Anthropogenic and Climatic Factors Enhancing Hypolimnetic Anoxia in a Temperate Mountain Lake ⇑ Javier Sánchez-España A, , M

Journal of Hydrology 555 (2017) 832–850

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Journal of Hydrology

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Research papers Anthropogenic and climatic factors enhancing hypolimnetic anoxia in a temperate mountain lake ⇑ Javier Sánchez-España a, , M. Pilar Mata a, Juana Vegas b, Mario Morellón c, Juan Antonio Rodríguez d, Ángel Salazar a, Iñaki Yusta e, Aida Chaos f, Carmen Pérez-Martínez g, Ana Navas h a Department of Geological Resources Research, Spanish Geological Survey (IGME), Calera, 1, 28760 Tres Cantos, Madrid, Spain b Department of Geological Resources Research, Spanish Geological Survey (IGME), Ríos Rosas, 23, 28003 Madrid, Spain c CITIMAC, Facultad de Ciencias, University of Cantabria, Avenida de los Castros s/n, 39005 Santander (Cantabria), Spain d Department of Geoscientiﬁc Infrastructure and Services, Spanish Geological Survey (IGME), Calera, 1, 28760 Tres Cantos, Madrid, Spain e Department of Mineralogy and Petrology, University of the Basque Country (UPV/EHU), Apdo 644, Bilbao, Spain f Faculty of Geological Sciences, Universidad Complutense de Madrid, 28040 Madrid, Spain g Department of Ecology, University of Granada, Campus Fuentenueva, 18071 Granada, Spain h Estación Experimental de Aula Dei (EEAD-CSIC), Avda Montañana 1005, 50059 Zaragoza, Spain article info abstract

Article history: Oxygen depletion (temporal or permanent) in freshwater ecosystems is a widespread and globally impor- Received 18 January 2017 tant environmental problem. However, the factors behind increased hypolimnetic anoxia in lakes and Received in revised form 19 October 2017 reservoirs are often diverse and may involve processes at different spatial and temporal scales. Here, Accepted 23 October 2017 we evaluate the combined effects of different anthropogenic pressures on the oxygen dynamics and Available online 24 October 2017 water chemistry of Lake Enol, an emblematic mountain lake in Picos de Europa National Park (NW This manuscript was handled by T. McVicar, Editor-in-Chief, with the assistance of Spain). A multidisciplinary study conducted over a period of four years (2013–2016) indicates that the Joshua R. Larsen, Associate Editor extent and duration of hypolimnetic anoxia has increased dramatically in recent years. The extent and duration of hypolimnetic anoxia is typical of meso-eutrophic systems, in contrast with the internal pro- Keywords: ductivity of the lake, which remains oligo-mesotrophic and phosphorus-limited. This apparent contradic- Anoxia tion is ascribed to the combination of different external pressures in the catchment, which have increased Cattle grazing the input of allochthonous organic matter in recent times through enhanced erosion and sediment trans- Human impact port. The most important among these pressures appears to be cattle grazing, which affects not only the Mountain lakes import of carbon and nutrients, but also the lake microbiology. The contribution of clear-cutting, runoff Sediment loadings channelling, and tourism is comparatively less significant. The cumulative effects of these local human Climate change impacts are not only affecting the lake metabolism, but also the import of sulfate, nitrate- and ammonium-nitrogen, and metals (Zn). However, these local factors alone cannot explain entirely the observed oxygen deficit. Climatic factors (e.g., warmer and drier spring and autumn seasons) are also reducing oxygen levels in deep waters through a longer and increasingly steep thermal stratification. Global warming may indirectly increase anoxia in many other mountain lakes in the near future. Ó 2017 Elsevier B.V. All rights reserved.

1. Introduction managers of aquatic systems must face is that of hypoxia/anoxia, which usually occurs as a natural response to increased organic Mountain lakes are greatly vulnerable to climatic changes and and/or nutrient loadings (Nürnberg, 2004), but which is also anthropogenic pressures (Parker et al., 2008). Their environmental affected by current climatic trends (e.g., Sahoo et al., 2013; North management relies on a delicate equilibrium between resource et al., 2014). The study of lakes in these areas is crucial to under- exploitation and ecosystem preservation (Debarbieux et al., stand the effects of human impacts, and provides the basis for 2014). This turns critical when applied to national parks, which their conservation and management (Lotter and Psenner, 2004). represent a reference in the conservation of natural heritage and In lakes affected by combined pressures at different spatial and biodiversity. A major important threat which environmental temporal scales, a precise evaluation of cause/effect relationships can only be achieved through multidisciplinary research including ecology, hydrology, limnology, geochemistry or soil science, ⇑ Corresponding author. among others. E-mail address: [email protected] (J. Sánchez-España). https://doi.org/10.1016/j.jhydrol.2017.10.049 0022-1694/Ó 2017 Elsevier B.V. All rights reserved. J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850 833

In this study, we evaluate the influence of different environ- gases –TDG–, photosynthetically active radiation –PAR–, and mental pressures on the water quality and oxygen dynamics of a chlorophyll-a –chl-a–) were taken with multi-parametric datason- mountain lake situated in a national park. Despite being in a pro- des (HatchÒ, YSI). ORP values are given as relative to the standard tected natural area, a number of anthropogenic activities in the hydrogen electrode (Eh) after temperature correction. PAR was lake surroundings (including cattle grazing, clear-cutting, alter- measured with a LI-COR sensor in the wavelength range 400– ations of the drainage network and intensive tourism) could be 1100 nm. The chl-a sensor measures relative fluorescence signal altering the ecological equilibrium of the lake, though the particu- and provides an estimate of chl-a concentration. The DO sensor lar effects of these different stressors are presently unknown. We measures oxygen concentration by luminescence (precision = 0.0 conducted a geochemical, limnological and microbiological study 1 mg/L O2). over a period of four years (2013–2016). The objectives of our T and DO data loggers (HOBO, Onset Computer Corporation) study were: (i) to obtain an accurate picture of the biogeochemical were installed at different depths in a mooring line (Fig. 1b). Five dynamics of the lake (including the extent and duration of anoxia, thermistors (at depths of 1, 5, 10, 15 and 19 m) and a DO logger phytoplankton dynamics and bacterial activity, nutrient and car- (at 19 m) recorded T and DO changes during the studied period bon concentration, trophic state) to identify possible indicators of at intervals of 1 h. Measuring ranges and resolution were 0–40 °C anthropogenic environmental pressures; (ii) to recognize spatial and 0.1 °C for the thermistors and 0–30 mg/L and 0.02 mg/L for chemical trends in basin soils and lake sediments which may help the DO logger. detect major fluxes of organic matter, nutrients or pollutants (e.g., metals) affecting the lake water quality and/or oxygen dynamics; 3.2. Sampling of waters and sediments and (iii) to evaluate the effects of climatic factors on the vertical distribution of dissolved oxygen in the water column by comparing Water samples for chemical analyses of major ion and nutrient inter-annual variations of lake stratification. These three objectives concentration and for microbiological analyses (phytoplankton, provide the structural sub-headings used in the Results and Dis- bacteria) were taken from different depths (at 0.5, 5, 10, 15 and cussion sections. 18 m below the lake surface) with a Van DornÒ sampling bottle The results and discussion related to local anthropogenic pres- (KC Denmark). Phytoplankton samples (3 replicates Â 125 mL) sures at the basin scale can be valuable for the future conservation were immediately fixed in 4% acetic Lugol’s solution. Samples for and management of this lake and many others situated in similar chemical analyses were filtered on site with 0.45 mm nitrocellulose mountain areas. The climatic effects on the extent of hypolimnetic membrane filters (MilliporeÒ), stored in polyethylene bottles anoxia, although based on local inter-annual oscillations of tem- (125–250 mL), acidified with HNO3 (metals), and cool-preserved perature and precipitation, may be useful to enlarge our under- during transport. standing of the impact of climate change (e.g., global warming) Sediment cores (15–50 cm in length) were taken with a gravity on oxygen dynamics in mountain lakes worldwide. corer (UWITEC) from shallow (4 m depth) to deep (21 m) areas for chemical and microbiological analyses (Fig. 1b). The cores were kept closed under dark conditions at 4 °C. The upper 10 cm of these 2. Study site cores were split lengthwise, sampled at intervals of 1–2 cm and later analysed in the laboratory by the methods described below. We centred our study in Lake Enol (1070 m a.s.l.; Picos de Two sediment traps (HYDRO-BIOS) were installed near the lake Europa National Park, NW Spain), which represents a paradigm of centre at depths of 5 and 19 m to collect suspended particulate environmental protection in Spain. Along with the adjacent Lake matter (SPM). These traps were sampled every two months; the Ercina, this lake forms an emblematic mountain landscape known collecting bottles (volume = 250 mL) were transported to the labo- as Covadonga Lakes (Fig. 1a) which was the first National Park ratory for chemical and mineralogical analyses of SPM and calcula- declared in Spain in 1918. At present, it is the second most visited tion of sedimentation rates. protected natural area in the nation with 630,000 visitors per year (OAPN, 2015). However, this lake is being subject to different 3.3. Microbiological analyses human pressures which threaten its future conservation (Fig. S1 in electronic Supplementary material). The number of tourists Phytoplankton (density, biomass and species composition) increased significantly in the 1980s, and cattle grazing has been were studied at the University of Granada. A volume of 50 mL of also intensified, with the number of cows amplified by a factor of each sample were allowed to settle in counting chambers before 3 since 2000 (OAPN, unpubl.). In addition, shrub clear-cutting is a examination with an inverted microscope. Identification was car- common practice in the area, and alterations in the drainage net- ried out to species level where possible. A minimum of 400 cells work (e.g., construction of roads and hiking trails around the lake) or setting units (colonies, filaments) were counted in order to get have increased soil erosion rates in recent times (Rodríguez et al., reliable abundance estimates. Individual biovolume was calculated 2016). The lake has a surface area of 133,000 m2, volume of 106 according to fitted geometric forms (Hillebrand et al., 1999), taking m3 and maximum depth of 22 m (Fig. 1b), and is regulated by an linear measures of 30 individuals. Biovolume data were converted outlet draining to the East. to biomass (in C) using regressions from Menden-Deuer and Lessard (2000). 3. Materials and methods The presence of enteric bacteria at different depths (including total coliforms, fecal coliforms and total enterococcus) was anal- 3.1. Hydrogeochemical profiling and T/DO recording ysed in a certified laboratory (Ensayos Microbiológicos, S.L.). Addi- tionally, semiquantitative analyses of bacterial metabolisms (iron- Geochemical profiles and sampling of waters and phytoplank- related and sulfate-reducing bacteria –IRB, SRB–, in addition to ton were conducted on a bimonthly basis from spring (May) to fall other bacterial groups including enteric bacteria and Pseudomon- (November) in 2013, 2014 and 2015, with additional data for ads) were carried out in waters and sediments by commercial bio- November 2016. Physico-chemical parameters (including logical activity reaction tests (BARTTM) from Hach (DBI, 2004). These temperature –T–, specific conductance –SpC–, pH, redox potential tests were conducted at room conditions and required between 6 –ORP–, dissolved oxygen –DO–, total pressure of dissolved and 10 days for each test. 834 J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850 a N

