Andrea G Bravo

Keywords: Mercury, methylmercury, streams, rivers, freshwaters, organic matter, EuroRun, fluorescence

Large-scale studies are needed to identify the drivers of total mercury (THg) and monomethyl-mercury (MeHg) concentrations in aquatic ecosystems. Studies attempting to link dissolved organic matter (DOM) to levels of THg or MeHg are few and geographically constrained. Additionally, stream and river systems have been understudied as compared to lakes. Hence, the aim of the study was to examine the influence of DOM concentration and composition, morphological descriptors, land uses and water chemistry on THg and MeHg concentrations and the percentage of THg as MeHg (%MeHg) in 29 streams across Europe spanning from 41°N to 64 °N. THg concentrations (0.06 -2.78 ng L -1 ) were highest in streams characterized by DOM with a high terrestrial soil signature and low nutrient content. MeHg concentrations (78 -160 pg L -1 ) varied non-systematically across systems. Relationships between DOM bulk characteristics and THg and MeHg suggest that while soil derived DOM inputs control THg concentrations, autochthonous DOM (aquatically produced) and the availability of electron acceptors for Hg methylating microorganisms (e.g. sulfate) drive %MeHg and potentially MeHg concentration. Overall, these results highlight the large spatial variability in THg and MeHg concentrations at European scale, and underscore the importance of DOM composition on Hg cycling in fluvial systems.

INTRODUCTION

Mercury (Hg) is a hazardous substance with the potential to produce significant adverse neurological and other health effects in severely exposed humans, with particular concerns on unborn children and infants [START_REF] Mason | Mercury biogeochemical cycling in the ocean and policy implications[END_REF]. Anthropogenic emissions and long-range transport of atmospheric-Hg have led to Hg concentrations above background levels in surface reservoirs (atmosphere, oceans or terrestrial) on a global scale [START_REF] Amos | Legacy impacts of all-time anthropogenic emissions on the global mercury cycle[END_REF]. Aquatic food webs are particularly vulnerable to increases in Hg, since its methylated form (monomethyl-mercury, MeHg) accumulates in organisms and biomagnifies in aquatic food webs [START_REF] Morel | The chemical cycle and bioaccumulation of mercury[END_REF], even at low concentrations. As a consequence, millions of people are readily exposed to harmful concentrations of MeHg via fish consumption [START_REF] Mason | Mercury biogeochemical cycling in the ocean and policy implications[END_REF].

In order to prevent human exposure to Hg, a global legally binding agreement, the Minamata Convention, was signed by 128 countries and entered into force in September 2017. Prior to the Minamata Convention, the European Water Framework Directive [START_REF] Wfd | Directive 2008/105/EC of 16 December 2008 on environmental quality standards[END_REF] classified Hg as one of 30 priority hazardous substances and set the maximum allowable concentrations of environmental quality standards to 70 ng L -1 . However, in aquatic systems, without any point anthropogenic or geological sources, total-Hg (THg) concentration typically ranges from less than 0.05 to 12 ng L -1 [START_REF] Dennis | Distribution patterns of mercury in Lakes and Rivers of northeastern North America[END_REF][START_REF] Driscoll | Mercury contamination in forest and freshwater ecosystems in the northeastern United States[END_REF][START_REF] Wiener | Ecotoxicology of mercury[END_REF]. Because the maximum allowable concentrations of environmental quality standards were much higher than naturally relevant concentration levels of THg, national monitoring programs did not have the incentive to detect THg at ambient concentrations. There is even less information about MeHg background levels, which generally are between one and two orders of magnitude lower as compared to THg levels [START_REF] Driscoll | Mercury contamination in forest and freshwater ecosystems in the northeastern United States[END_REF]. In this context, streams and rivers are of special concern because very few studies have reported THg and MeHg concentrations at a wide geographical scale [START_REF] Balogh | Episodes of elevated methylmercury concentrations in prairie streams[END_REF][START_REF] Brigham | Mercury cycling in stream ecosystems. 1. Water column chemistry and transport[END_REF][START_REF] Eklöf | Parsimonious model for simulating total mercury and methylmercury in boreal streams based on riparian flow paths and seasonality[END_REF][START_REF] Lescord | Optical Properties of Dissolved Organic Matter and Their Relation to Mercury Concentrations in Water and Biota Across a Remote Freshwater Drainage Basin[END_REF][START_REF] Marvin-Dipasquale | Production and Bed Sediment. 2. Pore Water Partitioning Mercury Cycling in Stream Methylmercury Production and Bed Sediment -Pore Water Partitioning[END_REF][START_REF] Tsui | Mercury bioaccumulation in a stream network[END_REF]. While rivers and streams have, however, been hypothesized to be important contributors to the global biogeochemical Hg budget [START_REF] Amos | Global biogeochemical implications of mercury discharges from rivers and sediment burial[END_REF][START_REF] Kocman | Toward an assessment of the global inventory of present-day mercury releases to freshwater environments[END_REF]Schartup et al., 2015a;[START_REF] Sunderland | Human impacts on open ocean mercury concentrations[END_REF], quantitative information on Hg levels is largely lacking, especially in the temperate zone. Filling this gap in knowledge is an important prerequisite in the ongoing quest to reduce uncertainties associated with the global biogeochemical cycling of Hg and for the development of remediation and risk management strategies within the context of the Minamata Convention.

