Metadata

Description
Natural water samples (16 to 50 L) were collected in Nalgene low density polyethylene carboys at the end of the dry (Mar-Apr) and the wet (Sep-Oct) season of 2002 and 2003 from all LTER sites. They were stored on ice and filtered through pre-combusted GF/F glass fiber filters upon arrival to the lab. The filtrate was processed through a tangential flow ultrafiltration system (TFF) equipped with 0.2um nominal molecular weight cutoff regenerated cellulose membranes, followed by a concentration with 1kDalton molecular weight cutoff membrane while samples were kept in iced water. Following the concentration procedure, the samples were diafiltered three times with 1-L of Milli-Q Water to eliminate salts. The concentrated UDOM samples were then freeze-dried and stored at room temperature in a dessicator. Before fluorescence or SEC-HPLC analysis, samples were re-hydrated with 0.05M Tris(hydroxymethl)aminomethane (THAM) (adjusted to pH at 7.0 with phosphoric acid) to a concentration of 20 mgCL-1 for Florida Bay samples (TS/Ph 9,10,and 11) and 5 mgCL-1 for all other samples.A series of emission fluorescence spectra of the samples were obtained at a ratio mode (emission signal-to-excitation lamp output) from lamda +10nm to lamda + 250nm at 1nm intervals, where lamda is the excitation wavelength. The excitation wavelength was scanned from 260 to 455 nm every 5 nm in a 1 cm quartz fluorescence cell at room temperature (20 degrees C). Fluorescence values were corrected for internal absorbance quenching following the procedures outlined in McKnight et al (2001). The Fluorescence spectra were also corrected for the internal instrument configuration using excitation and emission correction factors for the range of observations supplied by the manufacturer (e.g. Coble 1996; Kowalczuk et al. 2003). Fluorescence was reported as quinine sulfate units according to Maie et al. (2005). MilliQ-water was measured as the blank and subtracted from the sample spectra. UV-visible absorption spectra were measured with a UV-Vis scanning spectrophotometer using a 1 cm quartz cell. The molecular weight distribution of the UDOM was determined by size exclusion chromatography (SEC) (Maie et al. 2004; Scully et al. 2004). Analytical conditions were as follows: column, YMC-Pack Diol-120G (pore size 12 nm, I.D. 8.0mm x length 500mm), eluent, 0.05 M THAM (adjusted to pH at 7.0 with phosphoric acid); flow rate, 0.7 ml min-1; detection, fluorescence emission at 325 nm with an excitation at 280nm; injection volume, 200ul; temperature 22 degrees C. The void volume (VO; 13.8 min.) and void volume plus inner volume (VO+VI; 32.3 min.) were determined using Blue Dextran 2000 and tryptophan, respectively. The column was calibrated with several different dextran standards of know molecular weight. Total organic C and N content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer. Prior to analysis trace amounts of carbonate were removed by exposing the powered UDOM in a silver capsule to hydrochloric acid vapor for 4 hours (Hedges and Stern 1984). The samples were then dried under vacuum to eliminate any residual hydrochloric acid and sealed for later analysis.

Citation
Coble, Paula G 1996. Characterization of marine and terrestrial DOM in seawater using excitation-emission matrix spectroscopy. Marine Chemistry, 51: 325-346.

Instrumentation
Whatman GF/F glass fiber filters Nalgene polyethylene carboys Pellicon 2 Mini Flow Ultrafiltration System Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba Milan Italy).

