Sources of mercury in deep-sea sediments of the Mediterranean Sea as revealed by mercury stable isotopes

Mercury (Hg) and its stable isotope composition were used to determine the sources of Hg in deep-sea sediments of the Mediterranean Sea. Surface and down-core sediment δ202Hg values varied widely between −2.30 and +0.78‰, showed consistently positive values for mass independent fractionation of odd Hg isotopes (with average values of Δ199Hg = +0.10 ± 0.04‰ and Δ201Hg = +0.04 ± 0.02‰) and near-zero Δ200Hg values, indicating either multiple Hg sources or a combination of different Hg isotope fractionating processes before and after sediment deposition. Both mass-dependent and mass-independent fractionation processes influence the isotopic composition of Hg in the Mediterranean Sea. Positive Δ199Hg values are likely the result of enhanced Hg2+ photoreduction in the Mediterranean water column before incorporation of Hg into sediments, while mass-dependent fractionation decreases δ202Hg values due to kinetic isotope fractionation during deposition and mobilization. An isotope mixing model based on mass-dependent and mass-independent fractionation (δ202Hg and Δ199Hg) suggests at least three primary Hg sources of atmospheric deposition in the surface sediments: urban, industrial and global precipitation-derived. Industry is the main source of Hg in Algerian and Western Basin surface sediments and at two sites in the Adriatic Sea, while the urban contribution is most prominent at the Strait of Otranto (MS3) and in Adriatic surface sediments. The contribution from precipitation ranged from 10% in Algerian to 37% in W Basin sediments. Overall, results suggest that atmospheric Hg deposition to Mediterranean surface sediments is dominated by gaseous elemental mercury (58 ± 11%) rather than wet deposition.

often characterized by positive Δ 199 Hg [9][10][11] . In a similar fashion, MDF can improve our understanding of the processes that control Hg distribution and bioaccumulation.
Marine sedimentary Hg is often dominated by Hg of geogenic origin, but have also shown to retain anthropogenic contributions in surface layers 12,13 , while aqueous Hg is more exposed to photochemical and microbial reactions. Foucher et al. 12 , demonstrated that there is a well-resolved difference between the Hg isotopic composition of Adriatic Sea sediment (depleted in 202 Hg) and Hg in cinnabar derived originally from the Idrija region (enriched in 202 Hg), which drains into the Gulf of Trieste. On the other hand, no difference in Hg isotopic composition was observed between sapropels and historic sediments despite the 6-fold difference in Hg concentration in deep-sea Tyrrhenian Basin sediments 13 . This similarity may suggest that the marine sediments reflect the Hg isotopic composition of ambient seawater, or conversely may indicate that the isotopic composition of Hg in the ocean reflects that of Hg in the upper crust. Atmospheric deposition is the largest source of Hg to the global oceans. Pre-industrial atmospheric Hg originated primarily from volcanic and hydrothermal emissions. Recent measurements of volcanic and sedimentary deposits in California showed Hg isotopic compositions close to values reported for Pleistocene Mediterranean seawater 14 .
The aim of the present study is to investigate potential sources of Hg in Mediterranean deep-sea and Adriatic sediments. To our knowledge only a few studies report stable isotope compositions of Hg in deep-sea sediments and most of them were conducted close to estuaries or coastal region [15][16][17][18] . Ogrinc et al. 19 previously determined concentrations of Hg in sediments that were further characterized by C and N concentrations and isotopic composition. In this study, stable isotope ratios of Hg were utilized to better understand Hg sources and processing pathways in marine deep-sea sediments including depth profiles. Specifically, we suggest that the determination of δ 202 Hg, and Δ 199 Hg values in sediments can be used to develop a triple mixing model that provides estimates of the relative amounts of Hg derived from urban, industry and global atmospheric deposition.

Results and Discussion
Hg and its isotope composition in surface sediments. Organic carbon content (OC), concentrations and stable isotope data of Hg at selected locations in deep-sea sediments in Mediterranean and Adriatic Sea ( Fig. 1) are collected in Table 1. If we take into account sedimentation rates between 0.012 and 0.024 cm yr −1 20 determined in the Western and 0.003 cm yr −1 in the Eastern Basin 21 , only the first few cm correspond to the industrial period and have therefore the potential to show an influence of anthropogenic sources. The spatial variation of total Hg (HgT) in surface sediments ranged from 14 (MS2) to 153 ng g −1 (MS3 and MS4) in the Mediterranean and Adriatic Seas. HgT concentrations were normalized to OC content, because Hg often correlates closely with OC phases in sediments 22 . The degree of Hg enrichment relative to background values was defined as the enrichment factor (EF = (HgT/OC) sample /(HgT/OC) background ) , where the HgT background concentrations were taken from the average determined for the Levantine Sea sediments (MS2). The highest EF factor was observed at the surface ranging from 1.1 to 2.7 at AS1 and MS3, respectively. The EF > 1.5 at the surface sediments was observed at MS3, MS4, MS6 and AS3 indicating minor enrichment with Hg.
