EFFECT OF SOIL PROPERTIES ON LEAD BIOAVAILABILITY AND TOXICITY TO EARTHWORMS

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1 Environmentl Toxicology nd Chemistry, Vol. 25, No. 3, pp , SETC Printed in the US /06 $ EFFECT OF SOIL PROPERTIES ON LED IOVILILITY ND TOXICITY TO ERTHWORMS KREN D. RDHM,* ELIZETH. DYTON, NICHOLS T. ST, JCKIE SCHRODER, MRK PYTON, nd ROMN P. LNNO# U.S. Environmentl Protection gency, 109 T.W. lexnder Drive, Reserch Tringle Prk, North Crolin School of Nturl Resources, The Ohio Stte University, Columus, Ohio 43210, US Deprtment of Plnt nd Soil Sciences, Oklhom Stte University, Stillwter, Oklhom 74078, US Deprtment of Sttistics, Oklhom Stte University, Stillwter, Oklhom 74078, US #Deprtment of Entomology, The Ohio Stte University, Columus, Ohio 43210, US (Received 1 Novemer 2004; ccepted 28 Ferury 2005) strct Soil properties re importnt fctors modifying metl iovilility to ecologicl receptors. Twenty-one soils with wide rnge of soil properties (US; were mended with single concentrtion of P (2,000 mg/kg) to determine the effects of soil properties on P iovilility nd toxicity to erthworms. Erthworm mortlity rnged from 0 to % cute mortlity following exposure to the sme totl concentrtion of P (2,000 mg/kg) in mended field soils. Internl P concentrtions in erthworms rnged from 28.7 to 782 mg/kg, with men of 271 mg/kg. Pth nlysis ws used to prtition correltions in n ttempt to discern the reltive contriution of ech soil property. Results of pth nlysis indicted tht ws the most importnt soil property ffecting erthworm mortlity ( p 1) nd internl P ( p 5). Soil ws relted inversely to mortlity nd internl P, soil solution P, nd P iovilility. The most importnt soil property modifying reproduction ws morphous iron nd luminum oxides (FEL). ecuse FEL is rich in -dependent ction-exchnge sites, severl soil properties, including, FEL, nd ction-exchnge cpcity, hve cusl effect on P dsorption nd solule P. Pth nlysis is useful for ssessing contminted soils with wide rnge of soil properties nd cn ssist in ecologicl risk ssessment nd remedition decisions for contminted sites. Soil properties re importnt fctors modifying metl iovilility nd toxicity nd should e considered during the ecologicl risk ssessment of metls in contminted soils. Keywords Led Ecologicl risk ssessment iovilility Erthworm Soil properties INTRODUCTION Current routine prctices for investigting the nture nd extent of contmintion t metl-contminted sites involve determining totl metl content in soils s n estimte of exposure. The exposure point concentrtion (i.e., dose) in risk clcultion my e n ssumed percentge of the totl metl content, reflecting the metl iovilility, nd this often pproches %. Wheres this ssumption is conservtive in terms of eing protective of humn helth nd the environment, it my not e resonle estimte of site conditions, ecuse the ctul iovilility of metls my e much lower. The resulting risk my overestimte the true risk of exposure to site medi nd result in lengthy nd costly site remedition tht could e unwrrnted. Led toxicity is seldom relted directly to the totl soil P concentrtion ecuse of the modifying effects of soil physicochemicl properties (e.g.,, orgnic cron [], morphous iron nd luminum oxides [FEL], nd ction exchnge cpcity []) on P iovilility. Risk to soil invertertes sed on totl P concentrtion my not predict dverse effects ccurtely [1], ecuse exposure expressed s totl P does not consider the effect of these soil-modifying fctors on P iovilility, which often is much less thn % [2 4]. It is difficult to determine the effects of individul soil * To whom correspondence my e ddressed (rdhm.kren@ep.gov). Presented t the Symposium on Risk ssessment of Metls in Soils, 14th nnul Meeting, SETC Europe Meeting, Prgue, Czech Repulic, pril 18 22, properties on metl iovilility, ecuse mny soil properties re intercorrelted to some degree. Vrious regression