Groundwater fauna in an urban area: natural or affected?

In Germany 70 % of the drinking water demand is met by groundwater, whose quality is the product of manifold multiple physical-chemical and biological cleaning processes. As healthy groundwater ecosystems help to provide clean 10 drinking water, it is necessary to assess their ecological conditions of these ecosystems. This is particularly true for densely populated, urban areas, where faunistic groundwater investigations are still scarescarce. The aim of this study is therefore to provide a first-tier assessment of the groundwater fauna in an urban area. Thus, we assess examine the ecological condition status of an anthropogenically influenced aquifer by analysing the groundwater fauna in 39 groundwater monitoring wells in the city of Karlsruhe (Germany) and a nearby forest land. For classification, we apply the scheme from the Federal 15 Environmental Agency (UBA) groundwater ecosystem status index (GESI), in which a threshold of more than 70 % of Crustaceans and of less than 20 % of Oligochaetes serves as an indication for very good and good ecological conditions. In ourOur study reveals that only 35 % of the wells in the urban area residential, commercial and industrial areas, and 50 % of wells in the forested area fulfil these criteria, and even in the pristine forest land only 50 % of the wells indicate fine ecological conditions. While the assessment reveals that ecological conditions in the studied urban area are predominantly not in a good 20 ecological state, there is However, the study did not find no clear spatial patterns with respect to land use and other anthropogenic impacts, in particular groundwater temperature. HoweverNevertheless, there are noticeable differences in the spatial distribution of species in combination withand abiotic groundwater characteristics between wells in forest land and the urban areain groundwater of the different areas of the city, which indicates that a more comprehensive assessment methods areis required to fully capture evaluate the groundwater ecological status in more detailthe different effects on groundwater 25 fauna. In particular, more indicators, such as groundwater temperature, indicator species, delineation of site-specific characteristics and natural reference conditions should be considered.


Introduction
In Germany 70 % of the drinking water demand is met by groundwater, whose quality is the product of multiple physical-30 chemical and biological cleaning processes (German Environment Agency, 2018) (Avramov et al., 2010). Groundwater ecosystems are responsible for several services that help to provide clean drinking water, which is a vital resource for humanity (Griebler and Avramov, 2015). Bacteria and also fauna also play an important role in the biological self-purification of groundwater by the retention of organic matter, natural attenuation of pollutants, storing and buffering of nutrients as well as the elimination of pathogens. Organic matter and pollutants can be degraded and converted to valuable biomass or tied bound 35 by microbial activity. Protozoa and higher animals organisms can graze resulting biofilms, loosen the substrate and therefore stimulate the biological self-purification (Hancock et al., 2005;Boulton et al., 2008;Griebler and Avramov, 2015) (Avramov et al., 2010).
Yet, only healthyHealthy groundwater ecosystems can provide clean drinking water,. Groundwater ecosystems however, they are sensitive to external influences, such as chemical and thermal disturbances. The latter drives hydro-geochemical and 40 biological processes in groundwater systems, which are relatively typically isothermal (Brielmann et al., 2009;20011).
Groundwater fauna mainly consists of stygobiontte species, which spend their entire life in groundwater and are adjusted to this habitat (Hahn, 2006). Hence, in Central Europe they are assumed to be cold stenotherm, which means that they prefer cold temperatures. A variability in temperature tolerance among groundwater faunal groups and species is reported in various studies, which explains why the use of individual temperature thresholds is more useful to capture different preferences. 45 According to Spengler (2017) faunal diversity is generally declining at a temperature above 14 °C. Various authors reported species specific temperature preferences between 8 and 16 °C (for individuals of the species Niphargus inopinatus and Proasselus cavaticus (Brielmann et al., 2009(Brielmann et al., , 2011) and a specific temperature threshold of up to 19 °C (for Parastenocaris phyllura (Glatzel, 1990)). Above these thresholds the mortality of individuals raises until groundwater fauna is almost absent, for example at 22 °C in the study of Foulquier et al. (2011). However, temperature sensitivity is not only an issue at species 50 level but also for the communities as a whole. Spengler (2017) reported 12 °C to be a temperature threshold value indicated by a shift in community structure for faunal communities of groundwater of the Upper Rhine Valley. and can hardly persist water temperatures over 16 °C (Brielmann et al., 2009) or rather 14 °C (Spengler, 2017) for a longer time period.
Nevertheless, in German and European legislation, as in many countries globally, groundwater is not yet recognized as a protected habitat which is worthy of protection in the German and European legislation, as in many countries globally, and 55 there is no common ground understanding on the best practice of assessing the groundwater ecologyecological status of groundwater Spengler and Hahn, 2018). The assessment of surface water is typically based on biological, physical-chemical and supported by hydro-morphological criteria (European Water Framework Directive and German legislation article 5 of the 'Regulation on the Protection of Surface Water'). While groundwater quality is mostly assessed by physical-chemical and quantitative criteria, very few quantifiable ecological criteria are available for the assessment of 60 ecosystem the health of groundwater ecosystems. The availability of ecological criteria can only be increased by conducting a large number of studies dealing with the analyses of groundwater ecosystem health by investigating groundwater fauna.
Results from previous faunistic groundwater analyses are contained in a Germany-wide data record (data record by Hahn, 2005;Berkhoff, 2010;Stein et al., 2012;Gutjahr, 2013;Spengler, 2017;Spengler and Hahn, 2018). A closer look at the southwestern part of Germany, the German federal state Baden-Württemberg, is givenThe study by Hahn and Fuchs (2009). This 65 large-scaled study focuses on defining stygoregions , which extend over several square kilometres, and are based on different hydrogeological units located in Baden-Württemberg, Germany. They conclude that the observed patterns of groundwater communities reflect a high spatial and temporal heterogeneity of groundwater, with respect to hydrogeological aquifer types (with respect to habitat structure, food and, oxygen supply etc. Accordingly, stygobiotic biodiversity is likely to be underestimated Although there are various studies on this topic (e.g. Gibert and Deharveng, 2002;Malard et al., 2002;70 Deharveng et al., 2009;Dole-Olivier et al., 2009b) stygobiotic biodiversity is still likely to be underestimated at present.
