ISECNR activity: October 2015- March 2017 Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production ISECNR activity: October 2015- March 2017 B1. Analysis and characterization of remediated sediments (responsible) 01/01/2016-30/06/2016 .ISECNR involvement: Physical, chemical and biological characterization of sediments (as reported in the proposal) B2. Sediment treatment 01/01/2016-30/09/2016 .ISECNR involvement: Sediment landfarming C1. Monitoring and validation of treated sediments (during landfarming) 01/10/2015-30/06/2016 .ISECNR involvement: Physical, chemical and biological characterization of sediments (as reported in the proposal). C2. Monitoring and validation of the use of remediated sediments for horticultural production 01/04/2016-30/09/2017 .ISECNR involvement: chemical and biochemical characterization of the substrates (not planned) Firenze 30-31 March 2016

Site preparation: 9 December 2015 Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production B1 Analysis and characterization of remediated sediments B2 Sediment treatment Site preparation: 9 December 2015 Plant removal Previous Project:AGRIPORT

B2 Sediment treatment Landfarming Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production B2 Sediment treatment Landfarming Landfarming is a bioremediation technology in which contaminated sediment is spread on the upper soil zone or in biotreatment cells, mixed with nutrients and the material is periodically irrigated and aerated. LANDFARMING Contaminated sediment Organic contaminants are degraded by microorganisms and used as carbon source for growth and energy.

B2 Sediment treatment: Landfarming to: Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production B2 Sediment treatment: Landfarming to: -homogenize the substrate -increase the biological activities and as, a consequence, -further reduce the organic contamination Periodical (once per week) aeration by mechanically moving the sediments and turning them over : 3 months planned Covering during raining days

C1. Monitoring and validation of treated sediments Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production C1. Monitoring and validation of treated sediments 3 sampling points in about 40m2: A, B, C of about 1 m2 each 10 sub samples for each sampling point The results are the average between samples A, B and C A B C Campaign of sampling: T0, starting characterization (9 December 2015) T1, first sampling (13 January 2016) T2, second sampling (10 February 2016) T3, third sampling (10 March 2016)

Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production B1-C1 Action Sediment characterization and monitoring of treated sediments Parameters Physical (starting and end of experimentation) Texture, Bulk density Chemical (at each sampling time ) pH, Electrical Conductivity, Nutrients Total content and Availability, Microelements, Cation Exchange Capacity Water soluble carbon, Humic substances and degree of humification Biochemical: Enzyme activities: -Hydrolytic enzymes: b-glucosidase, phosphatase, sulfatase, Ossidoreductase: Dehydrogenase Toxicological analysis: -Total heavy metals and available heavy metals (Hg, Zn, Cd, Pb, Cu, Cr, Ni) - C<12, C>12, PAHs, PCBs, starting and end of experimentation -Ecotoxicological test (DISPAA) Plant analysis: Growth, heavy metal concentration (Hg, Zn, Cd, Pb, Cu, Cr, Ni) in roots, stems and leaves change slowly (static conditions) Very sensitive (dynamic conditions)

Decrease in C, N e P Organic matter mineralization Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production B1 and C1 Characterization and Monitoring: Main results Chemical analysis Bulk density from 1.29 to 1.08 g/cm3 No variations during the time Little decrease due to loss of salt with rain Slight increase: increase in the amount of exchange sites, probably for the qualitative changes in soil organic matter To: 9 December 2015 T3: 10 March 2016 Decrease in C, N e P Organic matter mineralization

Increase due to aerobic condition and organic matter mineralization Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Increase due to aerobic condition and organic matter mineralization Decrease due to the aerobic condition, as confirmed by the increase in nitrate Decrease due to a higher consumption by microorganisms with respect to the production with OM mineralization To: 9 December 2015 T3: 10 March 2016 Increase due to organic matter mineralization The increase in fulvic acids suggested the new formation of the more labile part of humic substances (fulvic acids ) considered precursors of the more stable part (humic acids), constant at the moment slightly decrease in time, confirming the mineralization process

Indicators of total microbial activity Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Biochemical analysis Indicators of total microbial activity Hydrolytic enzyme linked with C cycle Hydrolytic enzyme linked with P cycle very low toxicity of the sediment at the end of the landfarming process (T3, T100) and in the peat (TS 0) and in the mixture (TS 50) Responsible for the opening of aromatic rings and decomposition of recalcitrant organic molecules Increase in all the measured enzyme activities, indicative of the activation of the microbial metabolism and the increase of organic substrate mineralization including organic pollutants To: 9 December 2015 T3: 10 March 2016

