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Chief editor: F. Malard
UMR CNRS 5023 - Ecologie des Hydrosystèmes Fluviaux
Université Claude Bernard - Lyon 1 - Bat. Forel (403)
43, boulevard du 11 Novembre 1918
69622 Villeurbanne Cedex, France
e-mail: malard@univ-lyon1.fr

Associate editors: M.-J. Dole-Olivier, J. Mathieu, and F. Stoch
The following scientists contributed to the production of the manual: C. Boutin, A. Brancelj, A.I. Camacho, F. Fiers, D. Galassi, J. Gibert, T. Lefebure, P. Martin, B. Sket, and A.G. Valdecasas.

Forewords
This sampling manual is published within the framework of the European project PASCALIS. The project PASCALIS addresses a problem of growing concern in Europe, i.e. the biodiversity and ecosystem aspects of groundwater conservation. The main goal of this project is to establish a rigorous and detailed protocol for assessing groundwater biodiversity and to develop operational tools for its conservation. Knowledge on the distribution of groundwater animals in southern Europe is presently being organized in a large database and represented on maps. In addition, groundwater biodiversity is being surveyed at 6 selected regions in Europe following a standardized sampling procedure. Most taxonomic groups inhabiting a wide range of groundwater habitats are considered. The present manual provides a detailed description of the standard protocol that is used to assess groundwater biodiversity in the selected regions. This manual is made available below so that the project PASCALIS benefits for multiple end-users including scientists, public and civil society, groundwater managers, and policy makers. Comments regarding the content of this manual can be addressed to the public forum of the web site PASCALIS.
The project leader
Prof. Janine Gibert

Content of the manual (download here)

Introduction
Part I: Biodiversity in ground water
1.1. Diversity of groundwater habitats
1.2. Diversity of groundwater fauna
Part II: Objectives of the sampling manual
2.1. Objectives
2.2. Limitations
Part III: Hierarchical sampling scheme
3.1. Sampling strategy
3.2. Sampling procedure
3.3. Definition of sampling units and habitats
3.3.1. Region
3.3.2. Hydrogeographic basins
3.3.3. Karstic ground water
3.3.3.1. The unsaturated (vadose) zone
3.3.3.2. The saturated (phreatic) zone
3.3.4. Subsurface water in unconsolidated sediments
3.3.4.1. The hyporheic zone
3.3.4.2. Ground water in unconsolidated sediments
Part IV: Selection of sampling sites
4.1. Site selection
4.1.1. Sampling sites as access points to the different units of a region
4.1.2. Pre-selection of sampling sites
4.1.3. Field surveys
4.2. Register of sampling sites
4.3. Mapping of sampling sites
4.4. Environmental features of habitats
4.4.1. Site description
4.4.2. Land use and human activities
4.4.3. Physico-chemical measurements during sampling
Part V: Sampling methods and devices
5.1. Sampling in the hyporheic zone
5.1.1. Data sheet
5.1.2. Measurement of vertical hydraulic gradient
5.1.3. The Bou-Rouch pumping method
5.2. Sampling in springs
5.2.1. Data sheet
5.2.2. Drifting fauna
5.2.3. Sampling of habitats near springs
5.2.4. Artificial substrates and baited traps
5.3. Sampling in caves
5.3.1. Safety
5.3.2. Data sheet
5.3.3. Methods for sampling slowly infiltrating water in the vadose zone
5.3.3.1. Filtering water dripping from cave ceiling
5.3.3.2. Sampling in gours, puddles and pools
5.3.4. Methods for sampling subterranean brooks, rivers, lakes and siphons
5.4. Sampling in wells
5.4.1. Well type and design
5.4.2. Data sheet
5.4.3. Phreatobiological net
5.4.4. Baited traps
5.4.5. Pumping of well water
5.4.5.1. Sampling method
5.4.5.2. Permanent pump
5.4.5.3. Suction pumps for the sampling of shallow water-table aquifers
5.4.5.4. Pressure pumps for the sampling of deep water-table aquifers
5.5. Methods for physico-chemical measurements
5.5.1. Collection and preservation of samples
5.5.2. Field measurements
5.5.3. Laboratory analyses
5.6. Fixation, preservation, and processing of faunal samples
5.6.1. Fixation and preservation of most taxonomic groups
5.6.2. Specific fixation and preservation procedures
5.6.3. Processing of faunal samples
Conclusion
Acknowledgements
References
List of figures
List of tables
List of photos
Appendixes (not available on this site)



