POTENTIAL NITRIFICATION RATE AS A TOOL FOR SCREENING TOXICITY IN METAL-CONTAMINATED SOILS

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1 Environmentl Toxicology nd Chemistry, Vol. 0, No., pp , SETAC Printed in the USA /0 $ POTENTIAL NITRIFICATION RATE AS A TOOL FOR SCREENING TOXICITY IN METAL-CONTAMINATED SOILS ERIK SMOLDERS,* KRIS BRANS, FILIP COPPENS, nd ROEL MERCKX Lbortory of Soil Fertility nd Soil Biology, Ktholieke Universiteit Leuven, Kolsteelprk Arenberg, 0, 300 Leuven, Belgium (Received 3 December 000; Accepted April 00) Abstrct A potentil nitrifiction rte test (PNR) ws used to identify metl toxicity in field-contminted soils. The test ws pplied to metl slt spiked soils, to uncontminted soils, nd to 5 soils tht re contminted by former metl smelting ctivities. Four griculturl soils (ph 4.5 ) were spiked with vrious rtes of Cl (0 00 mg /kg dry wt) or Cl (0 3,000 mg /kg dry wt) nd were equilibrted more thn nine months prior to testing. The soil EC50s of the PNR were between 50 nd 350 mg /kg dry weight. No continuous decrese of the nitrifiction with incresing ppliction ws observed. The nitrifiction rte ws reduced by between 50 nd 80% t the highest ppliction in ll soils. The PNRs of uncontminted soils vried widely (0 mg N/kg/d), but most of this vribility is explined by soil ph (R 0.). The PNRs of the 5 contminted soils were 0 to 44% of the vlues predicted for n uncontminted soil t corresponding ph. Significnt toxicity in field-contminted soils ws identified if the PNR ws outside the 95% prediction intervl of the PNR for n uncontminted soil t corresponding ph nd ws found in seven soils. These soils contin 60 to 34,000 mg /kg dry weight nd 5 to 04 mg / kg dry weight nd hd ph 5.. No toxicity could be detected below ph, where even zero PNR vlue is within the 95%prediction intervl of uncontminted soils. It is concluded tht the nitrifiction is sensitive to metl stress but tht its power s soil biossy is low becuse of the high vribility of the endpoint between uncontminted soils. The ecologicl significnce of the ssy is discussed. Keywords Nitrifiction Cdmium Zinc Soil Soil solution INTRODUCTION Nitrifiction is the conversion of mmonium ( NH 4 ) to nitrite ( NO) nd then to nitrte ( NO3). Nitrifiction is performed by specific group of microorgnisms, tht is, Nitrosomons spp. for the oxidtion to NO nd Nitrobcter spp. for the oxidtion of NO to NO3. Nitrifiction is process tht is ffected by metls in soil [ 5]. For exmple, zinc pplictions s low s 00 mg /kg hve been shown to reduce the initil nitrifiction rte to bout 65% of tht in the unmended soil [5]. Becuse of its sensitivity to metl contmintion nd its key role in nitrogen cycling, nitrifiction is process tht cn be used in hzrd ssessment of chemicls in soil. The term nitrifiction potentil is commonly used for nitrifiction in soil t sturting substrte concentrtions, tht is, the nitrifiction observed immeditely fter dding NH 4 s the substrte [,3,5,6]. The soil nitrte concentrtions re subsequently mesured fter incubtion periods tht cn vry from few hours to 50 d [,6]. The ccumultion of NO -N is lso used s n endpoint, lthough this intermedite is found only in tests performed in suspensions [6] or in clcreous soils []. The Interntionl Orgniztion for Stndrdiztion (ISO, Genev, Switzerlnd) lists ISO 438 [] s 8-d soil incubtion test where the nitrifiction rte is used s n endpoint fter dding 00 mg NH 4 -N/kg soil s substrte. The nitrifiction rte strongly vries with soil properties in uncontminted soils. Lbortory tests show tht nitrifiction rte is slow in cid soils [,8]. The nitrifiction rte is lso reduced t elevted soil