant group. Here, we reconstruct the mitochondrial land plant phylogeny from a newly compiled data set. When among-lineage composition heterogeneity is accounted for in analyses of codon-degenerate nucleotide and amino acid data, the clade Setaphyta is recovered with high support, and hornworts are supported as the earliest-branching lineage of land plants. These new mitochondrial analyses demonstrate partial congruence with current hypotheses based on nuclear and chloroplast genome data, and provide further incentive for revision of how plants arose on land. UIDB/04326/2020, PTDC/BIA-EVF/1499/2014, EMBRC.PT ALG-01-0145-FEDER-022121, BIODATA.PT ALG-01-0145-FEDER-022231 info:eu-repo/semantics/publishedVersion

The diversity found among the Vitis vinifera L. species allows the production of wines with very different characteristics. The development of platforms suitable for food composition analysis is currently an emerging area. Among these, DNA biosensors have been developed for a wide variety of applications, ranging from food safety to authenticity. The main aim of this work was to study the detection capacity of the DNA-based optical biosensor using different V. vinifera matrices (leaf, must and wine). Genomic DNA was extracted from leaf, must and wine of three V. vinifera varieties and was tested on the long-period grating (LPG) DNA-based biosensor developed within our group. The biosensor was able to distinguish the varieties even using DNA extracted from complex matrices, revealing its potential to be applied in wine authenticity.

Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays ( Vv1 –705 bp; Vv2 –375 bp; and Vv3 –119 bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3 , produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes.

Introduction Graft incompatibility of Vitis spp is an unresolved worldwide problem with important economic consequences. Grafting comprises a complex set of morphological and physiological alterations, in which the phenolic compounds seem to be strongly involved. Therefore, a detailed analysis and recognition of structural phenolic compounds diversity in the two partners of a Vitis graft is of great importance to evaluate their role as markers of graft establishment. Objective To optimise a sample extraction method, and to develop and validate a high-performance liquid chromatography (HPLC) method for the simultaneous determination of phenolic acids and flavonols in the graft union so as to understand their behaviour in the metabolism of the scion–rootstock system, using compatible and incompatible combinations of a Syrah cultivar and two rootstocks (R110 and SO4). Methods Sixty extracts of Vitis grafting tissues were prepared and analysed by HPLC for the qualitative and quantitative determination of their phenolic profile. Results Among the phenolic compounds identified in the samples, one benzoic acid (gallic acid), three cinnamic acids (caffeic acid, ferulic acid and sinapic acid) and two flavonols (catechin and epicatechin) are potentially suitable as markers of graft incompatibility. Conclusion The method developed presents good performance and lends itself readily for application in routine analysis of the phenolic composition of Vitis grafting tissues to distinguish compatible and incompatible combinations in the graft callusing stage. Copyright © 2014 John Wiley & Sons, Ltd.

The wine sector is a billion euro business and therefore subjected to multiple attempts of fraudulent practices. This requires the development of rapid and reliable methods to detect such situations. Several methodologies have been developed based on the chemical profiles of the wines, but they are limited due to the environmental conditions that cannot be controlled. The use of DNA-based detection systems are an emergent research field that have been extended to a wide variety of food prod- ucts and are still the most reliable methods for varietal identification. However these methods are not suitable for geographical determination. Soil related fingerprints have a primary role considering that there is a relationship between the elemental compo- sition of wine and the composition of the provenance soil. WineBioCode is a project aiming to define the best strategy for wine authenticity based on a multidisciplinary approach. Two DNA-based strategies have been developed based on Real-time PCR and a label free optical biosensor platform. Both platforms enabled successful identification of specific DNA-targets when applied to Vitis vinifera L., and can be applied throughout the grape-wine chain. The methods are complementary and can be used in dif- ferent situations, according to the requirements. The geographical evaluation has been assessed by the strontium 87 Sr/86 Sr isotope ratio determination involving soil evaluation in the vineyards followed by its assay in the wine samples. The results are being integrated in order to establish the best procedure to be undertaken for wine fingerprinting, including varietal composition and geographical origin, therefore fulfilling the requirements of the geographical denominations in wine certification.