models hve een suggested to segregte the effects of vrious physicochemicl soil properties [1,4 6]. The rections etween cly minerls nd metls re ttriuted primrily to ction-exchnge or lignd-exchnge (i.e., specific dsorption) rections tht occur on FEL cly. For this reson, soil cly content typiclly is highly correlted with FEL content nd. Hydrous oxides of iron re contined in the cly frction of soils nd hve high sorption cpcity for P [7]. Hevy metls re ound strongly to soils tht re rich in orgnic mtter or cly [8]. However, ecuse mny soil prmeters hve more thn one inding mechnism nd re strongly intercorrelted with other soil prmeters, it is difficult to distinguish cusl effects from intercorreltion. The iovilility nd toxicity of metls to soil invertertes re ffected y mny fctors, including orgnic mtter content, cly, nd [4,9 13]. Jnssen et l. [5] studied the impct of soil properties on metl uptke y erthworms in 20 contminted (totl P concentrtions rnged from 0.34 to 4.09 mmol/kg) field soils. Using regression techniques, they found tht soil ws the most importnt soil property ffecting P prtitioning etween the soil solid phse nd the pore wter nd metl ccumultion in erthworms. Furthermore, their regression equtions for P ioccumultion fctors nd soil chrcteristics showed tht cly nd morphous iron oxides were the most importnt soil properties influencing ioccumultion. Peijnenurg et l. [1] studied the uptke kinetics of P in erthworms in the sme 20 soils used during 769

2 770 Environ. Toxicol. Chem. 25, 2006 K.D. rdhm et l. Tle 1. Txonomic clssifictions nd ckground metl levels of the 21 study soils Soil txonomic clssifiction Soil series Horizon ckground P levels (mg/kg) Fine-lomy, mixed, superctive, clcreous, mesic Typic Endoquoll Cnisteo Fine, mixed, thermic quic rgiudoll Dennis 13.7 Lomy, mixed, ctive, thermic renic Hplustlf Corse-lomy, mixed, superctive, mesic Cumulic Hpludoll Fine, mixed, superctive, thermic Udertic Pleustoll Dougherty Hnlon Kirklnd Fine, smectitic, mesic Typic Endoquert Luton 12.3 Fine-lomy, mixed, superctive, thermic ridic Clciustoll Mnsic Fine-silty, mixed, ctive, thermic Udic Pleustoll Norge 11.6 Fine, smectitic, thermic, Typic Epiqert Osge Fine-silty, mixed, superctive, thermic Pchic rgiustoll Pond Creek Sndy, mixed, mesic Lmellic Hplustlf Prtt Fine, smectitic, mesic ridic rgiustoll Richfield 12.6 Fine, smectitic, thermic Oxyquic Vertic rgiudoll Summit Fine, mixed, thermic Mollic lqulfs Fine-lomy, mixed, ctive, thermic Udic rgiustoll Tlok Teller Rnge Typicl rnge Typicl men ( Typicl P levels in uncontminted soil [7] the study y Jnssen et l. [5]. The min ojective of their study ws to quntify the modultion of iovilility s function of soil chrcteristics. Using multivrite expressions, Peijnenurg et l. [1] found tht soil ws the dominnt soil property tht contriuted most to explining the vrince in uptke rte constnts nd ioccumultion fctors. Correltion nd multiple-regression techniques customrily re used to exmine reltionships oth etween nd mong soil properties nd iologicl endpoints. However, these techniques provide little informtion regrding the reltive contriution of ech prmeter included in the eqution model. The reltive contriution of ech independent vrile is further msked y intercorreltions mong independent vriles. Pth nlysis, form of structurl eqution modeling [14], is technique tht cn e used to ugment trditionl regression nlysis nd provide quntittive vlue for the contriution of ech prmeter in explining the vrition of the dependent vrile. The focus of the present reserch ws to exmine the effect of soil properties on the iovilility nd toxicity of P to erthworms using field soils with wide rnge of soil properties tht were mended with the sme totl concentrtion of P (2,000 mg/kg dry wt). To our knowledge, little or no reserch is ville exmining the effect of soil properties on the iovilility nd soluility of P using the sme totl