Freshwaters receive Hg from a variety of sources including atmospheric deposition [START_REF] Fitzgerald | The case for atmospheric mercury contamination in remote Areas[END_REF], release from industrial discharge [START_REF] Bravo | High methylmercury production under ferruginous conditions in sediments impacted by sewage treatment plant discharges[END_REF][START_REF] Bravo | Extremely elevated methyl mercury levels in water, sediment and organisms in a Romanian reservoir affected by release of mercury from a chlor-alkali plant[END_REF], and lateral export from terrestrial sources (e.g. soils and wetlands) [START_REF] Brigham | Mercury cycling in stream ecosystems. 1. Water column chemistry and transport[END_REF][START_REF] Eklöf | Spatial and temporal variation of THg concentrations in run-off water from 19 boreal catchments, 2000-2010[END_REF]. Diffuse inputs from land management activities and remobilization of Hg previously accumulated in terrestrial ecosystems can be an important source comparable in magnitude to direct Hg anthropogenic inputs (e.g. industrial releases or mining activities) to surface waters [START_REF] Kocman | Toward an assessment of the global inventory of present-day mercury releases to freshwater environments[END_REF]. Long-term accumulation of Hg in terrestrial systems has been explained by the strong binding between atmospherically deposited Hg and sulfur sites within organic matter (OM) [START_REF] Skyllberg | Complexation of mercury(II) in soil organic matter: EXAFS evidence for linear two-coordination with reduced sulfur groups[END_REF][START_REF] Xia | X-ray absorption spectroscopic evidence for the complexation of Hg(II) by reduced sulfur in soil humic substances[END_REF]. In boreal ecosystems, characterized by OM-rich soils, the strong correlation between THg and dissolved organic carbon (DOC) found in several studies [START_REF] Brigham | Mercury cycling in stream ecosystems. 1. Water column chemistry and transport[END_REF][START_REF] Driscoll | The role of dissolved organic carbon in the chemistry and bioavailability of mercury in remote Adirondack lakes[END_REF][START_REF] Eklöf | Spatial and temporal variation of THg concentrations in run-off water from 19 boreal catchments, 2000-2010[END_REF][START_REF] Tsui | Mercury bioaccumulation in a stream network[END_REF] denotes that the mobilization of OM is an important vector for Hg transport from catchment soils to surface waters [START_REF] Eklöf | Spatial and temporal variation of THg concentrations in run-off water from 19 boreal catchments, 2000-2010[END_REF][START_REF] Grigal | Inputs and outputs of mercury from terrestrial watersheds: a review[END_REF]. Besides stream water DOC, the proportion of wetland area within the catchment and the discharge have been used to predict THg concentrations in surface waters (Brigham et al., 2009;[START_REF] Eklöf | Parsimonious model for simulating total mercury and methylmercury in boreal streams based on riparian flow paths and seasonality[END_REF][START_REF] Tsui | Mercury bioaccumulation in a stream network[END_REF]. In contrast, the relationship between DOC and MeHg remains poorly understood [START_REF] Brigham | Mercury cycling in stream ecosystems. 1. Water column chemistry and transport[END_REF][START_REF] Tsui | Mercury bioaccumulation in a stream network[END_REF]. The variation of MeHg concentrations in aquatic ecosystems is indeed more complex, as MeHg is potentially derived from numerous sources, not only terrestrial sources. In boreal streams, the wetland proportion of the catchment has also been linked to MeHg freshwater concentrations [START_REF] Brigham | Mercury cycling in stream ecosystems. 1. Water column chemistry and transport[END_REF]. Wetlands, characterized by oxygen-depletion and low to intermediate nutrient levels, are known to be important sites for MeHg production and a relevant source of MeHg to inland waters [START_REF] Schaefer | Detection of a key Hg methylation gene, hgcA, in wetland soils[END_REF][START_REF] Tjerngren | Potential Hg methylation and MeHg demethylation rates related to the nutrient status of different boreal wetlands[END_REF]. Besides the contribution of MeHg produced in wetlands to stream waters, in-stream MeHg formation might also be an additional key source of MeHg [START_REF] Balogh | Episodes of elevated methylmercury concentrations in prairie streams[END_REF][START_REF] Eklöf | Parsimonious model for simulating total mercury and methylmercury in boreal streams based on riparian flow paths and seasonality[END_REF][START_REF] Tsui | Mercury bioaccumulation in a stream network[END_REF], especially in catchments lacking extensive wetland coverage. The methylation of inorganic divalent Hg (IHg) to MeHg is primarily mediated by anaerobic micro-organisms [START_REF] Gilmour | Mercury methylation by novel microorganisms from new environments[END_REF] and is controlled by temperature, availability of electron acceptors (e.g.

SO 4

2-and Fe 3+ ) [START_REF] Bravo | High methylmercury production under ferruginous conditions in sediments impacted by sewage treatment plant discharges[END_REF] and the molecular composition of OM, which influences IHg-speciation [START_REF] Graham | Effect of dissolved organic matter source and character on microbial Hg methylation in Hg-S-DOM solutions[END_REF][START_REF] Jonsson | Differentiated availability of geochemical mercury pools controls methylmercury levels in estuarine sediment and biota[END_REF], IHg-availability (Chiasson-Gould et al., 2014;[START_REF] Mazrui | Enhanced availability of mercury bound to dissolved organic matter for methylation in marine sediments[END_REF][START_REF] Schaefer | High methylation rates of mercury bound to cysteine by Geobacter sulfurreducens[END_REF]Schartup et al., 2015b) and microbial activity [START_REF] Bravo | Molecular composition of organic matter controls methylmercury formation in boreal lakes[END_REF]. Specifically, IHg methylation processes are enhanced by the presence of algal OM [START_REF] Bravo | Molecular composition of organic matter controls methylmercury formation in boreal lakes[END_REF][START_REF] Schartup | Methylmercury production in estuarine sediments: Role of organic matter[END_REF] and fresh humic OM leached from organic rich soils [START_REF] Ortega | High methylmercury formation in ponds fueled by fresh humic and algal derived organic matter[END_REF]. Indeed, elevated MeHg concentrations have been linked to periods of high algal production [START_REF] Gascon Diez | Role of settling particles on mercury methylation in the oxic water column of freshwater systems[END_REF][START_REF] Soerensen | Organic matter drives high interannual variability in methylmercury concentrations in a subarctic coastal sea[END_REF] and leaf fall [START_REF] Balogh | Episodes of elevated methylmercury concentrations in prairie streams[END_REF]. However most studies investigating the role of OM as a transport vector for Hg and/or a key factor controlling MeHg formation have been carried out in the boreal biome. Consequently, there is still a high amount of uncertainty in identifying factors governing THg and MeHg levels in stream waters, particularly at a broader geographical scale.

The molecular composition of dissolved organic matter (DOM) is variable and can be an important factor in predicting THg and potentially, MeHg [START_REF] Bravo | Molecular composition of organic matter controls methylmercury formation in boreal lakes[END_REF][START_REF] Ortega | High methylmercury formation in ponds fueled by fresh humic and algal derived organic matter[END_REF][START_REF] Lescord | Optical Properties of Dissolved Organic Matter and Their Relation to Mercury Concentrations in Water and Biota Across a Remote Freshwater Drainage Basin[END_REF][START_REF] Noh | Potential contributions of dissolved organic matter to monomethylmercury distributions in temperate reservoirs as revealed by fluorescence spectroscopy[END_REF]Schartup et al., 2015b[START_REF] Schartup | Methylmercury production in estuarine sediments: Role of organic matter[END_REF]. A common fingerprinting approach used to characterize the bulk DOM composition in inland waters is fluorescence spectroscopy [START_REF] Fellman | Fluorescence spectroscopy opens new windows into dissolved organic matter dynamics in freshwater ecosystems: A review[END_REF]. The increasingly widespread use of fluorescence can be attributed to its wide accessibility, ease of use, and low costs. Three commonly used indexes (fluorescence index [START_REF] Mcknight | Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity[END_REF], freshness index [START_REF] Parlanti | Dissolved organic matter fluorescence spectroscopy as a tool to estimate biological activity in a coastal zone submitted to anthropogenic inputs[END_REF] and humification index [START_REF] Zsolnay | Differentiating with fluorescence spectroscopy the sources of dissolved organic matter in soils subjected to drying[END_REF]) are particularly useful for distinguishing between DOM derived from different sources [START_REF] Mcknight | Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity[END_REF], and degrees of freshness and humification [START_REF] Zsolnay | Differentiating with fluorescence spectroscopy the sources of dissolved organic matter in soils subjected to drying[END_REF]. Likewise, commonly known reoccurring peaks distributed across the fluorescence excitation-emission matrix (EEM) can provide insights into sample specific DOM characteristics. Fluorescence has indeed demonstrated the potential to reveal key DOM molecular characteristics associated with THg and MeHg concentrations [START_REF] Ortega | High methylmercury formation in ponds fueled by fresh humic and algal derived organic matter[END_REF][START_REF] Lescord | Optical Properties of Dissolved Organic Matter and Their Relation to Mercury Concentrations in Water and Biota Across a Remote Freshwater Drainage Basin[END_REF][START_REF] Noh | Potential contributions of dissolved organic matter to monomethylmercury distributions in temperate reservoirs as revealed by fluorescence spectroscopy[END_REF]Schartup et al., 2015b).

