Optimizing a remediation method on a tailing dam Iordache V.a, Dumitru A. L..a, Frâncu B. a, Iosif I. a, Catieşanu A..a Onete M..b, Neagoe A.a a University of Bucharest, Research Center for Ecological Services - CESEC b Bucharest Institute of Biology, Romanian Academy 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

Structure of the presentation Background: bioremediation as acceleration of the ecological succession. The importance of characterizing the temporal heterogeneitz Negative effects of the innoculation with commercial fungi due to expanded clay ? Experimental design, measured variables Preliminary pot experiment as a basis for field experimental design. Where we show that innoculation with 1% fungi is better than with 7% fungi. Field experiment. Where we show that innoculation with 2% fungi is better than with 1% fungi. Also, that addition of green manure beside top-soil makes a difference only in the short time scale. Conclusions 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

1 Background One expects during succession: A trend of increasing (plant) species richness with stronger inter-specific relations A minimization of abiotic fluxes going out of the system (greater system closure), and an increase in the internal stocks of macronutrients. (Neagoe et al. 2009) 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

1 Background Fungi (and bacteria) are succession accelerators in metal stressed environments Increased rezistance to toxic elements, increased uptake of macronutrients But: What is the effect of expanded clay support ? (Neagoe et al. 2014) (Neagoe et al. 2014) 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

1 Experimental design 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

1 Experimental design Soil: pH, electrical conductivity, nutrients, respiration, total metals, easily extractable metals. Plants: biomass by plant part / cover by vertical layer, N, P, pigments, metals concentrations. Oxidative stress variables to follow. Mycorrhization degree. Soil invertebrates sampled. 5 sampling times for substrate, 3 sampling times for plants. Work in progress 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Pot experiment The inoculation with 7% fungi did not increase the biomass of the plants compared to 1% fungi (grams dry weight, average, ANOVA). 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Pot experiment The inoculation with 7% fungi in one case decreased the P concentration in shoots compared to 1% fungi (µg/g d.w., average, ANOVA), and decreased the Cd in roots of plants (µg/g d.w.) grown without topsoil or green fertilizer. 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Pot experiment The inoculation with 7% fungi did not increase the N concentration in roots compared to 1% fungi (%, average, ANOVA). 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Pot experiment The inoculation with 7% fungi did not increase the relative abundance of arbuscules and the relative mycorrhizal frequency (%, average, ANOVA). There is no reason to apply in the field 7% fungi. These pot experimental results confirm previous results from Neagoe et al. 2014 with substrate from another tailing dam. We decided to compare in the field 1% fungi with 2% fungi. The operational hypothesis that no effects will not be observed, and that 1% will be the low cost option for accelerating the ecological succession. 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Field experiment The number of plant species by vegetation layer (0-2-5-10-20 > cm, and dead individuals. 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Field experiment Total cover by Agrostis capillaris and by cumulated other species in all layers (%). 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Field experiment Total cover by all species in two selected layers and in all layers (%). 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Field experiment Nitrogen concentrations in roots and shoots of A. capillaris (% d.w.). 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Field experiment Phosphorus concentrations in roots and shoots of A. capillaris (µg/g d.w.). 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Field experiment Assimilatory pigments concentrations in roots and shoots of A. capillaris (µg/g d.w.). 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Field experiment Pb and Cd in shoots of A. capillaris (µg/g d.w.). 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Field experiment Micorhization degree at sampling time 2: not conclusive. 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Field experiment Soil pH and conductivity (µS/cm). 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

3 Field experiment Soil respiration (mgCO2/g s.u. 2h), ammonium concentration in soil, bioavailable Cd and Pb in soil (µg/g d.w.). 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

4 Conclusions The pot experiment confirmed that inoculation with 7% fungi commercial inoculum did not led to a better plant development. This is probably due to the effect of expanded clay on properties of the substrate alone or in mixture with various amendments. The hypothesis that inoculation with 2% fungi in the field would not have an important effect compared to 1% fungi was rejected. There were important effects on species richness, plant variables and soil variables. The patterns of plant community and individual plant development are consistent under fungi inoculation are consistent with the idea that an acceleration of ecological succession occurs. Supplementary investigations are needed in order to assess the biogeochemical closure of the substrate – plants systems. The results from the field experiment pointed out the dynamic nature of the phenomena. Monitoring in the long term with seasonal sampling seem to be a must for assessing the most efficient bioremediation solutions. In our case the addition of green fertilizer beside top-soil could be useful only in the first months of plants development compared with simple top-soil amendment combined with 2% fungi inoculation. Some variables did not worth the effort to be measured each sampling time, such as mobile forms of N, P and toxic elements. It could be that their dynamic is much faster. Other monitoring techniques such as DGT technique or electrodes coupled with data loggers will be tested. 15th symposium on remediation ,From “expert knowledge” to basic science to application: 15 years of bio-geo interactions, October 13-14, 2016, Jena, Germany

Thanks for your attention ! Acknowledgements This research was performed in the project TIMMAR 98/2014 funded by UEFISCDI. We thank to Certeju de Sus town administration for logistic suport in the installation of the field experiment and to the Botanical Garden of Bucharest for support during the pot experiment. Our technician Liliana Lungu contributed to the lab and field activity, the colleagues Georgian Manuc, Costin Enache, Gabriel Burtea and Alexandru Răileanu helped us to install the field experiment, monitor the pot experiment and process the root samples, Dr. Minodora Manu determined the mycorhization degree. Thanks for your attention !