Total and Available Metals and Micronutrients Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Total and Available Metals and Micronutrients Total Metals and Micronutrients trend Total Hg, Cd and available Cr, Ni, Cd under limit of quantification As expected, no significant variations in Total Metals and Micronutrients (Hg, Cu, Cr, Zn, Ni, Pb, Al, Fe, V, Mn) Decrease in the available forms at T3 probably due to a loss in the leachate, or a bond with organic and inorganic matter All the metals, with the exception of Zn (about 250 mg/kg), are lower than the limit for urban use tab A (Dlg.s 152/2006) (Zn 150 mg/kg) and D.L. 161/2012. Available Metals and Micronutrients trend Organic contaminants Reduction of 28% in C>12 Little but not significant reduction for the more recalcitrant PCB and PAHs: PCB: from 0.040 to 0.039 mg/kg PAHs: from 53.8 to 49.2 mg/kg To: 9 December 015 T1: 13 January 2016 T2: 10 February 2016 T3: 10 March 2016 C>12 and PAHs still higher than the legal limit for the civil reuse (Dlg.s 152/2006) and D.L. 161/2012: 150 and 10 mg/kg, respectively

Conclusions on landfarming process Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Conclusions on landfarming process Increase in microbial activity (increase in the enzyme activities) Organic matter mineralization (decrease in C, N, P total forms and increase in soluble forms) Decrease in organic contamination Pollutants lower than the limit for urban use tab A. (Dlg.s 152/2006) and DL 161/2012, with the only exception of Zn, C>12 and PAHs The landfarming is successful Parameter Sediments at the end of landfarming D.L 161/2012 D lgs 152/2006 (Tab. A) D. lgs. 75/2010 Bulk density (g/cm3) 1,08 ±0,07 0,95 pH 8,1 ±0,01 4,5-8,5 Electrical conductivity (dS/m) 0,3 ±0,04 <1 TOC % 1,97 ±0,02 >4 TN % 0,13±0,01 <2,5 P2O5 % 0,11±0,02 <1,5 Cd (mg/kg) < L.Q 2 1,5 Cu (mg/kg) 34,3 ± 4,3 120 230 Hg (mg/kg) 0,075 ± 0,001 1 Ni(mg/kg) 34,6 ± 5,33 100 Pb(mg/kg) 35,2 ± 3,7 Zn (mg/kg) 248 ± 11 150 500 Physical and chemical characteristics comparable with those of an agronomic substrate (D.lgs 75/2010)

Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production ISECNR involved in: C2 Action: Monitoring and validation of the use of remediated sediments for plant nursing ISECNR and DISPAA Physical properteies Bulk density (dry), air volume, water volume, shrinking coefficient and total porosity Permeability, Substrate quantity   Chemical properties pH, Electrical conductivity, TN, Organic matter and ash contents, Water soluble nutrient concentration (NO3, NH3, Cl, P, F, K, Mg, Ca, Mn, Fe, Cu, Zn, B), Cation exchange capacity, Total concentration of Cd, Cr(VI), Cu, Ni, Pb, Zn, Hg Additional analyses due for the presence of sediments Total concentration of As, F, Mo, Se, Tl , Total hydrocarbons Biological properties Effects on plant growth using Chinese cabbage, Phytotoxicity test , Total biological activity Biotox Test Enzyme activities Human pathogens Escherichia coli enumeration Salmonella spp. enumeration

Action C2.Samples received and analyzed Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Action C2.Samples received and analyzed Italy Started Materials: 100% (100% Sediments) Strawberry and pomegranate: 0% (100% peat); 50% (50% peat and 50% sediments) Lettuce 0% (100% peat); 50% (50% peat and 50% sediments) Lettuce seedlings 0% (100% peat based substrate ); 25% (75% peat based substrate and 25% sediments); 50% (50% peat and 50% sediments) Strawberry (Camarosa and Monterey) 0%-50%-100%: -T1 after fertilization (1 week after plantation -T2 end of vegetative cycle (3 months after plantation) Pomegranate (Purple Queen and Mollar) 0%-50%-100%: -T1 after fertilization (1 week after plantation) -T2 after fall of the leaves (6 months after plantation) Lettuce : -T1 end of vegetative cycle (1 month after plantation)

Action C2.Samples received and analyzed Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Action C2.Samples received and analyzed Spain Started Materials: 100% (100% Sediments); 0% (100% peat); 50% (50% peat and 50% sediments). Strawberry (Monterey): -T1 end of vegetative cycle (6 months after plantation) Pomegranate (Purple Queen): -T1 after fall of the leaves (6 months after plantation) -T1 Strawberry Camarosa end of vegetative cycle (6 months after plantation) just arrived

