Relative Lability of Trace Metals Complexed in Aquatic Humic Substances Using Ion-Exchanger Cellulose-Hyphan

Neste estudo as frações metálicas, complexadas por substâncias húmicas aquáticas (SH) foram caracterizadas como lábeis/inertes em função do pH, tempo de complexação e concentração de SH. As SH foram extraídas por ultrafiltração e complexadas com íons metálicos bivalentes. Essas frações foram caracterizadas por processo de troca iônica utilizando resina celulósica Hyphan e procedimento em batelada. Os metais foram determinados por espectrometria de absorção atômica. Os resultados mostram diminuição dos coeficientes de distribuição (Kd) na presença de SH e que a labilidade relativa das frações metálicas complexadas pelas SH é influenciada pelo tempo de complexação, pH e concentração de SH. Até cerca de 15 min, a troca iônica en tre os metais complexados pela SH e a resina se comporta como reação de 2 ordem. Após esse período, o processo de troca é mais lento caracterizando uma cinética de 1 ordem. Os resultados permitem estabelecer a seguinte ordem de labilidade relativa para as frações metálicas complexadas por SH: Pb > Mn > Cd, Ni > Cu.


In tro duc tion
HS are a com plex mix ture of re lated macromolecules formed by the bi o log i cal de com po si tion of plants in soils, and trans ported to nat u ral wa ters by leach ing.These macromolecules ex hibit a great num ber of func tional groups, par tic u larly phe no lic and carboxylic 1,2 .Metal-organic in ter ac tions, in clud ing complexation re actions of metal ions and HS, are be com ing in creas ingly recog nized as im por tant fac tors in many nat u ral sys tems 3 .The struc ture and chem i cal be hav ior of these funcionalities have been stud ied over the last de cade.The high sta bil ity of metal frac tions when complexed with bi o log i cal or ar ti ficial ligand sources in ter fere di rectly in the trans por ta tion, ac cu mu la tion and bioavailability of met als in the en vi ronment 4 .There is con sid er able ev i dence that the free ionic forms of met als like cop per, lead, cad mium and alu mi num are usu ally the forms most toxic to aquatic biota, and that complexation by HS re duces the tox ic ity of these metal ions 5 .The lability/in ert ness of metal ions complexed with HS, and the way they are trans ported un der nat u ral con ditions, must be eval u ated 6 .
The low spe cies con cen tra tions in com plex ma tri ces, where there is also a great num ber of interferents, have been the ma jor prob lem in the de vel op ment of an a lyt i cal meth ods for the char ac ter iza tion of dif fer ent chem i cal species in the en vi ron ment, both qual i ta tively and quan ti tatively 7 .
Here, we de scribe how the che lat ing ion exchanger cellu lose-Hyphan can be used as an ef fec tive col lec tor for labile trace met als re main ing in aquatic HS.Using an op ti mized batch pro ce dure, the ki net ics, de gree of ion exchange re ac tion, and in flu ence of trans for ma tion pro cesses (age ing) can serve as pa ram e ters for the eval u a tion of metal lability in HS.

Chem i cals and re agents
The multi-element so lu tion of the metal and all ac ids and bases for the prep a ra tion of so lu tions and HS-isolation were highly pure.Deionized wa ter from a Millipore MilliQ sys tem was used.

Ion-exchange resin
The ion-exchanger used for the dif fer en ti a tion of labile/in ert metal frac tions was the resin of cel lu lose-based Hyphan (TM Riedel-de Haën AG), (1-(2-hydroxyfenylazo)-2-naftol) func tional amino group, C 6 H 3 (OH) N=NC 10 H 6 OH, with Kd  10 5 and an ex change ca pac ity of 0.2-0.4mmol Cu(II)/g of dry resin 8 .

Humic sub stance ex trac tion
Humic-rich wa ter was col lected from an eco log i cal reserve lake (Venner Moor) in Münster, Ger many.Af ter filtra tion through a 0.45 µm mem brane, 150 L of the sam ple was con cen trated by ultrafiltration (UF) to 0.5 L (keep ing the orig i nal pH of 4.5).A Millipore Pellicon sys tem was used (tan gen tial flow with a nom i nal mo lec u lar weight cut-off of  1000 Dal ton, a fil ter ing area of 0.42 m 2 , and a pen e trat ing flow of 25 mL/min).