concentrtion of P in field soils. This pproch provides wide rnge of exposures to vrious levels of ville P ecuse of the soil properties modifying the vilility of P. This pproch, in conjunction with pth nlysis, provides the ility to decompose simple correltions etween soil properties nd determine the importnce of individul soil properties on P iovilility nd iossy endpoints. In the present study, 21 soils with wide rnge of soil properties were mended with the sme totl concentrtion of P to determine the effect of soil properties on P iovilility nd toxicity. The present results cn e used to identify soil properties tht will modify site-specific ecologicl risks t sites with contminted soil. This specil issue contins compnion pper written y Dyton et l. [15] tht evlutes the contriution of soil properties to modifying phytovilility nd phytotoxicity of P to lettuce (Lctuc stiv). METHODS ND MTERILS Soil chrcteristics nd hndling Forty soils were collected from uncontminted sites, nd their physicochemicl properties (,, FEL, nd ) relted to metl iovilility were determined. suset of 21 soils (US; txonomy) ws chosen to provide wide rnge in physicochemicl properties (Tle 1). Soil ckground P levels were determined y microwve-ssisted, concentrted nitric cid digestion ccording to U.S. Environmentl Protection gency (U.S. EP) method 3051 [16] followed y P quntifiction using inductively coupled plsm tomic emission spectroscopy. Led levels were found to e within the typicl rnge for uncontminted soils [7] (Tle 1). Uncontminted soils were spiked with regent-grde P(NO 3 ) 2 t 2,000 mg/kg. The spike level ws chosen sed on initil rnge-finder erthworm iossys. For detiled description of the soil-spiking procedure, see Dyton et l. [15]. The finl soil P-spike concentrtion (2,000 mg/kg) ws confirmed y microwve-ssisted, concentrted nitric cid digestion ccording to U.S. EP method 3051 [16]. Soil nlysis Soil physicochemicl chrcteristics (,, FEL, nd ) relted to metl sorption were mesured in the 21 study

3 Modifying effect of soil properties Environ. Toxicol. Chem. 25, soils. For detiled description of soil chrcteriztion, see Dyton et l. [15]. Erthworm iossys iossys (28 d) using mture (clitellte) worms (Eiseni ndrei) were conducted in triplicte (10 worms/replicte) ccording to stndrd protocol [17]. mericn Society for Testing nd Mterils rtificil soil [17] nd unspiked control soils served s controls for qulity ssurnce with respect to survivl, cocoon production, nd growth. ll soils were mintined t 45% moisture, periodiclly weighed, nd rought ck to the initil moisture weight. Testing ws conducted in n environmentl chmer mintined t 20 1 C with constnt light. Erthworms were oserved dily for the first 8 d nd three times week therefter for the reminder of the test. Ded erthworms were frozen t 20 C for susequent nlysis. Cocoons were collected y hnd sorting when dily oservtions were mde for mortlity s indicted ove. t dy 28 of ech study, live erthworms were depurted for 24 h on moist filter pper, rinsed, weighed, nd stored s descried ove. Led concentrtions in erthworm tissue were determined s descried y Morgn nd Morris [18]. n individul worm from ech replicte (three replictes per soil P comintion) ws dried for 24 h t 80 C in preweighed, 10-ml glss eker nd then weighed. Next, individul worms were digested t 105 C in 5 ml of concentrted, trce metl grde HNO 3 nd llowed to evporte until pproximtely 0.5 ml of solution remined. Digests were then resoluilized in 3 ml of 0.5 M HNO 3, heted for 15 min t 60 C, nd diluted to finl volume of 10 ml with 0.5 M HNO 3. ll worm digests were stored in Nlgene (Nlge Nunc Interntionl, Rochester, NY, US) high-density polyethylene ottles until nlysis. Led concentrtions of erthworm digests were mesured using grphite furnce tomic sorption spectrometry (HG nlyst 700; Perkin-Elmer, Wellesley, M, US). The limit of detection for P in erthworm tissues ws 4 g/kg dry