INTRODUCTION – Common limits for phenotyping are costs in terms of equipment, specialized researchers, and time. Moreover, phenotyping requires specific methods in relation to the studied traits, and each method is generally designed to highlight very specific characteristics of the singular trait. Thus, the obtained results are not easily comparable between different institutions. However the data sharing is clearly the most powerful approach to limit the costs and efforts optimizing the obtained results in term of number of studied accessions. It should be stressed that funds are another great limit in research, especially in East countries, where the most interesting cultivar variability is preserved. 28 AIMS AND SCOPES – The main purpose of this work is the establishment of a phenotyping network able to produce comparable data among the Pan-European grapevine collections. MATERIALS AND METHODS – Two protocols were proposed: one for phenology and, the other, for enocarpological traits. Data were recorded in two consecutive vintages: 2012 and 2013. Concerning phenology, the extended BBCH scale (universal and flexible) was adapted to our objects by moving it into a chrono- phenological scale. Then, a cubic curve was selected for modelling the phenological courses. Thus, computing for each accession specific coefficients, it was possible to obtain the relative phenograms. Considering the different site climatic conditions, it was possible to spatialize the results, obtaining reference phenological maps. Concerning eno- carpological traits, the attention was focused on characteristics with a clear impact on the production quality, such as berry morphology (weight, diameters, number of seeds …) and composition (sugar, titratable acidity, anthocyanin and phenolic contents …). The protocol was organized to concatenate the data acquisition, with the objective to generate new derived variables. The methods were selected to be easily reproduced in all the involved collections, without the necessity of expensive facilities. RESULTS AND DISCUSSIONS – The first year of dataset was used to evaluate the methods. Two publications are already available confirming the protocols quality. Concerning phenology, both the variability between accessions grown into the same collection and the phenological shifts between the reference varieties cultivated in different areas were highlighted by the protocol. Matching climatic and phenological information of reference cultivars, phenophases maps were obtained. The performances obtained by the eno-carpological protocol were satisfactory both in a global point of view and in a singular collection evaluation. The project obtained an impressive participation, considering the volunteer contribution without any material funding to cover the work costs. Twenty-four institutions collaborate to the data collection. 1724 accessions were evaluated: 931 (583 in 2012 and 348 in 2013) for phenology and 793 (424 in 2012 and 369 in 2013) for eno-carpological descriptions. CONCLUSIONS AND POSSIBLE APPLICATIONS – The protocol sharing allows the data comparison among different grapevine collections. The possible future application for this dataset could be related to scientific studies of genetic associations. Moreover, the improved knowledge of minor and neglected cultivars could have a direct impact on the industrial production, giving information concerning new varieties for future introduction in viticultural areas.

Graft incompatibility of Vitis spp is an unresolved worldwide problem with important economic consequences. Grafting comprises a complex set of morphological and physiological alterations, in which the phenolic compounds seem to be strongly involved. Therefore, a detailed analysis and recognition of structural phenolic compounds diversity in the two partners of a Vitis graft is of great importance to evaluate their role as markers of graft establishment.To optimise a sample extraction method, and to develop and validate a high-performance liquid chromatography (HPLC) method for the simultaneous determination of phenolic acids and flavonols in the graft union so as to understand their behaviour in the metabolism of the scion-rootstock system, using compatible and incompatible combinations of a Syrah cultivar and two rootstocks (R110 and SO4).Sixty extracts of Vitis grafting tissues were prepared and analysed by HPLC for the qualitative and quantitative determination of their phenolic profile.Among the phenolic compounds identified in the samples, one benzoic acid (gallic acid), three cinnamic acids (caffeic acid, ferulic acid and sinapic acid) and two flavonols (catechin and epicatechin) are potentially suitable as markers of graft incompatibility.The method developed presents good performance and lends itself readily for application in routine analysis of the phenolic composition of Vitis grafting tissues to distinguish compatible and incompatible combinations in the graft callusing stage.

Grapevine (Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world. In the different Portuguese agro-ecosystems, grapevine plays an impor‐ tant role either as a border culture or as an extensive crop. The surface area used by vine‐ yards amounts to 4.9 % of the arable land [1], representing 240,000 ha, being the 7th largest area in the world and the 4th in the European Union [2]. In 2011 Portugal produced 5.9 mil‐ lion hectoliters of which 2.9 million hectoliters were exported, making the country the 12th world wine producer [2]. There are fourteen wine regions with Protected Geographical Indi‐ cation (Figure 1) and 31 wine areas with Designation of Origin status including Porto, estab‐ lished since 1756, the oldest legally established wine production region in the world. Each one of the wine regions has a particular set of grapevine cultivars adapted to its specific ter‐ roirs. Officially there are 343 cultivars allowed to be use in wine production in Portugal [3].