Dis solved or ganic car bon -DOC
The DOC value of the HS sam ple was determinated by ox i da tion to CO 2 and mea sure ment with an IR-analyzer (Shimadzu TOC 2000).The HS con cen tra tion was eval uated at 7 mg/mL, due to a DOC con cen tra tion of 14 mg/mL 9 .

Complexing ca pac ity (Cu(II)) of HS
The complexing ca pac ity (2.7 mmol Cu(II) per g DOC) of aquatic HS was de ter mined by a cop per(II) se lec tive elec trode (WTW Cu 500) 10 .

Prep a ra tion of stan dard ized HS so lu tions
The HS stan dards were pre pared from their con centrates by di lu tion to 1.0 mg/mL.In or der to re move the metals which oc cur nat u rally in the HS, 1 g (dry weight) of highly pure Hyphan resin was added and kept un der ag i tation for 72 h at pH 9.0.Af ter fil tra tion, the pH was ad justed to 4.5, and the HS stan dards were stocked in poly eth yl ene bot tles un der re frig er a tion at 4 °C.

Metal spik ing of HS
The spe cies formed be tween metal and humic substances (HS-M) were pre pared by add ing shares of multi-element so lu tion of the met als.The pH was ad justed by the ad di tion of NaOH or HCl and 1.0 mL of the cor respond ing buffer so lu tion.The vol umes were com pleted to 10.0 mL with highly pure wa ter, and the so lu tions were stirred for 24 h.The metal con cen tra tions in the HS so lutions were Cd 0.50, Ni 2.0, Mn 1.0, Cu 2.0, and Pb 5.0 mg/L.

Batch pro ce dure 11
Af ter the complexation equi lib rium (HS-M) was guaran teed (24 h), 20 mg Hyphan was added and stirred for 1 h.The ion-exchanger was fil tered through fil ter pa per (20 mm di am e ter) pre vi ously cleaned with 2 mol/L HCl.Pa per and resin were trans ferred to small flasks and ex tracted for 1 h with 3.0 mL of 2 mol/L HCl.Af ter centrifugation (4000 rpm), the analytes con tained in the ex tracts were deter mined by flame ab sorp tion atomic spec trom e try (FAAS).

Metal de ter mi na tions by FAAS
Trace met als con cen trated in the eluates were di rectly de ter mined by FAAS, ac cord ing to the man u fac turer's oper at ing in struc tions (Spec trom e ter Varian-Intralab-AA 1475).For the cal i bra tion curves, syn thetic metal stan dard so lu tions con tain ing 0 to 5.0 µg/mL of each metal in 2 mol/L HCl were used.

Dis tri bu tion co ef fi cient: ef fect of pH
The ef fi cient multi-element preconcentration by an ion ex change batch pro ce dure re quires col lec tors of fer ing high dis tri bu tion co ef fi cients 12 , Kd, pref er a bly of > 10 4 , cal culated by the fol low ing equa tion: where C col = con cen tra tion of met als in the col lec tor (mg/g), and C sol = con cen tra tion of met als in so lu tion (mg/mL).
The sep a ra tion char ac ter is tics us ing the ion-exchanger cel lu lose-Hyphan as the col lec tor for the re ac tive metal frac tions by means of metal dis tri bu tion co ef fi cient, Kd, in func tion of the pH is il lus trated in Fig. 1.Using batch pro cedure, these stud ies were car ried out in the pres ence of 1 mg/mL HS (HS-metal, equil i bra tion for 24 h) and with out HS (NaCl 1 g/L).In the ab sence of HS, the Hyphan col lector ex hib its Kd val ues as high as 10 4 to 10 5 (mL/g) for Cd, Pb, and Cu at a pH above 9.0, with prob a ble uni form be havior for all met als.In con trast, in the pres ence of HS the enrich ment of the met als on the col lec tor phase de creases, and the Kd val ues are also low ered, on or der of a fac tor of 100 in the pH range from 8 to 9. Thus, in slightly al ka line so lu tions there is a large change of met als from aquatic HS to ion-exchanger.
In gen eral, in the pres ence of HS and greater pH increases the ap par ent com plex sta bil ity by de creas ing the pro ton com pe ti tion with metal ions for weakly acidic binding sites 3 .This be hav ior was not fol lowed by the Cu fraction, in agree ment with re searches 13 which showed that the com plex sta bil ity for Cu-HS de creases with in creas ing pH.