Lake Enol

Lake Ercina

Covadonga Lakes

Outflow

100 m b

Fig. 1. Environmental setting of Lake Enol: (a) satellite image (2009) of lakes Enol and Ercina; (b) bathymetric map of Lake Enol with depth contours and geographic coordinates; the white point near the lake center in (b) indicates the approximate location of the mooring line (ML) holding the thermistors, DO sensors and sediment traps, and in which most physico-chemical depth profiles were taken; the orange points indicate the locations of sediment cores used in this study; the blue point indicates the sampling area of shore sediments (SS) used for microcosm experiments; the arrows indicate the position of inflows (in red) and outflow (in blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850 835

3.4. Chemical analyses of waters and sediments of pH are also observed during the stratification period (pHEpi =8. 4–8.6 vs.pHHyp = 7.1–7.3; Fig. 2c). The water chemistry was analysed by different techniques, including ion chromatography and continuous flow auto-analyser 4.1.2. Oxygen dynamics 2À À 3À À for major anions (e.g., SO4 ,NO3 ,PO4 , HCO3 ), total organic carbon A marked oxycline parallels the thermocline during the summer + (TOC), ammonia (NH4) and silica (SiO2), and atomic absorption and early autumn (Fig. 2d). This oxycline ranges from the metalim- spectrometry (AAS) and inductively coupled plasma-mass spec- netic oxygen maximum at 7–8 m (generated by photosynthetic trometry (ICP-MS) for major cations and trace elements. Total activity) to depths ranging from 18 m (July) to 13 m (September), phosphorus was also analysed by ICP-MS and UV–VIS spectropho- though the oxic/anoxic transition can reach even shallower depths, tometry after acid digestion in sulphuric acid at 220 °C. as discussed below. Thus, the thickness of the entirely anoxic (i.e., Sediment cores and SPM were analysed by ICP-AES (after previ- DO <0.1 mg/L or <1% sat.) bottom layer evolves from 3 m in July to ous acid digestion), elemental analyser (IR solid-state detector), X- 8 m in September, and can still be 5 m in November. During this ray diffraction (XRD) and X-ray fluorescence (XRF). TOC analyses period, the Eh of the hypolimnion gradually decreases from values were carried out at IPE-CSIC by infrared absorption of free CO2 after typical of oxidizing conditions in winter (400 mV) to near-zero combustion in a LECO analyzer. Total inorganic carbon was calcu- values indicative of strongly reducing conditions in November lated by difference between total and organic carbon. Total N was (Fig. 2e). The DO data recorded by the logger installed at 19 m indi- analysed with a VARIO MAX elemental analyser. The isotopic sig- cate that the lake bottom remains anoxic for as much as 6 months 13 15 nature of carbon (d CPDB) and nitrogen (d NAIR) in bottom sedi- and is not re-oxygenated until middle November during the lake ments and SPM was analysed at the Stable Isotopes Laboratory of overturn (Fig. 3b). The oxygen pulse observed in late July was anec- the Universidad Autónoma de Madrid (UAM). dotic and probably caused by heavy rainstorms occurred on those days. From November to May oxygen was present throughout the whole water column. Interestingly, both the oxygen drop experi- 3.5. Soil chemistry and reactivity tests enced in May 2014 (from 8 to 0 mg/L DO) and the re- oxygenation during the autumn turnover were very fast and hap- The chemistry of soils in the lake basin was studied along three pened within hours (Fig. 3b). transects from the catchment divides to the lake (T1, T2, T3) and an The TDG profile of 2014 (Fig. 2f) mimicked the photosynthetic additional transversal one (TT). Eighteen soil profiles (20–50 cm O peak at 6–7 m and the oxygen consumption across the oxycline. thickness, sectioned in depth intervals, totalizing 58 soil samples) 2 In the absence of O at depth, the increase of dissolved gases were collected to cover the different soil types recognized. The soil 2 towards the lake bottom which is commonly found suggests an samples were air-dried, ground and sieved (2 mm) in the labora- increase in the concentration of some other gases in the hypolim- tory. The analyses comprised, among others, pH and electrical con- nion, most likely CO by biologically driven organic matter miner- ductivity, carbonate, soil organic matter (SOM), soil organic carbon 2 alization. Based on the redox value (Eh = 50–150 mV, not so (SOC), total nitrogen, and trace metals. Chemical analyses were strongly reducing), organoleptic properties (e.g., lack of any odor), made by ICP-OES after total acid digestion with HF (48%) in a and chemical evolution (e.g., no change in color or turbidity upon microwave oven. storage) of the sampled deep anoxic waters, the content of other Qualitative reactivity tests and microcosm experiments with gases like H S, CH or NH is considered to be still negligible in shallow sediments were conducted to evaluate the reactivity of 2 4 4 comparison with CO , although they could be also present at trace different organic substrates taken from the lake shore and their 2 amounts. potential influence on the lake metabolism (details given in Sup- plemental material). 4.1.3. Phytoplankton dynamics The profiles shown in Fig. 4a show the variation of chlorophyll-a 4. Results concentration as a function of depth in 2013 and 2014. The chl-a concentrations were low and usually comprised between 0.1 and 4.1. Biogeochemical dynamics 1 mg/L chl-a, which is characteristic of oligotrophic lakes (<2.5 mg/ L; Wetzel, 2001). A relative concentration peak of 1.7 mg/L was 4.1.1. Seasonal stratification observed at around 8 m depth in July 2014, whereas the concentra- An example of seasonal evolution of lake stratification patterns tion maximum in 2013 was observed at depth (2.5 mg/L at 14–15 during 2014 is illustrated in Fig. 2. The temperature profiles shown m). The nature of this second peak was unclear and could corre- in Fig. 2a illustrate the progressive development of thermal strati- spond to either decaying phytoplankton biomass from upper levels fication as a response to continuous warming of the lake surface by or, alternatively, phototrophic microorganisms adapted to low solar radiation. An incipient stratification usually develops in late light conditions. The different vertical distribution of chl-a concen- May and increases during the summer, when a thermocline tration between July 2013 and July 2014 might have been influ- between 6 m and 14 m separates a warmer epilimnion (18–1 enced by local climatic factors, such as slight differences in 9 °C) from a cooler hypolimnion (7–8 °C). The thermal difference precipitation or mean ambient temperature during the spring decreases during the fall and the lake overturn takes place between and summer time of these two years, which were climatically dif- late October and middle November (Fig. 3a), depending on weather ferent (AEMET, unpublished; see Section 4.3 and Supplemental conditions. The temperature records obtained by the thermistors material). The extinction coefficients deduced from Fig. 4b are À1 indicate thermal homogeneity during the winter (with Tmin 2 0.2 m , which again indicate oligo-mesotrophic conditions °C in February) and initiation of stratification around April (Wetzel, 2001). (Fig. 3a), thus characterizing the lake as warm monomictic. The phytoplankton community of Lake Enol is dominated by The evolution in SpC indicates a progressive increase of solute Bacillariophyta, Chlorophyta, Dinoflagellata, Ochrophyta, Crypto- concentration in the hypolimnion during the summer (Fig. 2b) phyta and Cyanobacteria (62 and 50 different species identified along with a parallel decrease in the epilimnion. The difference in 2013 and 2014, respectively; Table S1 in Supplemental mate- in solute concentration between the two layers contributes to rial). The mean phytoplankton biomass per sampling day in 2014 the density difference and, along with the temperature gradient, was 57–312 mgCLÀ1. The profiles of phytoplankton cell density ensures stable density stratification. Important vertical gradients and biomass show a decrease from the lake surface to the 836 J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850