nitrte content [9] nd t high soil moisture content [0]. This vribility is excluded when testing * To whom correspondence my be ddressed (erik.smolders@gr.kuleuven.c.be). lbortory-contminted soils reltive to uncontminted control smples t stndrd environmentl conditions. For the purpose of screening soil qulity, however, need exists to know the expected vlue of the nitrifiction rte for uncontminted soils. This cn be chieved from survey of this process in uncontminted smples. Screening soil qulity in the field could be useful tool in risk ssessment or in monitoring remedition ctivities. This pper ddresses the use of the potentil nitrifiction rte (PNR) for screening toxicity in contminted soils. The PNR is mesured in wide series of uncontminted nd contminted soils nd tested t stndrd conditions in the lbortory. The contminted soils include soils tht re spiked in the lbortory with nd slts nd field-contminted soils (mixed metl pollution). The vribility of the PNR in uncontminted soils is relted to soil properties, nd this reltionship is used to determine if the PNR of contminted soil indictes toxicity. Soil smpling nd tretment MATERIALS A METHODS Two groups of soils were collected in this study: uncontminted soils nd 5 soils contminted by former smelter ctivities (field-contminted soils). The uncontminted soils were collected in Belgium in August 99 (soils 4) or in August 998 (soils 5 ). The soils include vriety of griculturl soils nd forest soils (Tble ). Totl nd concentrtions in these soils (Tble ) were below the bseline concentrtions of Flemish soils, which re defined s 90th percentiles of 450 soil smples collected in rble lnd, forest, nd fllow nd psture, well wy from point metl sources []. Soils nd 4 hve concentrtions bove the bse- 469

2 40 Environ. Toxicol. Chem. 0, 00 E. Smolders et l. Tble. Selected chrcteristics of the uncontminted soils Smple Lnd use ph CCl (0.0 M) % C % Cly CEC ph (cmol c /kg) Aqu regi soluble metls (mg/kg) Pine forest Lwn Riverside Riverside Grsslnd Pine forest Lwn Lwn Grsslnd Grsslnd b CEC ction exchnge cpcity. b not mesured. line concentrtions. These soils were smpled on riversides tht typiclly hve elevted metl concentrtions []. It is unknown if the elevted is from nthropogenic or from nturl origin. The PNRs of both soils re mong the highest mong the uncontminted soils t corresponding ph, indicting tht is not cusing n dverse effect (detils not shown). Soils to 4 were used for dose response nlysis nd were spiked with nd in the lbortory (see the following discussion). The field-contminted soils were collected in Belgium in September 998 nd include lso vriety of griculturl soils nd forest soils (Tble ). All soils were ir dried nd sieved ( mm) nd were stored t lest for one month prior to testing the PNR. The four soils used for dose response nlysis were rewetted with deionized Tble. The potentil nitrifiction rte (PNR) in 5 contminted soils, sorted by soil ph. The PNR is compred with the vlue expected for n uncontminted soil t corresponding ph (regression model Eqn. ) Smple Lnd use ph CCl (0.0 M) % C % Cly Aqu regi soluble metls (mg/kg dry wt) CEC ph (cmol c /kg) Observed PNR (mg N/kg d) Expected (95% PI) %of expected Bre Lwn (prk) Rodside Nturl grsslnd Nturl grsslnd Lwn (residentil) Industril site d ,00 3, (.5).0 (.4). (.4) 4.8 (.5) 6.8 (.5).6 (.4). (.4) 8.0 (.4) 9. (.4) 9.3 (.4) 9.5 (.4) 0.3 (.4) 0.8 (.4) 0.8 (.4). (.5) 44 b 33 b 0 b 0 b 3 b 4 b b 30 b c c 4 c 4 c c c c CEC ction exchnge cpcity. b Not significntly different from uncontminted soil t sme ph. c Vlue outside the 95% prediction intervl (PI) for uncontminted soils. d not mesured.