Measured δ 202 Hg values in surface sediments were variable and ranged from −2.30 at MS3 to −0.78‰ at MS6. The measured δ 202 Hg values fall between the average δ 202 Hg of −0.76 ± 0.16‰ established for background Mediterranean Sea sediment 14 and the average δ 202 Hg of −2.13 ± 0.46‰ for background Adriatic Sea sediments 12 . Positive MIF of odd Hg isotopes ( 199 Hg and 201 Hg) was observed in surface sediments with average values of Δ 199 Hg = +0.10 ± 0.04‰ and Δ 201 Hg = +0.04 ± 0.02‰ (Fig. 2). Similar Δ 199 Hg values of +0.10‰ were reported for modern Pacific deep-sea sediments 23 , Archean marine black shales (+0.15‰ 24 ,) and the South    www.nature.com/scientificreports www.nature.com/scientificreports/ China Sea (mean, +0.35 ± 0.09‰ 15 ,). The observed positive MIF in sediments could be a result of Hg settling from the water column, considering that photoreduction of Hg(II) typically leads to 199 Hg enrichment in the aqueous phase 6 . Another possible explanation for the observed positive Δ 199 Hg values could be atmospheric Hg deposition. A recent mass balance for the Mediterranean Sea suggested that as much as 45% of the Hg input is by atmospheric deposition 2 . This would be in accordance with global Hg rainfall observations, which display positive Δ 199 Hg (+0.37 ± 0.25‰, 1σ, n = 105) 11 . Positive ∆ 199 Hg values (0 to +1.0‰) have been reported for precipitation collected from different sites of the world 9,25-28 . In general, negative Δ 199 Hg values were reported in gaseous elemental Hg (GEM), while positive Δ 199 Hg values were reported in gaseous oxidized Hg (Hg 2+ ) and particulate/aerosol-bound Hg (Hg p ) species 7,26,29 . In this study, no MIF was observed for 200 Hg, which is consistent with previous studies in ocean sediments 12,14,22 . Although ∆ 200 Hg values different from 0 was frequently reported for atmospheric samples, ∆ 200 Hg values of 0 in sediments might be explained by the mixing of gaseous elemental mercury (GEM, with negative Δ 200 Hg values) with oxidized atmospheric Hg species in precipitation (with positive Δ 200 Hg values). A Δ 200 Hg isotopic mass balance using the above global rainfall end-member (+0.18 ± 0.15‰, 1σ, n = 105) and global GEM end-member (Δ 200 Hg = −0.05 ± 0.04‰, 1σ, n = 69) would suggest that 59 ± 11% (1σ) of Mediterranean surface sediment Hg was ultimately derived from GEM dry deposition, resulting in a net zero Δ 200 Hg.
The data from the Levantine basin (MS2) may represent the background of Mediterranean deep-sea sediments, having HgT concentrations as low as 14 ng g −1 . No influence of direct continental and/or volcanic sources could be identified, which is further supported by the sediment δ 13 C OC value of −21.6‰ 19 . The large amount of Ca found at MS2 reflects the presence of high concentrations of CaCO 3 , normally associated with biogenic pelagic deep-sea sediments (foraminiferal oozes).
At MS4, MS5 and AS2 the more positive δ 202 Hg values were associated with higher Hg concentrations, which may suggest a recent Hg source. Other studies suggested that industrial sources exhibit δ 202 Hg values closer to zero (−1 to 0‰) and insignificant MIF (Δ 199 Hg ∼ 0‰) 30,31 . The lowest δ 202 Hg value of −2.30‰ was observed at MS3 and is more similar to δ 202 Hg values (δ 202 Hg: −2 to −3‰) found in precipitation and atmospheric samples impacted by anthropogenic Hg emissions 12,15 . A recent study indicated that Hg in sediment at the Strait of Otranto (MS3) might originate from particulate Hg transported by the water currents from the Adriatic Sea 32 .
More negative δ 202 Hg values in the study area may also be explained by inputs from two others possible sources often characterized by low δ 202 Hg: (1) Fig. 3. Increasing concentrations of HgT were occasionally determined deeper in the cores, which may reflect changes or redistribution during diagenetic processes. In the Eastern Basin, the origin of the deeper HgT concentration variations could also be a consequence of natural variability caused by sea-level fluctuations or seismic activity. Down-core δ 202 Hg values do not show a clear pattern and were site specific implying either multiple sources, or varying amounts of microbial Hg reduction and loss, or a combination of both (Fig. 3).