The aim of this study was to identify the relationships between THg and MeHg concentrations and DOM composition (sources and quality) across a diversity of European streams. Unraveling the factors determining THg and MeHg background levels in the aquatic environment at a broad geographical scale is crucial for a better understanding of Hg cycling.

For this purpose, we measured THg and MeHg concentrations, DOM concentration and composition, anions and cations in filtered and unfiltered stream water at 29 sites with contrasting catchment characteristic across a wide latitudinal gradient across Europe.

MATERIAL AND METHODS

Organizational Framework

Water S1).

Sampling

The sampling procedure was explained and demonstrated to one lead representative from each team, who participated in a workshop performed in Sweden in September 2016.

All the material needed to collect water samples along with a detailed written protocol was provided to participants during the workshop (see SI). The sampling took place during 3 rd -14 th October 2016 and teams from Austria (AUT), Bulgaria (BGR), the Czech Republic (CZE), Germany (DEU), Spain (ESP), France (FRA), Great Britain (GBR), Portugal (PRT)

and Sweden (SWE) collected water samples for this study (Fig. 1). The 29 sampled sites were located across a wide latitudinal gradient (from 41°N to 64 °N) and altitude range (from 1 to 1560 m above sea level), and spanned a wide range of hydrological (discharge from 0.1 to 56 m 3 s -1; ) and catchment characteristics (Table S1). Each team provided hydrological (discharge, stream wetted width and stream order) and catchment characteristics data (land use in % and catchment area).

A total of 4 water samples were collected at each site. Amber borosilicate bottles (VWR, 250 mL) were used to store water and analyze THg and MeHg species in filtered and unfiltered fractions. A previous study determined the cleaning procedures suitable for THg and MeHg analyses with these bottles [START_REF] Cavalheiro | Assessment of background concentrations of organometallic compounds (methylmercury, ethyllead and butyl-and phenyltin) in French aquatic environments[END_REF] Unfiltered water samples were collected for determination of the total fraction ( T ) of THg T , MeHg T (acidified) and total organic carbon (TOC) concentration. Samples were kept at 4°C

and shipped to the laboratory by courier within 48 h. A subsample (10 mL) of unfiltered water was filtered in the laboratory, and stored frozen for later determination of anions and cations.

Because four of the vials broke during transport, these samples could not be determined (marked as not determined in Table 1 andS2).

Results for unfiltered water samples correspond to the total fraction (THg T , MeHg T and TOC), whereas the results for filtered water samples represent the dissolved fraction (THg D , MeHg D , DOC, DOM optical properties, anions and cations).

Chemical Analyses

Analysis of Inorganic-Hg and Methylmercury

Inorganic-Hg (IHg) and MeHg (monomethylmercury) concentrations (Table 1) were measured using species-specific isotope dilution and capillary gas chromatography (Trace GC Ultra, Thermo Fisher, Waltham, MA, USA equipped with a TriPlus RSH auto-sampler) hyphenated to inductively coupled plasma mass spectrometer (Thermo X Series 2) [START_REF] Cavalheiro | Assessment of background concentrations of organometallic compounds (methylmercury, ethyllead and butyl-and phenyltin) in French aquatic environments[END_REF][START_REF] Rodriguez Martín-Doimeadios | Using speciated isotope dilution with GC-inductively coupled plasma MS to determine and unravel the artificial formation of monomethylmercury in certified reference sediments[END_REF]. The detection limits of this method are 0.02 ng L -1 for IHg and 0.005 ng L -1 for MeHg. The measurement error (calculated by analyzing each sample three times) was less than 15% and 10% for IHg and MeHg concentrations, respectively. Based on previous studies [START_REF] Bravo | Extremely elevated methyl mercury levels in water, sediment and organisms in a Romanian reservoir affected by release of mercury from a chlor-alkali plant[END_REF]Monperrus et al., 2004;[START_REF] Rodriguez Martín-Doimeadios | Using speciated isotope dilution with GC-inductively coupled plasma MS to determine and unravel the artificial formation of monomethylmercury in certified reference sediments[END_REF], THg was calculated as the sum of IHg and MeHg.

TOC, DOC and DOM optical properties

TOC and DOC concentrations were determined on unfiltered and filtered samples, respectively. Prior to analysis, samples were acidified to a pH of 2 with 0.1 M HCl, and analyzed using a Shimadzu TOC-V CPH total carbon analyzer. Optical properties were determined on filtered water using an Aqualog spectrofluorometer (Horiba), which measures absorbance and fluorescence simultaneously. The fluorescence spectrophotometer has a routine protocol before running samples that involves i) instrument validation (ensuring the signal : noise ratio <20,000 counts ii) ensuring the excitation lamp is calibrated to a given wavelength (maximum intensity at 467 nm), iii) the emission energy is calibrated using pure water at an emission of 397 nm and an excitation of 350 nm. Fluorescence spectra were collected across excitation wavelengths (Ex λ ) 250 to 445 nm at 5 nm increments and across emission wavelengths (Em λ ) ranging from 300-600 nm. Bandpass and resolution were 5 nm.

All spectra were measured in a 1 cm quartz cuvette and automatically blank-subtracted, corrected for instrument biases and inner filter effects using the FluorEssence™ software (Horiba). Samples were measured for fluorescence at a 2 second integration time, with the exception of five more optical dense samples from Great Britain and Sweden, which were measured at 0.5 seconds. All intensities were normalized to 1 second integration time postanalysis and the excitation-emission matrixes (EEM) were expressed in fluorescence units (FU).