Regional investigations on the spatial variation of groundwater fauna, i.e. stygobiont occurrences, and corresponding environmental parameters, such as geological site characteristics and altitude, are rare (Dole-Olivier et al., 2009;Gibert et al., 2009). An approach to elucidate groundwater biodiversity patterns in six European and seven North-American regions was conducted in the PASCALIS project (Protocol for the AsSsessment and Conservation of Aquatic Life In the Subsurface) 75 , which aimed at mapping biodiversity and endemism patterns ) and. The study shows that regional processes, such as hydrological connectivity, in a specific habitat (e.g. river floodplains as in Ward and Tockner, 2001) have a much stronger influence on species composition than local habitat features such as permeability and saturation. Within a region, hydrogeology, altitude, palaeographical factors and human activities can interact in complex ways to produce dissimilar patterns of species compositions and diversity . Unfortunately, theThe PASCALIS 80 sampling protocol recommends selecting hydro-geographic basins that are not strongly affected by human activities such as groundwater pollutions (Malard et al., 2002), and does not biogeographically classify a groundwater system (Stein et al., 2012).
In urban areas, anthropogenic impacts, such as a dense building development, underground car parks, open geothermal systems and injections of thermal wastewater from industry result in local thermal alteration of groundwater up to several degrees (e.g. Taylor and Stefan, 2009;Zhu et al., 2011;Menberg et al., 2013b;Tissen et al., 2019). According to Brielmann et al. (2011) 85 annual temperature fluctuations in aquifers, caused by shallow geothermal energy systems, range between 4 °C in winter and ≤ 20 °C in summer. In 2000, the European Union (EU) (Water Framework Directive) defined the release of heat in the groundwater as a pollution, whereas the cooling of the groundwater is not particularly mentioned. Until now, there are no scientifically derived threshold values for groundwater temperature in the case of thermal (heat) pollution published, but none of these have been implemented in official regulations or water law (Hähnlein et al., 2010(Hähnlein et al., , 2013Blum et al., 2021) (Hähnlein 90 et al., 2010;2013). This results in a tension between conservation, exploitation and thermal use of groundwater. Yet,However, as seen in an aquifer ecosystem downstream of from an industrial facility in Freising (Germany), where groundwater is used for cooling resulting in a warm thermal plume, no relation between faunal abundance and groundwater temperature could be identified (Brielmann et al., 2009). Investigation of hydro-geochemical parameters, microbial activities, bacterial communities and groundwater faunal assemblages indicates that bacterial diversity clearly increaseds with temperature, while faunal 95 diversity usually decreasesdecreased with temperature (Brielmann et al., 2009). Similar results are provided by Griebler et al.( 2016), where potential impacts of geothermal energy use and storage of heat on groundwater are investigated. Temperature changes in groundwater correspond with changes in groundwater chemistry, biodiversity, community composition, microbial processes and function of the ecosystem. How exactly these groundwater communities react to changes in temperature and concentration of nutrients, dissolved organic carbon and oxygen, is not yet fully understood (Brielmann et al., 2009(Brielmann et al., , 2011100 Spengler, 2017;Sánchez et al., 2020).
Several approaches exist that allow a local assessment of the ecological state of groundwater based on different faunistic, hydro-chemical and physical parameters. Hose (2011, 2017) introduced the Groundwater Health Index (GHI), which is a tiered framework for assessing the health of groundwater ecosystems. Here, both biotic and abiotic attributes of groundwater ecosystems are used as benchmarks for ecosystem health. Their study shows that ecosystem health benchmarks 105 are probably more associated with aquifer typology than being applicable for local areas. This index is applied and tested by di Lorenzo et al. (2020) in unconsolidated aquifers in Italy located in nitrate vulnerable zones. They refined the index (wGHI N ) and demonstrated its applicability on shallow and deep aquifers and also revealed that this new index is limited due to low correlations between the indicators. The Commissioned by the Federal Environmental Agency of Germany (Umweltbundesamt, UBA), Griebler et al.( 2014), for example, developed a concept for an ecologically based assessment 110 scheme for groundwater ecosystems, which builds on the assessment of Hose (2011, 2017). This two-step scheme characterizes groundwater on two different levels by using the most important physico-chemical parameters, such as content of dissolved oxygen, as well as microbiological and faunistic characteristics such as amount of Oligochaetes and Crustaceans, and comparing these to reference values for natural, undisturbed and ecologically intact groundwater ecosystems (Griebler et al., 2014). Moreover,  introduced the Groundwater Health Index (GHI), which is a tiered framework 115 for assessing the health of groundwater ecosystems. The GHI uses biotic and abiotic attributes of groundwater ecosystem to set benchmarks, which provide an indication of ecosystem health. In fact, their study shows that ecosystem health benchmarks are probably more associate with aquifer typology, than being applicable for local areas. The common ground of both studies is the assessment of the ecological condition relative to a reference aquifer and the aim of classifying the locations (GHI: impacted or non-impacted groundwater). 120 Furthermore, the Groundwater-Fauna-Index (GFI), introduced by Hahn (2006), quantifies the ecological relevant ecological conditions in the groundwater as a result of hydrological exchange between surface and groundwater. It incorporates ecologically important groundwater parameters such as relative amount of detritus, variation of groundwater temperature and concentration of dissolved oxygen (Hahn, 2006).  used the GFI as part of a proposal for a groundwater habitat classification at on a local scale, which introduce five types of faunistic habitats as a result of surface water influence, 125 content of dissolved oxygen and amount of organic matter. Moreover, in the study of Berkhoff (2010) the GFI was used to examine the impact of the surface water influence on groundwater with the aim to develop a faunistic monitoring concept for hydrological exchange processes in the surrounding of waterside river bank filtration plants. Spengler and Hahn (2018) argued for the definition of a regional and ecological temperature threshold and an ecology based assessment of thermal stress in groundwater. 130 The objective of this study is to investigate specifically the groundwater fauna under beneath an urban arearesidential, commercial and industrial, i.e. urban areas in comparison to a natural forested area outside the build-up area of Karlsruhe land to determine whether land use has an impact on groundwater faunal communities. Hence, in 39 groundwater monitoring wells in Karlsruhe, Germany, the groundwater fauna is sampled, groundwater temperatures measured and as well as thermal and chemical properties are sampled analysedin 39 groundwater monitoring wells in Karlsruhe, Germany. In our study the 135 classification scheme by the Federal Environmental Agency of Germany (UBA)developed by Griebler et al. (2014) is applied.