The HgT concentrations at MS1 show an increase with depth together with a decrease in δ 202 Hg from −1.28 to −2.18‰ (Fig. 3). Assuming a sedimentation rate of 0.003 cm yr −1 for the Eastern Basin 21 , sediments at >5 cm depths likely correspond to AD 300 or older. Thus, these layers are pre-modern sediments and if the isotope signature was related to sources, the Hg may only originate from volcanic and/or hydrothermal emissions. More specifically, the low δ 202 Hg values could possibly reflect the isotope composition of Hg in ash originating from intensive volcano activity. This interpretation is further supported by previous studies indicating that these sediments contain the markers (tephroanalysis) of well-known historical eruptions (Pompei, AD 79, Pollena, AD 472, Ischia, AD 1301, dates that are all captured within the top 5 cm) 39  www.nature.com/scientificreports www.nature.com/scientificreports/ characterized. Nevertheless, the observed positive Δ 199 Hg data are broadly consistent with Hg as a product of photoreduction 5,43 .
In all other cores (MS3 to MS6) a decrease in Hg concentrations was observed with depth, while no clear pattern in δ 202 Hg values was discernible (Fig. 3). Low δ 202 Hg values of ∼ −2‰ were observed also deeper in the sediments at MS3 and MS5 (Fig. 3). Environmental processes such as microbial reduction 44 , photoreduction 3,44 , photodemethylation 3,44 , methylation 45 and evasion 46,47 always show a preferential loss of the lighter Hg isotope leaving the study system and the residual fraction enriched in the heavier isotope. On the other hand, mobilization causes the enrichment of Hg with the light isotope and cannot be excluded. The diagenetic remobilization It should be also mentioned that more negative δ 202 Hg values are usually associated with the highest ∆ 199 Hg values with a significant negative correlation of −14.28 (P < 0.001) (Fig. 4). Laboratory experiments on Hg 2+ photoreduction revealed δ 202 Hg/∆ 199 Hg of 0.83 49 , while for Tibetan Lakes the ratio was much higher with δ 202 Hg/∆ 199 Hg = 8.88 and 5.75 50 . Further no correlation between ∆ 199 Hg and HgT (P > 0.05), was observed, which may suggest that Mediterranean sediments may be more influenced by in-ocean processes. The increase of Δ 199 Hg is likely the result of enhanced Hg 2+ photoreduction in the Mediterranean water column before Hg is incorporated into sediments. For example, the importance of atmospheric transformation processes occurring in the marine boundary layer (MBL) may vary due to varying meteorological and climatic conditions in the Mediterranean Basin (i.e. warmer climate, high temperature and strong solar radiation) 1 , having a more or less pronounced effect on the fractionation of Hg isotopes. Possibly, (photo)processes created initially positive MIF and MDF, but subsequent stronger MDF-only reactions led to overall negative δ 202 Hg values, while conserving the initial Δ 199 Hg signature. This MDF fractionation can occur during settling or after sedimentation. Until now, the only known process generating a negative shift in δ 202 Hg is photoreduction of Hg complexed by thiols or Fe, Mn oxyhydroxides. Significant δ 202 Hg shifts are more likely to occur when only very small fractions of Hg are adsorbed relative to the total Hg in the system, which is actually the case in Mediterranean Sea 1,2 . Thus, further research on water column Hg processes in the Mediterranean Sea are needed to better understand the variations of Hg isotopes in this study.

Source apportionment model for mediterranean and adriatic surface sediments. Binary
and/or triple mixing models have previously been employed to estimate the relative contribution of sources of Hg 12,13,16,31,37,[51][52][53][54] . In this study, we propose that the relationship between concentrations and stable isotope data of Hg in Mediterranean and Adriatic surface sediments can be modelled using three sources, all three derived as emissions from the atmosphere (Fig. 4): industrial Hg (δ 202 Hg, −0.40‰; Δ 199 Hg, 0.05‰), urban Hg (δ 202 Hg, −2.23‰; Δ 199 Hg, 0.05‰), and global precipitation Hg (δ 202 Hg, −0.56 ± 0.24‰; Δ 199 Hg, +0.42 ± 0.25‰). We applied an adsorption shift of −0.6‰ 33 for our end-members, as a result of aquatic sediments particle adsorption during deposition, overall deposition shift of 0.4‰ and 0.05‰ for δ 202 Hg and Δ 199 Hg, respectively, as determined by Archer and Blum 55 . Fig. 4 also shows the global terrestrial end-member with δ 202 Hg of −1.3 ± 0.8‰ (1σ, n = 162) and Δ 199 Hg of −0.2 ± 0.2‰ (1σ, n = 163) 7,11,56 . It is evident that the terrestrial input was not a good fit as a potential source of Hg, which is also supported by recent mass balance for the Mediterranean Sea, estimating that rivers contribute only 14% of the total Hg inputs 2 . We are aware that these values may only be crude approximations at this point and the composition of Hg deposited onto sediments is likely a complex end product of a series of processes and pathways including photoreduction and photodemethylation, which influence Δ 199 Hg values post deposition/input to oceans.