Fig. 2. Vertical variation of temperature (T, in °C) (a), specific conductance (SpC, in mS/cm) (b), pH (c) dissolved oxygen (DO, in %. sat.) (d), redox potential (Eh, in mV) (e), and total dissolved gas pressure (TDG, in mmHg) (f) in Lake Enol as measured in different seasons in 2014. maximum depth sampled (15 m; Fig. 4c). These profiles do not p.] = 0.64), concluded that the fecal contamination in Lake Enol reveal a clear temporal pattern except for the higher values was basically of animal origin (Meléndez-Asensio et al., 2002). observed in the upper levels in September which were due to the Iron-related and sulfate-reducing bacteria have been also found proliferation of the green alga Desmodesmus cf. grahneisii. The at all depths (Table 1). These two bacterial groups usually show a observed biomass and composition of phytoplankton species reversed vertical trend, with iron-related bacteria increasing with correspond to oligo-mesotrophic conditions (Wetzel, 2001). depth and sulfate-reducing bacteria decreasing with depth. The BARTTM tests confirmed these findings and also detected enteric bacteria at most depths, including Pseudomonads at 19 m (Table 1). 4.1.4. Bacterial ecology of the water column Abundant enteric bacteria have been detected in the lake (Table 1), especially at the lake surface, where the numbers of total 4.1.5. Carbon and nutrient dynamics À and fecal coliforms were roughly coincident (e.g., >101 cfu/100 mL Vertical profiles of TOC, DIC (as HCO3 ), nitrate and ammonium in May 2014). The analysed inflows, which drain a small area of concentration for different seasons of 2013 and 2014 are given in pasture frequently occupied by livestock, were also rich in total Fig. 5. The concentration range of important nutrients in the epil- coliforms (>101–154 cfu/100 mL) of which 10–30% corresponded imnion, hypolimnion and selected inflows is also provided in to fecal coliforms (Table 1), and included an important presence Table 2. TOC was in the order of 3–4 mg/L in 2013 and around of total enterococcus (59–64 cfu/100 mL). Cow manure is the most 1–2 mg/L in 2014 (Fig. 5a, b). The concentration peak of 8 mg/L probable source for this microbiological pollution. This hypothesis measured in July 2014 at 10 m probably resulted from decaying is supported by previous studies which, based on the ratio between phytoplankton biomass settling from the photic zone. The evolu- fecal coliforms and fecal streptococcus found in the water (fec. co tion of DIC (which almost entirely consists in dissolved bicarbonate lif. = 290 cfu/100 mL, fec. strep. = 450 cfu/100 mL, ratio [colif./stre ion) is strongly regulated by calcite solubility, which is clearly J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850 837

24 by atmospheric deposition, as demonstrated in Lake Ercina by 22 a Arnaud (2009). 1 m 20 4.1.6. Sulfate and trace metal dynamics 18 Sulfate concentration tends to decrease during the summer and 16 fall (Fig. 5d). This trend has not been observed in other ions (e.g., 14 Turnover Na, Cl, Ca; not shown) and cannot be associated to dilution or mix- 12 2À ing. The sulfate content of the inﬂows (15–17 mg/L SO4 ; not 10 Turnover shown) indicates soil leaching in the catchment. 8 Dissolved iron and manganese were strongly enriched towards

Temperature (ºC) Temperature 6 the lake bottom during the summer and autumn (Fig. 6b, c). The 4 dissolved Fe content at depth entirely consisted of Fe(II), and largely exceeded the concentrations found by García and Martínez 2 Stratification Stratification (2010) (<0.1 mg/L Fe). Most trace metals, including many toxic 0 heavy metals considered to be poisonous for aquatic biota (e.g., Cd, Co, Cr, Hg, Ni, Mo, Pb, Sb, Se, Th, Tl, U) were usually below O N D J F M A M J J A S O N D J F M A M J J A detection limits (<0.05 to <0.5 mg/L). Among those which could m m 2013 2014 2015 be quantified, Zn (8–60 g/L) and Cu (0.2–5.5 g/L), in addition to 14 Fe, were usually enriched near the lake surface and are likely being b leached from the catchment and surrounding soils (Fig. 6d, e). 12 4.2. Spatial trends in basin soils and lake sediments 10 Oxygenic Oxygenic bottom bottom 4.2.1. Soil chemistry and cow manure reactivity 8 The investigated soils in the lake catchment are shallow and stony Leptosols over limestones and compacted Regosols (<60 6 DO (mg/L) No data cm) over glacial till (Fig. 7b). These soils are slightly acidic (pH = (sensor failure) Turnover Anoxic 5.9 ± 0.7), with low salinity (EC = 150 ± 14 mS/cm), low carbonate 4 bottom Turnover (0.18 ± 0.23%, range = bdl-1.27%), and sulfate-rich (130–1419 mg/ kg S, total sulfur). Iron (58,141 ± 18,873 mg/kg) and Al (32,605 ± 2 4301 mg/kg) were the most abundant metals. The soil transects T2 and T3 showed an increase of organic matter and nitrogen 0 (up to 0.7% N ) from the distal areas towards the lake shores O N D J F M A M J J A S O N D J F M A M J J A Kjheldal (Fig. 7c), which likely reflects the higher accumulation of livestock 2013 2014 2015 around the lake. All soil profiles showed a clear enrichment in organic matter (up to 22% SOC) and phosphate (up to 1025 mg/ Fig. 3. Temporal evolution of temperature (in °C) (a) and dissolved oxygen (DO, in kg) towards the upper horizon (Fig. 7d), which are influenced by mg/L) (b) in Lake Enol during the period from September 2013 to July (T) or March cattle activity. High contents of SOC and N were also found along (DO) 2015. Water temperatures displayed in (a) were recorded by thermistors transect T1 in the heavily grazed valley bottom. The C:N ratios of installed at different depths (1, 5, 10, 15 and 19 m) and attached to a mooring line in the deepest part of the lake (see Fig. 1). The DO data shown in (b) were recorded by these soils (19–23) match those of C3 land plants (Meyers et al., a DO logger emplaced at 19 m depth in the same mooring line. The turnover and 1984). stratification periods are indicated. Microcosm experiments showed a high reactivity of cow manure with potential effects for lake water quality. Among the substrates used, cow manure was the most reactive and the one pH-controlled (Fig. S2 in Supplemental material). Thus, DIC usually inflicting the most important geochemical changes in the overlying increases with depth as a response to calcite dissolution in the water (Fig. S3). This microcosm showed the lowest Eh and pH val- colder and anoxic hypolimnion (Fig. 5c). Inorganic nitrogen was ues and the highest conductivity values in sediment pore waters, À present in the form of either nitrate (108–413 mg/L NANO3 )or indicating a higher rate of organic matter decomposition and a + ammonium (16–558 mg/L NANH4), although ammonium was only more important release of organic acids and other dissolved sub- important in the anoxic hypolimnion during the summer (Table 2; stances. Chemical analyses of overlying water at the end of the Fig. 5e, f). Nitrate concentration decreased gradually during the experiments indicated that the main substances produced by À stratification period, and total nitrogen did not differ significantly anaerobic degradation of organic matter were nitrate (NO3 ), + À with depth (150–782 and 180–914 mg/L NT in the epilimnion and ammonium (NH4), and bicarbonate ion (HCO3 )(Table S2). The hypolimnion, respectively; Table 2). Phosphate phosphorus and microcosm amended with cow manure also resulted in very high total phosphorus in the epilimnion were usually below detection concentrations of organic carbon, silica, iron and manganese. The 3À levels (<6 mg/L PAPO4 and <10 mg/L Ptotal; Table 2). However, the presence of the latter two metals implied reduction of ferric and concentration of total phosphorus in the anoxic hypolimnion at manganese oxides coupled to organic matter oxidation. the end of the stratification period can reach notably high values

(e.g., 296 mg/L PT near the sediment/water interface in November 4.2.2. Composition of suspended particulate matter 2016; Table 2). Dissolved silica was present at very low contents The SPM collected at 5 m contained abundant algae (80–90%

(0.1–0.2 mg/L SiO2 at the surface and 0.2–2.9 mg/L SiO2 at depth; by volume), while that collected at 19 m was dominated by fine- Fig. 6a). The composition of inflowing waters (440–660 mg/L grained mineral particles plus decaying biomass. The contents of À NANO3 , 0.95–1.96 mg/L TOC) confirms that nitrate and organic TOC (8.1–22.4 wt%), total nitrogen (1.1–2.7 wt% NT) and resulting carbon are being leached from the surrounding soils and pastures [C/N]molar ratios (8.6–9.7) of the shallow SPM (Table 3) were diag- and enter the lake during rainstorms. In addition, some nitrogen nostic of lacustrine algae (Meyers and Ishiwatari, 1993). The car- (and phosphorus, in a minor extent) is probably entering the lake bon and nitrogen isotopic compositions of these sediments (d13C 838 J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850

Chl-a (µg/L) PAR (%) Phytoplankton biomass (µg C L-1) 0 0.5 1 1.5 2 2.5 0 10 20 30 40 50 60 70 80 90100 0 200 400 600 800 0 0 0

2 a 2 b 2 c

4 4 4

6 Metalimnetic peak 6 s.d. 6

8 8 ɳ~0.2 8

10 10 10 Depth (m) Depth (m) Depth 12 (m) Depth 12 12 Hypolimnetic peak May 2013 14 14 14 May 2014 Jul 2013 July 2014 Jul 2014 Sep 2014 16 16 16 Sept 2014 Nov 2014 18 18 18

20 20 20

Fig. 4. Vertical variation of chlorophyll-a concentration (Chl-a, in mg/L) (a), photosynthetically active radiation (PAR, in % with respect to incoming radiation at the surface; ɳ = Light extinction coefﬁcient; s.d., Secchi depth) (b), and phytoplanktonic biomass (in mgCLÀ1) (c) in Lake Enol in different seasons of 2013 and 2014.

Table 1 Total coliforms (TC), fecal coliforms (FC), total enterococcus (TE), Iron-related bacteria (IRB) and sulfate-reducing bacteria (SRB) found in Lake Enol at different depths (May 2014).

Depth TC FC TE IRB SRB(3) (m) (cfu/100 mL) (cfu/100 mL) (cfu/100 mL) (cfu/mL) (cfu/mL)

0 >101 >101 <1 3.5 Â 104 (1) 2.7 Â 104 5 n.a. n.a. n.a. 3.5–14 Â 104 (1) 75 10 n.a. n.a. n.a. 3.5–14 Â 104 1 15 n.a. n.a. n.a. 5.7 Â 105 (1) 1 19 56 <1 <1 5.7 Â 105 (2) 5 I-1 >101 10 59 n.a. n.a. I-2 >154 54 64 n.a. n.a.