The wells are characterized regarding the state of their the state of their ecosystem quality. Hence, we finally aim to distinguish areas with natural state of groundwater ecology from anthropogenically disturbed areas.

Study site 140
The study is performed in Karlsruhe, a city in the Upper Rhine Valley in south-western Germany. The urban region covers an area of 173 km 2 and has about 310,000 inhabitants (Amt für Stadtentwicklung -Statistikstelle, 2018). The Cenozoic continental rift valley is filled with Tertiary and Quaternary sediments, which are dominated by sands and gravels with minor contents of silt, clay and stones (Geyer et al., 2011). Sporadic layers with lower permeabilities lead to a separation of up to three aquifer levels (Wirsing and Luz, 2007). The upper aquifer is unconfined with a water table between 2 and 10 m below the ground. The 145 flow direction is towards northwest to of the Rhine River with groundwater flow velocities ranging between 0.5 and 1.5 m/d (Technologiezentrum Wasser, 2018).
Based on the land use plan of Karlsruhe, about 20 % of the study area (i.e. urban area, city centre, neighbouring districts, as well as parts of the Hardtwald forest and several outskirts) is covered by buildings. The rest is characterised by vegetation (~ 56 %) and artificial surface covers (~ 24 %), showing the complexity and heterogeneity of the urban environment. 150 According to Benz et al. (2016), the annual mean groundwater temperature (GWT) in Karlsruhe in the years 2011 and 2012 was 13.0  1.0 °C. Distinct temperature hotspots occur mainly below the city centre, where building densities are highest. In the north-western part of Karlsruhe, the increase of GWT with was about 3 K warmer than the annual mean land surface temperature (LST), which is mainly caused by several groundwater reinjections of thermal wastewater (Benz et al., 2016).
In general, groundwater in the region of Karlsruhe is of good quality so thatand the local drinking water supplier (Stadtwerke 155 Karlsruhe) only needs to removes oxidised iron and manganese from the pumped groundwater. However, two main contaminations, which affect groundwater quality, are known in the urban area (Stadt Karlsruhe, 2006). A contaminant plume, which contains a polycyclic aromatic hydrocarbons concentration of up to 500 µg/l, of 200 m length over the entire aquifer thickness is located at a former gas plant in the east of Karlsruhe ( Figure S1b) (Kühlers et al., 2012). in the east of Karlsruhe ( Figure S1b). This plume contains a polycyclic aromatic hydrocarbons concentration of up to 500 µg/l caused by the former gas plant in the east of Karlsruhe (Kühlers et al., 2012). Moreover, three parallel contamination plumes, of 2.5,500 km length each, can be found in the southeast of Karlsruhe (Figure S1b), where highly volatile chlorinated hydrocarbons (7 µg/l -26 µg/l) and their degradation products were detected (Wickert et al., 2006). In accordance with the suggestion made by , several integrative samplings (i.e. repeated samples taken over a period of time) (at least three) were conducted to achieve capture an ecologically representative samplingrepresentation of groundwater fauna, which also reflects the occurring species at a community level. Every well is 175 sampled at least three times. From 2011-2012, 22 measurement wells (mainly in the Hardtwald and the North-West of Karlsruhe) were sampled six times at a minimum interval of two months. In 2014, 17 measurement wells, mainly located in the south/inner city, were sampled three times (see Table S2). As the aim of this study is to provide a first-tier screening of the groundwater ecologyecological status, we sampled the fauna in the monitoring wells in accordance with the sampling manual of the European PASCALIS Project (Malard et al., 2002) and the procedure described by Hahn and Fuchs (2009), using a 180 modified Cvetkov net.

Material and sampling
Furthermore, the relative amount of sediment as an indication of the nutrient availability and the cavity system was measured.
Before the fauna sample from the net sampler was passed over a sieve with a mesh size of 74 μm, the sediment is separated and classified in different categories (sand, fine sand, ochre, detritus, silt). It should be noted that the detritus content is not recorded quantitatively but on the basis of estimated frequency classes. The estimation of the relative amounts of sediment per 185 sample is based on Table S1 in the supplement.
Mann-Whithney-tests (U-tests) were applied to detect potential impacts of groundwater characteristics (physical-chemical parameters), geology and well design on the groundwater quality as well as on groundwater fauna. Samples were regarded as significantly different if the p-value was < 5.0×10 -2 .
To better understand large-scale relationships as well as fine structures of high-dimensional biological data, the PHATE 190 (potential of heat diffusion for affinity-based transition embedding) analysis introduced by Moon et al. (2019) (https://github.com/KrishnaswamyLab/PHATE) was used. This dimensionality-reduction method generates a lowdimensional embedding specific for visualization, which provides an accurate, denoised representation of both local and global structures of a dataset without imposing strong assumptions on the structure of the data. The PHATE algorithm computes the pairwise distances from the data matrix and transforms the distances to affinities to encode local information by applying a 195 kernel function, which is developed to Euclidian distances. By using diffusion processes, global relationships are learned and encoded using the potential distance. Finally, the potential distance information is embedded into low dimensions for visualization by using metric Multi-Dimensional-Scaling (MDS) (Moon et al., 2019). Objects that are close to each other in the final graph therefore have similar characteristics.
The organism communities of the groundwater consist of microorganisms and invertebrates (in particular Crustaceans) 200 (Griebler et al., 2014). Crustaceans, especially Amphipods and Copepods, represent the majority of the groundwater fauna.
The identification keys from the following studies were used to identify the different groups in the samples: Einsle (1993), Janetzka et al. (1996), Meisch (2000), Schellenberg (1942) and Schminke et al. (2007). The sampled fauna for this study can be assigned to the subphylum Crustacea and four other subordinate taxa (Table 1).