Taking into account all three end-members the following ternary mixing model was used: where subscripts "glob", "ind", "urb" and "sam" are related to global precipitation, industrial, urban and sample, respectively. The uncertainty of the extracted F parameters was determined using stochastic Monte Carlo simulations. For each experimental parameter δ 202 Hg x and Δ 199 Hg x a normal distribution was created characterized by its mean and standard deviation values. For all parameters (i.e. δ 202 Hg glob , δ 202 Hg ind , δ 202 Hg urb , δ 202 Hg samp , Δ 199 Hg glob , …) a random value was later calculated from the corresponding distribution. These values were used to solve the equations (1-3). The results of different source contributions with standard deviation of calculated F numbers are presented in Table 1. We also estimate the strongest influence of experimental parameters on the extracted F parameters and found out that δ 202 Hg urb and Δ 199 Hg urb have the highest effect on all calculations. The model suggests that Hg pollution from the industry represents the main source of Hg at the surface at MS4, MS6, AS2 and AS3, while the urban contribution was the highest at MS3 and AS1. The contribution from global precipitation ranged from 10% at MS3 to 37% at MS6. We acknowledge the uncertainty of this model, as exact end-members have not been directly measured. However, the results represent the first rough estimates of the proposed sources to overall Hg distribution in surface deep-sea sediments in the Mediterranean and Adriatic Sea. It still contains several simplifications that require refinement in further studies. For example, the end members of the model are currently based only on a small dataset, relative to the size of the study area. Especially the high uncertainty for Hg isotope signatures in global precipitation should be addressed. Consequently, the model is to be used with caution, especially in the open ocean where secondary fractionation processes may impact the original source signatures. Addressing these uncertainties will strengthen the use of Hg isotope ratios as a tool to attribute sources of Hg in a complex system such as the Mediterranean Sea and eventually also other world oceans.

Materials and Methods
Study area and sampling. A site description and sampling protocol employed in collection of the samples studied here are provided in Ogrinc et al. 19 . Briefly, sediment samples were collected with a box corer from six sites during an oceanographic sampling campaign aboard the Italian research vessel Urania in August 2003 in the Eastern and Western basins of the Mediterranean and from four sites in October-November 2004. In addition, surface sediments were collected from four Adriatic sites, while profiles of up to 10 cm depths were taken from Mediterranean Sea locations. Sampling locations are presented in Fig. 1. organic carbon (oc) content. OC content in sediments was determined by a Carlo Erba elemental analyzer (model EA 1108) after acidification with 1 N HCl to remove carbonate material and is expressed in wt.%. The precision of measurements was ± 3%.
Mercury isotope ratio analysis. Typically, 0.2 g of dried sediment was digested with 10 ml of a concentrated acids mixture (HNO 3 /HCl, 7:3 v/v) in open glass vessels. Digestion was performed on a hot plate at 120-140 °C for approx. 6 h and then diluted to 40 ml with Milli-Q water. Where necessary, the mass of sediment was increased to achieve a final concentration of Hg of at least 1 µg l −1 . Concentrations for the bracketing standard (NIST 3133) were adjusted to match Hg concentrations in sediment digests to within 10%. Hg isotopic compositions were determined using a continuous Hg° vapour generation method and analysis using a multiple-collector inductively coupled plasma mass spectrometer (MC−ICP/MS) equipped with nine Faraday cups (Neptune, Thermo Fisher Scientific, Bremen, Germany). A more detailed description of the overall instrumental setup and analytical conditions used in this study can be found elsewhere 12,57 . Results for Hg isotope ratios are reported as the deviation from a common Hg standard solution (NIST 3133 Hg) using the customary δ-notation expressed in per mil (‰) 58  where β is the scale factor of the theoretical MDF law and is equal to 0.2520 for 199 Hg, 0.5024 for 200 Hg, and 0.7520 for 201 Hg 58 . Data uncertainties reported in this study reflect the larger values of either the external precision of the replication of the UM-Almadeń standard solution or the measurement uncertainty of repeated sample analysis. The overall measured average and uncertainty for UM-Almadeń was δ 202 Hg = −0.52 ± 0.09‰; Δ 199 Hg = −0.01 ± 0.05‰; Δ 200 Hg = 0.00 ± 0.03‰; and Δ 201 Hg = 0.00 ± 0.05‰, for 2σ level. These results agreed well with previous studies 58 .