1. Speciation of Th(IV) in marine systems Peter H. Santschi Laboratory for Oceanographic and Environmental Research (LOER) Depts. Of Oceanography and Marine Sciences Texas A&M University Galveston, TX

2. Outline Introduction: Chem. Ocng. vs. Mar. Chem. ThO 2 solubility Solution and surface speciation: inorganic Solution and surface speciation: organic Uniqueness of sticky macromolecular ligand Relationship to POC/ 234 Th ratio Family of marine sticky spiderweb- like ligands with fractal and amphiphilic properties, persistence Lengths of 100s nm, contour lengths of 100s - 1000s of nm and thickness of 1-2 nm TEM picture of stained marine colloids in nanoplast (Santschi et al., 1998). 500 nm

3. Th(IV) as an oceanographic tracer ( at [Th] ≤ 10 -12 M) New Production by [POC/ 234 Th p ]* 234 Th-flux Particle and Coagulation residence times Colloidal Pumping Particle sources Deep water ventilation rates Boundary scavenging YET, WE TAKE TH(IV) SPECIATION FOR GRANTED

4. Myths about Th(IV) Reversibility of adsorption to solids at the molecular level. Silica and Carbonates as major adsorbents. Th(IV) sorbs to almost anything, and thus, works well as an oceanographic tracer. Therefore, we do not need to know more about its marine chemistry.

5. Particle-water partition coefficients, K d : K d = f p /{(1-f p )C p, with f p =fraction on particles, and C p =particle concentration; Kd {ml/g or L/kg} 2) 1) 2) 2) Simulating natural conditions; Guo et al., 2002. 1) Abstracting natural conditions; Quigley et al., 2002, and references therein; => 1) ∆ clean/unclean; 2) Kd(Th(IV))~Kd(Pa(IV,V)); Kd(PS) is max.

6. Predicted f p for Th(IV) using lab-based K d & field C p (compared to observed f p = 0.1 - 0.5)* *) f p = K d *C p /(1+K d *C p )

7. What do we need to know about Th(IV) speciation? Because of relatively low abundance of Fe-oxides and medium to low Kd values for more abundant SiO 2 and CaCO 3, we need to concentrate more on surface speciation/sorption to COM/POM than to inorganics. Likely organic phases need more focus, especially the acid polysaccharide-rich rigid exopolymers aggregated as “marine spiderwebs” acting as “sticky ligands”.

8. Fibrillar exopolymers (“TEP”) as marine snow and sticky ligands (“marine spiderwebs”) with fractal properties (voids, scaling invariant) TEM of spiderweb-like fibrils [nm-µm; Santschi et al., 1998, Wilkinson, unpublished] Marine Snow [mm to cm; Alldredge and Gottschalk, 1989] Dissolved matter Colloidal matter Small aggregates Sedimenting aggregates 500nm

9. 234 Th deficiencies in the water column as a measure of particle scavenging intensity in surface and deep waters (Santschi et al., 1999. CSR, 19, 609) Aggregates ≥ 1.5 mm diameter

10. Solution and surface speciation: inorganic speciation Solution ThO 2 solubility Iron oxide and silica surface Sorption reversibility Presence or absence of organic matter

11. Th(IV) complexation in pure water Langmuir and Herman, 1980. GCA 44, 1753  Th-hydroxo species dominant at pH=8, even at high phosphate or EDTA concentration => ThO 2 solubility ~ 10 -13 M

12. ThO 2 solubility ~ 10 -8 M, regardless of crystallinity and size at pH=8 (Fanghaenel and Neck, 2002. Pure Appl. Chem., 72, 1895) -8 -15

13. Murphy et al., 1999. Coll. Surfaces A, 157, 47 => Importance of hydroxy-carbonate complexes

14. Th(IV) sorbs more strongly on Iron oxides in presence of marine COM U(VI) Th(IV)

15. Th(IV) forms inner-sphere complexes on hematite surface

16. No detectable desorption from hematite ( ) and COM ( ) colloids within 3 days after resuspension of tagged colloids into artificial seawater solutions (Quigley et al., 1996, 2001). predicted 0 sorption desorption

17. Disaggregation mascarading as “desorption” when clusters of 70 nm hematite particles are 0.4 µm filtered, but [Th] = 0 when 0.03µm filtered (Quigley et al., 1996. Aquat. Geochem. 1, 277)