Description
Natural water samples (16 to 50 L) were collected in Nalgene low density polyethylene carboys at the end of the dry (Mar-Apr) and the wet (Sep-Oct) season of 2002 and 2003 from all LTER sites. They were stored on ice and filtered through pre-combusted GF/F glass fiber filters upon arrival to the lab. The filtrate was processed through a tangential flow ultrafiltration system (TFF) equipped with 0.2um nominal molecular weight cutoff regenerated cellulose membranes, followed by a concentration with 1kDalton molecular weight cutoff membrane while samples were kept in iced water. Following the concentration procedure, the samples were diafiltered three times with 1-L of Milli-Q Water to eliminate salts. The concentrated UDOM samples were then freeze-dried and stored at room temperature in a dessicator. Before fluorescence or SEC-HPLC analysis, samples were re-hydrated with 0.05M Tris(hydroxymethl)aminomethane (THAM) (adjusted to pH at 7.0 with phosphoric acid) to a concentration of 20 mgCL-1 for Florida Bay samples (TS/Ph 9,10,and 11) and 5 mgCL-1 for all other samples.A series of emission fluorescence spectra of the samples were obtained at a ratio mode (emission signal-to-excitation lamp output) from lamda +10nm to lamda + 250nm at 1nm intervals, where lamda is the excitation wavelength. The excitation wavelength was scanned from 260 to 455 nm every 5 nm in a 1 cm quartz fluorescence cell at room temperature (20 degrees C). Fluorescence values were corrected for internal absorbance quenching following the procedures outlined in McKnight et al (2001). The Fluorescence spectra were also corrected for the internal instrument configuration using excitation and emission correction factors for the range of observations supplied by the manufacturer (e.g. Coble 1996; Kowalczuk et al. 2003). Fluorescence was reported as quinine sulfate units according to Maie et al. (2005). MilliQ-water was measured as the blank and subtracted from the sample spectra. UV-visible absorption spectra were measured with a UV-Vis scanning spectrophotometer using a 1 cm quartz cell. The molecular weight distribution of the UDOM was determined by size exclusion chromatography (SEC) (Maie et al. 2004; Scully et al. 2004). Analytical conditions were as follows: column, YMC-Pack Diol-120G (pore size 12 nm, I.D. 8.0mm x length 500mm), eluent, 0.05 M THAM (adjusted to pH at 7.0 with phosphoric acid); flow rate, 0.7 ml min-1; detection, fluorescence emission at 325 nm with an excitation at 280nm; injection volume, 200ul; temperature 22 degrees C. The void volume (VO; 13.8 min.) and void volume plus inner volume (VO+VI; 32.3 min.) were determined using Blue Dextran 2000 and tryptophan, respectively. The column was calibrated with several different dextran standards of know molecular weight. Total organic C and N content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer. Prior to analysis trace amounts of carbonate were removed by exposing the powered UDOM in a silver capsule to hydrochloric acid vapor for 4 hours (Hedges and Stern 1984). The samples were then dried under vacuum to eliminate any residual hydrochloric acid and sealed for later analysis.

Citation
Hedges, J I 1984. Carbon and nitrogen determinations of carbonate-containing solids. Limnology and Oceanography, 29: 657-663.

Instrumentation
Whatman GF/F glass fiber filters Nalgene polyethylene carboys Pellicon 2 Mini Flow Ultrafiltration System Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba Milan Italy).