Introduction
Although research on biological diversity in subsurface water typically lags behind that of surface freshwater, considerable efforts by scientists over the second half of the 20th century have revealed the unexpectedly high diversity of living forms in ground water (Botosaneanu 1986, Juberthie and Decu 1994, 1998). During the 1990s, there have been more than 10 books dealing with the biology and ecology of ground water, a publication rate probably unequalled in many other research fields (Botosaneanu 1986, Camacho 1992a, Juberthie and Decu 1994, 1998, Gibert et al. 1994a, 1997, Culver et al. 1995, Wilkens et al. 2000, Jones and Mulholland 2000, Griebler et al. 2001). Whereas earlier research works were essentially restricted to the study of animals living in caves, faunal investigations carried out in a variety of subterranean habitats have demonstrated that groundwater animals can potentially be found wherever subsurface water exists (Rouch 1986, Danielopol 1989). Despite a long tradition of species inventory in subterranean ecology, groundwater ecologists have only recently begun to synthesise and map the vast amount of existing data on the distribution of groundwater species (Juberthie and Decu 1994, 1998). Mapping biodiversity is emerging as an essential task for understanding biodiversity patterns and the development of conservation strategies. Culver et al. (2000) mapped the distribution of 927 obligate cave-dwelling species in the 48 contiguous states of the United States (www.karstwaters.org). In Europe, the research project PASCALIS “Protocols for the ASsessment and Conservation of Aquatic Life In the Subsurface” is the first attempt to provide large-scale distribution patterns of specific richness and endemism in ground water. The first phase of the project is devoted to the compilation and mapping of existing data on the distribution of subterranean aquatic taxa across a European gradient (Belgium, France, Spain, Italy, and Slovenia).

In addition to the mapping of groundwater biodiversity, the project PASCALIS also aims to develop several validated methods for:
1) determining the reliability of biodiversity patterns revealed by the mapping of existing data;
2) using a standard field sampling protocol to obtain an unbiased estimate of groundwater biodiversity in areas for which no data exist;
3) predicting overall species richness based on biodiversity indicators in regions with incomplete data sets.
This second phase of the project is essential because the assessment of biodiversity within a given area strongly depends on:
1) the number of observations (i.e. sampling sites);
2) the number of habitats (e.g. historically, sampling was mainly carried out in caves);
3) the amount of information available on various taxonomic groups (e.g. the macrofauna has received considerably more attention than the meiofauna).
These three important issues have been largely overlooked in the field of groundwater biodiversity, thereby severely restricting the usefulness of biodiversity maps for basic and conservation purposes. Within the framework of the project PASCALIS, these issues will be addressed based on the results of an extensive sampling survey in 6 regions distributed in southern Europe: the Walloon karst (Belgium), the meridional Jura (Eastern France), the Roussillon region (France), the Cantabrica (Spain), the Lessinian mountains (Italy), and the Krim massif (Slovenia). Faunal sampling within these regions will be carried out following a standard protocol, the objective of which is to provide a data set as complete as possible on the distribution of groundwater species.

The ensuing material provides a detailed description of the sampling protocol that will be applied in the selected regions. This protocol has been established within the framework of the research project PASCALIS based on the results of:
1) discussions among partners of the project;
2) an electronic forum (11 February - 4 March 2002) among 37 scientists from 11 countries;
3) a sampling workshop held in Lyon, France (22-26 April 2002) during which 41 participants from 6 European countries learnt about methods and devices for sampling groundwater fauna.
The first part of the manual gives a brief account of the diversity of habitats and animals in subsurface water. Secondly, we define the objectives of the sampling protocol and specify the geomorphologic features of areas where this protocol can be applied. Thirdly, we present the rationale of a stratified sampling scheme and define the different hierarchical units to be sampled. Fourthly, we propose a general procedure for selecting and mapping sampling sites and managing environmental data. The last part of the manual outlines the recommended methods and devices for the sampling of groundwater fauna in springs, caves, wells, and the hyporheic zone of rivers.



List of figures
Figure 1: Three-dimensional view of different aquifers. Modified after Gibert (2001).