Abbreviations: I-1, Inﬂow-1; I-2, Inﬂow-2 (see Fig. 1); n.a., not analysed; cfu, colony-forming units. (1) Detection of enteric bacteria; (2) Detection of Pseudomonads ssp.; (3) Including aerobic and anaerobic SRB.

= À28.5 to À31.6‰, d15N = 4.1–5.5‰) also matched those of lake 4.2.3. Biogeochemistry of bottom sediments algae (Table 3; Fig. 8). However, the high C:N:P relation A clear chemical and microbial stratification was observed in (190:18:1–580:56:1; not shown) deviates notably from the Red- the studied sediment cores (Table 5). All cores exhibited a clear field ratio (106:16:1) and suggests a significant contribution of enrichment in Fe and Mn in the upper 2 cm, indicating accumula- plant-derived, terrestrial organic matter to the lake. The SPM col- tion of freshly formed iron and manganese oxides/oxyhydroxides lected in the deeper trap consisted in detrital quartz, illite- by oxidative precipitation of Fe(II) and Mn(II/III) during re- muscovite and calcite (Table 4), but a significant content of Fe oxygenation of the anoxic hypolimnion after the fall overturn. (4.2–5.4 wt%) and Mn (0.1–2.0 wt%) suggested the presence of Besides, IRB capable of Fe(II) oxidation and/or Fe(III) reduction amorphous and/or colloidal iron and manganese oxides. were remarkably more abundant in the upper cm, indicating a Mean annual sedimentation rates of 12 mm yearÀ1 have been preferential iron cycling where more bioavailable iron exists. IRB calculated. These values are considerably higher than those were far more abundant in the deeper sediments where iron is calculated by radiometric methods like 210Pb and 239À240Pu more abundant. SRB could only be detected near the sediment/ (López-Merino et al., 2011; Casas et al., 2015) which yielded aver- water interface in the shallow sediments, being present at much age values 1.9 mm yearÀ1 for the period 1800–2007 CE. In terms higher concentrations than the IRB (Table 5). In the deep sedi- of sediment mass, however, the annual rates of sediment accumu- ments, SRB were concentrated in a narrow layer at 1 cm below lation (820–900 g mÀ2 yrÀ1) match those deduced by López- the water/sediment interface (Table 3). The TOC content was very Merino et al. (2011) for the last decades (1980–2007 CE). The similar in the shallow and deep sediments, and in both cases disparity between present-day and historical sedimentation rates increased towards the sediment/water interface (Table 5). This ver- is likely due to compaction and early diagenesis. Considering the tical trend points to an increase in the organic matter loadings to chemical composition of these sediments (Tables 3 and 4), the the lake in modern times as a response to (i) enhanced lake pro- reported sediment fluxes account for annual inputs of 57–93 g C ductivity, (ii) increased allochthonous input, or (iii) a combination mÀ2 yrÀ1, 7–12 g N mÀ2 yrÀ1 and 0.8–0.9 g P mÀ2 yrÀ1, in addition of both. Given the recent nature of these uppermost sediments to 34–48 g Fe mÀ2 yrÀ1 and 2–18 g Mn mÀ2 yrÀ1 to the lake basin. (around 25 years for sediments at 5 cm below the sediment/water Despite a significant contribution of terrestrial organic matter to interface, according to Casas et al., 2015), artifacts introduced by the SPM, the carbon annual input is still characteristic of diagenetic shifts (e.g., Meyers & Ishiwatari, 1993) are considered oligotrophic conditions (4–180 g mÀ2 yrÀ1; Wetzel, 2001). to be negligible. J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850 839

- TOC (mg/L) TOC (mg/L) HCO3 (mg/L) 1 2 3 4 5 6 0 2 4 6 8 70 80 90 100 110 120 130 0 a 2 b c

6 may-13 8 jul-13 10 sep-13

Depth (m) 12 nov-13

14 May 2014 16 July 2014 Sep 2014 18 Nov 2014

+ - 2- NH4 (µg/L) NO3 (mg/L) SO4 (mg/L) 0 200 400 600 800 0.0 0.5 1.0 1.5 2.0 0 5 10 15 0 d e 2 f

6 jul-13 summer/fall 8 sep-13 Summer/fall trend trend 10

Depth (m) 12

Fig. 5. Vertical evolution of the concentration of major ions in Lake Enol as measured in four different seasons (May, July, September and November) in 2013 (only TOC, a) and À + À 2014 (rest of parameters, b–f): (a), (b) total organic carbon (TOC, in mg/L), (c) total dissolved bicarbonate (HCO3 , in mg/L), (d) ammonium (NH4,inmg/L), (e) nitrate (NO3 ,in 2À mg/L), and (f) total dissolved sulfate (SO4 , in mg/L).

Table 2 Nutrient concentrations (minimum and maximum values) in Enol Lake (period 2013–2016).

À + 3À Depth TIC TOC NANO3 NANH4 Ntotal PAPO4 Ptotal Chl-a SiO2 mg/L mg/L lg/L lg/L lg/L lg/L lg/L lg/L mg/L Epilimnion Min 17.50 1.72 145 16 150 <6 <10 0.44 0.1 Max 24.80 3.84 413 35 782 <6 <10 0.55 0.2 Hypolimnion Min 19.70 1.13 108 <35 180 <6 <10 0.96 0.2 Max 30.67 3.96 315 558 914 <6 296 1.80 2.9 Inﬂows (n = 2) Min 40.72 0.95 440 <35 – <16 – – 1.5 Max 42.30 1.96 660 <35 – <16 – – 1.7

The plot of d13C vs. [C/N] for the lake sediments shows a clear d13C composition with increasing proximity to the shores. Hence, trend of the organic matter composition across the lake basin, with it can be concluded that the organic matter accumulated in the an increasing terrestrial inﬂuence towards the lake shores (Fig. 8). central part of the lake mostly derives from primary productivity, The bottom sediments at the deepest point of the lake (21 m) show whereas the shallow sediments show a higher proportion of C/N ratios (7 À9) and carbon isotopic compositions (d13C=À31 to organic matter derived from surrounding soils and terrestrial À29‰) which are very similar to those found in SPM and match plants. The few points of shallow (4 m) sediments plotting in the values typically found in lake algae (Meyers et al., 1984; Fig. 8). ﬁeld typical of lacustrine algae correspond to areas colonized by The sediments of shallow areas show increasing C/N ratios and benthic macroalgae (Charophyceae). 840 J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850

SiO2 (mg/L) Fe (μg/L) Mn (μg/L) 0 1 2 3 4 5 0 500 1000 1500 2000 2500 0 500 1000 1500 0 2 a b c 4 Mayo-14May 2014 6 Julio-14July 2014 sep-14Sep 2014 8 nov-14Nov 2014 10 12 Depth (m) 14 16 Summer/fall trend Summer/fall trend 18 20 Zn (μg/L) Cu (μg/L) Ba (μg/L) 0 10 20 30 40 50 60 0 1 2 3 4 5 6 0 5 10 15 0 2 d e f 4 6 8 10 12 Depth (m) 14 16 18 20

Fig. 6. Vertical variation of dissolved silica (a) and trace metal (Fe, Mn, Zn, Cu, Ba) (b–f) concentration in Lake Enol during different seasons in 2014.

4.3. Inter-annual patterns of lake stratification the thermal stratification and may advance the beginning of the anoxic period. This is well illustrated in Fig. S5 (Supplemen- The climatic fluctuations observed during the study period tal material). A warmer winter period in 2014 with respect to had an immediate impact on the lake dynamics and stratifica- 2013 resulted in a warmer epilimnion in May (Fig. S5a). Two tion. The temperature of the epilimnion at the end of the sum- months later, and despite the thermal stratification in 2014 mer period (September) was up to 3 °C higher in years with was less pronounced than in 2013 (Fig. S5b), the thickness of warmer and drier summers (e.g., 2014, Tepi =19°C) with respect the anoxic layer was much more important (4 m in 2014 vs. to years with colder or more humid summers (e.g., 2010, Tepi = 2 m in 2013; Fig. S5c). As discussed below, this inter-annual 16 °C; Fig. 9a). Besides, the stratification pattern and physical variability of oxygen distribution in the lake is directly related structure of the lake at the beginning of November was also with oscillations of the temperature-driven stratification/mixing highly dependent on climatic conditions. Years in which the regime (Fig. 9d), which is in turn affected by local climatic water column was completely homogenized as a result of factors. P colder and more humid climate (e.g., 2013) alternate with Anoxic factors (AF = [ tiai/A0], where t is the number of days others in which the water column was still strongly stratified of anoxia, ai is the hypolimnetic sediment area covered by after a warmer and drier autumn (e.g., 2014 or 2016; Fig. 9d– anoxic water and A0 is the lake surface area; Nürnberg, 1995) f). Differences in the vertical evolution of other parameters of 17–53 daysÁ were calculated for the period 2013–2016 measured in the lake (SpC, pH, TDG) during the study period (Table 6). According to these AF, Lake Enol would be classified are also shown in the Supplemental material (Fig. S4). Such as mesotrophic (20–40 days) to eutrophic (40–60 days) variability of the lake stratification has direct implications in (Nürnberg, 2004). The area of sediment surface overlaid by the oxygen dynamics within the lake. This is especially relevant anoxic water (Anoxic Surface Area, ASA) ranged from a lowest as regards to the depth of the oxic/anoxic boundary in late value of 6671 m2 (5% of the total lake surface area) measured summer (September), and especially, in autumn (October- in July 2014 to the maximum value of 40,865 m2 (31%) in November), which displayed differences between years September 2015 (Table 6; Fig. 10c, and Fig. S6 in Supplemental (Fig. 9b, e). The oxic/anoxic boundary at the end of the summer material). The corresponding anoxic hypolimnetic volume was at depths around 15 m in 2010 and 2013, but the anoxic (AHV) represented 15.3% of the lake volume in September front was at only 11 m depth in 2015 (Fig. 9b). This variability 2015. The relative areal oxygen deficit (RAOD, hypolimnetic is even larger when comparing different autumn periods oxygen depletion rate relative to the lake surface during the (Fig. 9e): the anoxic layer reached depths of 15–16 m in 2010 stratification period; Hutchinson, 1957; Cornett and Rigler, À2 À1 and 2014, 13 m in 2016, while the lake was fully oxygenated 1980) ranged between 0.15 and 0.26 g O2 m day (Table 6), in 2013. The climatic variability can also affect the onset of which indicates mesotrophic conditions. J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850 841

Distal T3 Lake bSoil transects a T1-TT N b N Lake Enol kjheldahl (%) SOC (%) SOC SOM (%) c

Fig. 7. (a) Aereal view of Lake Enol with indication of soil transects (T1-TT) carried out in this study; (b) Picture of a typical soil profile in T1; (c) geochemical transect T3 showing the spatial variation of soil organic carbon (SOC), soil organic matter (SOM), and nitrogen (Kjheldahl) in the top 10 cm of the soils along the eastern slope (see enrichment in both carbon and nitrogen in the close proximity of the lake); (d) Vertical profile of total phosphorus (P), total sulfur (S), and soil organic carbon (SOC) in a typical soil profile of T1 transect.