Description
Natural water samples (16 to 50 L) were collected in Nalgene low density polyethylene carboys at the end of the dry (Mar-Apr) and the wet (Sep-Oct) season of 2002 and 2003 from all LTER sites. They were stored on ice and filtered through pre-combusted GF/F glass fiber filters upon arrival to the lab. The filtrate was processed through a tangential flow ultrafiltration system (TFF) equipped with 0.2um nominal molecular weight cutoff regenerated cellulose membranes, followed by a concentration with 1kDalton molecular weight cutoff membrane while samples were kept in iced water. Following the concentration procedure, the samples were diafiltered three times with 1-L of Milli-Q Water to eliminate salts. The concentrated UDOM samples were then freeze-dried and stored at room temperature in a dessicator. Before fluorescence or SEC-HPLC analysis, samples were re-hydrated with 0.05M Tris(hydroxymethl)aminomethane (THAM) (adjusted to pH at 7.0 with phosphoric acid) to a concentration of 20 mgCL-1 for Florida Bay samples (TS/Ph 9,10,and 11) and 5 mgCL-1 for all other samples.A series of emission fluorescence spectra of the samples were obtained at a ratio mode (emission signal-to-excitation lamp output) from lamda +10nm to lamda + 250nm at 1nm intervals, where lamda is the excitation wavelength. The excitation wavelength was scanned from 260 to 455 nm every 5 nm in a 1 cm quartz fluorescence cell at room temperature (20 degrees C). Fluorescence values were corrected for internal absorbance quenching following the procedures outlined in McKnight et al (2001). The Fluorescence spectra were also corrected for the internal instrument configuration using excitation and emission correction factors for the range of observations supplied by the manufacturer (e.g. Coble 1996; Kowalczuk et al. 2003). Fluorescence was reported as quinine sulfate units according to Maie et al. (2005). MilliQ-water was measured as the blank and subtracted from the sample spectra. UV-visible absorption spectra were measured with a UV-Vis scanning spectrophotometer using a 1 cm quartz cell. The molecular weight distribution of the UDOM was determined by size exclusion chromatography (SEC) (Maie et al. 2004; Scully et al. 2004). Analytical conditions were as follows: column, YMC-Pack Diol-120G (pore size 12 nm, I.D. 8.0mm x length 500mm), eluent, 0.05 M THAM (adjusted to pH at 7.0 with phosphoric acid); flow rate, 0.7 ml min-1; detection, fluorescence emission at 325 nm with an excitation at 280nm; injection volume, 200ul; temperature 22 degrees C. The void volume (VO; 13.8 min.) and void volume plus inner volume (VO+VI; 32.3 min.) were determined using Blue Dextran 2000 and tryptophan, respectively. The column was calibrated with several different dextran standards of know molecular weight. Total organic C and N content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer. Prior to analysis trace amounts of carbonate were removed by exposing the powered UDOM in a silver capsule to hydrochloric acid vapor for 4 hours (Hedges and Stern 1984). The samples were then dried under vacuum to eliminate any residual hydrochloric acid and sealed for later analysis.

Citation
Kowalczuk, P 2003. Characterization of CDOM in an organic-rich river and surrounding coastal ocean in the South Atlantic Bight. Aquatic Science, 65: 384-401.

Instrumentation
Whatman GF/F glass fiber filters Nalgene polyethylene carboys Pellicon 2 Mini Flow Ultrafiltration System Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba Milan Italy).

Description
Natural water samples (16 to 50 L) were collected in Nalgene low density polyethylene carboys at the end of the dry (Mar-Apr) and the wet (Sep-Oct) season of 2002 and 2003 from all LTER sites. They were stored on ice and filtered through pre-combusted GF/F glass fiber filters upon arrival to the lab. The filtrate was processed through a tangential flow ultrafiltration system (TFF) equipped with 0.2um nominal molecular weight cutoff regenerated cellulose membranes, followed by a concentration with 1kDalton molecular weight cutoff membrane while samples were kept in iced water. Following the concentration procedure, the samples were diafiltered three times with 1-L of Milli-Q Water to eliminate salts. The concentrated UDOM samples were then freeze-dried and stored at room temperature in a dessicator. Before fluorescence or SEC-HPLC analysis, samples were re-hydrated with 0.05M Tris(hydroxymethl)aminomethane (THAM) (adjusted to pH at 7.0 with phosphoric acid) to a concentration of 20 mgCL-1 for Florida Bay samples (TS/Ph 9,10,and 11) and 5 mgCL-1 for all other samples.A series of emission fluorescence spectra of the samples were obtained at a ratio mode (emission signal-to-excitation lamp output) from lamda +10nm to lamda + 250nm at 1nm intervals, where lamda is the excitation wavelength. The excitation wavelength was scanned from 260 to 455 nm every 5 nm in a 1 cm quartz fluorescence cell at room temperature (20 degrees C). Fluorescence values were corrected for internal absorbance quenching following the procedures outlined in McKnight et al (2001). The Fluorescence spectra were also corrected for the internal instrument configuration using excitation and emission correction factors for the range of observations supplied by the manufacturer (e.g. Coble 1996; Kowalczuk et al. 2003). Fluorescence was reported as quinine sulfate units according to Maie et al. (2005). MilliQ-water was measured as the blank and subtracted from the sample spectra. UV-visible absorption spectra were measured with a UV-Vis scanning spectrophotometer using a 1 cm quartz cell. The molecular weight distribution of the UDOM was determined by size exclusion chromatography (SEC) (Maie et al. 2004; Scully et al. 2004). Analytical conditions were as follows: column, YMC-Pack Diol-120G (pore size 12 nm, I.D. 8.0mm x length 500mm), eluent, 0.05 M THAM (adjusted to pH at 7.0 with phosphoric acid); flow rate, 0.7 ml min-1; detection, fluorescence emission at 325 nm with an excitation at 280nm; injection volume, 200ul; temperature 22 degrees C. The void volume (VO; 13.8 min.) and void volume plus inner volume (VO+VI; 32.3 min.) were determined using Blue Dextran 2000 and tryptophan, respectively. The column was calibrated with several different dextran standards of know molecular weight. Total organic C and N content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer. Prior to analysis trace amounts of carbonate were removed by exposing the powered UDOM in a silver capsule to hydrochloric acid vapor for 4 hours (Hedges and Stern 1984). The samples were then dried under vacuum to eliminate any residual hydrochloric acid and sealed for later analysis.