Peak picking was used to define the relative intensity of four common peaks across the EEM including the Peak A (λ ex : 260 nm, λ em : 400-460 nm), Peak C (λ ex : 320-360 nm, λ em : 420-460 nm), and Peak M (λ ex : 290-310 nm, λ em : 370-410 nm) associated with soil terrestrial DOM, as well as a protein-like peak T (λ ex : 275 nm, λ em : 340-350 nm) that is considered to be of algal/bacterial origin (i.e. autochthonous to the aquatic environment) [START_REF] Fellman | Fluorescence spectroscopy opens new windows into dissolved organic matter dynamics in freshwater ecosystems: A review[END_REF][START_REF] Huguet | Properties of fluorescent dissolved organic matter in the Gironde Estuary[END_REF]. The summed area of total fluorescence (TF)

under the EEMs was used to normalize each of the four peaks, which were expressed as a percent of TF (i.e. %Peak A = Peak A/ TF x 100%). We also calculated three common fluorescence indexes: i) the fluorescence index (FI, calculated as the ratio between emission at wavelengths of 470 and 520 nm, at an excitation of 370 nm), an indicator of source being more microbial (≈1.8) or terrestrial (≈1.2) [START_REF] Cory | Chemical characteristics of fulvic acids from Arctic surface waters: Microbial contributions and photochemical transformations[END_REF][START_REF] Mcknight | Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity[END_REF], ii) the freshness index (calculated as the ratio of emission at 380 nm, divided by the emission maxima between 420 and 435 nm, at an excitation of 310 nm) which reflects the age of OM, with higher values representing more recently produced OM [START_REF] Parlanti | Dissolved organic matter fluorescence spectroscopy as a tool to estimate biological activity in a coastal zone submitted to anthropogenic inputs[END_REF] and iii) the humification index (HIX, ratio of peak areas across an emission spectra [435-480 nm / 300-345 nm]), indicating the degree of humification [START_REF] Zsolnay | Differentiating with fluorescence spectroscopy the sources of dissolved organic matter in soils subjected to drying[END_REF]. The absorbance at 254 nm (A 254 ) was used to calculate carbon specific absorbance (SUVA 254 ), a commonly used metric of the degree of aromaticity, as A 254 /DOC concentration normalized to a 1 m path length (L mg C -1 m -1 ) [START_REF] Weishaar | Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon[END_REF].

Anions and Cations

Inorganic analyses were conducted on a Metrohm IC system (883 Basic IC Plus and 919 Autosampler Plus). Anions (F -, Cl -, NO 3 -and SO 4 2-

) were separated on a Metrosep A Supp 5 analytical column (150 x 4.0 mm) fitted with a Metrosep A Supp 4/5 guard column at 0.7 mL min -1 using a carbonate eluent (3.2 mM Na 2 CO 3 + 1.0 mM NaHCO 3 ). A Metrosep C4 column (250 x 2.0 mm) with a Metrosep C4 guard column was used for separation of the cations (Na + , NH 4 + , K + , Ca 2+ , Mg 2+ ) with an eluent of 1.7 mM nitric acid and 0.7 mM dipicolinic acid at a flow rate of 0.2 mL min -1 .

Statistical Analyses

Kolmogorov-Smirnov test and quantile-quantile plots were used to evaluate the normality of the data. Accordingly, the samples with incomplete information (DEU1_1, DEU1_2, GBR2_2, FRA1_2, PRT1_1, CZE1_1, GBR2_1 and ESP2_3) were excluded from the PCA analysis. All data were log-transformed, scaled and centered prior to PCA analysis. PCA analysis were carried out using the prcomp function of the package stats in R 3.2.4 (R Core Team, 2016).

Partial least squares (PLS) regression models were subsequently applied to predict THg concentration (Y response variable) from DOM optical properties, water chemistry and catchment characteristics (X predictor variables, Table S5 and Table S6). A second PLS, excluding the sites with high THg concentrations (Great Britain, GBR1_1, GBR1_2), was also tested (Supplementary information, Table S7). The PLS model performance was examined based on the cumulative goodness of fit (R 2 Y, explained variation), and the cumulative goodness of prediction (or Q 2 , predicted variation). The variable influence on projections (VIP scores) provided a means of assigning the importance of X variables on the overall PLS model. X-predictor variables with an influence score ≥1 were considered highly influential, between 0.8 and 1.0 moderately influential, and <0.8 less influential predictors [START_REF] Eriksson | Introduction to Multi-and Megavariate Data Analysis Using Projection Methods[END_REF]. The PLS models were performed using the plsr function of the pls package in R [START_REF] Mevik | The pls Package: Principal Component and Partial Least Squares Regression in R[END_REF]. Graphics were built in R 3.2.4 (R Core Team, 2016) and modified to fit the journal requirements with Inkscape 0.92 (https://inkscape.org/es/).

THg and MeHg loads of both total and dissolved fractions (Table S3) were calculated with the stream discharge value provided by the participants (Table S1) and the concentrations measured in this study (Table 1).

RESULTS

Mercury Levels (THg, IHg, MeHg and %MeHg)

THg concentrations were consistently low among sites (Table 1). Median values for total (THg T ) and dissolved fractions (THg D ) were 0.46 ng L -1 (interquartile range, IQR: 0.28 -0.8) and 0.24 ng L -1 (IQR: 0.28 -0.49), respectively. Streams in Great Britain (GBR) and Austria (AUT) had the highest THg T and THg D concentrations (Fig. 2a, Table 1). THg T concentrations were generally higher than THg D (p = 0.004), although both were significantly correlated (R 2 =0.62, p = 0.04; Table S3). IHg T was also slightly higher than IHg D (Fig. 2b).

MeHg T concentrations ranged between 7.8 and 159 pg L -1 and were similar (p = 0.07) to MeHg D (6.6 -134 pg L -1 ; Fig. 2c). The highest MeHg concentrations, for total and dissolved fractions, were found at sites from Sweden (SWE2_1), Great Britain (GBR1_1) and Spain (ESP1_3). The %MeHg was very similar in both the total (%MeHg T ) and dissolved (%MeHg D ) fractions (Fig. 2d) but varied among sites, ranging from 1 to 43%. The highest %MeHg T was found at a Spanish site (ESP1_3). The median of THg T and MeHg T loads were 3.9 kg year -1 (IQR: 0.6 -6.8) and 0.3 kg year -1 (IQR: 0.1 -0.5), respectively.

Significant correlations were found between the total and dissolved fraction of THg, IHg and MeHg (Table S3). As total and dissolved fractions were correlated, we used the total fraction (THg T , MeHg T , %MeHg T ) for all the following statistical analyses (PCA and PLS).

Water Chemistry

DOC and TOC concentrations ranged between 0.9 and 18.5 mg L -1 and 0.9 and 22.2 mg L -1 , respectively. DOC and TOC were significantly correlated (R 2 = 0.99, p < 0.01). The highest concentrations were found in Great Britain (GBR1_1, GBR1_2) and Sweden (SWE1_1) and the lowest in Spain (ESP1_2, ESP2_1) and Austria (AUT2_1, AUT2_2, AUT2_3; Table S2). The concentrations of sulfate, a well-known electron acceptor for Hg methylating microorganisms, varied largely and ranged from 1.3 (AUT1_1) to 731 (ESP2_3) mg L -1 . About 50% of the studied sites presented sulfate concentrations lower than 12.6 mg L -1 (IQR: 6.8 -66 mg L -1 ). Spanish sites presented the highest sulfate concentrations (Table S2). Nitrate concentration varied between 0.04 (GBR1_1) and 23 (ESP2_2) mg L -1 . The median value for nitrate concentration was 1.9 mg L -1 (IQR: 0.7 -2.7).