Strawberry and pomegranate Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Total parameters Italy Spain Italian Regulation Sediments Strawberry and pomegranate Lettuce D. lgs. 75/2010 Started Materials 100% 0% 50% Ash (%) 95.9 74.7 88.4 16.5 89.2 96.3 12.1 84.5 - Volatile solids (%) 4.10 25.3 11.6 83.5 10.8 3.74 87.9 15.5 TOC % 1.81 15.2 5.76 35 4.56 1.70 38.1 6.81 >4 TN % 0.13 0.51 0.17 1.64 0.24 1.30 0.30 <2.5 As (mg/Kg) 9.54 2.34 13.7 0.814 8.66 13.2 <0.5 7.9 Mo (mg/Kg) 2.24 1.58 12.0 2.9 2.96 2.55 Se (mg/Kg) 1.3 0.85 118 1.22 2.05 1.25 Cr (VI) (mg/Kg) <2.0 <0.2 Ti (mg/Kg) 233 175 264 139 226 225 9.03 203 Tl (mg/kg) 0.618 C>12 (mg/Kg) 174 114 <50 82 <30 31.9 Cd (mg/Kg) 0.96 1.5 Cu (mg/Kg) 51.7 9.28 35.0 17.5 48.4 55.4 14.1 43.7 230 Ni (mg/Kg) 44.3 2.47 28.9 36.9 42.1 38.9 0.15 32.5 100 Pb (mg/Kg) 46.1 33,6 8.56 42.9 68 1.35 33.7 Zn (mg/Kg) 237 11.3 133 32 219 194 13.5 170 500 Hg (mg/Kg) 0.073 0.021 0.029 0.017 0.085 0.070 0.024 0.066

Strawberry and pomegranate Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Extractable parameters Italy Spain Sediments Strawberry and pomegranate Lettuce Started Materials 100% 0% 50% pH 8.64 6.01 7.22 5.62 7.95 8,02 6.16 8.08 E.C. (mS cm) 282 105 176 682 525 353 1129 596 NO3 (mg/l) 12 26 15 255 148 304 190 NH3 (mg/l) 0.18 0.13 0.10 0.50 0.22 0.08 1.03 0.25 Chloride (mg/l) 6.52 5.19 5.22 7.21 7.70 11.4 20.5 12.4 P (mg/l) <0.4 12.7 16.8 <04 F (mg/l) 2,36 0,50 0,57 <0,05 1,05 0.91 0,08 1,09 K(mg/l) 14.0 11.1 9.06 35.1 24.9 9.35 64.7 18.8 Mg (mg/l) 4.17 1.65 4.35 17.7 9.70 8.44 22.1 Ca(mg/l) 23.6 68.3 52.3 31.2 114 67.1 Mn(mg/l) 0.00 0.035 0.008 0.061 0.0 0.071 Fe (mg/l) 0.086 0.224 0.097 0.023 0.089 Cu(mg/l) 0.001 0.031 0.002 0.009 Zn(mg/l) B(mg/l) 0.365 0.324 0.404 0.336 0.322 0.309 0.295 0.321

Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Italian site Higher values in the sediments (100%) and in the mixture (50% ) No significant variation Increase with fertilization (T1) and decrease at T2 As expected, higher values in the peat (0%) No significant variation Same trend of E.C.

Italian site Enzymatic activities Slightly increase over time Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Italian site Enzymatic activities Slightly increase over time Higher values for peat as expected Butyrate esterase as indicator of overall microbial activity Increase over time: root development? Arylsulfatase higher in 50% and over time in all treatments

Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Total heavy metals Higher values in the sediments (100%) and in the mixtures (50% Slight decrease in 100% over time

N and P soluble compounds Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production N and P soluble compounds Very low values Higher values in the sediments (100%) Increase with fertilization (T1) relevant decrease at T2 Higher values in the peat (0%) No variation with fertilization At T2 decrease in 0% and increase in 50% and 100% Soluble P < 0.4 mg/l

Soluble macro and micro elements Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Soluble macro and micro elements Significant decrease over time Same trend for K and Mg In pomegranate, decrease at T1 and increase at T2 in 50% and 100% In strawberry, no great variation at T1 and decrease at T2 for fructification Decrease over time, especially in Strawberry

Soluble micro elements Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Soluble micro elements In Pomegranate increase at T1 (Fe and Cu) and T2 (Fe, Cu, Zn, Mn) In strawberry, different behavior of elements: Fe increase at T1 and decrease at T2

Spanish site Higher values in the sediments (100%) Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Spanish site Higher values in the sediments (100%) No significant variation Higher values in the peat (0% ) Increase at T1 As expected, higher values in the peat (0%) No significant variation Same trend of E.C.