Table 3 Carbon and nitrogen isotopic composition of suspended particulate matter (SPM) sampled at depths of 5 and 19 m in Lake Enol during 2014.

13 15 Sample d CPDB TOC d Nair NT [C/N]m ‰ % ‰ % SPM sampled at 5 m May À31.0 8.5 4.9 1.2 8.6 July À28.5 14.7 5.4 1.8 9.3 September À30.5 8.1 5.0 1.1 8.9 November À29.1 22.4 5.1 2.7 9.6 SPM sampled at 19 m May À31.5 10.1 4.1 1.3 9.3 July À31.6 11.3 4.4 1.4 9.1 September À31.1 7.5 4.4 1.0 8.6 November À30.3 7.0 5.5 0.9 9.3

5. Discussion in surface lake waters and runoff entering the lake (Table 1) represents an important degree of bacterial pollution and, in the absence 5.1. Biogeochemical trends and proxies of anthropogenic pressure of waste water discharge, is directly ascribed to the presence of cattle. The influx of these bacteria is both direct (deposition of A schematic diagram illustrating the major fluxes of biogeo- feces in water) and indirect (leaching of soils) (Fig. 10a). chemically important elements in the lake is given in Fig. 10a. During the stratification period, microbial respiration rates lar- Leaching, erosion and transport of basin soils represents an impor- gely exceed the diffusion rate of dissolved oxygen from upper tant input of organic carbon, nitrogen and sulfate, which enter the levels, and a strong O2 depletion occurs soon after the formation lake mostly by runoff. Additional nitrogen and phosphorus is likely of the thermocline. Even though the primary production of entering the lake by atmospheric deposition, though the latter autochthonous carbon in the phototrophic zone is still low and pathway has not been evaluated. The detection of fecal coliforms typical of oligo-mesotrophic conditions, the combination of 842 J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850

a geogenic source (e.g., mining), the abundance of this element in -23 Lacustrine the lake and surrounding soils is assumed to be related to traffic algae of vehicles and atmospheric deposition, as discussed below. -25 Shallow sediments However, the drop of nitrate and sulfate might have been also Increasing terrestrial influence C3 influenced by microbial consumption by algae (nitrate) and bacte- -27 land plants ria (nitrate and sulfate). The redox conditions are compatible with sulfate reduction, and this process may take place in the first cm of the sediments. Apparent sulfate reduction daily rates around 0.5 ‰ ) -29 ( 4 m À3 À1

C nmol cm d have been deduced from Fig. 5d, which are in line

13 12 m

δ with those found in eutrophic lakes (e.g., Ingvorsen et al., 1981). 21 m -31 The bacterial sulfate reduction activity does not result, however, SPM in the accumulation of H2S, since this biological byproduct reacts -33 Deep sediments with metals (Fe(II), Mn(II)) to form insoluble sulfides (Fig. 10b). Autochthonous producon Ferrous iron, manganese and ammonia released from the sediments are vertically diffused towards the oxycline, where these -35 0 5 10 15 20 25 30 substances may be re-oxidized. The enrichment of SRB in the lake C/N surface suggests the presence of species able to survive under aerobic conditions (Dilling & Cypionka, 1990; Jonkers et al., 2005). Fig. 8. Carbon isotopic composition (d13C) vs. C/N molar ratio of sediments taken With respect to nitrate-respiring microorganisms, various species from different depths, including SPM and sediment cores from depths of 4, 12 and of Pseudomonads (detected in the water column) are able to carry 21 m (upper 10 cm; Fig. 1b). Fields for lacustrine algae and land plants after Meyers out denitrification and dissimilatory nitrate reduction under et al. (1984). anoxic conditions, in addition to iron reduction (Aguado et al., 2012). autochthonous and allochthonous inputs provide sufficient fresh The lake had been previously classified as oligotrophic (Pons & Niembro, 1986; García & Martínez, 2010; Moreno et al., 2011), organic carbon to consume the available hypolimnetic O2 by het- erotrophic bacterial metabolisms. This situation leads to a contin- though the higher maximum concentrations of nitrate nitrogen, uous increase of sediment area covered by anoxic water during the total nitrogen and total phosphorus found in this work with stratification period (Fig. 10c and S6). In the anoxic bottom, miner- respect to previous studies (Fig. S7) suggest that the lake is likely alization of organic carbon takes place via iron and manganese at an early stage of eutrophication. Despite the biological produc- reduction, both in the sediments and in the water column tivity of the lake is still at moderate levels, it may have increased (Fig. 10b). This combined biogeochemical cycling of carbon, iron with respect to pristine (strictly oligotrophic) conditions due to and manganese, provokes the progressive increase of dissolved an increased input of nutrients from the catchment. This conclu- solids (Fe(II), Mn(II)/(III)) and dissolved carbon dioxide, which are sion is supported by recent studies of sediment cores (e.g., reflected in corresponding increases of SpC and TDG, respectively. López-Merino et al., 2011; Ortiz et al., 2016), which have reported These parameters are thus related to the intensity of anaerobic a significant increase in phytoplankton productivity and microbial microbial respiration taking place in the sediments, and their mea- activity in the upper cm of the lake sediments corresponding to the surement in deep waters during the autumn provides an estimate last three decades (ca. 1980–2013 CE). The only limiting factor pre- of the duration and severity of hypolimnetic anoxia. The extent of venting Lake Enol from conspicuous algal blooms in the summer anaerobic microbial respiration coupled to iron cycling in the sed- time seems to be the scarcity of phosphorus in the productive zone. iments (as deduced from the high Fe(II) concentrations found at The concentration of bioavailable phosphorus is probably being depth) is much more important than what has been considered limited by several factors. Firstly, the shallow sediments of Lake to date. This is probably related to the longer duration of hypolim- Enol are densely colonized by charophytes, which have a high netic anoxia in this lake in recent years, as discussed below. potential for phosphorus uptake and can outcompete planktonic The decrease of nitrate and sulfate concentration in the water algae (Kufel and Ozimek, 1994). Secondly, the formation of differ- column during the summer and early autumn (Fig. 5d, e), as well ent mineral phases may also be sequestering this nutrient at sub- as the drop of metal content in the epilimnion during the same stantial extent. Phosphate may coprecipitate with calcite (Murphy period, likely denotes a decline of inputs from the catchment due et al., 1983), a mineral that has been frequently observed in sus- to diminished rainfall and runoff. Among the trace metals, zinc is pended and bottom sediments (Table 4), and geochemical calcula- specially enriched during the rainy period and its maximum con- tions (e.g., saturation index, defined as SI = log [IAP/Ksp], where IAP centration in surface water (60 mg/L Zn; Fig. 6d) largely surpasses is the ion activity product and Ksp is the solubility product con- the average concentrations found in surface natural waters and stant; data not shown) indicate solubility saturation with respect lakes of the world (1.5–12.9 mg/L Zn; Wetzel, 2001). Zinc concen- to certain phosphate minerals. Besides, SEM-EDS analyses of Fe tration, therefore, appears to be a good indicator of environmental (III) and Mn oxy-hydroxides formed in the lake during the winter pollution and anthropogenic activity in this lake. In the absence of overturn (which yielded 0.3 wt% P on average and up to 3.5 wt%

Table 4 Metal content (measured by ICP-AES) and dominant mineralogy (XRD) of suspended particulate matter collected in the deep sediment trap (19 m) in Enol Lake in four different periods (Nov 2013, May 2014, July 2014, and Sept 2014).

Al Ca Fe K Mg Mn P Mineralogy (% wt) (% wt) (% wt) (% wt) (% wt) (% wt) (% wt) (XRD) 6.30 2.43 5.37 2.11 0.42 0.56 0.10 Qtz, Mc, Cc 6.30 4.10 4.58 2.02 0.39 0.38 0.09 Qtz, Mc, Cc 5.94 1.11 4.88 1.85 0.37 1.97 0.11 Qtz, Mc, Cc 6.18 1.64 4.17 1.90 0.38 0.08 0.11 Qtz, Mc, Cc

Abbreviations: Qtz, quartz; Mc, muscovite; Cc, calcite. J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850 843

Table 5 Biogeochemical parameters measured in the topmost ﬁve cm of two sediment cores from Lake Enol.