DOM Optical Characteristics

Very distinct fluorescence signatures were observed between study sites, with four illustrative EEMs representing contrasting signatures displayed in Figure 3. Sites from Great

Britain showed a strong optical signature representing terrestrially derived DOM, as indicated by the high intensity of Peaks A and C, while in contrast, sites from Spain (ESP) and the Czech Republic (CZE), with high Peak T intensities, represented DOM optical signatures of waters typically enriched in autochthonous DOM (Fig. 3, Table S2). The wide range of bulk DOM optical characteristics captured in this dataset can be attributed to the broad geographical scale of this study.

Analyzed water samples also captured a wide range of DOM characteristics based on each of the measured optical indexes. The SUVA 254 , which is associated with the presence of aromatic [START_REF] Weishaar | Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon[END_REF] and high molecular mass compounds [START_REF] Chowdhury | Trihalomethanes in drinking water: Effect of natural organic matter distribution[END_REF] typically spans from <1 to a maximum of 6 L mg C -1 m -1 [START_REF] Weishaar | Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon[END_REF], and was found to range from 2.4 (CZE1_2) to 5.2 (GBR1_1, GBR1_2) L mg C -1 m -1 . HIX values spanned from 2.2 to 25 (Table S2). The highest HIX values were measured in Austria (AUT1_2) and Great Britain (GBR1_1, GBR1_2). Some of the lowest values were observed in the Czech Republic (CZE1_2) and Austria (AUT2_2). While high HIX values (> 10) have been linked to the presence of complex molecules and strongly humified organic material, mainly of terrestrial origin, low values (<4) have been associated with autochthonous OM [START_REF] Huguet | Properties of fluorescent dissolved organic matter in the Gironde Estuary[END_REF]. The fluorescence index (FI) covered almost the full range of this index, from a value of 1.3, representing terrestrial sources (degraded plant and soil OM; lower values), in GBR1_2, to a value of 1.8, indicating microbial sources (including extracellular release and leachate from bacteria and algae) in ESP2_2, ESP3_3 [START_REF] Cory | Chemical characteristics of fulvic acids from Arctic surface waters: Microbial contributions and photochemical transformations[END_REF][START_REF] Mcknight | Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity[END_REF]. The freshness index, typically associated with the contribution of recently produced microbial-derived DOM [START_REF] Parlanti | Dissolved organic matter fluorescence spectroscopy as a tool to estimate biological activity in a coastal zone submitted to anthropogenic inputs[END_REF], also spanned a wide range from 0.34 to 0.91 and was positively correlated to the FI and negatively correlated to the HIX and SUVA 254 (Fig. 4).

The wide distribution of DOM composition between sites indicated differences in the source, processing pathways and biological reactivity of the DOM, as shown by the PCA analysis (Fig. 5). The first principal component (PC1) explained 41% of the total variance across 21 European sites (Fig. 5a). Positive loadings on PC1 were associated with high [START_REF] Ohno | Fluorescence inner-filtering correction for determining the humification index of dissolved organic matter[END_REF]. Running waters from Great Britain (GBR1_1; GBR1_2), Sweden (SWE1_1, SWE2_1) and Austria (AUT1_1, AUT1_2) were characterized by low anions, cations and high concentrations of terrestrial humidified DOM (positive scores in PC1 Fig. 5b). In contrast, negative scores (Fig. 5b) of running waters from Spain (ESP) and the Czech Republic indicated an enrichment in nutrients and fresh autochthonous OM. Also, negative loadings for the percentage of urban and agricultural areas in the studied catchments suggest that anthropogenic activities might enrich running waters in nutrients and enhance primary production (Fig. 5a). The second component (PC2) explained 14% of the variance. Positive loadings on PC2 were found for the fluorescence Peak A and the ratio of the Peaks A to C (A/C). Both Peaks A and C have been linked to the presence of humic substances [START_REF] Fellman | Fluorescence spectroscopy opens new windows into dissolved organic matter dynamics in freshwater ecosystems: A review[END_REF]. However, an increase in the relative abundance of the peak A compared to Peak C (A/C peak ratio) has been related to DOM processing, as Peak C is lost preferentially to Peak A under both photo-and biodegradation processes [START_REF] Kothawala | Selective loss and preservation of lake water dissolved organic matter fluorescence during long-term dark incubations[END_REF]. Therefore, positive loadings of PC2 indicated terrestrial DOM that had been processed (Fig. 5a). On the opposite end, negative loadings on PC2 were associated with aromatic compounds (SUVA 254 ) [START_REF] Weishaar | Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon[END_REF] and terrestrial DOM (Peak C) yet not strongly degraded [START_REF] Kothawala | Selective loss and preservation of lake water dissolved organic matter fluorescence during long-term dark incubations[END_REF]. predictive components (R 2 X, 50%), the variance of THg concentrations was well explained (R 2 Y, 83.2%) and relatively well predicted (Q 2 , 60%) by the PLS model I (Fig. 6, Table S5, Table S6). PLS model I indicates that while TOC and A 254 presented the highest positive correlation with THg, freshness index, FI and agriculture were negatively correlated. When not including sites highly influenced by terrestrial DOM inputs, GBR1_1 and GBR1_2, a second PLS (PLS model II), with two predictive components (R 2 X, 40%), explained the variance of THg T concentrations relatively well (R 2 Y, 82%) but could not predict them (Q 2 , -2%) (Table S7).

Predictors of THg, MeHg and %MeHg Concentrations

It was not possible to predict a significant proportion of the variability in MeHg concentrations using a PLS model with the measured variables. Nevertheless, the concentration of MeHg was significantly correlated to %MeHg (Fig. 4). Additionally, the %MeHg was significantly and positively correlated to Peak M (%), FI and sulfate concentration and negatively related to HIX and THg T (Fig. 4).

Discussion

Hg levels in European running waters

Our results provide new information on THg and MeHg concentrations in streams at a European scale. THg concentrations across the studied European running waters were in the same range as those measured in pristine streams of northern Sweden [START_REF] Eklöf | Spatial and temporal variation of THg concentrations in run-off water from 19 boreal catchments, 2000-2010[END_REF], USA (Oregon, Wisconsin and Florida, Brigham et al., (2009); California, [START_REF] Tsui | Mercury bioaccumulation in a stream network[END_REF] and Minnesota, [START_REF] Balogh | Episodes of elevated methylmercury concentrations in prairie streams[END_REF]) and France [START_REF] Cavalheiro | Assessment of background concentrations of organometallic compounds (methylmercury, ethyllead and butyl-and phenyltin) in French aquatic environments[END_REF] and were lower than those reported for rivers typically contaminated by point sources [START_REF] Baptista-Salazar | Grain-size dependence of mercury speciation in river suspended matter, sediments and soils in a mercury mining area at varying hydrological conditions[END_REF][START_REF] Bravo | Extremely elevated methyl mercury levels in water, sediment and organisms in a Romanian reservoir affected by release of mercury from a chlor-alkali plant[END_REF]. Therefore, none of the study sites appeared to be affected by point sources of Hg.