Classification scheme by the Federal Environmental Agency of GermanyGriebler et al. (2014)
Commissioned by the Federal Environmental Agency of Germany (UBA), Griebler et al. (2014) developed a two-step ecologically based classification scheme for the Federal Environmental Agency of Germany (UBA) for characterization of 210 groundwater ecologyecosystems and. They also defined spatially dependent reference values of ecologically intact groundwater ecosystems. In order to enable a statement about the exposure (organic, chemical, structural) of the groundwater at a specific site, biotic and abiotic parameters, which are determined and compared with reference values, are used to distinguish locations with ecological conditions, which arevery good or good O.K., i.e. very good or good ecological conditions or locations which fail these criteria, i.e. affected areas (Figure 1Figure 1). If an ecological assessment of 215 groundwater ecosystems, which is based on the groundwater fauna analysis, takes place, some faunistic criteria must be considered. Invertebrates avoid habitats that are ochred or have a low content of dissolved oxygen. Thus, unstressed or natural habitats are defined as Five criteria, of which at least three biological/ecological indicator parameters are to be selected, are taken as basis for a reliable assessment. The measured indicator parameters of any location have to be compared with the reference values provided by Griebler et al. (2014). Unstressed or natural groundwater habitats are defined as areas with a 220 content of dissolved oxygen > 1.0 mg/l, that are not ochred and have an existing fauna, i.e. an amount of > 50 % of Stygobites, of > 70 % of Crustaceans and of < 20 % of Oligochaetes (Figure 1Figure 1). This results inallows a qualitative interpretation of the ecological condition of the groundwater system. If the results indicates affected ecological conditions, which means thati.e. one or more biological/ecological indicators are out of the reference range, an assessment according to the Level 2 scheme is necessary. This requires a determination of reference values at local reference locations which are protected and 225 have a weak surface influence as well as a subsequent comparison of these values with measured data. to obtain also a qualitative and quantitative interpretation of the ecological conditions. As our aim is a first-tier screening of an urban area, we only apply Level 1 in our study. *Stygophile organisms are found primarily in surface water, but they can survive in shallow groundwater for a while (Preuß and Schminke, 2004).   As expected, measured GWT at the bottom of the wells, in 8.5 to 39.0 m depth, are mainly constant over the repeated 250 measurements. The lowest GWT ranging between 10.5 and 10.9 °C were measured in the eight wells of the forested area (Table S1S2). In contrast, the highest average GWT with 17.5 °C was measured in a well near the city hospital (T113) (Figure   2Figure 2a). The mean value of all wells is 13.5 ± 2.1 °C, which is similar to the results from Benz et al. (2015) with 13.0 ± 1.0 °C. According to Benz et al. (2017), annual shallow GWT vary between 6 and 16 °C in the area of Karlsruhe, which is in line with the temperatures measured during fauna sampling (Figure 3Figure 3a). For the urban area in the north-western 255 part of the city, Figure 2Figure 2a shows a clear warming trend, which was also observed by Menberg et al. (2013a,b). The increased GWT in this area can be traced back to effects of urban infrastructures and industries, which use groundwater for cooling purposes. The content of dissolved oxygen acts as a limiting factor for groundwater fauna, since groundwater is usually under-saturated 265 with a varying oxygen content between 0 and 8 mg/l (Griebler et al., 2014;Kunkel et al., 2004). In this study, the average content of dissolved oxygen in all wells is between 1.0 and 12.8 mg/l (Figure 3Figure 3b and Figure S1a). As expected, the monitoring wells, located in the forested area (Hardtwald) show the highest content, while the lowest values are found in urban areas, which and is likely linked to aquifer contamination and other anthropogenic effects (content of dissolved oxygen of forested vs. urban area: U-test: p-value = 5.3×10 -3 , n = 8; 31). Urban water can be polluted in multiple ways, which affects the 270 chemical and biological oxygen consumption in the groundwater. The higher the pollution and/or biological activity, the lower the dissolved oxygen. (Kunkel et al., 2004;Griebler et al., 2014). Moreover, it seems that with a greater depth of the measurement wells the content of dissolved oxygen is increasing (U-test: p-value = <10 -13 , n = 39). This can be explained by the fact that shallow wells can have a low water column in which oxygen can rapidly be consumed by groundwater microorganisms, chemical reactions and/or groundwater fauna. In the upper unscreened part of deeper wells, dissolved oxygen 275 can be consumed while in the lower screened part oxygen is continuously refilled by oxic groundwater from the surrounding (Malard et al., 2002). Furthermore, reducing conditions in the overlaying soil can result in a low content of dissolved oxygen in groundwater.

Physical and chemical parameters
Nitrate is often named as an important pollutant in groundwater. The natural and geogenic concentrations of nitrate in groundwater is usually under 10 mg/l (Griebler et al., 2014). In our study area, the average nitrate contents of all wells varies 280 vary between 1.3 and 14.7 mg/l. In the urban area average nitrate concentrations are generally higher and correlate inversely with the content ofthe dissolved oxygen (U-test: p-value = 4.0×10 -3 , n = 39) showing the link between pollution nitrate content and oxygen consumption. Wells with a content of dissolved oxygen below 1.5 mg/l have an average content of nitrate of 1.5 mg/l, most likely caused by nitrate reduction under anoxic conditions. Groundwater with reducing conditions (< 5 mg/l dissolved oxygen) has an average nitrate content of about 7 mg/l in contrast to groundwater with oxidising conditions with 285 9 mg/l, which promotes the oxidation of ammonium to nitrate. The lowest nitrate concentrations are found in the forested area ( Figure 3Figure 3c and Figure S1c), where atmospheric nitrogen is hold held back by forest soils (U-test: p-value = 1.7×10 -3 , n = 8) and fertilization is prohibited due to water protection regulations in the forested area. At the same time, anthropogenic impact is minimal as fertilization is forbidden due to the presence of water protection areas in the forest area (Aber et al., 1998;Schönthaler and von Adrian-Werburg, 2008). Within the study, theMoreover, the average contents concentration of iron and 290 phosphate are low and in most cases below the detection limit of the test ( Figure S1d, e) and also below the natural and geogenic concentrations (phosphate: 0.05 mg/l (Griebler et al., 2014) and iron: 3.3 mg/l (Kunkel et al., 2004)) within the study site.