Depth TOC Fe2O3 MnO IRB SRB (cm) (wt.%) (wt.%) (wt.%) (cfu/mL) (cfu/mL) Shallow sediment (10 m) 0 6.5 3.5 2.4 9000 28,000 1 5.8 3.4 1.8 5000 28,000 2 5.0 3.0 1.1 2000 n.d. 3 4.6 1.0 0.4 2000 n.d. 4 4.2 0.9 0.2 2000 n.d. 5 4.1 0.5 0.1 n.d. n.d. Deep sediment (21 m) 0 6.0 6.0 5.5 140,000 n.d. 1 5.4 5.5 5.0 70,000 25,000 2 4.5 3.8 2.5 10,000 25,000 3 3.7 3.7 2.0 10,000 n.d. 4 3.4 3.6 1.0 n.d. n.d. 5 3.3 3.5 0.9 n.d. n.d.

Abbreviations: TOC, total organic carbon; IRB, Iron-related bacteria; SRB, sulfate-reducing bacteria; n.d., not detected.

T (⁰C) DO (%) Eh (mV) 4 6 8 10 12 14 16 18 20 0 25 50 75 100 125 150 0 100 200 300 400 0 a b c 2

Depth (m) 12

14 Sep 2010 16 sep-13Sep 2013 18 sep-14Sep 2014 sep-15Sep 2015 20

T (ºC) DO (%) Eh (mV) 6 8 10 12 14 0 20 40 60 80 100 0 100 200 300 400 500 0 d 2 e f

6 Oct 2010 nov-13Nov 2013 8 nov-14Nov 2014 10 nov-16Nov 2016

Fig. 9. Inter-annual variation of temperature (T, in °C), dissolved oxygen (DO, in %. sat.) and redox potential (Eh, in mV) in Lake Enol between different years: Comparison of late summer (2013, 2014 and 2015) (a–c) and mid autumn (2013, 2014 and 2016) (d–f). Additional data for 2010 in (a, b) and (d, e) taken from (García & Martínez, 2010). 844 J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850

Table 6 Different indicators of anoxia calculated for Lake Enol between 2013 and 2016.

Date Anox. depth(1) ASA(2) AHV(3) RAOD(4) m % % May 2014 – 0 0 – July 2014 18 5.11 0.90 0.15 Nov 2014 16 13.10 3.67 – Sept 2013 15 14.62 4.28 0.18 Sept 2014 13 22.80 8.84 0.21 Sept 2015 11 31.30 15.35 0.26 Nov 2016 13 22.80 8.84 0.21

(5) Anoxic Factor (AF) =[Rtiai/A0] AF Summer 2013 = 17 days AF Summer 2014 = 33 days AF Summer 2015 = 46 days AF Summer 2016 = 53 days

(1) (2) Reference depth for the oxic/anoxic boundary (in meters below the lake surface), considering anoxia as <0.1 mg/L dissolved O2; Anoxic surface area (% with respect to 2 (3) 3 (4) À2 À1 (5) total lake area of 133,000 m ); Anoxic hypolimnetic volume (% with respect to total lake volume of 1.06 Mm ); Relative areal oxygen deﬁcit (in g O2 m day ); Where t is the period of anoxia (in days), a is the hypolimnetic area and A0 is the lake surface area (after Nürnberg, 1995).

Atmospheric Sediment input deposition (N, P) Sediment input C, N, P, S, bacteria 2- - - SO4 O2 HCO3 NO3 Phytoplankton Diffusion Re-oxidation Cycling Oxic Precipitation Fe2+ Mn2+ CO2 Settling + Anoxic NH4 Fe2+ Mn2+ Organic matter a Chemical sediment

Sediments (C, N, P, Fe, Mn) P Dead algal Oxic CO Biomass 2 Fe2+ (C, N, P) 2+ Mn Anoxic + NH4

SH 100 m 2 FeS2 June (ASA=3-5%) July (ASA=5-10%) IRB Nov (ASA=15-30%) b c Sept (ASA=30-35%)

Fig. 10. Schematic drawing representing the dynamics of several important substances in Lake Enol during a typical stratiﬁcation period; (b) Detail of metal and nutrient exchange between bottom sediments and anoxic deep waters of the hypolimnion (IRB, Iron-related bacteria; SRB, Sulfate-reducing bacteria) (not to scale); (c) Temporal evolution of hypolimnetic anoxia during a stratiﬁcation period (June to November), with estimation of the anoxic surface area (ASA) in % with respect to the total lake surface area.

P; not shown) suggest that phosphate adsorption on these minerals 5.2. Influence of local (basin-scale) factors on hypolimnetic anoxia represents an important P-sequestering process. The high content of total phosphorus observed in the anoxic hypolimnion at the Oxygen depletion in lakes and reservoirs is a widespread and end of the stratification period (Table 2) suggests a release of this globally important environmental problem (e.g., Charlton, 1980; element from the sediments by desorption and/or redissolution Gordon, 1993; Diaz, 2001). Studies by Livingstone and Imboden processes. (1996), Rippey and McSorley (2009),orMüller et al. (2012) have J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850 845 helped improve the understanding of oxygen depletion processes addressed an increase of essential nutrients (nitrogen and phos- in lakes. Hypoxia and anoxia usually occur as a response to phate) in mountain lakes by direct deposition of feces and urine increased nutrients (eutrophication) or allochthonous inputs of (e.g., Ruggiero et al., 2004; Carpenter et al., 2005; Declerck et al., organic matter (e.g., dystrophic lakes). 2006; Butler et al., 2008). These changes may lead to strong The long periods of hypolimnetic anoxia observed in Lake Enol increases of the biological oxygen demand (BOD) and consequently (Figs. 2 and 3; see also Fig. S6 in the Supplemental material) are not in sharp decreases of the available dissolved oxygen in lake waters typical in mountain lakes of the Iberian Peninsula, which usually (e.g., Hendry et al., 2006). Other deleterious effects derived from show either short anoxic periods or no anoxia at all (e.g., fertilization are related with an increase in micronutrients, which Morales-Baquero et al., 1992, Toro et al., 2006, Velasco & Álvarez, may also lead to algal blooms (e.g., Downs et al., 2008), or with 2000). The strong oxygen depletion occurring in this lake during negative impacts on invertebrates (e.g., Schmutzer et al., 2008). the summer and autumn, as quantified by anoxia-related parame- Direct trampling by cattle in lake shores may also cause important ters (AF, ASA, AHV, RAOD; Table 6) is usually characteristic of remobilization of sediment substrate and benthic biomass (Toro eutrophic lakes (Wetzel, 2001), being apparently contradictory et al., 2006). with most other biogeochemical parameters measured in this A number of evidences obtained in the present study (e.g., study (e.g., low phosphorus and chlorophyll concentration, low widespread observation of abundant cattle in lake waters and phytoplanktonic biomass, low annual input of organic carbon) shores, heavily remobilized soils and sediments in cattle- which indicate oligo-mesotrophy. However, the results obtained impacted areas, detection of fecal bacterial species in the water suggest that the hypolimnetic anoxia of Lake Enol is increasing in column, significant ammonium concentrations in the hypolim- extension and duration over the last years (Fig. S7 in Supplemental nion; Figs. S1a-b and S3; Tables 1–2 and S2) suggest that the pres- material): strict anoxia did not exist in the summer of 1986 (2.3– ence of livestock is negatively affecting the water quality and 3.7 mg/L DO at the lake bottom; Pons & Niembro, 1986), it affected oxygen consumption in Lake Enol through different processes: (i) the deepest 5–6 m of the lake in the same period of 2010 (García & firstly, the intense remobilization of lake shore sediments and Martínez, 2010), and presently affects a half of the water column adjacent soils by trampling is increasing significantly the natural (e.g., 11 m in 2016). This trend draws a worrying scenario for the erosion rate, which indirectly enhances oxygen consumption ecological state of the lake in the future. The intensification of through the input of allochthonous sediments rich in organic mat- hypolimnetic anoxia seems to be partly provoked by the aforemen- ter and nutrients; (ii) secondly, the deposition of cow manure and tioned increased input of organic carbon, which has been recog- urine in soils and lake shores represents an important and direct nized as a major factor causing strong oxygen depletion in lakes input of organic carbon and bioavailable nitrogen to the lake; and reservoirs worldwide (e.g., Jansson et al., 2003; Marcé et al., and (iii) thirdly, the enteric bacteria present in cow feces are being 2008). In the case of Lake Enol, the strong oxygen depletion permanently transported to the water column and sediments, appears to result from the combination of several factors related altering somehow the original microbial ecology and the natural with the basin management. These factors (summarized in rates of microbial respiration in the lake. Table S3; Supplemental material) are discussed below. In relation to the latter aspect, cow manure has been shown to be highly reactive under aqueous conditions and these depositions have the capacity to create strongly reducing microenvironments, 5.2.1. Cattle grazing release soluble organic compounds and ammonium nitrogen, and An important threat for the ecological preservation of lakes catalyze the reduction of iron and manganese minerals in the sed- worldwide derives from cattle activity (e.g., Hendry et al., 2006; iments. This has been confirmed by the microcosm experiments Declerck et al., 2006; Butler et al., 2008). The combined effects of (e.g., Table S2 and Fig. S3 in Suppl. material). This chemical reactiv- intense treading of adjacent soils (i.e., enhanced input of sediment ity is not only derived from the labile nature of the organic com- loads) and increased input of nutrients (via direct and indirect pounds present in cow feces, but is also related with the high transport of organic matter, nitrogen and phosphorus to the lake) concentration and metabolic activity of anaerobic bacteria which may inflict severe deterioration on the lake ecology and water are usually present in cow manure and which are efficient decom- quality. There is, however, a considerable scarcity of studies deal- posers (e.g., Willey et al., 2016). Ortiz et al. (2016) have recently ing with the effects of livestock activity on lake ecosystems, and detected fecal stanols (e.g., 24-ethylcoprostanol from herbivores) the ultimate processes leading to water quality and ecosystem in sediments of Lake Enol deposited since the 17th century, with deterioration are still unclear. A summary of selected studies deal- an increased concentration during the last 20 years. Using the co ing with cattle grazing-derived effects on lake ecosystems is given prostanol/24-ethylcoprostanol ratio as a proxy for distinguishing in Table 7. As deduced from this table, the relation between cattle human vs. herbivore fecal pollution, these authors concluded that activity and lake ecosystems is controversial and often difficult to the main source of fecal material was derived from cattle. Although predict due to the existence of many controlling factors, including the lack of precise data on numbers and distribution of cows in the size and type of livestock, geology, topography, hydrology and soil lake area does not allow quantification of the cattle-derived com- type, or size and depth of the affected lake. Some researchers have ponent on oxygen consumption, the increased cattle activity seen no effects on lake ecology or water quality which could be around the lake in recent times is evidenced in the water column directly attributed to cattle activity (e.g., Steinman et al., 2003; and lake sediments, and appears to be a major factor behind the Hundey et al., 2014) while others have even reported benefits of increased hypolimnetic anoxia in Lake Enol. Nitrogen in manure traditional livestock activity for the preservation of lake ecosys- occurs mainly in organic forms (e.g., proteins) and as ammonium + tems (e.g., Marty, 2005). However, the effects of agriculture and (NH4) which can then be converted to nitrate by water and/or sed- cattle activity on water quality have been well established in dif- iment microbes (Pettygrove et al., 2009). Thus, in addition of ferent lakes (e.g., Clausen and Meals, 1989; Hall et al., 1999). Graz- releasing labile organic compounds upon degradation in the lake ing can modify the environment in a number of ways, including: (i) sediments, cow manure may also contribute indirectly to increase alteration of species composition, (ii) disruption of nutrient oxygen consumption through fertilization and enhanced primary cycling, (iii) reduction of litter cover, (iv) compaction of soils, (v) production. Considering the abundance of cow manure in the lake, reduction of infiltration, (vi) increase of runoff, and (vii) increase and the high reactivity of these wastes as compared to other of soil erosion, and through all these mechanisms, a resulting organic substrates (e.g., grass, macrophytes, characeans), an impor- increase of production in aquatic systems. Different studies have tant portion of organic matter and nitrogen (also phosphorus in a 846 J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850