Equi lib rium fun da men tals
The ion-exchangers of cel lu lose, par tic u larly the Hyphan resin, pres ent high dis tri bu tion co ef fi cients on the or der of 10 5 for some metal ions in salt so lu tions with out HS 14 .Ac cord ing to pre vi ous stud ies 15 , cel lu lose-based ion-exchangers can be used as ef fec tive col lec tors for la bile trace met als re main ing in HS so lu tion.Ap plying an op timized batch pro ce dure, both the ki net ics and the de gree of metal re moved from HS can be use ful for char ac ter iz ing the lability/in ert ness of heavy met als bound to HS.The exchange of metal ions be tween res ins and la bile macromolecular metal com plexes like HS-M can be described by the fol low ing equi lib rium: so lu tion exchanger (charges omit ted for sim plic ity) where HS = humic sub stances, HS-M = spe cies formed between metal, Ex= ion-exchanger, solid phase, and M-Ex= spe cies formed be tween the metal and the col lec tor.Thus, the con cen tra tions of spe cies HS-M and M-Ex, gov erned by the equi lib rium con stants K HS and K Ex , are shifted de pend ing on im por tant pa ram e ters like pH, concen tra tion of humic sub stances, complexation time, and the ra tio of metal ions to HS.

Lability or der
Ta ble 1 shows the grad ual sep a ra tion of all metal ions complexed by HS.The ion Cu reaches the ex change equilib rium be fore the other metal ions stud ied (about 15 min).In gen eral, a con tact time of more than 60 min is re quired to at tain the ex change equi lib rium as given by Eq. 1. Af terwards, the ef fi ciency sep a ra tion does not change.The sep ara tion of metal ions from HS so lu tions, pre-conditioned for 24 h, ex hib its the typ i cal lability or der of Pb > Mn > Cd, Ni > Cu.In par tic u lar, Cu is known to form strong HS complexes 16 .

Half-life time
Ac cord ing to Ta ble 1, if 120 min is con sid ered the time in which the max i mum con cen tra tion of met als is re covered by the resin, the sep a ra tion ki net ics has a half-life time (t 1/2 ) in the range of 1 to 5 min (Cu) and 5 to 15 min (Pb, Mn, Cd, and Ni).

Re ac tion or der
Fig ure 1. Metal dis tri bu tion co ef fi cient, Kd, as a func tion of pH in the pres ence of salt (NaCl 1 g/L) and complexing HS (1 mg/mL).Con ditions: 24 h complexation time, 20 mg Hyphan.
Ta ble 1. Re cov ery of metal ions pre-complexed by HS in func tion of con tact time with the ion-exchanger cel lu lose-Hyphan.Con di tions: 1 mg/mL HS, pH 9, 24 h complexation time, 20 mg Hyphan.

Metals
Con tact time (min)/re cov ery % The ki net ics and the re ac tion or der of this ion-exchange pro cess can be de rived from Fig. 2 (drawn from the data in Ta ble 1), which ex hib its the sep a ra tion of the ex change labile met als Pb, Cd, and Cu (con cen tra tion C L ) plot ted as a func tion of the con tact time.Ini tially, < 15 min, the la bile metal frac tions complexed by aquatic HS are sep a rated rela tively quickly.In this case, the metal ex change be tween HS so lu tions and the solid ion-exchanger oc curs, fol low ing a 2 nd or der re ac tion.Thus, the metal ions complexed with the HS re act di rectly with the im mo bi lized groups in the Hyphan.Later, the re main ing metal frac tions in the HS react fol low ing a slow 1 st or der ki net ics, com pared to metal ex change by Hyphan from aque ous salt so lu tions (t 1/2 of about 20 s 14 ).The change of ki netic re ac tion oc curs because the func tional groups of the ion-exchanger re act prefer en tially with metal frac tions bound to the ex ter nal sphere of HS.The rate of re ac tion de creases ow ing to the dif fi culty of the exchanger in in ter act ing with metal ions complexed in less ac ces si ble sites in the in ner sphere of HS.