Considering these findings, clear differences in the spatial distribution patterns of abiotic groundwater characteristics are noticeable. The forested area shows lower average GWT than the urban area (U-test: p-value = 3.3×10 -5 , n = 8; 31), lower 295 nitrate concentrations (U-test: p-value = 4.1×10 -3 , n = 8; 31) and higher dissolved oxygen concentrations (U-test: pvalue = 5.3×10 -3 , n = 8; 31), which indicates a correlation between abiotic groundwater characteristics and land use in the study area. Moreover, no impact of groundwater originating from the urban area on the wells in the forested area is observed, as the groundwater flow direction in Karlsruhe is northwest (see Chapter 2.1 and Figure 2c). Further investigations demonstrated that besides one larger and two smaller contamination sites (however, still with concentrations below the 300 threshold values, Figure S1b), only minor groundwater pollution is documented in Karlsruhe (see Supplement). The chemical and physical parameters considered in the long-term monitoring system are within the range of local background and below threshold values of the drinking water ordinance of Germany (see Supplement for more information). Thus, the main documented impacts on groundwater quality in the study area are related to temperature and oxygen. The rural forest area shows a lower average GWT, lower nitrate concentrations and higher dissolved oxygen concentrations, which indicates a 305 correlation between abiotic groundwater characteristics and land use in the study area.

Groundwater fauna
The biotic communities of the groundwater consist of microorganisms and invertebrates (in particular Crustaceans) (Griebler et al., 2014). In the entire pool of samples 3,633 666 individuals were detected in 37 of 39 wells, which means that 95 % of the wells are colonised (Table S2S3). With 2,0142,047 individuals, the group of Crustacea was found to be the most abundant 310 group (55 56 %). 976 individuals (27 %) of the order of Cyclopoida dominated this group, followed by the genus Parastenocaris with 599 individuals (17 16 %), by the order of Bathynellacea (371), Amphipoda (66), Harpacticoida (33) and Nauplia. The communities of the monitoring wells also frequently contained Oligochaetes (1,343 individuals, 37 %).

Furthermore, individuals of the phylum Nematoda (228 individuals) and Microturbellaria (46 individuals) were also often present. 315
Overall, there is a noticeable difference in the spatial distribution of species within the study area. Individuals of the subphylum Crustacea were found in larger numbers, with regard to the number of wells, in the monitoring wells in the forested area (660 690 individuals in eight wells) than compared to those in the urban area (1,3541,357 individuals in 31 wells). Furthermore, no individuals of the order Bathynellacea and only 135 individuals of the genus Parastenocaris were found in the forested area.
In contrast, larger numbers of the latter species as well as of Oligochaetes are characteristics characteristically found in of the 320 wells in the urban area. However, in contrast to the abiotic characteristics, no clear pattern of faunal diversity and land use was observed, as Crustaceans and individuals of other subordinate taxa were found both in the rural forested and in the urban area.

330
Stygobiotic Amphipods, i.e. large-bodied invertebrates that predominantly live within wellswhich due to their size have a habitat preference for open spaces such as wells (Table 1) (e.g. Hahn and Matzke, 2005;, were found only in only three wells (Figure 2cFigure 2b). 46 individuals of this order were detected in the forest and 20 individuals in the urban area (Figure 4a,b). Although statistical analysis showed no clear differences between the abundance of Amphipods and land use (U-test: p-value = 1.5×10 -1 , n = 8; 31), the higher number of individuals in the forest area could support the hypothesis 335 thatAs mentioned above, Amphipods indicate healthy groundwater ecosystems, as they react most sensitive to disturbances such as pollutants (Korbel and Hose, 2011) as well asand groundwater temperature. (11 ± 5 °C (Brielmann et al., 2011) up to 17 °C ). In laboratory experiments with a thermal tank, Brielmann et al. (2011) found that 77 % of the individuals of the studied Amphipods (Niphargus inopinatus) preferred areas with a temperature between 8 and 16 °C. In addition, Spengler (2017) and Issartel et al. (2005) observed maximum temperatures up to 17 °C. The lack of a statistically significant correlation might also 340 be related to the low number of wells (n = 8 in the forested area) and individuals (n = 46). Amphipods They are important ecosystem service providers in terms of bioturbation and organic decomposition (Boulton et al., 2008)., As observed in laboratory experiments (Smith et al., 2016),as they show an active movementactively move, with possible migration speeds between 1.7 and 3.5 ×x 10 4 m per year observed in laboratory experiments (Smith et al., 2016). If In most cases when Amphipods were found, in most cases higher amounts concentrations of individuals of the order Cyclopoida were also 345 identified (Abundance Amphipoda vs. Cyclopoida: U-test: p-value = 9.6×10 -5 , n = 39). Individuals of the latter order were mostly generally be found in larger quantities in the majority of the wells (479 in the forested area and 497 in the urban area), as they are the largest group of Crustaceans in this environment (Fuchs et al., 2006) and can tolerate a wide temperature range (e.g. upper thermal limit of 26.9 ± 0.2 °C in laboratory tests by Sánchez et al. (2020)) (Spengler, 2017).
The order Harpacticoida, which includes the genus Parastenocaris, have an elongated body shape and a stem-chiselling 350 movement, which is why they are predestined for living in cavities and groundwater ( Hahn, 1996;Fuchs, 2007), preferring sand and gravel as a substrate . Larger numbers of Parastenocaris (464 individuals), which can tolerate GWT from 8 to > 20 °C (Fuchs et al., 2006) (e.g. Parastenocaris phyllura up to 22.5 °C in laboratory tests; (Glatzel, 1990)), were found in the urban area, especially in the northwest area (Figure 2Figure 2b). This area is characterised by GWT between 16 and 18 °C, the highest at the study site. This observation is comparable with previous studies (Hahn, 2006;Hahn et al., 355 2013;Spengler, 2017), which showed that the genus Parastenocaris is particularly non-competitive and can often be found isolated in structurally burdened and physico-chemically altered areas. Accordingly, only 135 individuals were detected in the forested area.