18. Silica: Östhols, 1995. GCA, 59, 1235

19. Th(IV) sorption to SiO 2 in presence of Humic Acids (Moulin et al., 2004. In: Humic Substances, Taylor and Francis, Inc., p.275)

20. Enhanced Partitioning Coefficients (K c ) to Polysaccharide Enriched Colloidal Organic Matter (COM) over Unpurified COM (Quigley et al., 2002. L&O, 47, 367)

21. Increased Colloid-Water Partition Coefficient (Kc) of 234 Th(IV) as a fct. of Polysaccharide Content (Quigley et al., 2002. L&O, 47, 367) Enrichment through alcohol precipitation (g-PS/g-OM) K c = K c (o)*10 (2.2f PS ) =>Increase in Kc and ∆ 14 C

22. Consequences for POC/[ 234 Th] ratio The POC/[ 234 Th p ] ratio is a function of the particle-water partition coefficient, K d = K d (o)*10 (2.2f PS ) (cm 3 g -1 ), [ 234 Th d ] (in dpm/l) = dissolved 234 Th concentration, [SPM] = the suspended particulate matter concentration ( in g/L), [OC] = organic carbon (µmol-OC/mg particles), f OC and f PS = fractions of OC (OC/SPM) and polysaccharides, CHO (PS/OC), respectively, POC/[ 234 Th p ] = ([POC]/SPM])/([ 234 Th d ]  K d ) or = [f OC ]/([ 234 Th d ]  K d ) => Log{[POC]/[ 234 Th p ]} = log(f OC ) – log[ 234 Th d ] – logK d (o) – 2.2 f PS ≈ constant - 2.2 f PS [POC/ 234 Th]  1/f PS (if f PS is small) Or [ 234 Th/POC]  f PS

23. Relationship between 234 Th/POC ratio and POC-normalized APS and Carbohydrate Concentrations [Guo et al., 2002, Mar. Chem. 78, 103-119; Santschi et al., GRL.30,C2, 1044] 2001 cruise to Gulf of Mexico: Filled circles: sinking particles Collected at 65, 90, 120 m depth; Open circles: suspended particles (sum of 0.5, 1, 10, 53 µm fractions) 2000 cruise to Gulf of Mexico: Open circles: suspended particles CHO/OC~0.1 URA,APS/OC~0.01 => Need for lab experiments

24. “Sticky” macromolecular Th(IV)-binding ligand (Quigley et al., 1996, 2001, 2002; Alvarado, 2004) Sticky coefficient of 0.9 Low pK a of 2-3 Low pH IEP of 2-3 Molecular weight of ~ 10 kDa Functional groups: R-COO -, R-OPO 3 -, R- OSO 3 -, alone or in concert Apparent irreversibility of sorption

25. 2D PolyAcrylamide Gel Electrophoresis of Gulf of Mexico COM radiolabeled with 14 C and 234 Th (Quigley et al., 2002, L&O 47, 367; Alvarado-Quiroz, 2004, PhD Dissertation, TAMU, College Station, TX) 234 Th labeled COM, with similar distribution as polysaccharide-enriched COM, and 14 C-labeled sugar OH groups of COM (Quigley et al., 2002) (Santschi et al., 2003, GRL 30(2), 1044). -> Th(IV)- binding molecule contains Phosphate and Sulfate (Alvarado, 2004)

26. COM from GOM St. 4 – 72m: IC – PO 4 & SO 4 (Alvarado-Quiroz, 2004, PhD Dissertation, TAMU, College Station, TX) pH IEP ≈ 2-3 => Family of ligand systems with varying MW and fct groups from COM & EPS harvested from marinephytoplankton and bacteria Phosphate, SulfateTh(IV)

27. Model Acid Polysaccharides with RCOO -, ROSO 3 -, or R 2 OPO 3 - binding sites - Carrageenan Teichoic Acid Note: Carrageenans act as blood anticoagulants, while alginates act as blood coagulants, but both are used as emulsifiers and stabilizers in the food and pharaceutical industry

