3 Materials and Methods

3.1 Materials and Methods

Benthic diatom samples were taken yearly at the Ebro river basin, monthly for the experimental setup and bi-monthly at the temporary river sites (Table 2). For the period investigated in this work, different persons took care of sampling and preparation. The sampling followed the European and Spanish norm (AENOR 2014, 2004) and the Ebro river sampling protocol (Cambra et al. 2005). Thus, diatom communities were scrubbed with either a toothbrush or knife from five randomly collected stones from the river bottom (Kelly et al. 1998). The resulting scrub was put into a mixture of river water and either 4 % formaldehyde (until 2012) or 70 % ethanol. In the laboratory, an aliquot of the samples was digested according to the 30% hydrogen peroxide (110 ºC for 12 h)-hydrochloric acid (1M) protocol and washed with distilled water (Cambra et al. 2005). From the suspension, one drop was dried on a coverslip, which was subsequently mounted on a microscopic slide using Naphrax® (Brunell microscopy) mounting medium. 

Table 2 Total number of sampling sites and samplings for each of the studies, including main basin.

Study	Sampling periodicity	Basin	Number of sites	Number of samples
3. Macro-scale	yearly	Ebro	50	356
4. Meso-scale	yearly	Segre (Ebro)	16	60
6.1 Micro-scale I (experiment)	monthly	Llobregat	3	15
6.2 Micro-scale II (temporary rivers)	bi-monthly	Catalan rivers	10	47
	bi-monthly	Ebro	5	25
	bi-monthly	Júcar	9	39
6.3 Micro-scale III (cell size distributions)	yearly	Ebro	8	48

 

All samples were investigated using a Zeiss Jenaval (Carl Zeiss Jena) microscope using a 100x objective (GF Planochromat PhV HI 100x/ 1,30 ∞/0.17-A) and Differential Interference Contrast (DIC), except for the samples for size measurements that followed a different protocol, explained in Chapter 6.3. Diatoms were identified following the Spanish and European norms (AENOR 2005; AENOR 2014b). During preparation, diatom frustules usually separate, thus, only valves are used for identification and calculation of diatom biotic indices. For the identification to the lowest taxonomical level possible, general bibliography was used (Krammer and Lange-Bertalot 1986, 1988, 1991a, 1991b; Hofmann et al. 2011; Bey and Ector 2013). If needed, monographs were used to increase the taxonomic resolution (Levkov et al. 2013; Williams 1990, 1985; Romero and Jahn 2013; Trobajo et al. 2013). Nomenclature was updated using Diatombase (Kociolek et al. 2015). To increase statistical significance, a minimum of 400 valves per sample was identified. For the micro-scale experiment 1,000 valves were counted whenever possible. All inventories were standardized to the proportional abundance (percentage of each species on the total count) and transformed [log(x+1)]. The transformation was made because of value disparity, to increase the visibility of rare diatom taxa or life forms.

The resulting diatom inventories were inserted into Omnidia software (Lecointe et al. 1993) to calculate index values. Spanish legislature established the Specific Pollution-sensitivity Index, IPS in its French initials (Coste 1982), to be the standard index for diatom biomonitoring (Ministerio de Agricultura 2008; Ministerio de Agricultura Alimentación y Medio Ambiente (MAGRAMA) 2013). Other indices used were the Biologic Diatom Index, IBD in French (Prygiel et al. 2006; Prygiel et al. 2002) and the CEE index (Descy and Coste 1990). More information regarding diatom indices is given in Chapter 1.1.4.

In the experimental setup, diatoms were observed in vivo to gather the main life form of the different taxa (Berthon et al. 2011). The database established by Rimet and Bouchez (2012) was used to translate the taxonomical data into life forms and ecological guilds for the temporary rivers (AnnexTable 1). Ecological guilds were also subdivided in size categories, following B-Béres et al. (2017). The life forms were similarly modified from the database using the Riato et al. (2017) methodology. More information on life forms and ecological guilds is presented in Chapter 1.1.3.1.

Physico-chemical data were obtained from various sources. The Ebro data came from 3-monthly samples taken according to the WFD norm and made available through the website of the authorities (Confederación del Ebro 2015). Llobregat data were obtained in situ and contrasted with the River Authorities data (Agència Catalana de l’Aigua (ACA) 2014). Finally, bi-monthly physico-chemical analyses were made during the sampling efforts of the temporary rivers. The data consisted of chemical (ammonium, oxygen), nutritional (nitrate, nitrite, phosphate, N/P ratio, silica concentrations), conductivity, alkalinity, hardness, temperature and pH. In some studies, the parameter altitude was added, although it also influencing nutrient levels and water temperature.