Figure 2: A classification of groundwater fauna (modified after Gibert et al. 1994b).

Figure 3: The interstitial habitat and some of the subterranean-dwelling organisms. After Danielopol et al. (1994).

Figure 4: Hierarchical sampling scheme used within the framework of the European project PASCALIS (Protocols for the ASsessment and Conservation of Aquatic Life In the Subsurface).

Figure 5: Different steps of the sampling procedure used within the framework of the European project PASCALIS (Protocols for the ASsessment and Conservation of Aquatic Life In the Subsurface).

Figure 6: Location of the 6 regions selected for sampling groundwater fauna within the framework of the European project PASCALIS (Protocols for the ASsessment and Conservation of Aquatic Life In the Subsurface).

Figure 7: Location of the 4 hydrogeographic basins selected for sampling groundwater fauna in the Walloon Karst (Belgium).

Figure 8: Location of the 4 hydrogeographic basins selected for sampling groundwater fauna in the Meridional Jura (France).

Figure 9: Location of the 4 hydrogeographic basins selected for sampling groundwater fauna in the Roussillon Region (France).

Figures 10.1 and 10.2: Location of the 4 hydrogeographic basins selected for sampling groundwater fauna in the Cantabrica Region (Spain).

Figure 11 : Location of the 4 hydrogeographic basins selected for sampling groundwater fauna in the Lessinian Mountains (Italy).

Figure 12: Location of the 4 hydrogeographic basins selected for sampling groundwater fauna in the Krim Massif (Slovenia).

Figure 13: Idealised view of a karst aquifer. Modified after Mangin (1974/1975).

Figure 14: The epikarst. A – after Mangin (1974/1975). B – after Williams (1983).

Figure 15: Conceptual groundwater flow models in karst aquifers. Modified after Gibert et al. (1994 c).

Figure 16: Surface-subsurface hydrological exchanges in the hyporheic zone induced by spatial variation in streambed topography and sediment permeability. Modified after Malard et al. 2002.

Figure 17: Conceptual cross-sectional models of surface channels and beds showing relationships of channel water to hyporheic, groundwater, and impermeable zones. Modified after Malard et al. (2000).

Figure 18: Spatial and temporal domain of hyporheic interactions and relation to roughness features in channels. After Harvey and Wagner (2000).

Figure 19: Geomorphologic units generating hyporheic flow paths.

Figure 20 (not available yet): Map showing the location of sampling sites in the Walloon karst (Belgium).

Figure 21: Map showing the location of sampling sites in the Meridional Jura (France).

Figure 22 (not available yet): Map showing the location of sampling sites in the Roussillon region (France).

Figure 23: Map showing the location of sampling sites in the Cantabrica Region (Spain).

Figure 24 : Map showing the location of sampling sites in the Lessinian Mountains (Italy).

Figure 25: Map showing the location of sampling sites in the Krim Massif (Slovenia).

Figure 26: Description of the T-bar (left box) and measurement of pressure differential between surface water and hyporheic water (right box).

Figure 27: Description of the Bou-Rouch pumping method for sampling invertebrates in the hyporheic zone of rivers. Modified after Bou (1974).

Figure 28: Description of the peristaltic pump for pumping water in the Bou-Rouch stand pipe for the measurement of physico-chemical parameters and collection of water samples.

Figure 29: Description of the vacuum pump for sampling invertebrates in the hyporheic zone of rivers. A manual diaphragm pump is used to create vacuum in the jar.

Figure 30: Drift net for the sampling of groundwater invertebrates in caves and springs. Modified after Vervier (1988).

Figure 31: Pond net, Hess sampler, and Surber sampler for the sampling of groundwater invertebrates in the benthic layer of springs. Modified after Niederreiter (2002) and Camacho (1992).

Figure 32: Device for filtering water dropping from cave ceiling. Animals are trapped in the bottle.

Figure 33: The Karaman-Chappuis method for sampling groundwater invertebrates in the bank sediments of surface and underground rivers and lakes. Modified after Delamare Debouteville (1960).

Figure 34: Artificial substrate for the sampling of invertebrates in water bodies of caves and limnocrene springs. After Vervier (1990).