28. Surface water vs. Deep water: Middle Atlantic Bight 2600 m 2 m Fibrils in 1-200 nm COM documented by Atomic Force Microscopy (AFM, horizontal distance 10 µm) [Santschi et al., 1998. L&O 43, 896] -> Forms and shapes of colloids: pearls on necklace most common colloidal form -> “spiderweb”; -> fibrils in surface and bottom waters, but not in mid-depth waters. -> modern radiocarbon ages of pure fibrils (e.g., ~100% CHO) 10 µm

29. Fibrils on cell surface (Leppard, 1995, Sci. Total. Environ. 165:103) TEM of bacterial exopolymers used for experiments (without cell lysis, bacterial and dust contamination); scale bar: 200nm Origins of fibrillar EPS Functions and roles of EPS Floc formers (“marine snow”, “lake snow”), Form matrix components of biofilms, Play roles in colloid scavenging Facilitate microbial adhesion to surfaces. Bind extracellular enzymes in their active forms, as well as nutrients, Scavenge trace metals from the water, Templates for FeOOH, MnO 2, CaCO 3 and SiO 2 growth Can immobilize toxic substances, Can alter the surface characteristics of suspended particles Modify the solubility of associated molecules.

30. Where can Th(IV) go? - substitute for Ca 2+ with similar ionic radius. Role of Metals: Stabilization of  -helices through Ca 2+ bridging -> biosorbent for trace metals by, e.g., Ca substitution => Rigidity of polymer through Ca 2+ stabilized alpha-helices

31. Characterization of Exopolymeric fibrils from Sagitulla st. (Hung and Santschi, unpublished) TEM Spectrophotometric Methods GC-MS (#1,2,6,8,9 : unknown peaks, 3: glucose, 4: galactose, 5: galacturonic acid, 7: mannuronic acid). Scale bare 200 nm

32. Lab: Problem of colloidal impurities in all reagents and tracers used in laboratory experiments: Results ~ fct(purity of chemicals) Colloids are everywhere in laboratory reagents and tracers, at concentrations 10 -8 to 10 -10 M. Possible Sources: atmospheric dust, leaching from glassware. Possible compounds: Fe and Al hydroxides, silicates, bacterial EPS.

33. Importance of Experimental Conditions in Lab Experiments Clean-up of tracer and reagents Clean-up of mineral phases, e.g., SiO 2 Neutralization method of acid: buffer vs. base (e.g., NaOH) Phase separation: Ultrafiltration vs. centrifugation for particle or colloid separation

34. Solution conditions: 0.1 M NaClO4, pH of 7-8, using Th(V) tracer (K. Roberts, TAMUG) ProblemConditionLog Kd Neutralization of tracer acid, SiO 2 NaOH NaHCO 3 4.35 3.74 Clean conditions (teflon, clean reagents, clean- room), SiO 2 Reagent-grade reag. TM-grade reagents 5.5 2.5)* Acid/base clean-up of SiO 2 Clean silica Silica not precleaned 5.5 5.8* Phase separation (Xanthan, alginate, carrageenan), clean Centrifugation 1 kDa ultrafiltration ~ 4.5 6 - 7 Colloidal form of tracer ion Only 1-8 % of tracer ion ≤ 1 kDa (pH 8) - *) In clean solutions there is greater wall loss => Know about, or swamp colloidal impurities!?

35. Oceanographic Perspective

36. Guo et al., 1995. EPSL, 133, 117 Is 234 Th representative for Th(IV)? particle-water partitioning same for short-lived 234 Th as for long-lived 230 Th in seawater particle concentration effect for Th-isotopes due to the presence of colloidal macromolecular ligands in seawater

37. => Particle concentration effect across sizes indicates Th-binding ligands down to small (≤ 10 kDa) sizes

38. Guo et al., 1997. Coll. Surfaces A, 120, 255. => 234 Th(IV) partitions between solution, colloid and particle phases broadly similar to organic carbon

39. “Th-complexing capacity” Hirose and Tanoue, 1998. Mar. Chem. 59, 235 Valid for pH of 1 Hirose and Tanoue (2004. Sci.World J., 4, 67): Constant ratio of [ 232 Th p ] to [“Strong Organic Ligand”] Profile shape similar to that of POC, PON, Proteins

40. Hirose and Tanoue, 2001. Mar. Env. Res., 59, 95. (L/C in SPM in surface Ocean: ~ 1.5, deep Ocean: ~ 4 mmol/mol-C) => Ligand site:carbon (L/C) of ~ 0.001, and proportional to surface area:volume ratios {mmol/ mol-C}

