Image analysis from TEM and AFM micrograph, Table S1, S2, S3, Figure S1, Figure S2

We report on metal enrichment along a natural pH gradient owing to increased CO2 degassing at cold, shal- low seeps of Vulcano Island in the Mediterranean Sea, off Sicily. We assessed composition of unfiltered and filtered seawater (b100 nm) along acidic zones ranging between ambient and pH 5, and showed that most seep derived elements are present as nanoclusters which then aggregate into larger colloids while mixing with ambient seawater along a pH gradient. Size and elemental composition of such naturally occurring nanoparticles assessed by modern characterisation methods were in good agreement with the results from conventional analytical methods. We provide analytical evidence for the presence in the water column of a large fraction of seep derived ele- ments (e.g. approximately 50% of iron, over 80% of Mn, 100% of Cr, S and Zn) in the form of nano sized par- ticles (e.g. b100 nm) even at typical open ocean pHs. We launch in situ sampling protocols and sample preparation procedures for multi-method suitable to obtain accurate measurements on nanoparticles from environmental samples. Based on our results a first insight to the formation of natural nanoparticles at cold CO2 seeps is presented and the persistence of such nano-clusters in the surrounding seawater is stipulated.

(Table S3) Benthic foraminiferal Mg/Ca, Li/Ca and stable isotope ratios of ODP Site 122-761

The sensitivities of benthic foraminiferal Mg/Ca and Li/Ca to bottom water temperature and carbonate saturation state have recently been assessed. Here we present a new approach that uses paired Mg/Ca and Li/Ca records to calculate simultaneous changes in temperature and saturation state. Using previously published records, we first use this approach to document a cooling of deep ocean waters associated with the establishment of the Antarctic ice sheet at the Eocene-Oligocene climate transition. We then apply this approach to new records of the Middle Miocene Climate Transition from ODP Site 761 to estimate variations in bottom water temperature and the oxygen isotopic composition of seawater. We estimate that the oxygen isotopic composition of seawater varied by ~1 per mil between the deglacial extreme of the Miocene Climatic Optimum and the glacial maximum following the Middle Miocene Climate Transition, indicating large amplitude variations in ice volume. However, the longer-term change between 15.3 and 12.5 Ma is marked by a ~1°C cooling of deep waters, and an increase in the oxygen isotopic composition of seawater of ~0.6 per mil. We find that bottom water saturation state increased in the lead up to the Middle Miocene Climate Transition and decreased shortly after. This supports decreasing pCO2 as a driver for global cooling and ice sheet expansion, in agreement with existing boron isotope and leaf stomatal index CO2 records but in contrast to the published alkenone CO2 records.