Table 7 Literature review of selected studies dealing with the effects of cattle grazing and climate change on lake ecosystems.

Study Findings Studies dealing with effects of cattle grazing Steinman et al. Study of wetlands in Florida, USA. Cattle stocking density had few significant effects on water-column nutrient concentration or invertebrate (2003) community structure Ruggiero et al. Limnological study of an Apennine shallow lake, covering a period of 4 years. Cattle grazing provoked sharp increases in nutrient concentration (2004) and phytoplankton biomass, as well as deterioration of lake community structure Marty (2005) Study of vernal pools in Central California. Livestock does not necessarily damage native communities. Grazing helps maintain native plant and aquatic diversity Carpenter et al. Review of non-point pollution of surface waters in the United States. High density of livestock cause an exceeding manure production and (2005) nutrient (phosphorus, nitrogen) flows to aquatic ecosystems Declerck et al. Study of 126 ponds in Belgium. Trampling by cattle seriously affects water turbidity (2006) Hendry et al. (2006) Detailed water quality investigations over a period of 28 years in Tamar Lakes (UK). The source of nutrient enrichment that fuels the algal blooms corresponds with a substantial increase in livestock farming. Increase of organic contaminants (ammonia), biochemical oxygen demand and suspended solids during rainfall events Toro et al. (2006) Revision of major limnological characteristics of high mountain lakes in the Spanish Central Range. Discussion of the effects produced by tourism, cattle, lake damming, wastewater inflow, watershed erosion, or environmental warming Schmutzer et al. Study of 7 wetlands in Tennessee, USA. Cattle farming may have direct or indirect negative effects on larval amphibians by decreasing water (2008) quality through deposition of nitrogenous waste. They recommend excluding livestock from aquatic environments Downs et al. (2008) Study of two New Zealand lakes with literature review. Micronutrient enrichment (e.g., B, Co, Cu, Mo; commonly applied as agricultural fertiliser in the catchment of lakes) can significantly increase phytoplankton productivity and eutrophication in lakes. No evident effects of N and P increases Butler et al. (2008) Study of manured riparian grasslands with contrasting drainage and simulated grazing pressure. Results indicate that livestock heavy-use areas in the riparian zone may export substantial suspended solids and nutrients, especially on poorly drained soils Hundey et al. Multi-proxy study from lake sediment records of six remote alpine lakes in the Uinta Mountains, Utah, USA. Local free-range grazing is not (2014) directly responsible for changes in primary production. Enhanced atmospheric deposition of nitrogen and phosphorus may explain the recent and more prominent increase in production This study (2017) Study of a temperate mountain lake in NW Spain with intensive cattle grazing over a period of four years. Direct negative impact on lake water quality and oxygen consumption rate due to the combination of different processes (enhanced erosion of lake shores by trampling, direct deposition of feces and urine, both causing import of carbon and nutrients). Importance of alterations of the natural lake metabolism due to bacterial pollution derived from manure. High reactivity of feces under aqueous conditions Studies dealing with effects of climate change Verburg et al. Evolution of Lake Tanganika in the past century. A sharpened density gradient has slowed vertical mixing and reduced primary production. (2003) Increased warming rates during the coming century may continue to slow mixing and further reduce productivity in deep tropical lakes Parker et al. (2008) Long-term study of 4 lakes in Canadian Rockies. Alpine lakes highly sensitive to climatic changes, which affect to lake physics, chemistry and biology. Forecasts of increased climatic variability in the future pose serious treats for the biodiversity and ecosystem of high-elevation lakes Vincent (2009) Revision on the effects of climate change on lakes. Climate change affects lakes through many different physical, chemical and biological processes. Some effects impact directly on the water bodies, while others are indirect and affect firstly to the basin Foley et al. (2012) Impact of climate change in a small lake in the UK over a long time period (1968–2008). The establishment of thermal stratification strongly related to the onset of DO depletion in the lower hypolimnion. A progressively earlier onset of stratification and later overturn increases the duration of stratification. The negative effects of eutrophication on hypolimnetic DO are likely to be exacerbated in the future by changes in lake thermal structure brought about by a warming climate Sahoo et al. (2013) Numerical model to predict mixing conditions in the future in Lake Tahoe, USA. Deep mixing will progressively increase in the future due to global warming. When the lake fails to completely mix, the bottom waters will not be replenished with dissolved oxygen and will turn anoxic. The results indicate that continued climate changes can pose serious threats to the lake ecology North et al. (2014) 39 year-study of temperature, dissolved oxygen and phosphorus concentration profiles in Lake Zurich. The increase in hypoxia during the study period was not a consequence of a change in trophic status (oligotrophic), but directly related with changes in the lake’s mixing regime, which caused an abrupt decrease in deep-water oxygen concentration and an expansion of the hypoxic zone in autumn O’Reilly et al. Extensive study of 235 lakes worldwide over a period of 25 years (1985–2009). Surface water warming rates are dependent on combinations of (2015) climate and local characteristics. The most rapidly warming lakes are widely geographically distributed. Ice-free lakes tend to experience increases in air temperature and solar radiation Schwefel et al. Comparison of observational findings and modeling on the effects of climate change on deepwater oxygen and winter mixing in Lake Geneva. (2016) Simulations predict a decrease in the maximum winter mixing depth and hypolimnetic oxygen concentration in response to predicted increase of atmospheric temperatures. The results demonstrate that changes in deep mixing can have stronger impact than eutrophication on the deepwater oxygen levels of oligomictic lakes Snortheim et al. Study of meteorological drivers of hypolimnetic anoxia in a eutrophic, north temperate lake. Among the studied factors, air temperature was (2016) found to have the greatest impact on the anoxic factor, followed by wind speed and relative humidity This study (2017) 4-year study of the effects of climatic variations on a temperate mountain lake in NW Spain. Results suggest that local climatic variations related with climate change are causing a warmer epilimnion and a longer lake stratification which is driving longer and more pronounced periods of hypolimnetic anoxia. The effects are not only related with warming, but also with decreased frequency of precipitation and storm events