41. Fibrillar exopolymers (“TEP”) as marine snow and sticky ligands (“marine spiderwebs”) with fractal properties (voids, scaling invariant) TEM of spiderweb-like fibrils [nm-µm; Santschi et al., 1998, Wilkinson, unpublished] Marine Snow [mm to cm; Alldredge and Gottschalk, 1989] Dissolved matter Colloidal matter Small aggregates Sedimenting aggregates 500nm

42. “Sticky coefficient” of 0.9 for polysaccharide-enriched colloidal macromolecular organic matter, and 0.8 for COM (Quigley et al., 2001. Mar. Chem., 76, 27) Amphiphilic Properties of EPS; Surface Activity of Hydrocolloid by smaller amounts of covalently bound hydrophobic proteins (e.g., gum arabic; Dickinson, 2003, Food Hydrocolloids, 17, 25) or lipids

43. Th(IV) sorbed to natural particles and colloids with same end- state, even when at different initial conditions (tagged colloids, or tagged particles) 234 Th-tagged OM: LMW≤10kDa HMW≥10kDa Particles ≤0.4µm, ≥0.1 µm P≥0.1µm P>0.1µm Sorption kinetics: k 1 a Cp   ≈ 0.3) observed in bot lab (Nyffeler et al., 1984; Honeyman and Santschi, 1989; Stordal et al., 1996; Wen et al., 1997; Quigley et al., 2001) and field (e.g., Honeyman and Santschi, 1989; Baskaran et al., 1992)

44. Results from Field Experiments: Sampling stations in the Gulf of Mexico * S6 * S4 Warm Core Rings (red), Cold Core Rings (blue)

45. Importance of Prymnesiophytes in 2001 and Cyanobacteria in 2000 [Santschi et al., 2003, GRL 30, 1044] Different phytoplankton species appear, at times, to control acid polysaccharide (APS) e.g., uronic acid (URA), production and 234 Th(IV) complexation Abundance in ocean: CHO/POC ~ 0.1, APS/POC ~ 0.01, URA/POC ~ 0.01 (all: Santschi et al., 2003; Hung et al., 2003), L/POC ~ 0.001 (Hirose, 2004).

46. Relationship between bacterial production (BP) and a) total APS concentration (µg-C/L), and b) 234 Th/POC ratios (May 2001, Gulf of Mexico) [Santschi et al., 2003, GRL 30(2), 1044] =>Interplay between sorptive, aggregating and enzymatic Processes; -> Microbial APS production and degradation coupled to coagulation of more recalcitrant APS fragments provides a steady conveyor belt for 234 Th to and from Particles, with the “ligand soup” being regulated by the microbial community in the water, as a self-regulating (autoporetic) system.

47. Summary and Conclusions Experimental Lab Results with Th(IV) tracers at environmentally relevant (low) concentration levels depend on experimental (ultra- clean vs. ambient impurities) conditions, with tracers likely present as pseudo-colloids at neutral pH, with environmental significance only when colloidal impurities are known or controlled. Family of Th(IV)-binding surface-active macromolecular ligands with varying functional groups and molecular weights, produced by phytoplankton and bacteria, partly degraded by bacterial enzymes but re-aggregating as smaller fibrillar units on their way to bottom, with aggregation pathway dominating for TEP and Th(IV), degradation pathway dominating for OC. EPS might act as “colloid trap”, like a marine spiderweb, sinking at speeds controlled by its fractal dimensions, and in proportion to the mineral ballast (SiO 2, CaCO 3, Al 2 O 3 ).

48. Where do we go from here? => Constrain variability in POC/[ 234 Th] ratios We need an improved relationship between the POC/[ 234 Th] ratio and the ligand, CHO or OC content, whereby CHO/OC~0.1, APS(URA,LPS)/OC~0.01, L/OC~0.001. More insight into molecular mechanisms of Th(IV) “scavenging” needed; => coupling of complexation to colloids/particle aggregation into sinkable particles important. Importance of hydrophobic-lipophilic balance (HLB numbers) for parameterizing “stickiness”?

49. In summary, what is needed is: Better insight into the molecular mechanisms of the physical, chemical and biomolecular mechanisms of Th(IV) binding to a “sticky” macromolecular ligand family of compounds requires a paradigm shift.