Fluvial systems are an important transport pathway and ultimately deliver Hg originating from catchment soils to the marine environment. At a broader scale, we attempted to provide a rough estimate discharge of THg T and MeHg T of European rivers considering median values obtained in this study and the total European river discharge (3100 km 3 ) [START_REF] Shiklomanov | World Water Resources at the Beginning of the Twenty-First Century[END_REF]. Without considering point source of contamination, such as chlor-alkali or mining activities, known to increase stream Hg concentrations, the estimated European river discharge of THg T and MeHg T would be 1426 kg year -1 (IQR: 868 -2494)

and 97 kg year -1 (IQR: 60 -254), respectively. Particles are an important vector of Hg transport in streams [START_REF] Schuster | Mercury Export from the Yukon River Basin and Potential Response to a Changing Climate[END_REF] and can influence hydrologically mediated processes such as sedimentation and/or remobilization (e.g heavy rain events) of Hg previously deposited within river systems, which can largely affect THg T and MeHg T loads [START_REF] Baptista-Salazar | Grain-size dependence of mercury speciation in river suspended matter, sediments and soils in a mercury mining area at varying hydrological conditions[END_REF][START_REF] Moreno-Brush | Is mercury from small-scale gold mining prevalent in the southeastern Peruvian Amazon?[END_REF][START_REF] Sunderland | Human impacts on open ocean mercury concentrations[END_REF].

Measured THg and MeHg concentrations were notably higher in the total fraction compared to the dissolved fraction, highlighting the role of particles in the transport and fate of Hg in the studied fluvial systems. As precise hydrological data were unavailable here, and our calculations are based on a single sampling campaign, and the discharge of THg and MeHg only provides an initial rough estimate to be considered with care. In order to improve upon current estimates of European Hg discharge into the oceans [START_REF] Kocman | Toward an assessment of the global inventory of present-day mercury releases to freshwater environments[END_REF], greater monitoring of THg and MeHg is required across fluvial systems, particularly beyond the boreal ecozone.

Terrestrial DOM: An Important Source of THg in European Running Waters

Using a multivariate approach comparing 21 streams spanning a wide geographical area across Europe, we were able to discriminate between the relative influence of bulk DOM optical characteristics and environmental variables on THg and MeHg concentrations.

PC1 of the PLS model showed positive correlations between the highly influential predictors TOC and A 254 with THg concentrations in the studied sites and suggests that terrestrial DOM is an important driver of THg in streams (Fig. 6). Sites characterized by humic-rich soils, such as those in Sweden and Great Britain [START_REF] Jones | Estimating organic carbon in the soils of Europe for policy support[END_REF], presented the highest THg T and THg D concentrations. The positive correlation between terrestrial DOM and THg concentration in boreal rivers and streams has been previously reported in the literature [START_REF] Brigham | Mercury cycling in stream ecosystems. 1. Water column chemistry and transport[END_REF][START_REF] Eklöf | Spatial and temporal variation of THg concentrations in run-off water from 19 boreal catchments, 2000-2010[END_REF]. As atmospheric Hg deposition during the industrial era have led to the accumulation of Hg in organic rich soils [START_REF] Johansson | Impact of atmospheric long range transport of lead, mercury and cadmium on the Swedish forest environment[END_REF], the remobilization of Hg-enriched soil OM is an important process controlling THg concentrations in streams [START_REF] Baptista-Salazar | Grain-size dependence of mercury speciation in river suspended matter, sediments and soils in a mercury mining area at varying hydrological conditions[END_REF][START_REF] Brigham | Mercury cycling in stream ecosystems. 1. Water column chemistry and transport[END_REF][START_REF] Driscoll | The role of dissolved organic carbon in the chemistry and bioavailability of mercury in remote Adirondack lakes[END_REF][START_REF] Eklöf | Spatial and temporal variation of THg concentrations in run-off water from 19 boreal catchments, 2000-2010[END_REF][START_REF] Kirk | Multiyear total and methyl mercury exports from two major sub-arctic rivers draining into Hudson Bay, Canada[END_REF][START_REF] Tsui | Mercury bioaccumulation in a stream network[END_REF] and lakes [START_REF] Bravo | Molecular composition of organic matter controls methylmercury formation in boreal lakes[END_REF].

It is noteworthy to highlight the lack of a significant correlation between TOC and THg T concentration (p = 0.6) when the sites with the highest TOC and THg T concentrations (SWE2_1, GBR1_1 and GBR1_2) were excluded from the statistical analyses. Also, a poor predictability was obtained in a PLS model (PLS model II) built for THg T excluding the sites highly influenced by terrestrial DOM (GBR1_1 and GBR1_2, Table S7). Therefore, TOC, and DOM components associated with terrestrial DOM, failed to predict THg T concentrations in southern parts of Europe (FRA, ESP, CZE, AUT, BGR and DEU). This suggests that other Hg sources are relevant for the studied southern streams. Direct inputs of Hg deposited by wet and dry atmospheric depositional processes [START_REF] Brigham | Mercury cycling in stream ecosystems. 1. Water column chemistry and transport[END_REF][START_REF] Domagalski | Comparison of mercury mass loading in streams to atmospheric deposition in watersheds of Western North America: Evidence for non-atmospheric mercury sources[END_REF][START_REF] Lee | Modelling atmospheric mercury transport and deposition across Europe and the UK[END_REF] could contribute to THg T concentrations in streams with low inputs of terrestrial DOM. Besides atmospheric deposition, catchment characteristics and changes in runoff may be relevant in determining THg T in streams and rivers [START_REF] Baptista-Salazar | Grain-size dependence of mercury speciation in river suspended matter, sediments and soils in a mercury mining area at varying hydrological conditions[END_REF][START_REF] Domagalski | Comparison of mercury mass loading in streams to atmospheric deposition in watersheds of Western North America: Evidence for non-atmospheric mercury sources[END_REF]. Therefore, our results confirm the role of terrestrial OM as an important source of Hg for streams but highlight the uncertainties on the drivers of THg concentrations at broader global scale when considering systems with low loads of terrestrial OM.