Citation
Maie, Nagamitsu 2004. Chemical characteristics and potential source of fulvic acids leached from the plow layer of paddy soil.. Geoderma, 120: 309-323.

Instrumentation
Whatman GF/F glass fiber filters Nalgene polyethylene carboys Pellicon 2 Mini Flow Ultrafiltration System Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba Milan Italy).

Description
Natural water samples (16 to 50 L) were collected in Nalgene low density polyethylene carboys at the end of the dry (Mar-Apr) and the wet (Sep-Oct) season of 2002 and 2003 from all LTER sites. They were stored on ice and filtered through pre-combusted GF/F glass fiber filters upon arrival to the lab. The filtrate was processed through a tangential flow ultrafiltration system (TFF) equipped with 0.2um nominal molecular weight cutoff regenerated cellulose membranes, followed by a concentration with 1kDalton molecular weight cutoff membrane while samples were kept in iced water. Following the concentration procedure, the samples were diafiltered three times with 1-L of Milli-Q Water to eliminate salts. The concentrated UDOM samples were then freeze-dried and stored at room temperature in a dessicator. Before fluorescence or SEC-HPLC analysis, samples were re-hydrated with 0.05M Tris(hydroxymethl)aminomethane (THAM) (adjusted to pH at 7.0 with phosphoric acid) to a concentration of 20 mgCL-1 for Florida Bay samples (TS/Ph 9,10,and 11) and 5 mgCL-1 for all other samples.A series of emission fluorescence spectra of the samples were obtained at a ratio mode (emission signal-to-excitation lamp output) from lamda +10nm to lamda + 250nm at 1nm intervals, where lamda is the excitation wavelength. The excitation wavelength was scanned from 260 to 455 nm every 5 nm in a 1 cm quartz fluorescence cell at room temperature (20 degrees C). Fluorescence values were corrected for internal absorbance quenching following the procedures outlined in McKnight et al (2001). The Fluorescence spectra were also corrected for the internal instrument configuration using excitation and emission correction factors for the range of observations supplied by the manufacturer (e.g. Coble 1996; Kowalczuk et al. 2003). Fluorescence was reported as quinine sulfate units according to Maie et al. (2005). MilliQ-water was measured as the blank and subtracted from the sample spectra. UV-visible absorption spectra were measured with a UV-Vis scanning spectrophotometer using a 1 cm quartz cell. The molecular weight distribution of the UDOM was determined by size exclusion chromatography (SEC) (Maie et al. 2004; Scully et al. 2004). Analytical conditions were as follows: column, YMC-Pack Diol-120G (pore size 12 nm, I.D. 8.0mm x length 500mm), eluent, 0.05 M THAM (adjusted to pH at 7.0 with phosphoric acid); flow rate, 0.7 ml min-1; detection, fluorescence emission at 325 nm with an excitation at 280nm; injection volume, 200ul; temperature 22 degrees C. The void volume (VO; 13.8 min.) and void volume plus inner volume (VO+VI; 32.3 min.) were determined using Blue Dextran 2000 and tryptophan, respectively. The column was calibrated with several different dextran standards of know molecular weight. Total organic C and N content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer. Prior to analysis trace amounts of carbonate were removed by exposing the powered UDOM in a silver capsule to hydrochloric acid vapor for 4 hours (Hedges and Stern 1984). The samples were then dried under vacuum to eliminate any residual hydrochloric acid and sealed for later analysis.