weight. ll tissue nlyses included procedurl lnks, spikes, nd certified reference mteril (Loster Heptopncres, TORT-2; Ntionl Reserch Council, Ottw, ON, Cnd) conducted in triplicte. Digested lnks contined P concentrtions elow the limit of detection. Men (% reltive stndrd devition) recoveries of P from spike nd certified reference mteril (Loster Heptopncres) were 97% (2.8%) nd 98% (1.2%), respectively. Dt nlysis Sttisticl nlyses were performed using PC SS Version 8.2 [19]. efore nlysis, ll dt were normlized (studentized), llowing the dt to e expressed in stndrd form with men of zero nd stndrd devition of one [20]. ecuse the simple or comined reltionships etween the iologicl endpoints nd soil properties my not e liner, ll sttisticl nlyses were performed on the studentized (R) nd the studentized nd then linerized dt sets [21] to find the est-fit model. Dt set trnsformtions used to linerize the studentized dt sets were squre root (SR). Only the iologicl endpoints, not the soil properties, were trnsformed, using the SR trnsformtion. The reltionship etween SR erthworm iologicl endpoints (P ioccumultion, mortlity, nd cocoon production) nd soil properties (,, FEL, nd ) were exmined using simple liner regression. The intercorreltion etween independent vriles lso ws determined using simple liner regression [21]. The comined reltionships mong ech iologicl endpoint nd soil properties were exmined further using multipleliner-regression nd pth nlysis [14,21]. Pth nlysis ws used to prtition simple correltions into two types of reltionships: The effect of n exogenous vrile (i.e., soil property) on the iologicl endpoint, referred to s direct effect, nd the effect of exogenous vriles on the response vrile s medited y their effect on, referred to indirect effects. The significnce levels for the direct effects (pth coefficients) re derived from the p vlues ssocited with the prtil slopes from the multiple regressions. The significnce levels for the pth totl re the correltion coefficients (r) from the simple correltions. Pth nlysis my provide insight regrding the reltive contriution of ech independent vrile (i.e., individul soil property) included in the model towrd explining the vrition in iologicl endpoints. For dditionl informtion concerning the pth nlysis model used here, see Dyton et l. [15]. Soil nlysis RESULTS ND DISCUSSION Txonomic clssifictions nd ckground P concentrtions of study soils (temperte climte soils including three soil orders) re listed in Tle 1. Soil physicochemicl properties of the 21 selected soils re summrized in Figure 2 of Dyton et l. [15]. The for Pspiked soils rnged from 3.8 to 7.8, with men of 5.8. Soil rnged from 3.01 to 32.4 cmol c /kg, with men of 16.8 cmol c /kg. Orgnic cron rnged from 5.00 to 3 g/kg, with men of 15.1 g/kg. The FEL content rnged from 9 to mol/kg, with men of 57 mol/kg. Erthworm iossys Erthworm mortlity rnged from 0 to 6%, with men of 1.4% in the control (i.e., unspiked) soils (dt not shown). Erthworm mortlity in ll rtificil soil ws less thn 10%. Erthworm mortlity in P-mended (2,000 mg/kg) soils rnged from 0 to %, with men of 36% (Tle 2). This suggests tht P mendments sustntilly ffected lethlity reltive to the controls, nd the wide rnge of responses indictes tht the iovilility of P ws modified y soil properties. Mortlities oserved in mny of the soils tested suggest tht P detoxifiction mechnisms were exceeded. The min detoxifiction pthwy for P is thought to e sequestrtion within inorgnic mtrices or inding to orgnic lignds [22]. The min site of storge is the chlorgogenous tissue tht contins grnules of phosphte-rich complexes contining C nd Zn [23]. These grnules re involved in inding orderline metls, such s P, y exchnging mtrix-ssocited C [23,24]. dverse effects cn e expected only when the cpcity of detoxifiction mechnisms is exceeded, occurring t very high internl concentrtion (IC) when metl is slowly sequestered [25]. The men ICs of E. ndrei exposed to the control (i.e., unspiked) soils were 8 mg/kg of P (dt not shown). Erthworm IC fter exposure to P-mended soils rnged widely, from 28.7 to 782 mg/kg, with men of 271 mg/kg (Tle 2). 27-fold difference in erthworm IC of P fter exposure to the sme totl P soil content in 21 soils indictes tht the