Statistical analyses were performed using R software (R Development Core Team 2016). The main packages used were “vegan” (Oksanen 2013a, 2014, 2013b) for multivariate analyses, “FactoMineR” (Husson et al. 2015) for PCA, “PerformanceAnalytics” (Peterson et al. 2018) for Spearman and Pearson correlations, “cluster” (Maechler et al. 2015), “MASS” (Venables et al. 2015), and through “labdsv” (Roberts 2015) and “indicspecies” (De Cáceres 2013) for indication values (Dufrêne and Legendre 1997). For the Macro-scale division, the “diatSOM” package was used (Bottin, et al. 2014). The life form studies (Chapter 6.1 and Chapter 6.2) were compared using permanova and anosim calculations (both with“vegan”), and proved using Tukey Honest Significant Distance (HSD) test. Significance (henceforth p) was shown with asterisks, which are also re-stated at the necessary tables (0 ’***’ 0.001 ’**’ 0.01 ’*’ 0.05, non-significant values showed no asterisk).

References

AENOR. 2014a. EN 13946: 2014 Calidad Del Agua. Guía Para El Muestreo de Rutina y El Pretratamiento de Diatomeas Bentónicas de Ríos y Lagos.

AENOR. 2014b. EN 14407: 2014 Calidad Del Agua. Guía Para La Identificación y Recuento de Muestras de Diatomeas Bentónicas de Ríos y Lagos. Normas Para Los Invertebrados Bentónicos.

AENOR. 2005. “Norma Española UNE-EN 14407:2005 Calidad Del Agua. Guía Para La Identificación, Recuento e Interpretación de Muestras de Diatomeas Bentónicas de Ríos.” AENOR 16pp.

AENOR. 2004. “UNE-EN13946:2004 Water Quality - Guidance Standard for the Routine Sampling and Pretreatment of Benthic Diatoms from Rivers.” AENOR 20 pp.

Agència Catalana de l’Aigua (ACA). 2014. “SDIM 2.0 -Programa de Seguiment i Control.” 13.

B-Béres, V. et al. 2017. “Ecological Background of Diatom Functional Groups: Comparability of Classification Systems.” Ecological Indicators 82:183–88.

Berthon, V., A. Bouchez, and F. Rimet. 2011. “Using Diatom Life-Forms and Ecological Guilds to Assess Organic Pollution and Trophic Level in Rivers: A Case Study of Rivers in South-Eastern France.” Hydrobiologia 673(1):259–71. Retrieved July 22, 2014 (http://link.springer.com/10.1007/s10750-011-0786-1).

Bey, M. Y. and L. Ector. 2013. Atlas Des Diatomées Des Cours d’eau de La Région Rhône-Alpes.

Bottin, M., J. L. L. Giraudel, S. Lek, and J. Tison-Rosebery. 2014. “DiatSOM : A R-Package for Diatom Biotypology Using Self-Organizing Maps.” Diatom Research 29(1):5–9. Retrieved (http://www.tandfonline.com/doi/abs/10.1080/0269249X.2013.804447).

De Cáceres, M. 2013. “How to Use the Indicspecies Package (Ver. 1.7.1).” R Project 29.

Cambra, J., L. Ector, and S. Sabater. 2005. Metodología Para El Establecimiento El Estado Ecológico Según La Directiva Marco Del Agua: Protocolos de Muestreo y Análisis Para Fitobentos (Microalgas Bentonicas). edited by C. del Ebro. Confederación Hidrográfica del Ebro. Retrieved October 30, 2014 (http://scholar.google.com/scholar?hl=en&btnG=Search&q=intitle:Metodología+para+el+establecimiento+el+Estado+Ecológicosegún+la+Directiva+MARCO+del+Agua-+Protocolos+de+muestreo+y+análisis#0).

CEN. 2014. UNE-EN 13946:2014 Water Quality - Guidance for the Routine Sampling and Preparation of Benthic Diatoms from Rivers and Lakes.

CEN, EN13946. 2003. “Water Quality Guidance Standard for the Routine Sampling and Pre-Treatment of Benthic Diatoms from Rivers.” Comité Européen de Normalisation.