In addition, quantities of Bathynellacea (371 individuals) were found in five monitoring wells all located in the urban area in a depth of 9 to 13.5 m and by medium ,at a GWT of 12-15 °C all located in the urban area (371 individuals), 360 respectively ( Figure 4b). This order typically inhabits the interstitial groundwater, which is characterised by a dominant exchange with the surface water and high variations in GWT, and can tolerate temperatures up to 18 °C (Stein et al., 2012).
Interestingly, one location in the southern city area with 272 individuals is characterised by a high fluctuation in GWT (standard deviation of 3.4 °C) and a rather high nitrate content (8.3 mg/l) compared to wells in the forested area, which are both indications for a disturbed and stressed habitat. 365 Besides the group of Crustaceans, Oligochaetes, which can tolerate a wide temperature range, were also found in large abundance in the study site. A significant amount of the subclass Oligochaeta (996 individuals) was found in the urban area ( Figure 4b), compared to an overall number of 1,343 individuals. In general, the number of Oligochaetes is larger in locations with high GWT (12.6 --17.3 °C) and high nitrate concentrations (up to 14 mg/l, which is above the geogenic concentration of 10 mg/l and higher compared to wells in the forested area). 370 Finally, Nematodes and Microturbellarians were found at locations with unfavourable living conditions, such as a low content of dissolved oxygen, or a high amount of fine substrates, as also reported by Hahn et al. (2013), . Bboth can tolerate high temperature ranges (Turbellaria: 2 -20°C (Herrmann, 1985), Acari: 9.1 -18.5 °C (Więcek et al., 2013)). Here, both were found in larger quantities in the urban area of Karlsruhe (Figure 4b). This area has the lowest content of dissolved oxygen, high relativerelatively higher amount of detritus (> 2) and the highest nitrate concentrations (> 6 mg/l). 375 Eventually, correlation analysis between groundwater fauna and the chemical parameters showed that Stygobites are only slightly affected by groundwater qualitychemistry (Hahn, 2006;Schmidt et al., 2007;Stein et al., 2010). Only the Spearman's rank correlation coefficient between the number of taxa and the content of dissolved oxygen is significant with a value of  = 0.53 (p-value = 3.0×10 -4 , n = 39p-value = 0.0005, n = 39). Moreover, it is assumed that groundwater fauna can usually cope well with short-term changes of chemical-physical parameters (Griebler et al., 2016). Previous studies showed that some 380 species can even benefit from pollutants (Matzke, 2006;Zuurbier et al., 2013). In case of nitrate, numerous studies underline that nitrate at concentrations below 50 mg/l does not directly affect groundwater fauna (Fakher el Abiari et al., 1998;Mösslacher and Notenboom, 2000;Di Lorenzo and Galassi, 2013;Di Lorenzo et al., 2020). As the highest average nitrate content per well is below 15 mg/l in this study, a direct negative effect of the nitrate concentration on the groundwater fauna is unlikely. Thus, nitrate is only mentioned as one measured parameter and is not discussed as a potential anthropogenic impact 385 in this study.
The natural influence on porosity, groundwater flow and nutrient delivery were also discussed as primary influence on natural Stygobites distribution by previous studies (Hahn, 2006;Korbel and Hose, 2015). One important natural influence is the local geology, as fine sands and silts are typically rather harsh environments, resulting in an impoverishment of specific groundwater fauna such as Crustacea (Hahn, 1996). The city of Karlsruhe is located on carbonate ('Würm') gravel and river terrace sands, 390 pervaded by bands of drifting sand and inland dune sands. These sediments are highly water-permeable and show almost exclusively vertical seepage of water movement. Flood sediments (on top of river gravel) and bog formations, are located in the east and west of Karlsruhe (Regierungspräsidium Freiburg, 2019). This local geology limits the cavity size and therefore has impacts on the habitat of the groundwater fauna (Wirsing and Luz, 2007). For example, individuals of the genus Parastenocaris typically inhabit small-scale cavity systems (Spengler, 2017). Individuals of this genus can be found both in 395 the wells drilled in gravel (4 wells) and in drifting sand sediments (3 wells) (abundance Parastenocaris vs. geological units: U-test: p-value = 1.4×10 -9 , n = 39). Amphipods are predominantly found in measurement wells located in the 'Würm' gravels (in 5 of 7 wells) (abundance Amphipoda vs geological units: U-test: p-value = 9.0×10 -11 , n = 39). Moreover, it seems that differences in the geological units have an influence on the total amount of individuals (U-test: p-value=1.7×10 -9 , n = 39) and the relative amount of detritus (U-test: p-value = 3.0×10 -3 , n = 39). As these results show, regional geology seems to have an 400 influence on the occurrence of specific groundwater taxa and on the number of individuals as well as on food supply, in terms of available organic matter. However, it is not possible to give a reliable estimate of the strength of the anthropogenic impacts, e.g. if they are strong enough to overrule the regional selective forces. Hence, this should be investigated in more detail in future studies. Some Llimitations regarding the sampling method have tomust be considered when interpreting the faunistic results. In this 405 study, a simple basic screening of well water was conducted, using a net sampler and bailer, to assess examine conditions in the groundwater monitoring wells (39 wells with an average diameter of 132.5 mm, which corresponds to an area of 0.003 ‰ of the total urban area). According to the sampling manual of the PASCALIS Project 'the use of a phreatobiological net alone is considered as a satisfactory method for sampling groundwater fauna in large diameter wells' (Malard et al., 2002). Yet, several studies (e.g. Scheytt, 2014) report that scooped samples of wells are not representative, and therefore the water 410 remaining in a well has to be purged and discarded before sampling. Nevertheless, pumping can result in the selection of the taxa, especially in the presence of very fine sediments, and can result in changes of the sediment composition in the surrounding of wells and therefore in changes of habitat conditions. Other studies, on the other hand, found no significant differences in hydro-chemical values (temperature, pH, dissolved oxygen, etc.) between the surrounding groundwater and the standing water in a well (Hahn and Matzke, 2005;. The sampled groundwater fauna of corresponding wells and aquifers 415 were also shown to be similar with respect to the types of faunal communities. However, in terms of total abundance however, as well as the numbers of individuals per litre, monitoring wells appear to exhibit larger numbers, caused by filtration effects (Hahn and Matzke, 2005;. As the aim of this study is to provide an overview of the groundwater fauna community (assess biodiversity) and to receive a first impression of groundwater ecology, sampling the fauna by using a net sampler is sufficient. In order to achieve a representative sampling of groundwater fauna in the aquifer 420 and to reflect the occurring species at a community level, a more comprehensive sampling method is required, e.g. the use of a defined standard sampling method using a pump to collect animals (Malard et al., 2002). Care should also be taken when interpreting faunistic results of sites that are sampled in different years. To improve comparison of the biotic communities, a consistent sampling period of every well is necessary in the future.