4 772 Environ. Toxicol. Chem. 25, 2006 K.D. rdhm et l. Tle 2. Mortlity, internl P, nd reproduction of erthworms exposed to 21 P-mended (2,000 mg/kg) soils Soil series Horizon Mortlity (%) Internl P (mg/kg dry wt) Reproduction (no. of cocoons) Reltive reproduction (%) Cnisteo Dennis Dennis Dougherty Hnlon Kirklnd Luton Mnsic Mnsic Norge Osge Osge Pond Creek Pond Creek Prtt Prtt Richfield Summit Summit Tlok Teller 690 Men Minimum Mximum ( Reproduction of Eiseni ndrei in P-mended soils re presented reltive to the control soils. iovilility of P is eing modified y soil properties. This finding is consistent with those of Peijnenurg et l. [1], indicting tht soil properties hve significnt impct on P uptke y E. ndrei. Reproduction in the P-mended soils is expressed reltive to the control vlue to determine the effect of P on reproduction. Results show tht reltive reproduction of E. ndrei decresed with incresing internl P concentrtions (Fig. 1). This result for P is the sme trend tht Lock et l. [25] oserved for Cd in their study of Cd-contminted soils using Eiseni fetid. The oserved vriility in P iovilility ws the result of differences in P interctions with the physicochemicl mtrix of the soil. Reltionships etween soil properties nd iologicl endpoints Erthworms exposed to soils in which % mortlity occurred were removed from erthworm IC sttisticl models to void comprison of depurted nd nondepurted worms. This ws done to void comprison of differences in the soil content of the erthworm gut nd differences in P uptke cused y the physiologicl effects of cutely toxic P exposure. The est-fit model (linerized) for ech reltionship ws provided y the SR trnsformtion of the iologicl endpoint. Soil properties were not trnsformed. Simple liner correltion coefficients (r) for the reltionship etween the erthworm iossy endpoints (normlized nd then linerized) nd soil properties were determined (Tle 3) to scertin which soil properties my ct s modifiers of P toxicity nd iovilility. significnt negtive reltionship ws oserved etween mortlity nd (r 0.695, p 1) nd etween mortlity nd (r 0.467, p 5). No significnt Tle 3. Simple liner correltion coefficients for reltionships etween the squre root of iossy endpoints nd soil properties for erthworms exposed to 21 P-mended (2,000 mg/kg) soils Simple liner correltion coefficients (r) Soil property Mortlity Internl P Reltive reproduction FEL 0.695** * 0.506* ** 0.631* 0.716** 0.661** 0.874*** Fig. 1. Reltive reproduction versus internl P concentrtions of Eiseni ndrei fter 28-d exposure to P-mended (2,000 mg/kg) soils. Soil txonomy nd series re listed in Tle 1. * p 5, ** p 1, *** p 1. ction exchnge cpcity; FEL morphous iron nd luminum oxides; orgnic cron.

5 Modifying effect of soil properties Environ. Toxicol. Chem. 25, Tle 4. Intercorreltions etween soil properties for 21 P-mended (2,000 mg/kg) soils Soil prmeter FEL Simple correltion coefficients (r) FEL ** ** 0.70** 0.55** Soil txonomy nd series re listed in Tle 1. ** p 1. ction exchnge cpcity; FEL morphous iron nd luminum oxides; orgnic cron. Tle 6. Results of pth nlysis including direct nd indirect effects nd simple correltion coefficients (r) for erthworms exposed to Pspiked (2,000 mg/kg) soils Soil property Direct effect Squre root mortlity 0.750** 73 FEL Squre root internl P FEL 0.502** Squre root reltive reproduction FEL 0.402* Indirect effect N c N N Correltion coefficient 0.695** * 0.506* ** 0.631* 0.716** 0.661** 0.874*** Soil txonomy nd series re listed in Tle 1. * p 5, ** p 1, *** p 1. ction exchnge cpcity; FEL morphous