Autochthonous DOM and Nutrients: Important Drivers of MeHg Formation in

European Running Waters

In this study, MeHg concentrations measured in running waters were in the same range as those previously reported for Sweden [START_REF] Skyllberg | Distribution of mercury, methyl mercury and organic sulphur species in soil, soil solution and stream of a boreal forest catchment[END_REF], USA (California, Tsui et al., (2009); Florida, [START_REF] Brigham | Mercury cycling in stream ecosystems. 1. Water column chemistry and transport[END_REF]) and France [START_REF] Cavalheiro | Assessment of background concentrations of organometallic compounds (methylmercury, ethyllead and butyl-and phenyltin) in French aquatic environments[END_REF]. Similarly, the %MeHg measured in samples collected in Sweden and Great Britain were similar to those previously reported in the literature [START_REF] Brigham | Mercury cycling in stream ecosystems. 1. Water column chemistry and transport[END_REF][START_REF] Skyllberg | Distribution of mercury, methyl mercury and organic sulphur species in soil, soil solution and stream of a boreal forest catchment[END_REF]. In contrast to THg, MeHg concentrations were not correlated to TOC, A 254 or HIX (Fig. 4), suggesting that terrestrial sources are not the main vector for MeHg in the studied streams. MeHg concentrations have been related to the concentration of autochthonous OM in reservoirs, lakes and in the Baltic sea [START_REF] Gascon Diez | Role of settling particles on mercury methylation in the oxic water column of freshwater systems[END_REF][START_REF] Noh | Potential contributions of dissolved organic matter to monomethylmercury distributions in temperate reservoirs as revealed by fluorescence spectroscopy[END_REF][START_REF] Soerensen | Organic matter drives high interannual variability in methylmercury concentrations in a subarctic coastal sea[END_REF].

The %MeHg has been considered a proxy of net MeHg formation [START_REF] Drott | Do potential methylation rates reflect accumulated methyl mercury in contaminated sediments?[END_REF].

In the studied European streams studied here, the positive correlation between %MeHg to Peak M and the FI indicated that autochthonous OM is important for MeHg formation. Peak M, as well as Peak A and Peak C, have been generally described as humic-like peaks [START_REF] Fellman | Fluorescence spectroscopy opens new windows into dissolved organic matter dynamics in freshwater ecosystems: A review[END_REF]. Peaks A and C have been linked to DOM derived primarily from vascular plant sources, aromatic and with a higher-molecular-weight fraction of the DOM pool [START_REF] Coble | Characterization of marine and terrestrial DOM in seawater using excitationemission matrix spectroscopy[END_REF]. In contrast, Peak M represents compounds less aromatic and of lower molecular weight as compared to peaks A and C [START_REF] Fellman | Fluorescence spectroscopy opens new windows into dissolved organic matter dynamics in freshwater ecosystems: A review[END_REF]. In a previous study performed in freshwaters, strong correlations between Peak M and Peak T (protein-like)

were reported, but weaker relationships were observed between Peak M and Peaks A and C [START_REF] Kothawala | Controls of dissolved organic matter quality : evidence from a large-scale boreal lake survey[END_REF]. Indeed, Peak M was originally used as a marker for autochthonous DOM from planktonic production [START_REF] Fellman | Fluorescence spectroscopy opens new windows into dissolved organic matter dynamics in freshwater ecosystems: A review[END_REF]. A positive correlation of the %MeHg with Peak M and the FI thus suggests that net MeHg production is linked to fresh autochthonous OM. In contrast, the negative correlation between %MeHg and HIX suggests that high concentrations of highly humified terrestrial DOM hampered Hg methylation processes most likely by decreasing Hg bioavailability [START_REF] Chiasson-Gould | Dissolved organic matter kinetically controls mercury bioavailability to bacteria[END_REF][START_REF] Soerensen | Organic matter drives high interannual variability in methylmercury concentrations in a subarctic coastal sea[END_REF] and/or bacterial activity [START_REF] Bravo | Molecular composition of organic matter controls methylmercury formation in boreal lakes[END_REF]. High Hg methylation rates have been associated with algal derived OM [START_REF] Bravo | Molecular composition of organic matter controls methylmercury formation in boreal lakes[END_REF][START_REF] Gascon Diez | Role of settling particles on mercury methylation in the oxic water column of freshwater systems[END_REF][START_REF] Ortega | High methylmercury formation in ponds fueled by fresh humic and algal derived organic matter[END_REF][START_REF] Mazrui | Enhanced availability of mercury bound to dissolved organic matter for methylation in marine sediments[END_REF]. Our study provides further evidence of the potential role of algal and/or microbial DOM in the in situ formation of MeHg in streams.

The methylation of IHg to MeHg in aquatic systems has been primarily attributed to the activity of sulfate-reducing bacteria, iron-reducing bacteria (FeRB), methanogens and Firmicutes [START_REF] Gilmour | Mercury methylation by novel microorganisms from new environments[END_REF] in oxygen limiting conditions of sediments [START_REF] Bravo | Molecular composition of organic matter controls methylmercury formation in boreal lakes[END_REF], hypolimnion [START_REF] Eckley | Determination of mercury methylation potentials in the water column of lakes across Canada[END_REF] and settling particles [START_REF] Gascon Diez | Role of settling particles on mercury methylation in the oxic water column of freshwater systems[END_REF]. As sulfate can be used as an electron acceptor by Hg methylating microorganisms [START_REF] Pak | Mercury methylation and demethylation in anoxic lake sediments and by strictly anaerobic bacteria[END_REF], the positive significant correlation found between the %MeHg and the sulfate concentration, suggests an important role of SRB in MeHg formation in running waters and provides further evidence for the relevance of in situ MeHg formation.

Stream sulfate mainly originates from atmospheric deposition and/or geogenic sources [START_REF] Ledesma | Towards an Improved Conceptualization of Riparian Zones in Boreal Forest Headwaters[END_REF]. Because sulfate release is accompanied by cation leaching [START_REF] Prechtel | Response of sulphur dynamics in European catchments to decreasing sulphate deposition[END_REF], high concentrations of both sulfate and cations (e.g. calcium, Table S2) in Spanish sites indicate an effect of geological substrate of the river basin on the stream water chemistry. Although it was not the focus of this work, these results underline the relevance of geology and catchment characteristics on stream water properties and ultimately also for Hg cycling.

Conclusion

Our findings indicated that streams enriched in terrestrial DOM, as typical of Northern study sites, revealed the highest THg concentrations, suggesting the source was from organic rich catchment soils. In contrast, Southern European streams were enriched in nutrients and autochthonous DOM and although inputs of THg and terrestrial DOM from the catchment were lower, we propose that MeHg levels were regulated by. We propose that the elevated MeHg concentrations in some of these sites could be due to a higher in situ MeHg formation, likely related to enhanced microbial activity, which is boosted by the availability of autochthonous algal/microbial derived DOM and sulfate. Our results show the interplay between DOM components and THg and MeHg concentrations in streams and provide new insights into Hg cycling in aquatic systems at a broad geographical European scale.