Classification scheme by the Federal Environmental Agency of GermanyGriebler et al. (2014) 425
In three wells, evaluation with the UBA classification scheme by Griebler et al. (2014) was not possible due to ocherous conditions in two monitoring wells and low content of dissolved oxygen (<1 mg/l) in the third well. According to the UBA classification scheme by Griebler et al. (2014), unstressed (meaning no natural or anthropogenic stressors), or natural groundwater habitats have an amount of more than 70 % of Crustaceans and less than 20 % of Oligochaetes. In 36 % of the sampled wells, i.e. 14 out of 39, these criteria are were fulfilled, indicating O.K.very good or good ecological conditions or in 430 other words a natural groundwater habitat (Figure 4c). These natural areas tend to contain more individuals of the orders Amphipoda, Cyclopoida and Bathynellacea. Monitoring wells, which do not fulfil these criteria and are accordingly defined as affected areas not having natural ecological conditions, contain more Oligochaetes and also Nematodes, which is partly explained by the used criteria of this classification scheme (Figure 4d). Surprisingly, only 50 % of the wells in the rural forest, which is also the catchment area of the drinking water supply of 435 Karlsruhe, are described as natural groundwater habitats. An identical number of wells yielded habitats with affected ecological conditions. The main difference between natural and affected wells in the forested area arises from the occurrence of specific species. 86 to 100 % of species found in natural wells are Crustaceans Natural wells have an amount of Crustaceans of 86 to 100 %, in contrast to affected wells with only 33-67 % (Table S1 S2 and Table S2S3). However, the abiotic parameters scarcely differ between natural and affected wells (average values for GWT: 10.8 and 10.6 °C, dissolved oxygen: 7.1 and 8.8 mg/l, 440 nitrate: 2.5 and 3.0 mg/l), indicating that there are other processes or parameters that influence the groundwater fauna in these wells. One reason could be the varying local geology , as mentioned above. Moreover, food supply is one of the most limiting parameters for the survival of groundwater fauna (Datry et al., 2005;Hahn, 2006). If the organic carbon supply varies on a small scale, this can influence microbiology and therefore groundwater fauna as well, although, short-term changes in nutrient supply can be compensated by groundwater fauna.as fine sands and silts are typical rather harsh environments resulting in an 445 impoverishment of specific groundwater fauna such as Crustacea (Hahn, 1996).
In contrast to the forest land, the majority of wells (65 %) in the urban area are categorised as affected habitats. As expected, this indicates anthropogenically influenced groundwater ecosystems beneath the studied urban area. Once more, no significant differences between the abiotic parameters of natural and affected wells are observed (e.g. median of dissolved oxygen: 4.7 and 5.8 mg/l, median of nitrate: 7.2 and 7.8 mg/l). On the other hand, the remaining 35 % of the wells in the urban area show 450 natural ecological conditions, even though some of them are located in areas with anthropogenic impacts such as increased groundwater temperatures. Hence, no distinct spatial pattern of the ecological condition with respect to land use could be identified.
In future, a further subdivision of a study area in more land use categories could be useful to specifically look at typical anthropogenic impacts. Furthermore, the integration of more biological criteria is useful to improve the results of the 455 assessment according to Griebler et al. (2014). Because of heterogeneous groundwater ecosystems in Germany it is likely that reference values provided by Griebler et al. (2014) do not reflect the situation in Karlsruhe correctly. Considering site-specific characteristics and reference values would lead to a more robust assessment. Other assessments, like the similarly structured GHI or wGHI N Di Lorenzo et al., 2020b) can additionally be used. Moreover, there are a couple of newly developed indexes, like the D-A-C-Index, which is based on microbiological indicators and shows whether groundwater 460 reserves deviate from natural references (Fillinger et al., 2019), which can be used in the future. As mentioned in the introduction, another way to quantify the relevant ecological conditions in the groundwater is the GFI. During the preparation of this study, the GFI was tested on the data (see Supplement), however, it did not provide any additional information or valuable insights and was therefore excluded. The influence of multiple stressors, such as the pollution of the groundwater through industrial plants etc., and their effects on the governing parameters can bias the GFI. In general, the GFI seems to be 465 suitable only for unpolluted and anthropogenically undisturbed groundwater with sufficient oxygen concentrations (> 1 mg/l).
Moreover, under urban areas changes in GWT are caused by anthropogenic heat inputs (Menberg et al., 2013b(Menberg et al., , 2013aBenz et al., 2014;Tissen et al., 2018), rather than being related to surface water influences. Hence, the GFI appears to be unsuitable for the assessment of the groundwater fauna in an urban setting. The same outcome emerges for the Shannon diversity index, which was also tested during the preparation of the study and showed no clear distribution pattern according to faunal diversity 470 and was therefore not considered further.
This observed spatial heterogeneity in ecological conditions and heat anomalies in an urban area therefore also offer the potential to use groundwater for heating and cooling, and even to locally store energy in form of aquifer thermal energy storage (ATES) systems (e.g. Fleuchaus et al., 2018).

PHATE analysis 475
A PHATE analysis is conducted using the following 15 input parameters: depth, GWT, nitrate and phosphate content, relative amount of detritus, geological unit, numbers of taxa, number of individuals, Shannon diversity, amount of Crustaceans and Oligochaetes (according to Griebler et al., 2014) and the abundance of Amphipods as well as of individuals of the order Cyclopoida, Bathynellacea and the genus Parastenocaris. The content of dissolved oxygen is not considered in this analysis, since it was always above the limit of 1 mg/l, except for in one case. Thus, dissolved oxygen is not expected to have an 480 influence on the groundwater fauna in our study area.  (Table S4). However, diversity and abundance was found to be low in Group III.
