1. LIFE12 ENV/IT/352 Determination of trace elements in commercial dyes, crude chromophore, purified dyes and naturalized dyes Alessandro D’Ulivo BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I) Action C5 - Monitoring of the benefits from dyeing leather with naturalised colorants.

2. LIFE12 ENV/IT/352 Crude Chromophore Commercial Dye [Crude Chromophores + Additives ] Purified Chromophore [ From commercial Dyes ] Naturalized Dyes [ from Purified Dyes] YELLOW Disperse Yellow 42 Teratop Brown GWL Disperse Yellow 42 DY42 Nat ORANGE Disperse Orange 30 Foron Brown Yellow S-2RFL 150 Disperse Orange 30 D030 Nat RED Disperse Red 202 Foron Brilliant Red S-RGL 200 Disperse Red 202 DR202 Nat BLUENA* Teratop Blue GLF Disperse Blue 27DB27 Analized Dyes & Chromophore BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I) (*) Not commercially available

3. Trace element Determination Pressurized MW Digestion, HNO 3 + H 2 O 2 Determination by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) Perkin Elmer Optima 8000 Elements: Hg, Cd, Pb, Tl Mn, Cr, V, Co, Ni, Cu, Zn B, As, Sb, Se Be, Ba Al, Fe LIFE12 ENV/IT/352 BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I)

4. Direct Determination of Mercury Direct determination of Hg in solid and liquid samples Digestion not required No Hg losses No Hg contamination Typical Sample size 100 - 500 mg L.O.Q < 20 pg Hg (< 0.2 ppb) DMA-80: fully compliant with US EPA method 7473 and with ASTM method D-6722-01. Mercury Determination at sub p.p.b. Level LIFE12 ENV/IT/352 BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I)

5. Direct Determination of Mercury Results LIFE12 ENV/IT/352 Detected Elements in at least one of the Dyes Hg, Se, Pb, Cr, Cd, Pb, V, Be, Ni, Mn, Zn, Al, Cu, Fe, B, Ba Above Limit of quantification L.O.Q. = 10 s Elements Non-Detected in any dyes Tl  0.17 ; Co  0.17 (mg/kg) Below the reported L.O.Q. Indicators of potential hazard level BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I)

6. Commercial Dyes Commercial «Crude» Chromophores Naturalised Dyes Purified Chromophores Additives Lactose LIFE12 ENV/IT/352 Soxhlet Extraction BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I)

7. Commercial DyesCrude ChromophoresLOQ m Element Teratop Brown GWL (mg/kg) Foron Brown Yellow S-2RFL150 (mg/kg) Foron Brilliant Red S-RGL200 (mg/kg) Teratop Blue GLF (mg/kg) Disperse Yellow 42 (mg/kg) Disperse Orange 30 (mg/kg) Disperse Red 202 (mg/kg) (mg/kg) Al 32.749.847.626.164.3--0.83 As 8.1-a7.5-0.94--0.83 B ---64.13.6-29.00.83 Ba 0.840.634.83.01.7--0.02 Be -----0.30-0.07 Cd 0.100.13--0.15--0.08 Co -------0.17 Cr -3.83.32.80.59 0.600.17 Cu ---11.40.40 1.40.33 Fe 21.549.944.5112.429.21.833.70.08 Hg 0.00700.460.00170.660.0600.0100.090 8.5  10 -5 Mn 11.218.312.959.10.910.250.340.08 Ni -2.4 2.10.42- Pb ---3.52.01.51.30.83 Sb 2.11.9-----1.70 Se 3.92.22.0-2.4 -1.70 Tl -------1.70 V 9.814.322.11.80.731.1 0.42 Zn 2.9--10.64.60.621.60.17 Total b 97.7148.6147.1299.4114.813.275.1[10.4] c a - concentrations that are not reported are below LOQ m. b - sum of concentrations includes LOD m whenever the determined values cannot be quantified. c - sum of LOQ m. LIFE12 ENV/IT/352 BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I)

8. LIFE12 ENV/IT/352 Purified Chromophores from Commercial Dyes Naturalised DyesLOQ m Element Disperse Yellow 42 (mg/kg) Disperse Orange 30 (mg/kg) Disperse Red 202 (mg/kg) Disperse Blue 27 (mg/kg) DY42 Nat (mg/kg) D030 Nat (mg/kg) DR202 Nat (mg/kg) DB27 Nat (mg/kg) (mg/kg) Al-----0.906.6-0.83 As8.2-3.9-----0.83 B-------- Ba0.37----0.030.170.190.02 Be----0.310.290.27-0.07 Cd0.13-------0.08 Co--------0.17 Cr---1.10.850.454.52.10.17 Cu---1.0----0.33 Fe---2.01.71.049.72.10.08 Hg0.000150.00120.00010.00300.0100.0200.0100.020 8.5  10 -5 Mn-----0.190.570.250.08 Ni---0.50--2.11.20.42 Pb---3.72.2---0.83 Sb3.71.7- --- 1.70 Se6.8-------1.70 Tl--------1.70 V----1.21.31.21.00.42 Zn---1.717.72.86.415.50.17 Total b 25.310.412.617.631.013.071.497.7[10.4] c a - concentrations that are not reported are below LOQ m ; b - sum of concentrations includes LOD m whenever the determined values cannot be quantified; c - sum of LOQ m BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I)

9. LIFE12 ENV/IT/352 Non quantifiable Elements (Conc ≤ LOQ m ) BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I)

10. Direct Determination of MercuryTotal Element Content LIFE12 ENV/IT/352 BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I)

11. Direct Determination of Mercury Mercury LIFE12 ENV/IT/352 BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I)

12. LIFE12 ENV/IT/352 In general total elements content follow the order: Commercial Dyes > Crude Chromophore ≥ Naturalized Dyes > Purified Dyes Similar trend is found for mercury. Contamination of commercial dyes can be addressed to the use of additives with low degree of purity. Naturalized dyes, presents some problems of contamination by Zn, Fe and Al, probably arising from the apparatus employed in synthesis/purification. Conclusion BIONAD 24 Month Meeting January 27th, 2016, UNIFI Premises, Florence (I)