minor extent) entering the lake is considered to be directly related input to the lake, and thus, is partly responsible for the intensifica- with cattle activity. tion of soil-derived organic carbon and nutrients loadings. How- ever, the volume of soil eroded and transported along these 5.2.2. Modification of the drainage network artificial channels is still small as compared to the total lake vol- Artificial modifications of the natural drainage network around ume, so that the impact of this factor on the spread of hypolimnetic a lake may also have important effects on sediment and nutrient anoxia in the lake is considered to be of minor importance as com- loadings. In Lake Enol, the construction of a road and associated pared to, e.g., cattle grazing. infrastructure has severely modified the drainage network, con- centrating water flow in specific points where soil erosion is signif- 5.2.3. Clear-cutting icantly increased with respect to natural conditions (Fig. S1e, f). Clear-cutting is a common practice in the area to increase grass- This increased erosion has contributed to increase the sediment land surface. This activity has been recently observed in adjacent J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850 847 slopes around the lake (Fig. S1d). Mowing of shrubs helps increase on climate and land uses, being the input of OM considerably soil erosion and runoff flow, and this may also represent an addi- higher in cold-humid stages as a result of more abundant run off tional, indirect factor contributing to increase organic carbon and with terrestrial organic matter from the catchment. The study of nutrient influx to the lake. However, there’s no evidence to support the relations between local variations of climate conditions (tem- that deforestation around the lake is now more important than in perature, precipitation) and lake water parameters during the per- previous times, and therefore, this practice is not considered to iod covered by our study (2013–2016) may help evaluate the play a relevant role in the observed expansion of hypolimnetic influence of climatic factors on the stratification and oxygen anoxia. dynamics of this lake. We have found evidence supporting a climatic control on 5.2.4. Tourism hypolimnetic anoxia in Lake Enol. In particular, both the mean In recreational or touristic lakes, the pressure of visitors and/or air temperature and precipitation in the month of November urbanization may also cause important increases in sediment load- (where the autumn overturn usually takes place), seem to be con- ings (e.g., Toro and Granados, 2002) and/or elevated phosphorus trolling factors on the extent and duration of thermal stratifica- release from the sediments (e.g., Garrison and Wakeman, 2000; tion, and therefore, on the magnitude of hypolimnetic anoxia Köster et al., 2005; Toro et al., 2006). The touristic activity around (Fig. 11). With respect to the first year of our study (2013), which Lake Enol (e.g., high numbers of hikers walking along poorly can be considered a more climatically standard year (www.aemet. designed and badly maintained trails or in adjacent slopes) may es), the autumn period of the following years has been increas- also increase soil erosion rates (Sutherland et al., 2001; Olive & ingly warmer and drier (as observed in two different weather sta- Marion, 2009). However, the influx of tourists and vehicles is lar- tions located nearby; Fig. 11a). This climatic trend matches gely concentrated in specific lake areas and periods of the year predictions provided by the Spanish Agency of Meteorology (Fig. S1c, e), being also regulated and controlled by park rangers, (AEMET) which foresee progressive increases of temperature so that the indirect impact of tourism on soil erosion and lake oxy- and drops in precipitation in Spain during the 21st century gen dynamics is considered to be of minor importance. The recre- (Morata Gasca, 2014). The years 2014, 2015 and 2016 have all ational use of the lake (e.g., swimming, kayaking, etc.) is been climatically anomalous at a global scale (e.g., Gómez- prohibited, and there is no discharge of waste water to the lake, Cantero, 2015), with different temperature records registered in so a direct entrance of nutrients by these pathways is also consid- the area (www.aemet.es). The increased temperature has resulted ered negligible. in epilimnetic temperatures warm enough to prevent mixing and The traffic of vehicles, however, is probably affecting the lake sustain stratification in a period of the year at which the water chemistry to some extent through emissions of NOx, SO2 or metals. column should be fully homogenized under typical autumn con- The higher concentrations of Zn and Cu in the lake surface during ditions (Fig. 9d). A good correlation has been found between rainy periods (Fig. 6d, e) could be partly related with washing of the thickness of the anoxic layer at the end of the stratification roads and transport of traffic-related dust to the lake (Zhang period and the mean monthly data of both parameters (ambient et al., 2012), though this contribution would be difficult to distin- temperature and precipitation measured in the area in Novem- guish from that of atmospheric deposition, which can also repre- ber; Fig. 11c). sent an important input of these metals (Sharma et al., 2008; A similarly good correlation is observed between the climatic Camarero et al., 2009). variables and the duration of the anoxic period (as defined by the anoxic factor; Fig. 11d). Although microbially driven oxygen 5.3. Climatic influence on anoxia development consumption in the lake (as defined by the RAOD) has also experienced increases during some years (e.g., 2013–2015), the evolution The relation between physical, chemical and biological effects of RAOD is not fully consistent with the general trend of increasing of climate change and direct human pressures at basin scale are anoxia (e.g., 2016; Fig. 11b). This suggests that the climatic varia- often hard to disentangle (Vincent, 2009). A selection of studies tions experienced in the area (i.e., more sunny days with higher focused on climate change effects on lakes is also given in Table 7. average temperature along with less rainy days with lower average After an extensive study in 235 lakes around the world between precipitation during the autumn) are probably helping to extend 1985 and 2009, O’Reilly et al. (2015) found that a rise in surface the duration of the anoxic period. A longer exposure to solar radi- water temperature is occurring as a response to climate change, ation and a diminished entrance of rain water both tend to keep though regional characteristics or morphometric aspects were also the epilimnetic temperature, which helps maintain the vertical seen to influence this temperate increase. These data are in agree- density gradient and ensures a stable stratification which lasts ment with previous studies that showed that alpine lakes are longer. A sharper density gradient diminishes downwards oxygen highly sensitive to climatic variations (Parker et al., 2008). The diffusion. At the same time, the solubility of O2 is reduced in the effects of global warming on the duration and severity of hypolim- warmer epilimnion, which further reduce oxygen concentration netic anoxia have been documented in different lakes of the world in the upper levels. (e.g., Verburg et al., 2003; North et al., 2014; Jenny et al., 2016; Rain and runoff entering the lake during storm events (which Snortheim et al., 2016). The impact on climate change has been have been less frequent during the autumn period of the studied investigated in small lakes (e.g., Foley et al., 2012), as well as in years) affects the density stratification in two major ways: (i) if many big lakes (e.g. Sahoo et al., 2013, Schwefel et al., 2016). From the inflowing water is colder, it favors the cooling of the epilimnion this research, it is generally assumed that a warmer epilimnion and thus the density homogenization of the lake, and (ii) if this results in a lower solubility of dissolved oxygen, which decreases water is more dense (e.g., having a higher dissolved solids content, in concentration. Further, a higher density contrast with the cooler which is commonly the case; Table 5) it sinks to deeper levels and hypolimnion makes mixing more difficult and reduces the diffu- helps mixing the epilimnion. Thus, both factors, temperature and sion of dissolved oxygen to deeper levels. precipitation, play a role in maintaining the lake stratification, The observation of significant changes in the organic matter although they are obviously related (autumn fronts and storms content found in the sedimentary record of Lake Enol suggests that often bring colder temperatures and abundant precipitation). The this lake is very sensitive to environmental and climatic changes. reduction of the frequency and intensity of these fronts during Paleolimnological studies conducted by López-Merino et al. the autumn season is likely causing a longer stratification and a (2011) showed a strong dependence of the organic matter input longer anoxic period. 848 J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850

14 60 27 a b

260 (mm) 50 25

13 ) -1 d

40 -2 210 12 23 cm RAOD 2 30 AF 11 160 21 20

110 O (mg RAOD

Precipitation November 19

10 days) (AF, Factor Anoxic 10 Mean Temperature November (ºC) 9 60 Mean 0 17 2013 2014 2015 2016 2013 2014 2015 2016 Year Year

Mean Precipitation November (mm) Mean Precipitation November (mm) 0 100 200 300 0 100 200 300 400 7 60 c d 6 50

5 40 4 30 3 20 2 Anoxic Factor (days) Factor Anoxic 10 1 Thickness anoxic layer Thickness anoxic layer (m)

0 0 9 10 11 12 13 9 10 11 12 13 Mean Temperature November (ºC) Mean Temperature November (ºC)

Fig. 11. (a) Mean data of ambient temperature (in °C) and precipitation (in mm) measured by two weather stations located in Amieva and Gijón (Asturias) during the month of November in the four consecutive years covered by this study (2013–2016) (data downloaded from www.aemet.es); (b) Anoxic factor (AF, in days) and relative aereal À2 À1 oxygen deﬁcit (RAOD, in mg O2 cm d ) calculated for Lake Enol in the same periods (Table 6) (dashed-lined crosses represent the standard error); (c) Linear regression lines showing the correlation between mean ambient temperature (circles) and precipitation (triangles) measured in the area (Amieva) during November (2013–2016) and the corresponding thickness of the anoxic hypolimnetic layer found in Lake Enol on those periods; (d) Linear regression lines showing the correlation between mean ambient temperature (circles) and precipitation (triangles) measured in the area (Amieva) during November (2013–2016) and the corresponding anoxic factor calculated for Lake Enol in those periods (Table 6).

The duration of anoxia, however, can also be extended by an often associated with episodes of strong wind, their scarcity earlier onset of the stratification period in spring, as illustrated in or diminished frequency can also help maintain stratification Fig. S5. Temperature fluctuations in the spring time may cause longer due to a reduced wind-driven epilimnetic circulation. changes in the phytoplankton dynamics and, consequently, on Wind speed data recorded in a nearby meteorological station the rate of oxygen consumption. In this regard, the hypolimnetic indicate that the month of October was generally more windy chlorophyll maxium observed in 2014 (and absent in 2013; in 2013 as compared to the following years (Fig. S8 in Supple- Fig. 4a) likely represents sinking planktonic microalgae, and this mental material). This was especially visible in late October, senescent biomass may have helped increase the rate of oxygen which is close to the turnover period and to our autumn sam- consumption in the hypolimnion with respect to previous years, pling. The effect of climatic changes on lake stratification and thus accelerating the progression of the anoxic front. The impor- oxygen dynamics should therefore be studied in full and con- tance of these springtime climate fluctuations on the spread of sidering different climatic variables, instead of just a single hypolimnetic anoxia could increase in the future if global warming parameter. keeps advancing. In this sense, the spring of 2017 has been recently Despite the still limited set of climatic and limnological data, known to be the warmest and drier spring time recorded in Spain which must be taken with caution, the observations reported here since 1965 (official report available from Aemet in www.aemet.es), are in line with recent studies reporting the influence of global so Lake Enol and similar mountain lakes could represent unrivalled warming on the spread of hypoxia and anoxia in lakes and fresh- observatories to follow the effects of climate change on oxygen water ecosystems worldwide (Foley et al., 2012; Jenny et al., dynamics. 2016; North et al., 2014; Table 7). Under a scenario of global cli- In addition to temperature and precipitation, wind is mate change, in which ambient temperature is expected to experi- another important factor controlling the occurrence of the lake ence a gradual increase and precipitation will continue to decrease turnover, as wind-driven convection cells have an importance (Morata Gasca, 2014), it is envisaged that hypolimnetic anoxia will influence on vertical motion and lake density stratification be further increased in the future in many other mountain lakes of (e.g., Wetzel, 2001). Because storms and depressions are also the area. J. Sánchez-España et al. / Journal of Hydrology 555 (2017) 832–850 849

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