Citation
Maie, Nagamitsu 2005. Chemical characteristics of dissolved organic matter (DOM) in an extremely oligotrophic subtropical wetland.. Limnology and Oceanography, 50: 23-25.

Instrumentation
Whatman GF/F glass fiber filters Nalgene polyethylene carboys Pellicon 2 Mini Flow Ultrafiltration System Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba Milan Italy).

Description
Natural water samples (16 to 50 L) were collected in Nalgene low density polyethylene carboys at the end of the dry (Mar-Apr) and the wet (Sep-Oct) season of 2002 and 2003 from all LTER sites. They were stored on ice and filtered through pre-combusted GF/F glass fiber filters upon arrival to the lab. The filtrate was processed through a tangential flow ultrafiltration system (TFF) equipped with 0.2um nominal molecular weight cutoff regenerated cellulose membranes, followed by a concentration with 1kDalton molecular weight cutoff membrane while samples were kept in iced water. Following the concentration procedure, the samples were diafiltered three times with 1-L of Milli-Q Water to eliminate salts. The concentrated UDOM samples were then freeze-dried and stored at room temperature in a dessicator. Before fluorescence or SEC-HPLC analysis, samples were re-hydrated with 0.05M Tris(hydroxymethl)aminomethane (THAM) (adjusted to pH at 7.0 with phosphoric acid) to a concentration of 20 mgCL-1 for Florida Bay samples (TS/Ph 9,10,and 11) and 5 mgCL-1 for all other samples.A series of emission fluorescence spectra of the samples were obtained at a ratio mode (emission signal-to-excitation lamp output) from lamda +10nm to lamda + 250nm at 1nm intervals, where lamda is the excitation wavelength. The excitation wavelength was scanned from 260 to 455 nm every 5 nm in a 1 cm quartz fluorescence cell at room temperature (20 degrees C). Fluorescence values were corrected for internal absorbance quenching following the procedures outlined in McKnight et al (2001). The Fluorescence spectra were also corrected for the internal instrument configuration using excitation and emission correction factors for the range of observations supplied by the manufacturer (e.g. Coble 1996; Kowalczuk et al. 2003). Fluorescence was reported as quinine sulfate units according to Maie et al. (2005). MilliQ-water was measured as the blank and subtracted from the sample spectra. UV-visible absorption spectra were measured with a UV-Vis scanning spectrophotometer using a 1 cm quartz cell. The molecular weight distribution of the UDOM was determined by size exclusion chromatography (SEC) (Maie et al. 2004; Scully et al. 2004). Analytical conditions were as follows: column, YMC-Pack Diol-120G (pore size 12 nm, I.D. 8.0mm x length 500mm), eluent, 0.05 M THAM (adjusted to pH at 7.0 with phosphoric acid); flow rate, 0.7 ml min-1; detection, fluorescence emission at 325 nm with an excitation at 280nm; injection volume, 200ul; temperature 22 degrees C. The void volume (VO; 13.8 min.) and void volume plus inner volume (VO+VI; 32.3 min.) were determined using Blue Dextran 2000 and tryptophan, respectively. The column was calibrated with several different dextran standards of know molecular weight. Total organic C and N content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer. Prior to analysis trace amounts of carbonate were removed by exposing the powered UDOM in a silver capsule to hydrochloric acid vapor for 4 hours (Hedges and Stern 1984). The samples were then dried under vacuum to eliminate any residual hydrochloric acid and sealed for later analysis.