Ef fect of HS con cen tra tion on the dis tri bu tion co ef fi cients
HS ex hibit con sid er able complexing ca pac i ties for metal ions, due to the broad spec trum of po ten tial bind ing sites.Thus, the aquatic HS con cen tra tion could be an impor tant fac tor af fect ing the avail abil ity of met als in the aquatic en vi ron ment.Ac cord ingly, the dis tri bu tion co ef ficients, Kd, of metal ions on cel lu lose-Hyphan as a func tion of the HS con cen tra tion was de ter mined by batch pro cedure.Fig ure 3 shows that all met als were better re cov ered in so lu tions with lower con cen tra tions of HS.The complexation ca pac ity in creases at high humic con cen tra -tions be cause there are more bind ing sites avail able for metal ion complexation, and the equi lib rium is shifted towards the HS-M spe cies.Thus, humic-rich wa ters are impor tant as nat u ral "buff ers" for heavy met als in the en vi ron ment.The in flu ence of age ing pro cesses In the en vi ron ment, the HS/met als in ter ac tions oc cur over a rel a tively long time (age ing pro cess).Thus, this is an im por tant fac tor that may af fect the lability of metal fractions complexed by HS.The in ter ac tion time be tween aquatic humic sub stances and metal ions was stud ied.Fig -  ure 4 shows that if the complexation time is in creased, the lability of the metal frac tions bound in aquatic HS decreases.This be hav ior is not the same for all met als.For instance, the man ga nese ion shows a re mark able de crease in lability, from 70% of re cov ery at 0.1 h to 10% af ter 10 days.In gen eral, met als complexed with the most ex ter nal groups of the HS macromolecule re act pref er en tially with the func tional groups of the resin.Con se quently, the metal re mains complexed for a long time, prob a bly due to the trans for ma tion pro cess and in ner re ar range ments.As time in creases, less ac ces si ble bind ing sites of HS may be oc cupied, and a change of metal ions from weaker to stron ger bind ing sites may take place 17 .

Con clu sions
The pres ent study dem on strates that the dis tri bu tion coef fi cients, Kd, of metal ions are strongly in flu enced by pH and HS con cen tra tion.The in flu ence of con tact time between the ion-exchanger and the metal-HS so lu tion char acter izes, at first, a 2 nd or der re ac tion with a half-life (t 1/2 ) of 1 to 5 min for Cu ion and 5 and 15 min for Pb, Mn, Cd, and Ni.An other im por tant fac tor that in flu ences the lability of metal frac tions bound to aquatic HS is the age ing pro cess.This trans for ma tion pro cess showed a grad ual de crease of the over all lability of metal ions in dis solved HS.This behav ior is prob a bly due to slow metal ex change pro cesses caused by an in ner re ar range ment in the bind ing sites within the HS mol e cules.Thus, it can be con cluded that accu mu la tion, trans por ta tion, hydrogeochemical cy cles, and bioavailability of trace met als in nat u ral wa ters fre quently de pend on the lability/in ert ness of met als bound to humic sub stances.ure 4 shows that if the complexation time is in creased, the lability of the metal frac tions bound in aquatic HS decreases.This be hav ior is not the same for all met als.For instance, the man ga nese ion shows a re mark able de crease in lability, from 70% of re cov ery at 0.1 h to 10% af ter 10 days.In gen eral, met als complexed with the most ex ter nal groups of the HS macromolecule re act pref er en tially with the func tional groups of the resin.Con se quently, the metal re mains complexed for a long time, prob a bly due to the trans for ma tion pro cess and in ner re ar range ments.As time in creases, less ac ces si ble bind ing sites of HS may be oc cupied, and a change of metal ions from weaker to stron ger bind ing sites may take place 17 .

Con clu sions
The pres ent study dem on strates that the dis tri bu tion coef fi cients, Kd, of metal ions are strongly in flu enced by pH and HS con cen tra tion.The in flu ence of con tact time between the ion-exchanger and the metal-HS so lu tion char acter izes, at first, a 2 nd or der re ac tion with a half-life (t 1/2 ) of 1 to 5 min for Cu ion and 5 and 15 min for Pb, Mn, Cd, and Ni.An other im por tant fac tor that in flu ences the lability of metal frac tions bound to aquatic HS is the age ing pro cess.This trans for ma tion pro cess showed a grad ual de crease of the over all lability of metal ions in dis solved HS.This behav ior is prob a bly due to slow metal ex change pro cesses caused by an in ner re ar range ment in the bind ing sites within the HS mol e cules.Thus, it can be con cluded that accu mu la tion, trans por ta tion, hydrogeochemical cy cles, and bioavailability of trace met als in nat u ral wa ters fre quently de pend on the lability/in ert ness of met als bound to humic sub stances.

Fig ure 2 .
Fig ure 2. Sep a ra tion of ex change-labile metal frac tions (con cen tration C L) from HS as a func tion of con tact time.Con di tions: see Ta ble

Fig ure 3 .Fig ure 4 .
Fig ure3.Metal dis tri bu tion co ef fi cients as a func tion of HS con centra tion.Con di tions: pH 9, 24 h complexation time, 20 mg Hyphan.