1. Response of Agrostis capillaris grown on soil contaminated with toxic elements to different nitrogen and phosphorus supply Aurora Neagoe, Virgil Iordache, Radu Lăcă t usu, Paula Constantinescu University of Bucharest, Romania (aurora.neagoe@unibuc.ro(aurora.neagoe@unibuc.ro) University of Bucharest Research Centre for Ecological Services 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014, Jena, Germany

2. Introduction A. Objectives B. Methods C. Results 1. GLM of biomass and nutrition variables 1.a Potassium nutrition and mycorrhization 1.2 GLM of other response variables 3. Nitrogen and phosphorus supply and balance 4.a Multiple regressions for bioaccumulation 4.b Multiple regressions for oxidative stress 5. Metadata analysis D. Discussions E. Conclusions Outline 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany

3. A. Objectives Assessing the effect of N and P on the accumulation of toxic elements in A. capillaris L. Assessing the effect of N and P on the oxidative stress variables in A. capillaris L. pH (H 2 O) Moistur eEC Soil resp. N- NH 4 + N- NO 3 - P- PO 4 3- Variable % µS/c m mgCO 2 /g d.wmg/kg d.w. average3.29.7525.117.849.115.5121.86 SD0.131.0314.753.965.173.627.1 n=13AsCuFeKMnPbZn Element smg/kg d.w. average81.75314.92516211852502.1532.3248.6 SD (n=13)52.07106.937212598179268.759.4 B. Methods 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany pH = 5.4 (CaCO 3 - 5%)

4. No. treatment Lab codeExperimental treatmentNo. pots 1N(-), P(-)450 g polluted soil + 60 ml water + 22.5 g CaCO 3 + 0.3g seeds5 2N(+), P(-)450 g polluted soil + 10ml sol. 1 N + 50 ml dist.water + 2 2.5g CaCO 3 + 0.3g seeds5 3N(-), P(+)450 g polluted soil + 10 ml sol. 1 P + 50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 4N(+), P(+)450 g polluted soil + 5ml sol. 1 N + 5ml sol. 1 P + 50 ml dist.water + 22.5 g CaCO3 + 0.3g seeds5 5N(++), P(-)450 g polluted soil + 10 ml sol. 2 N + 50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 6N(-), P(++)450 g polluted soil + 10 ml sol. 2 P + 50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 7N(++), P(++)450 g polluted soil + 5 ml sol. 2N + 5 ml sol. 2 P + 50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 8N(+), P(++)450 g polluted soil + 5 ml sol. 1 N + 5 ml sol. 2 P + 50 ml dist.water + 22.5 CaCO 3 + 0.3g seeds5 9N(++), P(+)450 g polluted soil + 5 ml sol. 2 N + 5 ml sol.1 P +50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 10N(+++), P(-)450 g polluted soil + 10 ml sol. 3 N + 50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 11N(-), P(+++)450 g polluted soil + 10 ml sol. 3 P + 50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 12N(+++), P(+++)450 g polluted soil + 5 ml sol. 3 N + 5 ml sol. 3 P +50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 13N(+++), P(+)450 g polluted soil + 5 ml sol. 3 N + 5 ml sol. 1P + 50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 14N(+), P(+++)450 gpolluted soil + 5 ml sol 1 N + 5 ml sol. 3 P + 50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 15N(+++), P(++)450 g polluted soil + 5ml sol 3 N + 5 ml sol. 2 P + 50 ml dist.water + 22.5 g CaCO 3 + 0.3g seeds5 16N(++), P(+++)450 g polluted soil + 5 ml sol. 2 N + 5 ml sol. 3 P + 50 ml dist.water + 22.5 CaCO 3 + 0.3g seeds5 Total pots = 80 1N = echiv. of 15 2N = echiv. of 30 kg N/ha as NH 4 NO 3 3N = echiv. of 60 1P = echiv. of 15 2P = echiv. of 30 kg P/ha as K 2 HPO 4 3P = echiv. of 60 Bifactorial design 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany

5.  Soil respiration - incubation and titration method;  Mycorrization degree (Trouvelot et al.,1986) method using a Carl Zeiss Axio Imager 2 microscop;  Nitrogen in plants - Kjeldahl method;  Other elements in soil and plants - wet digestion followed by ICP-MS;  Extractable elements in soil - ammonium nitrate method 1M followed by ICP- MS;  Nitrate in soil - sulfosalicylic acid method;  Ammonia in soil - Na nitroprusside method;  Phosphate in soil - malachite green method;  Proteins in aboveground and root parts - Lowry et al.( 1951) method;  Superoxide dismutase - McCord and Friedovic (1969) method ;  Peroxidase - Lagrimini (1991) guaiacol peroxidase method;  Lipid peroxides - involved malonaldehyde determination by using thiobarbituric acid Hodges et al. (1999) method;  Assimilating pigments - Schopfer (1989) method. B. Methods - measured variables 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany B. Methods - derived variables Conc. of elem. in roots Conc. of elem. in above - AF = and TF = Conc. of elem. in soil Conc. of elem. in roots - Pool of elements - B:B - Biomass above:Biomass roots [Low ratio - low nutrition; high ratio - high nutrition] - P:P - P in roots:P in above [Low ratio - high P; high ratio - low P] - N:P - N in above:P in above - N in roots:P in above [ 20 P imbalanced] - N:K - N in above:K in above - N in roots:K in roots [N:K < 2.1 exclude limitations of K] - P:K - P above:K above [P:K >3.4 exclude limitations of K] - P roots:P roots - Statistical processing using Statistics and Excell softwares (multiple regression and GLM).

6. 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany

7. 1.1 GLM of biomass and nutrition variables C. Results *B = Biomass **Above = aboveground of plant part GLM testN fertilizerP fertilizerN*P Variable ppp Biomass - roots NS0.025NS Biomass - above NS Biomass - total NS0.019NS B above/B roots NS0.0450.013 N above 0.0010.0000.006 N roots NS0.000 P above 0.0160.000NS P roots NS0.000NS N/P above0.000 NS N/P roots NS 0.000 NS P roots/P above NS - Low ratio - low nutrition - High ratio - high nutrition - N/P <10 N imbalanced - N/P >20 P imbalanced 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany

8. Results - N/P Discussion 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany High ratio - low P Low ratio - high P

9. 1.1a GLM of K nutrition and mycorrhization N/K < 2.1 exclude limitations of K P/K > 3.4 exclude limitations of K GLM test N fertilizer P fertilizer N*P Variableppp K above0.002NS0.003 K rootsNS0.001NS N/K above P/K above N/K roots P/K roots <2.1>3.4<2.1>3.4 Average0.69224.551.4428.828 Min.0.37414.340.5863.894 Max.1.11880.592.70720.61 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany

11. GLM test N fertilizerP fertilizerN*P Variable ppp pH NS 0.011 EC NS0.007NS Soil moist. NS Soil resp. NS0.001NS N-NH 4 + NS0.000NS N-NO 3 - 0.0000.040NS N-NO 2 - NS0.008NS P-PO 4 3- NS0.000NS P bioav. 0.000 As bioav. 0.000 Cr bioav. 0.000 Cu bioav. 0.000 Mn bioav. 0.0040.000NS Ni bioav. 0.000 NS Pb bioav. 0.000 Zn bioav. 0.000 1.2 GLM of other response variables 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany

12. 1.2 GLM of other response variables (continuation) GLM test N fertilizerP fertilizerN*P Variable ppp As above 0.000NS Cr above 0.0010.0020.006 Cu above 0.000 Mn above NS Ni above NS0.006NS Pb above 0.0010.0070.029 Zn above 0.002NS As roots NS0.012NS Cr roots NS0.024NS Cu roots NS Mn roots 0.000NS0.000 Ni roots NS Pb roots 0.000NS0.000 Zn roots NS 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany

13. GLM testN fertilizerP fertilizerN*P Variableppp Cu AF totalNS Pb AF total0.000NS0.000 Zn AF totalNS Cu AF bioav.0.000 NS Pb AF bioav.0.000 NS Zn AF bioav0.011NS0.047 Cu TF0.0000.0020.013 Pb TF 0.000 Zn TF0.0000.015NS 1.2 GLM of other response variables (continuation) 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany

14. 1.2 GLM of other response variables (continuation) GLM test N fertilizerP fertilizerN*P Variable ppp Protein above 0.0000.0090.018 Protein roots NS0.0010.000 SOD above 0.000 0.005 SOD roots NS0.000 POD above 0.0190.0010.002 POD roots NS0.000 LP above 0.0000.0040.000 LP roots NS Chl.a NS0.0020.009 Chl.b NS0.0150.034 Carot. 0.0370.0070.015

15. 3. N and P supply and balance 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany Establishing of supply classes

16. N supply P supply N*P ANOVA ppp pH 0.000NS0.002 EC NS 0.006 Soil resp. 0.003NS Pool of Cu 0.003NS0.006 Pool of As 0.0190.010 Pool of Zn 0.077NS0.016 Cu AF 0.0000.0410.006 Zn AF 0.000NS Cu TF 0.003NS Pb TF NS Zn TF NS Cu AF bioav. 0.001NS Pb AF bioav. 0.0020.001NS Zn AF bioav. NS N supply P supplyN*P ANOVA ppp Biomass total 0.0010.0210.025 SOD above0.000NS SOD roots0.000NS POD above0.000NS POD roots0.0000.0010.000 Chl. a0.010NS Chl. b0.005NS Carot.0.025NS N/P above0.001NS N/P roots0.0000.002NS 3. N and P supply and balance (ANOVA) 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany Groups of N & P supply

17. The effect of N and P supply and balance on biomass 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany P supply P supply 0 N supply 0 Supply of N and P

18. -Imbalance P (_P) -Balance of N & P (O) -Imbalance N (_N) Group 1_ PGroup 2O ANOVA pp Cu AF totalNS Pb AF total0.044NS Zn AF totalNS Cu TFNS Pb TFNS Zn TFNS Cu AF bioav.0.077NS Pb AF bioav.NS Zn AF bioav.NS Group 1_ PGroup 2 _N ANOVApp Cu FA0.001NS Pb FANS Zn FANS Cu TFNS Pb TFNS Zn TFNS Cu AF bioav.0.030NS Pb AF bioav.NS Zn AF bioav.NS 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany The effect of balance classes

19. 4.a Predictive statistical models for accumulation of metals in A. capillaris Regression summary for dependent variables 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany Cu AF total Pb AF total Zn AF total Coef.p-levelR2R2 Coef.p-levelR2R2 Coef.p-levelR2R2 Intercept 0.0000.409 Intercept 0.0000.358 Intercept 0.0000.202 Log N-NO 3 - 0.4160.000Lg EC0.3650.002pH-0.4360.000 pH0.2790.005pH-0.3040.010Lg P Sol-0.2130.047 Log N-NO 2 - 0.2430.011 LgN-NO 3 - 0.2920.011 Lg Cu AF bioav. Lg Pb AF bioav. Lg Zn AF bioav. Coef.p-levelR2R2 Coef.p-levelR2R2 Coef.p-levelR2R2 Intercept 0.380.781 Intercept 0.2230.594 Intercept0.0000.52 Lg P soil-0.490.000 Lg P soil-0.320.002 Lg P soil-0.610.000 Lg P-PO 4 3- 0.5030.000 Lg P-PO 4 3- 0.4490.000 pH-0.420.000 Lg N-NO 3 - 0.2230.003 Soil moist.0.3350.000 Lg EC0.3540.000 Lg N-NO 2 - -0.240.019 pH0.1980.011 Lg N-NO 3 - 0.1880.040