Spanish site Enzymatic activities Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Spanish site Enzymatic activities Same pattern for all activities over time Increase in pomegranate at 50% and 100%

Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Total heavy metals Higher values in the sediments (100%) and in the mixtures (50% ) Same trend of Italy No significant variation

N and P soluble compounds Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production N and P soluble compounds Very low values Higher values in the peat (0%) Higher values in the peat (0%) Decrease over time in 50%, No significant variation in 100% Soluble P < 0.4 mg/l

Soluble macro elements All elements higher in the peat (0%) Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Soluble macro elements All elements higher in the peat (0%) Increase in 50 % and 100%, different from Italy Successful fructification in strawberry? Same trend Mg and Ca – Reduction over time

Soluble micro elements Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Soluble micro elements Very low values for Mn and Fe, traces only in few samples Very low values, increase at T1 Decrease over time, especially in Strawberry

Lettuce Increase at T1 Higher values in the sediments (100%) Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Lettuce Increase at T1 Higher values in the sediments (100%) Little variation As expected, higher values in the peat (0%) No significant variation Similar trend of E.C.

No substantial changes, with the exception of arylsulfatase Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Lettuce Enzymatic activities No substantial changes, with the exception of arylsulfatase

Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Total heavy metals Higher values in the sediments (100%) and in the mixtures (50% ) No significant changes over time

N and P soluble compounds Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production N and P soluble compounds Very low values, Higher values in the peat at T0 ( 0%) Significant decrease at T1 Higher values in the peat (0%) Increase in 100% Soluble P < 0.4 mg/l 12 mg/l only in the peat of lettuce

Soluble macro elements Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Soluble macro elements increase at T1 in 50% and 100%

Soluble micro elements Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Soluble micro elements Little decrease Very low values, traces at T1 in 50% and 100% Very low values for Mn, found only in few samples Very low values. Differently from the others, higher in the sediments (100%) Little increase in 0% and 50% and decrease in 100%

Lettuce seedlings 0% 25% 50% Not volatile solids % 15,2 81,6 88,2 Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Lettuce seedlings 0% 25% 50% Not volatile solids % 15,2 81,6 88,2 Volatile solids 84,8 18,4 11,8 pH 5,71 7,62 7,64 EC dS/cm 575 412 566 NO3 mg/l 147 42 130 NH3 0,19 0,27 0,29 Chloride 8,46 6,50 7,63 P 10,53 < 0,4 Cu mg/kg 16,5 39,5 47,5 Zn 30,2 203,8 179,5 Cd nd Ni 27,3 40,3 Pb 10,8 42,3 39,3 K 32,1 25,7 22,4 Mg 13,9 8,8 11,5 Ca 47,3 34,1 49,6 Mn 0,05 Fe 0,31 0,43 0,14 0,04 B 0,30 0,32 Bglucosidase umol MUB/gh 697 543 397 Phosphatase 2496 652 550 Butyrate esterase 2703 1681 1367 Arylsulfatase 51,3 55,8 61,8

Soluble macro elements (+) Arylsulfatase (-) Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production PC 1 (32.6%) Organic matter (-) Enzyme activities (-) Total heavy metals (+) PC 2 (23.1%) Soluble macro elements (+) Arylsulfatase (-) 0%, 50% and 100% are well discriminated 50% has an intermediate characteristics between 0% and 100% with good levels of metabolic activity and soluble elements for plant growth

Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Conclusions Similar performance of the substrates has been demonstrated in Italy and Spain, for both tested plant species. Regardless of the fertilization, it generally seems that a low content in soluble elements is present However, a fertilization plan seems to be necessary (due to the probably low absorption level in Spain) The element contents seems to be related to plant species: increase of soluble elements in pomegranate in Italy (considering that vegetation cycle is still in progress) lower elements in the strawberry trials both in Italy and Spain Increase in lettuce at the end of the vegetation cycle In the 50% mixture, good level of overall microbial activity is maintained over time, guaranteeing an acceptable plant growing environment Afterwards, these results should be related to plant growth and physiology A better interpretation of the results will be performed after the analysis of the next samples or in the second vegetative cycle

Pistoia Giovani e Ricerca Scientifica: Bando 2016 Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Progetto finanziato 2017-2019 Pistoia Giovani e Ricerca Scientifica: Bando 2016 Posidonia oceanica e sedimenti per la produzione di substrati per la vivaistica e orticoltura Responsabile scientifico: Dr Grazia Masciandaro Ricercatore: Dr. Eleonora Peruzzi Ente proponente: CNR – ISE Partner: Azienda Zelari Finanziamento: 50000 euro (Costo totale 80000 euro)

Thanks for your attention and GOOD WORK Project LIFE14 ENV/IT/113 - Demonstration of the suitability of dredged remediated sediments for safe and sustainable horticulture production Next activities: Analyze the just arrived samples (Strawberry Camarosa of Spain) Analyze the next samples -Second cycle for strawberry (Italy and Spain) -Further samples for pomegranate: after fructification (T3) and second fall of the leaves (T4) (Italy and Spain) -Samples of cutting trials (Italy and Spain) Thanks for your attention and GOOD WORK