Table 1. Concentrations of inorganic-Hg (IHg), methylmercury (MeHg), total-Hg (THg = IHg+ MeHg) and the percentage of MeHg as THg (%MeHg) in the dissolved (filtered 0.45 μm: D ) and total (unfiltered: T ) fraction of the studied streams. Fluorescence Units

Country Stream ID IHg D MeHg D THg D MeHg D IHg T MeHg T THg T MeHg T ng L -1 pg L -1 ng L -1 % ng L -1 pg L -1 ng L -1 % Austria AUT1_1 1.
Fluorescence Units

A

  PLS model (PLS model I, Fig. 6) identified key factors driving THg T concentrations in running waters. From the 29 available sites, only 21 sites were included in the PLS model which contained a complete set of required variables. To interpret the PLS loading plots, the distance and positioning of Y variables (THg, MeHg or %MeHg) relative to X variables indicated how well, or poorly, they related to each other. The greater the distance a variable (X or Y) lies from the origin, the greater its overall influence. Variables situated close together on the PLS plot are positively correlated, while variables opposite to each other are negatively correlated. The proportion of variance in the X variables (i.e. DOM composition, anions, cations and environmental factors) explained by the model is indicated by the R 2 X. The R 2 Y value indicates the proportion of variance in THg explained by the model. With two

Figure 2 .

 2 Figure 2. Box and whisker plots of the distribution of a) total Hg (THg) in the unfiltered (total) fraction THg T and the filtered (dissolved) fraction THg D (n = 27), b) inorganic Hg (IHg) in the total IHg T and dissolved fractions IHg D (n = 27), c) monomethyl-Hg (MeHg) concentrations (n = 27) in the total (MeHg T ) and dissolved (MeHg D ) fractions and d) the %MeHg (n = 27).Boxes represent 25 th (Q1) and 75 th (Q3) percentile. The median is indicated within the box. The lower whiskers represent the lowest value within 1.5 IQR of the Q1, and the highest within 1.5 IQR of Q3.

Figure 3 .

 3 Figure 3. Excitation-emission matrixes from sites presenting contrasted DOM characteristics a) and b) show samples with an eminently terrestrially derived DOM rich in humic peaks A and C. Plots c) and d) display samples with a marked microbial or algal signature as indicated by the high intensities in the peak T region. Intensities are shown in fluorescence units.

Figure 4 .

 4 Figure 4. Significant (p < 0.05, n=21) correlations between THg T , MeHg T , the %MeHg T , DOM optical properties and stream characteristics. The ellipses have their eccentricity parametrically scaled to the Pearson correlation value (narrower ellipses represent higher correlation values). The orientation of the ellipses indicate negative (red) or positive (blue) correlations (scale on the right).

Figure 5 .

 5 Figure 5. Principal Component Analysis (PCA) of European streams (n = 21) showing the explained variability arising from principle components 1 (PC1) and 2 (PC2) based on bulk optical characteristics of dissolved organic matter (DOM) and environmental variables including stream order and catchment cover, as presented in (a) the loadings plot, and (b) the corresponding scores plot displaying the 21 study sites.

Figure 6 .

 6 Figure6. Partial least squares loadings plot predicting the variability of total mercury (THg) concentration (Y variable, red) with several DOM optical properties and geographical and water chemistry characteristics as predictors (X variables). Fluorescence peaks are expressed as percentage of total fluorescence. Variables in blue were identified to be highly influential predictors of THg (with a variable influence on projection of ≥ 1.0) Graphical Abstract. Conceptual framework of (a) streams characterized by high inputs of terrestrial DOM and high concentrations of THg and (b) streams enriched in microbial/algal DOM depicting high MeHg formation (%MeHg).

  Figure 1 Click here to download high resolution image

  Figure3

  by the median and the inter-quartile range (IQR). DOM optical parameters, MeHg and %MeHg met normality after being log-transformed. Parametric tests were used to analyze linear correlations (Pearson) between DOM optical parameters,

	THg and MeHg concentrations, and %MeHg and to compare Hg concentrations in the total
	and dissolved fractions (t-test). All statistical tests were carried out in R 3.2.4 (http://www.R-
	project.org/) (R Core Team, 2016).
	Principle component analysis (PCA) was used as an initial diagnostic tool to examine
	key relationships and identify strongly correlated variables including optical measurements
	(i.e. %Peak A, %Peak C, %Peak M and %Peak T, SUVA 254 , fluorescence index, freshness

The distribution of THg concentrations were slightly skewed due to the high concentrations measured at sites in Great Britain. The center and dispersion of THg concentrations were summarized index, humification index), TOC, sulfate, nitrate and calcium concentrations, land use and stream order. This complete information was available for 21 of the 29 studied sites.

  Distribution of the 29 sampling sites across nine European countries (see exact coordinates in Table 1).

	Figure 1.
			04	12	1.05	1.2	0.96		0.97	1.6
		AUT1_2	0.8	19	0.82	2.3	0.93		0.95	2.0
		AUT2_1	0.14	38	0.18	21.1	0.52		0.57	9.5
		AUT2_2	0.06	7	0.06	10.4	0.05	8	0.06	13.0
		AUT2_3	0.2	9	0.21	4.4	0.34		0.37	7.0
	Bulgaria	BGR1_1	0.19	24	0.21	11.4	0.26		0.28	9.8
		BGR1_2	0.2	39	0.24	16.4	0.23		0.26	12.0
	Czech Rep	CZE1_1	0.45	53	0.51	10.4	nd	nd	nd	nd
		CZE1_2	0.24	7	0.25	2.8	0.64	112	0.75	15.0
	Germany	DEU1_1	0.17	67	0.23	28.7	0.3		0.31	3.9
		DEU1_2	0.14	36	0.17	20.8	0.34		0.4	15.0
		DEU2_1	0.14	10	0.15	6.7	0.4		0.46	12.2
		DEU2_2	0.11	26	0.13	20.1	0.55		0.6	8.7
	Spain	ESP1_1	0.3	67	0.37	18.2	0.37		0.46	19.3
		ESP1_2	0.22	16	0.24	6.7	0.13		0.14	9.7
		ESP1_3	0.16	122	0.28	43.0	0.21	159	0.37	43.1
		ESP2_1	0.14	38	0.17	21.8	0.1		0.13	23.5
		ESP2_2	0.53	52	0.58	9.1	0.86		0.94	8.1
		ESP2_3	0.5	53	0.56	9.6	0.14		0.2	32.5
	France	FRA1_1	0.47	15	0.48	3.1	0.49		0.51	2.9
		FRA1_2	0.39	17	0.41	4.2	0.86		0.88	2.2
	Great Britain	GBR1_1	2.17	134	2.3	5.8	2.66	124	2.78	4.5
		GBR1_2	nd	nd	nd	nd	2.31		2.33	1.0
		GBR2_1	0.19	45	0.24	19.2	0.33		0.42	20.9
		GBR2_2	0.14	17	0.16	11.1	0.27		0.28	5.4
	Portugal	PRT1_1	0.25	20	0.27	7.5	nd	nd	nd	nd
	Sweden	SWE1_1	nd	nd	nd	nd	0.44		0.53	16.8
		SWE2_1	0.48	135	0.61	21.9	0.7	153	0.86	17.8
		SWE2_2	0.13	26	0.16	16.2	0.15		0.18	16.4

Acknowledgements

KA and PB. The rest of the co-authors carried out the sampling and contributed to the writing of the MS.