An even higher average GWT of 15.0 °C was found for Group II, which mostly consists of wells drilled in drifting sand 490 sediments. Surprisingly, these wells also show the highest diversity (≥ three Taxa per well), the highest Shannon diversity (see Supplement), highest amount of individuals in total, as well as of individuals of the genus Parastenocaris. Individuals of this genus are often found isolated in altered areas (Spengler, 2017). Moreover, in five wells of Group II individuals of the order Bathynellacea, which can tolerate temperatures up to 18 °C and typically inhabit interstitial groundwater (Stein et al., 2012), were found. The presence of individuals of the genus Parastenocaris and the order Bathynellacea in Group II suggests that 495 they may act as type species for urban situations. The observation that Group II shows the highest GWT and the highest Shannon diversity is in contrast to findings of previous studies that noticed decreased diversity at elevated temperatures (Brielmann et al., 2009). These diverging observations suggest that faunal quantities, such as diversity or abundance, are not always suitable indicators for changes within organism communities. For example, if species disappear due to increased temperatures and are substituted by more tolerant species, the difference in diversity may be marginal and the change in the 500 community may not be noticeable.
Wells of Group I (blue) are drilled predominantly in Würm gravel (geological unit of Group I vs. Group II: U-test: pvalue = 8.2×10 -3 , n = 13; 14), while having the lowest GWT (GWT of Group I vs. Group II: U-test: p-value = 2.0×10 -5 , n = 13; 14). These wells show a moderate diversity and amount of individuals, yet the highest average amount of Crustaceans as well as the highest amount of Amphipods and individuals of the order Cyclopoida. Considering these findings and the U-505 Test results (see Table S5), the grouping of the measurement wells seems to be influenced by the composition of the groundwater organism communities, the faunal diversity (numbers of taxa and amount of individuals), as well as the geological unit and the GWT ( Figure S3-S4).
Considering the spatial distribution of the grouped wells in the study area, it becomes apparent that all wells in the forested area fall within Group I ( Figure 5). Those wells which are located outside the forested area are in locations with nearby green 510 areas (parks, recreational areas, etc.). In contrast, the wells of the other three groups are heterogeneously distributed within the urban area. Many of the measurement wells of Group III and IV are associated with suspected or known contaminated sites ( Figure S1b). Overall, a spatial pattern of abiotic groundwater characteristics (GWT, nitrate content) and occurrence of particular species (Parastenocaris) within the study area is apparent in the PHATE analysis, which confirm the classification according to land use. Yet again, no clear spatial pattern regarding faunal diversity in the study area could be identified. 515 Although, a tendency of clustering of wells from Group III with higher diversity and amount of individuals can be seen in the northwest city area.

Conclusion
The aim of this study is to provide a first-tier assessment of the ecological state of groundwater in an urban area and to 520 distinguish areas with a natural state of the groundwater ecology from anthropogenically affected areas. To achieve this, we examine the groundwater fauna, as well as abiotic parameters in 39 groundwater monitoring wells in the urbanresidential, commercial and industrial areas (31 wells) and a forested area (eight wells) outside the built-up area of Karlsruhe, Germany, and a nearby forest land using the simple UBA classification scheme by Griebler et al. (2014) to characterise the sampled monitoring wells. 525 We found a noticeable difference in the spatial distribution of abiotic groundwater characteristics and special species within the study area. The rural forested area shows lower GWT, lower nitrate concentrations and higher dissolved oxygen concentrations, which indicates a correlation between abiotic groundwater characteristics and land use. Moreover, Amphipods are more abundant in wells in the forested than in urban area. However, both in the rural forested and in the urban area Crustaceans and individuals of other subordinate taxa were widely found and therefore no clear spatial pattern regarding faunal 530 diversity and land use was found, as both in the rural forest and in the urban area Crustaceans and individuals of other subordinate taxa were widely found. In terms of faunal quantity, Crustaceans were found in larger numbers, with respect to the number of wells, in the monitoring wells in the forested area than compared to those in the urban area. Larger amounts of the genus Parastenocaris as well as of Nematodes and Oligochaetes were found to be characteristics for wells in the urban area. 535 Furthermore, no clear spatial pattern of ecological groundwater conditions according to the UBA classification scheme by Griebler et al. (2014) could be observed. Surprisingly, only 50 % of the sampled wells in the rural forested area were described as natural (undisturbed) groundwater habitats, while the other four were characterised as habitats with affected ecological conditions. Yet, the majority of wells (65 %) in the urban area were classified as affected locations, which suggest, suggesting that there are noticeable differences in the groundwater ecosystems between the surrounding rural forested areas and urban 540 areas. The Level 2 assessment from Griebler et al. (2014) can help to achieve a more reliable and quantitative ecological assessment of urban aquifers as it divides groundwater ecosystems in ecological grades according to the intensity of anthropogenic disturbance. It is based on the use of local reference values and the collaboration with experts, however, is challenging to apply. Therefore, further studies with large-scale and repeated measurement campaigns are needed to verify our findings. This should also include other cities and the determination of undisturbed local reference values which are required 545 for a more reliable but also quantitative ecological assessment of urban aquifers. Moreover, a wider range of indicators should be considered in a classification scheme, such as temperature, porosity of the aquifer, groundwater flow, pollutants and nutrient supply, especially when investigating urban areas. In addition, an important adaptation for an improved evaluation method is the determination of fauna at species level which will provide more information (i.e. about Stygobionts, Stygophiles, Stygoxenes) and also consider the endemism of stygobiotic species. In this context, classification schemes should pay more 550 attention to the different groundwater species and their potential use as indicator species.. Thus, further studies with largerscale and repeated measurement campaigns are needed to verify our findings also in other cities, and to provide undisturbed local reference values which are required for a more reliable and also quantitative ecological assessment of urban aquifers.
Finally, city and also energy planning should seriously consider urban groundwater ecosystems as they provide valuable information for a sustainable use of the subsurface.