CEN, EN14407. 2004. “Water Quality Guidance Standard for the Identification and Ennumeration of Benthic Diatom Samples from Rivers, and Their Interpretation.” Comité Européen de Normalisation.

CHE and Confederación Hidrográfica del Ebro. 2015. “Web de Consulta de Datos de Calidad de Agua Superficiales.” Retrieved (http://www.datossuperficiales.chebro.es:81/WCASF/?rvn=1).

Coste, M. 1982. “Etude Des Méthodes Biologiques d’appréciation Quantitative de La Qualité Des Eaux.” Rapport Cemagref 218.

Descy, J. P. and M. Coste. 1990. “Utilisation Des Diatomeés Benthiques Pour La Mesure de La Qualité Des Eaux Du Bassin Artois-Picardie: Bilan et Perspectives.” Annales de Limnologie 29(3–4):255–67.

Dufrêne, M. and P. Legendre. 1997. “Species Assemblages and Indicator Species: The Need for a Flexible Assymetrical Approach.” Ecological Monographs 67(3):345–66.

Hofmann, G., M. Werum, and H. Lange-Bertalot. 2011. Diatomeen Im Süßwasser-Benthos von Mitteleuropa. Bestimmungsflora Kieselalgen Für Die Ökologische Praxis. Über 700 Der Häufigsten Arten Und Ihre Ökologie. ARG Gantner.

Husson, F., J. Josse, S. Le, and J. Mazet. 2015. “Package ‘ FactoMineR .’” R Topics Documented 1–95.

Kelly, M. G. et al. 1998. “Recommendations for the Routine Sampling of Diatoms for Water Quality Assessments in Europe.” Journal of Applied Phycology1 10:215–24.

Kociolek, J. P. et al. 2015. “DiatomBase.” Retrieved (http://www.diatombase.org/).

Krammer, K. and H. Lange-Bertalot. 1988. Süßwasserflora Vom Mitteleuropa. Bacillariophyceae 2. Bacillariaceae, Epithemiaceae, Surirellaceae 2/2. edited by H. Ettl, J. Gerloff, H. Heynig, and D. Mollenhauer. G. Fischer Verlag. Jena.

Krammer, K. and H. Lange-Bertalot. 1986. Süßwasserflora von Mitteleuropa. Bacillariophyceae. 1. Teil: Naviculaceae, Vol 2/1. edited by H. Ettl, J. Gerloff, H. Heynig, and D. Mollenhauer. G. Fischer Verlag. Jena.

Krammer, K. and H. Lange-Bertalot. 1991a. Süßwasserflora von Mitteleuropa. Bacillariophyceae. 3. Teil: Centrales, Fragilariaceae, Eunotiaceae. 2/3. edited by H. Ettl, J. Gerloff, H. Heynig, and D. Mollenhauer. G. Fischer Verlag. Jena.

Krammer, K. and H. Lange-Bertalot. 1991b. Süßwasserflora von Mitteleuropa. Bacillariophyceae. 4. Teil: Achnanthaceae, Kritische Ergänzungen Zu Navicula (Lineolatae) Und Gomphonema. edited by H. Ettl, J. Gerloff, H. Heynig, and D. Mollenhauer. G. Fischer Verlag. Jena.

Lecointe, C., M. Coste, and J. Prygiel. 1993. “‘Omnidia’: Software for Taxonomy, Calculation of Diatom Indices and Inventories Management.” Hydrobiologia 269(1):509–13.

Levkov, Z., D. Metzeltin, and A. Pavlov. 2013. Luticola and Luticolopsis. Diatoms of. edited by H. L.- Bertalot.

Maechler, M. et al. 2015. “Package ‘ Cluster .’” R Topics Documented 79. Retrieved (https://cran.r-project.org/web/packages/cluster/cluster.pdf).

Ministerio de Agricultura, Alimentación y Medio Ambiente (MAGRAMA). 2008. “ANEXO III. ORDEN ARM/2656/2008, de 10 de Septiembre, Por La Que Se Aprueba La Instrucción de Planificación Hidrológica.” Boletín Oficial Del Estado (BOE) 229:38561–73.

Ministerio de Agricultura Alimentación y Medio Ambiente (MAGRAMA). 2013. Protocolo de Cálculo Del Índice de Polusensibilidad Específica: Código IPS-2013.

Oksanen, J. 2013a. “Multivariate Analysis of Ecological Communities in R : Vegan Tutorial.”