Figure 35: A - Design of a drilled well in unconsolidated sediments. B – Vertical change in dissolved oxygen concentration in the screened and unscreened sections of the well.

Figure 36: The Cvetkov phreatobiological net for sampling groundwater invertebrates in large diameter wells. Modified after Bou (1974).

Figure 37: Examples of baited traps for sampling groundwater invertebrates in cave water bodies and wells. Modified after Boutin and Boulanouar (1983).

Figure 38: Inertial pump for sampling water and invertebrates in small-diameter wells. Modified after Malard et al. (1997).

Figure 39: Air-lift pump for sampling invertebrates in wells. Modified after Malard et al. (1997).

Figure 40: Pneumatic piston pump for sampling water and invertebrates in wells. Modified after Niederreiter (2002).

Figure 41: Ejector pump for sampling invertebrates in wells. Modified after Malard et al. (1997).



List of tables
Table 1: Comparison of aquatic subterranean habitats based on the size of the voids, degree of interconnectedness between voids, and strength of hydrologic linkages with the surface environment.

Table 2: Species richness among different taxonomic groups in ground water. After Gibert and Deharveng (2002).

Table 3: Species richness among different taxonomic groups in European ground water. Extracted from Sket (1999), Table I.

Table 4: Ranges of specific yield (i.e. effective porosity) for different geological formations. Modified after Castany (1982).

Table 5: Ranges of permeability for different geological formations. Modified after Freeze and Cherry (1979).

Table 6: Structure of the register of sampling sites used within the framework of the European project PASCALIS (Protocols for the ASsessment and Conservation of Aquatic Life In the Subsurface).

Table 7: Data sheet for sampling invertebrates in the hyporheic zone.

Table 8: Data sheet for sampling invertebrates in springs.

Table 9: Data sheet for sampling invertebrates in caves.

Table 10: Data sheet for sampling invertebrates in wells.

Table 11: Land use categories as defined by the Corine Land Cover nomenclature.

Table 12: Comparison of several pressure pumps for sampling invertebrates in wells. Modified after Malard et al. (1997).

Table 13: Example of data sheet for sorting faunal samples collected in the Meridional Jura (France).



List of photos
Photo 1: The T-bar for measuring vertical hydraulic gradient between the surface stream and the hyporheic zone.

Photo 2: Hammering of mobile pipe into the river bed for sampling the hyporheos.

Photo 3: The Bou-Rouch pumping method for sampling invertebrates in the hyporheic zone of rivers.

Photo 4: Sampling of hyporheic water with the peristaltic pump for physico-chemical measurements.

Photo 5: The vacuum pump for sampling invertebrates in the hyporheic zone of rivers.

Photo 6: Drift net for the sampling of invertebrates in karstic springs.

Photo 7: Filtering of water dropping from cave ceiling.

Photo 8: A hand pear-shaped pump for emptying small water bodies in caves. Water is then filtered through a plankton net.

Photo 9: A hand peristaltic pump for emptying small water bodies in caves. Water is then filtered through a plankton net.

Photo 10: Hand nets for collecting invertebrates from water bodies in caves.

Photo 11: Filtering of cave water bodies with a hand net.

Photo 12: Baited trap for collecting animals in water bodies of caves.

Photo 13: Collection of macro-invertebrates with a suction device in puddles of caves.

Photo 14: Sampling of interstitial invertebrates with the Karaman-Chappuis method.

Photo 15: Artificial substrate for sampling groundwater invertebrates in water-bodies of caves.

Photo 16: A manual device for installing piezometers in areas where the groundwater table is very shallow (i.e. < 2 m below the soil surface).

Photo 17: A multi-parameter sonde for measuring vertical changes in temperature, specific conductance, dissolved oxygen, pH, and redox potential in wells.

Photo 18: The Cvetkov phreatobiological net for sampling groundwater invertebrates in large diameter wells.

Photo 19: The Bou-Rouch pump for sampling groundwater invertebrates in shallow water-table aquifers (i.e. groundwater table not deeper than about 8 m below the ground surface).

Photo 20: The inertial pump for sampling water and invertebrates in small diameter wells.

Photo 21: The air lift for sampling groundwater invertebrates in wells.

Photo 22: The pneumatic piston pump for sampling water and invertebrates in wells.

Photo 23: The ejector pump for sampling groundwater invertebrates in wells.