Wine quality and market value greatly depend on the grapevine varietal composition, which may be characteristic of specific regions. In order to defend the distinct regions, Denominations of Origin were defined to protect against fraudulent practices. In this study, we evaluated the efficiency of two microsatellite-based systems (microsatellite (SSR) and intermicrosatellite (ISSR)) for must varietal composition determination and their potential role in certification purposes. Eleven Vitis vinifera L. varieties from leaf and monovarietal must DNA samples were screened with six SSR and 14 ISSR primers to discriminate polymorphisms. Principal coordinates analysis was performed with DCENTER on the resultant data using unweighted pair group mathematical average and revealed that ISSRs markers were not suitable for certification procedures, whereas nuclear SSR markers presented a complete correspondence between leaf and must samples, demonstrating that they were adequate for traceability purposes.

To assess the different origins of Portuguese grapevine varieties, we used six nuclear and four chloro- plastidal microsatellites as molecular markers, in order to compare the genetic structure of native wild-vines with native grapevine varieties. Both native subspecies have a great diversity, and a high interrelationship across the six nuclear microsatellites. Although identical numbers of alleles were found in each population, their distribu - tion was different in the vinifera and sylvestris subspecies. Portuguese wild-vines have only c hlorotypes A and B; A being the most frequent. The fifty-seven analysed Portuguese varieties have chlorotypes A, B, C, and D. The most frequent was the chlorotype A (75%), followed by D (21%). The results obtained reinforced the idea of Western Europe as having been one of the domestication centres for the grapevine, with contributions from the Eastern European gene pool. The observed genetic structure is a starting point from which to clarify the high number of native cultivars found in Portugal, and reinforces their probable origin in the Iberian Peninsula.

In the framework of the COST Action FA1003 – ‘GRAPENET: East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding’ – common phenotyping methods have been proposed to characterize germplasm accessions. The protocols aimed at the description of traits of interest for the cultivars exploitation. In particular, phenological and eno- carpological methods were selected. This paper will focus on the phenological records. The extended BBCH scale was selected and adapted to our scope. Methodological details, including agrometeorological data process are presented together with an overview of the first year results. Phenograms were obtained for each observed accession. Replications among the collections and in different years will be necessary to better define the cultivars characteristics. In 2012, the phenological protocol was applied by 20 institutions spread in 16 countries (Azerbaijan, Chech Republic, Croatia, Cyprus, Georgia, Germany, Greece, Hungary, Italy, Latvia, Luxembourg, Moldova, Portugal, Spain, Switzerland, Ukraine). 562 accessions were monitored. The large application of the protocol underlines the determination to collaborate among the European research institutions involved in grapevine germplasm evaluation.

Doutoramento em Restauro e Gestão Fluviais - Instituto Superior de Agronomia / Faculdade de Arquitetura / Instituto Superior Técnico. Universidade de Lisboa Freshwater ecosystems are influenced by diverse anthropogenic stressors and natural disturbances producing interacting effects (synergies and antagonisms) often unpredicted based on their independent effects, and thereby represent a major concern to predict ecological changes and develop effective management and restoration strategies. The aim of this Thesis was to assess different responses of key stream biological elements (i.e., biofilms and macroinvertebrates) to the impact of main stressors acting in concert in Mediterranean rivers related with water scarcity and pollution (i.e., flow velocity reduction/stagnation and sewage contamination/hypoxia), using mesocosm approaches and evidences of a case study. In a first part, the effects of flow reduction and oxygen depletion conditions in two seasons were evaluated in macroinvertebrates behavioural and functional responses, conducting to evidences of changes in drift patterns and loss of trait diversity with potential impacts on ecosystem functioning and services. In a second part was investigated how single and combined effects of water flow stagnation and sewage contamination affect primary producers and primary consumers, showing that while stagnant water had an overall inhibitory effect, sewage inputs increased biological responses. Also, stressors combined effect revealed to be unfavourable for some biofilm assemblages (decreasing bacteria and algae diversity) while advantageous to other (increasing fungi diversity). In the last part, flow and water quality variations were followed over time in a Mediterranean urban stream, leading to observations of an impoverished community with low diversity and high abundance of tolerant taxa and resistance traits, which was associated with multiple-stressors patterns of urban ecosystems. Overall it was showed that the effects of representative multiple-stressor scenarios on Mediterranean stream biota are dependent on water scarcity conditions and that reductions on sewage inputs needs to be given priority by stream managers to prevent further degradation of these aquatic ecosystems N/A