Oksanen, J. 2013b. “Multivariate Analysis of Ecological Communities in R.” R Topics Documented 1–40.

Oksanen, J. 2014. “Vegan : An Introduction to Ordination.” 1–11.

Peterson, B. G. et al. 2018. “Package ‘ PerformanceAnalytics ’ R Topics Documented :” R Topics Documented 46–47.

Prygiel, J. et al. 2002. “Determination of the Biological Diatom Index ( IBD NF T 90 – 354 ): Results of an Intercomparison Exercise.” Journal of Applied Phycology 14:27–40.

Prygiel, J., M. Coste, and J. Tison. 2006. “A Short History of the Use of Diatoms and Diatom Indices in French Networks, and IBD Update According to Water Framework Directive Requirements.” Diatomededelingen 30:16–25.

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Riato, L., V. Della Bella, M. Leira, J. C. Taylor, and P. J. Oberholster. 2017. “A Diatom Functional-Based Approach to Assess Changing Environmental Conditions in Temporary Depressional Wetlands.” Ecological Indicators 78:205–13.

Rimet, F. and A. Bouchez. 2012. “Life-Forms, Cell-Sizes and Ecological Guilds of Diatoms in European Rivers.” Knowledge and Management of Aquatic Ecosystems (406):01. Retrieved June 26, 2014 (http://www.kmae-journal.org/10.1051/kmae/2012018).

Roberts, D. W. 2015. “Labdsv: Ordination and Multivariate Analysis for Ecology.” R Topics Documented 59. Retrieved (http://ecology.msu.montana.edu/labdsv/R/labs/).

Romero, O. E. and R. Jahn. 2013. “Typification of Cocconeis Lineata and Cocconeis Euglypta (Bacillariophyta).” Diatom Research 28(2):175–84.

Trobajo, R. et al. 2013. “Morphology and Identity of Some Ecologically Important Small Nitzschia Species.” Diatom Research 28(1):37–59. Retrieved (http://www.tandfonline.com/doi/abs/10.1080/0269249X.2012.734531).

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Williams, D. M. 1990. “Cladistic Analysis of Some Freshwater Araphid Diatoms (Bacillariophyta) with Particular Reference to Diatoma and Meridion.” Plant Systematics and Evolution 171:89–97.

Williams, D. M. 1985. Morphology, Taxonomy and Inter-Relationships of the Ribbed Araphid Diatoms from the Genera Diatoma and Meridion (Diatomaceae: Bacillariophyta). A. R. G. Gantner Verlag K. G.

3.2  Studied sites

Rivers with north-eastern river mouths are influenced by Mediterranean or Continental climates. The main studied rivers, Ebro and Llobregat flow into the Mediterranean (Figure 5). The TRivers project Rivers additionally included other Catalan rivers, located in the north east, but also included some rivers going down the shoreline of the Mediterranean, too (1.3.3, Figure 5). 

Figure 5 Sites studied in this thesis marked for each of the chapters. Map elaborated with QGIS software. 

Following the WFD, Spanish rivers were subdivided into catchments, each with its own authority to bio-monitor and control. Ebro is one of the largest transborder basins in Spain. Spanish Ebro river Authorities have treaties with France and Andorra because of the shared Ebro tributaries. An example for one of such treaties is the Segre basin legislation (Communauté de Communes et al. 2007; Pyrenées-Cerdagne and Communauté de Communes 2008; Préfet des Pyrénées-orientales and Direction Départamentale des Territoires et de la Mer 2014). Other Mediterranean rivers studied in this thesis contain are the Llobregat and other Catalan catchments, managed by the Catalan river authorities (Agència Catalana de l’Aigua, ACA). This administration regulates several river basins, torrents and temporal streams, not yet regularized in the WFD, as do the authorities of the Ebro and the Júcar Rivers (Confederación Hidrográfica del Ebro and C. H. Júcar). 

References

Communauté de Communes, et al. Contrat de Rivière Du Sègre En Cerdagne. 2007.

Préfet des Pyrénées-orientales, and Direction Départamentale des Territoires et de la Mer. Arrête Préfèctoral N^o 2014275-0001 Portant Modification Du Comité de Rivière Sur Le Bassin Versant Du Sègre (Français). Vol. 33, no. 0, 2014.

Pyrenées-Cerdagne, and Communauté de Communes. Le Contrat de Rivière Transfrontalier Du Segre 2008 - 2012. 2008, p. 7.