iron nd luminum oxides; orgnic cron. c N not pplicle (no cusl reltionship of on exogenous vriles). correltion ws found etween mortlity nd or FEL (Tle 3). Significnt negtive reltionships were found etween internl P nd (r 0.668, p 1) nd (r 0.506, p 5). The reltionships etween internl P nd nd FEL were not significnt. Cly nd hve een reported in regression models s eing importnt soil properties modifying P uptke rtes [1,5]. Significnt correltions were found etween reltive reproduction nd the soil properties: (r 0.716, p 1), (r 0.874, p 1), (r 0.631, p 5), nd FEL (r 0.661, p 1) (Tle 3). Intercorreltions etween soil properties re summrized in Tle 4. For the 21 soils used in the present study, significnt correltion ws found etween nd (r 0.69, p 1), wheres no significnt reltionship ws found etween nd or FEL (Tle 4). Orgnic cron ws significntly relted to (r 0.70, p 1) nd to FEL (r 0.61, p 1) (Tle 4). Soil orgnic mtter contriutes to the nd increses metl dsorption, therey reducing metl concentrtions in solution [8]. dditionlly, FEL ws significntly correlted with (r 0.55, p 1) for the study soils (Tle 4). To determine if the comined effects of soil properties my modify soil P toxicity, multiple liner-regression model ws used to exmine the reltionship etween ech erthworm iossy endpoint (normlized nd then linerized) nd the comined soil properties. The model included,, FEL, nd s exogenous vriles. The est-fit model for ech reltionship ws with the SR trnsformtion, nd ll re shown in Tle 5. significnt reltionship ws found etween erthworm mortlity nd soil properties (r , p 1). The reltionship etween erthworm internl P concentrtion nd soil properties lso ws significnt (r , p 1). The reltionship etween reltive reproduction nd soil properties ws highly significnt (r , p 1) (Tle 5). Multiple-regression equtions lone my not e useful for predicting metl iovilility or for mking remedition decisions. This is ecuse they my not provide sufficient insight regrding the reltive contriution of ech independent vrile (i.e., individul soil property) included in the models towrd explining the vrition in iologicl endpoints. Strong intercorreltions etween independent vriles cn result in pprent correltions with the endogenous vrile tht my not e well sustntited. Prtitioning correltions into direct nd indirect effects using pth nlysis my provide dditionl informtion concerning the reltive contriution of ech soil property. Results of pth nlysis (Tle 6) showed tht the highly significnt correltion (r 0.695, p 1) etween nd mortlity (Tle 3) ws cused entirely y the direct effect (Tle 6) of on mortlity (r 0.750, p 1). lthough the simple correltion (Tle 3) etween nd mortlity (r 0.467, p 5) ws significnt, pth nlysis showed tht the direct effect of on mortlity ws negligile (r 67) (Tle 6). Therefore, the pprent correltion of with mortlity ws cused y strong intercorreltions (Tle 4) etween nd (r 0.69, p 1), FEL (r 0.55, p 1), nd (r 0.70, p 1) nd not y n effect of on mortlity. Pth nlysis showed no significnt contriutions from either or FEL (Tle 6) to explining vritions in mortlity. Pth nlysis shows tht the simple correltion (Tle 3) etween nd internl P (r 0.506, p 5) ws cused entirely y the direct effect of on internl P (r 0.502, p 5) (Tle 6). This finding is consistent with the results for mortlity nd is resonle ecuse ffects the solu- Tle 5. Multiple-regression equtions nd coefficient of determintion (R 2 ) for the reltionship etween the squre root (SR) of iossy endpoints nd soil properties for erthworms exposed to 21 P-mended (2,000 mg/kg) soils Endpoint Multiple liner regression R 2 SR mortlity 0.75() 73() 0.40(FEL) 67() 0.60** SR internl concentrtion 0.502() 64() 0.429)(FEL) 0.177() 0.60** SR reltive reproduction 0.273() 0.176() 0.402(FEL) 0.414() 0.89*** Soil txonomy nd series re listed in Tle 1. ** p 1, *** p 1. ction exchnge cpcity; FEL morphous iron nd luminum oxides; orgnic cron.