20. Lg Cu TF Lg Pb TF Zn TF Coef. p-levelR2R2 Coef. p-levelR2R2 Coef. p-levelR2R2 Intercept0.003 0.386 Intercept0.0000.0190.229Intercept0.000 0.161 Lg P soil0.5110.000Lg P soil0.4580.000 Lg P soil0.4150.000 Lg N-NO 3 - -0.2350.020 Soil moist.-0.2580.019 Regression summary for dependent variables 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany 4.a Predictive statistical models for accumulation of metals in A. capillaris

21. 4.b Predictive statistical models for oxidative stress in A. capillaris 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany Regression summary for dependent variables P imbalance, n = 16Balanced P and N, n=43P Inbalanced, n=14 Coef.p-levelR2R2 Coef.p-levelR2R2 Coef.p-levelR2R2 SOD above Intercept 0.4100.762Intercept 0.0000.474 Intercept 0.0000.784 As0.6040.000 N/P above -0.5610.000 N/P above -0.8830.000 Zn0.3910.030 Cu 0.2840.023 Coef.p-levelR2R2 Coef.p-levelR2R2 Coef.p-levelR2R2 POD above Intercept 0.9210.773Intercept 0.0000.362 Intercept 0.0000.854 Cr0.6710.000 N/P above -0.4620.000 N/P above -0.6610.000 Zn0.3520.032 Pb 0.3130.024 As 0.3630.041

22. P ImbalanceAsCrCuMnPbZnN/P above POD aboveR 2 =0.814R 2 =0.817R 2 =0.814R 2 =0.028R 2 =0.790R 2 =0.630R 2 =-0.1585 n=16p=0.000 p=0.920p=0.000 p=0.558 Balanced N & PAsCrCuMnPbZnN/P above POD aboveR 2 =0.448R 2 =0.326 R 2 =0.212R 2 =0.382R 2 =0.276R 2 =-0.510 n=43p=0.000 p=0.172p=0.011p=0.073p=0.000 N ImbalancedAsCrCuMnPbZnN/P above POD aboveR 2 =0.742R 2 =0.359R 2 =0.050R 2 =0.757R 2 =0.494 R 2 =- 0.007 R 2 =-0.870 n=13p=0.004p=0.229p=0,872p=0.003p=0.086p=0.982p=0.000 4.b Predictive statistical models for oxidative stress in A. capillaris 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany

23. D. Discussions 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014, Jena, Germany

24. 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany D. Discussions (continuation) Differences between lab and field Low ratio - high P; high ratio - low P10 N imbalanced; >20 P imbalanced

25. E. Conclusions - The amendments with N and P significantly change the variables of the contaminated soil. This in turn, change the N and P concentrations and their ratios in plants, the biomass of roots and aboveground, as well as the concentration of toxic elements and the oxidative stress variables. - In our experimental conditions the plants moved from P deficiency and imbalance to all other variants of P and N nutrition and balance/imbalance. - As a result the AF and TF of toxic elements could be significantly modelled with multiple regressions having as independent variables the soil variables changed by the additional amendments. - The oxidative stress variables were best modelled by splitting the data in three classes of balance/ imbalance. The oxidative stress significantly depends both of N/P ratio and of the concentration of toxic elements. 13 th Symposium on remediation, sedimentary pore space cementation: role of microbes, September 25 th - 26 th 2014 Jena, Germany

26. - In the context of N and P nutrition from the whole basin and from the other contaminated area, our models described only discrete part of the whole eco-physiological space. The models allowed an understanding of the basic causes controlling accumulation factors and oxidative stress but, by no means can be extrapolated to the A. capillaris populations in the whole catchment and in other contaminated area. - In the future, we will compare the behavior of A. capillaris in field samples covering the full N and P gradients with the behavior in an experiment manipulated in order to cover the full field gradient. E. Conclusions (continuation)

27. Thank you for attention! Acknowledgments: -This research was support by the partnership projects: ASPABIR no. 50 - 2012 and TIMMAR no. 98 -2014. -Thanks to MSc Roxana Gheorghe for mycorrhization degree determination and to MSc Andrei C ă ld ă ru for their help in caring the potted plants