Citation
McKnight, D M 2001. Spectrofluorometric characterization of dissolved organic matter for the identification of percursor organic material and aromaticity.. Limnology and Oceanography, 46: 38-48.

Instrumentation
Whatman GF/F glass fiber filters Nalgene polyethylene carboys Pellicon 2 Mini Flow Ultrafiltration System Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba Milan Italy).

Description
Natural water samples (16 to 50 L) were collected in Nalgene low density polyethylene carboys at the end of the dry (Mar-Apr) and the wet (Sep-Oct) season of 2002 and 2003 from all LTER sites. They were stored on ice and filtered through pre-combusted GF/F glass fiber filters upon arrival to the lab. The filtrate was processed through a tangential flow ultrafiltration system (TFF) equipped with 0.2um nominal molecular weight cutoff regenerated cellulose membranes, followed by a concentration with 1kDalton molecular weight cutoff membrane while samples were kept in iced water. Following the concentration procedure, the samples were diafiltered three times with 1-L of Milli-Q Water to eliminate salts. The concentrated UDOM samples were then freeze-dried and stored at room temperature in a dessicator. Before fluorescence or SEC-HPLC analysis, samples were re-hydrated with 0.05M Tris(hydroxymethl)aminomethane (THAM) (adjusted to pH at 7.0 with phosphoric acid) to a concentration of 20 mgCL-1 for Florida Bay samples (TS/Ph 9,10,and 11) and 5 mgCL-1 for all other samples.A series of emission fluorescence spectra of the samples were obtained at a ratio mode (emission signal-to-excitation lamp output) from lamda +10nm to lamda + 250nm at 1nm intervals, where lamda is the excitation wavelength. The excitation wavelength was scanned from 260 to 455 nm every 5 nm in a 1 cm quartz fluorescence cell at room temperature (20 degrees C). Fluorescence values were corrected for internal absorbance quenching following the procedures outlined in McKnight et al (2001). The Fluorescence spectra were also corrected for the internal instrument configuration using excitation and emission correction factors for the range of observations supplied by the manufacturer (e.g. Coble 1996; Kowalczuk et al. 2003). Fluorescence was reported as quinine sulfate units according to Maie et al. (2005). MilliQ-water was measured as the blank and subtracted from the sample spectra. UV-visible absorption spectra were measured with a UV-Vis scanning spectrophotometer using a 1 cm quartz cell. The molecular weight distribution of the UDOM was determined by size exclusion chromatography (SEC) (Maie et al. 2004; Scully et al. 2004). Analytical conditions were as follows: column, YMC-Pack Diol-120G (pore size 12 nm, I.D. 8.0mm x length 500mm), eluent, 0.05 M THAM (adjusted to pH at 7.0 with phosphoric acid); flow rate, 0.7 ml min-1; detection, fluorescence emission at 325 nm with an excitation at 280nm; injection volume, 200ul; temperature 22 degrees C. The void volume (VO; 13.8 min.) and void volume plus inner volume (VO+VI; 32.3 min.) were determined using Blue Dextran 2000 and tryptophan, respectively. The column was calibrated with several different dextran standards of know molecular weight. Total organic C and N content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer. Prior to analysis trace amounts of carbonate were removed by exposing the powered UDOM in a silver capsule to hydrochloric acid vapor for 4 hours (Hedges and Stern 1984). The samples were then dried under vacuum to eliminate any residual hydrochloric acid and sealed for later analysis.

Citation
Scully, N M 2004. Early diagenesis of plant derived dissolved organic matter along a wetland, mangrove, estuary ecotone.. Limnology and Oceanography, 49: 1667-1678.

Instrumentation
Whatman GF/F glass fiber filters Nalgene polyethylene carboys Pellicon 2 Mini Flow Ultrafiltration System Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba Milan Italy).