6 774 Environ. Toxicol. Chem. 25, 2006 K.D. rdhm et l. ility of P, mking it more or less ville for uptke y n orgnism. Soil ws the most importnt soil property for uptke nd ioccumultion in E. ndrei exposed to 20 field contminted soils [1]. lthough highly significnt, the simple correltion (Tle 6) etween nd internl P (r 0.668, p 1) ws cused primrily y the intercorreltions etween nd the other exogenous vriles. Pth nlysis (Tle 6) showed tht direct effect of on internl P ws not strong (r 0.177), so the pprent correltion of with internl P ws cused primrily y significnt intercorreltions etween nd (r 0.69, p 1), FEL (r 0.55, p 1), nd (r 0.70, p 1) (Tle 4) nd not y n effect of. Pth nlysis showed no significnt reltionships etween internl P nd either or FEL (Tle 6). Soil hs the potentil to modify metl soluility nd vilility y controlling metl dissolution nd precipittion. Soil regultes the ioniztion of -dependent ion-exchnge sites on orgnic mtter nd metl oxide cly minerls ffecting ctionic metl vilility. Soil is relted inversely to P dissolved in soil solution. The present results clerly show the inverse reltionship etween soil nd mortlity nd soil nd internl P concentrtion (i.e., tissue P). The simple correltion etween nd reltive reproduction (r 0.631, p 5) ws significnt. The totl effect of on reproduction ws prtilly cused y the direct effect of on reproduction (r 0.273), strongly supported y the indirect effect of vi (r 0.244). The reminder of the simple correltion ws cused y intercorreltions mong the exogenous vriles. Soil is relted directly to ecuse of the dependence of mny ction-exchnge sites. The totl correltion etween nd reproduction (r 0.759, p 1) ws highly significnt. However, pth nlysis showed tht the contriution of, ecuse the direct effect of (r 0.176) nd the indirect effect of vi (r 0.128), ccounted for less thn hlf the pprent totl correltion. The reminder of the totl correltion ws cused y intercorreltions mong exogenous vriles (Tle 4). The totl correltion etween FEL nd reproduction (r 0.661, p 1) ws significnt, nd pth nlysis (Tle 6) indicted tht the effect of FEL on reproduction ws cused primrily y the significnt direct effect of FEL (r 0.402, p 5) on reproduction, finding tht is supported, to some extent, y the indirect contriution of FEL vi (r 0.150). Mny studies hve suggested tht P cn form stle complexes with [7,26 29] nd with metl oxide surfces [7,26 28,30]. The totl correltion etween nd reproduction (r 0.874, p 1) ws highly significnt. However, pth nlysis showed tht only pproximtely hlf the totl effect of on reproduction ws cused y the direct effect of (r 0.414), with the reminder cused y intercorreltions mong the exogenous vriles (Tle 6). The most importnt soil property modifying reproduction ws FEL (Tle 6). morphous iron nd luminum oxides re lrge sinks for P nd other trce elements [31]. ecuse FEL is rich in -dependent ction-exchnge sites, severl soil properties, including, FEL, nd, hve cusl effect on P dsorption [15]. dsorption of P y FEL decreses soil solution P. The effect of ws weker for reproduction compred to its contriution to mortlity nd internl P. However, is importnt, ecuse reproduction is relted to internl P concentrtions, s shown in Figure 1. CONCLUSIONS The rnges of responses oserved in the present study were the result of differences in P iovilility cused y P interctions with the physicochemicl mtrix of the soil. Soil properties mitigted contminnt iovilility nd toxicity to ecologicl receptors. Soil ws relted inversely to erthworm mortlity nd internl P. Results of pth nlysis indicted tht ws the most importnt soil property ffecting mortlity nd internl P. Wheres simple correltion nlysis (Tle 3) suggested tht ws correlted significntly with mortlity nd internl P, pth nlysis ws useful in showing tht the effect of, either directly or indirectly, on mortlity nd internl P ws negligile. Therefore, the pprent correltion of with mortlity nd internl P ws cused y significnt intercorreltions mong nd other exogenous vriles. Ction exchnge cpcity, however, did contriute to explining some of the vrition in reltive reproduction. The effect of nd FEL on reproduction ws prtilly cused y the indirect effects of these vriles vi. Increses in FEL content lso were relted significntly to incresed cocoon production, s demonstrted y the significnt direct effect of FEL. Pth nlysis is useful sttisticl tool, ecuse it provides insight regrding the reltive contriution of ech independent vrile, either directly nd indirectly, towrd explining vrition in iologicl endpoints. lthough pth nlysis cnnot prove cusl reltionship, it cn refute one. Pth nlysis my e useful for ecologicl studies involving soils with wide rnge of physiochemicl properties nd cn ssist in site ecologicl risk ssessment nd remedition decisions. Results from the present study clerly show tht soil properties cn gretly reduce contminnt iovilility nd risk. Soil properties should e considered when ssessing the risk of P contminted soils to ecologicl receptors. Soil properties re importnt fctors modifying metl iovilility nd should e considered in the ecologicl risk ssessment process so s not to overestimte contminnt risk. cknowledgement We would like to thnk Derrick Chppell for ssistnce with the iossys. We re grteful to Shron Hrper for her ssistnce nd review. The U.S. EP, through its Office of Reserch nd Development, funded nd mnged or prtilly funded nd collorted in the reserch descried here under contrct CR to R.P. Lnno nd N.T. st. It hs een sujected to gency review nd pproved for puliction. REFERENCES 1. 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