3 Nitrifiction in metl-contminted soils Environ. Toxicol. Chem. 0, 00 4 Tble 3. Effect of soil properties on the potentil nitrifiction rte (PNR, mg N/kg d) in uncontminted soils. Results of stepwise multiple regression; ll vribles re significnt t the 0.05 level Step Soil vrible Prtil R Model R 3 ph %C CEC (cmol c /kg) PNR ph.0%c 0.6 CEC CEC ction exchnge cpcity. wter contining pproprite mounts of nd s their chloride slts to obtin rnge of soil metl concentrtions (control nd six tretments). The metl slts were pplied t rte of, 0, nd 00 mg /kg, or 300, 600, nd 3,000 mg /kg dry weight. The control soils were rewetted with deionized wter only nd were further treted similrly s the metl-spiked soils. In soil 4, we included three dditionl ph tretments (the originl ph nd two lower ph vlues) using 35 nd 60 mmol H /kg bsed on H SO 4. These three ph tretments were pplied to ll seven metl tretments. The finl moisture contents of the soils were % (soil ), % (soil ), 0% (soil 3), nd % (soil 4). All soils were mixed fter spiking, nd the smples were stored in drkness for dt 5C followed by drying/rewetting cycle during d (plnt growth chmber). The soils were subsequently stored t 4C. The toxicity tests were performed between 9 nd 9 months fter spiking the soils. Soil chrcteristics The soil texture ws determined with the pipette method fter soil smple pretretment nd snd removl [3]. Soil ph ws mesured in CCl 0.0 M in solid/liquid (S/L) rtio of :.5 fter overnight equilibrtion. The ction exchnge cpcity (CEC) ws mesured using silver thioure s the index ction in 0.-M NH 4 OAc buffer t ph.0 [4]. Totl crbon ws mesured by dry combustion (solid smpler nlyzer CA 00, Sklr, Bred, The Netherlnds). The soil metl concentrtions were mesured fter n qu regi digest nd either flme tomic bsorption spectroscopy (FAAS), grphite furnce tomic bsorption spectroscopy (GFAAS, deuterium bckground correction nd.4-mm H 3 PO 4 s modifier), or inductively coupled plsm/opticl emission spectroscopy. The wter holding cpcity ws determined by submerging soil-filled (50-g) cylinder with perforted bse (covered with filter pper) in wter bth for 3 h t room temperture nd subsequently llowing the wter to drin overnight on try of snd. The wter content ws determined fter drying t 05C. All soil properties re expressed on n oven-dried (05C) weight bsis (Tbles nd 3). Soil solution ws seprted by centrifugtion (,00 g, h) nd filtered (0.45 m). The metl concentrtions in soil solution nd in soil digests were mesured with FAAS. The concentrtions below 0. mg/l were mesured using GFAAS. Soil properties of the spiked soils were mesured fter the spiking nd 4-week equilibrtion period. Potentil nitrifiction rte Soil smples (00 g) were incubted in drkness t 5C for d in closed.5-l continers prior to substrte ddition. The soils were mended with 00 mg NH 4 -N/kg dry weight Fig.. Soil nitrte concentrtion fter ddition of 00 mg NH 4 -N/kg dry weight in soil 4 t ph nd t three ddition rtes. control; 300 mg /kg dry weight; 600 mg /kg dry weight; 300 mg /kg dry weight. [s (NH 4 ) SO 4 ] through stock solution contining 80 mg (NH 4 ) SO 4 /ml. Additionl deionized wter ws dded to djust soil moisture content to 60% of wter holding cpcity. The soil ws subsequently mixed nd incubted s 00-g liquots in.5-l closed continers t 5C. Subsmples were tken t 0,, nd 4 d fter substrte ddition (0 g, three replictes). The soil nitrte ws mesured colorimetriclly in filtered soil extrct ( M KCl, :5 solid:liquid rtio, h end-over-end shking). Sttistics The potentil nitrifiction rte ws clculted s the slope of the regression of soil nitrte to time. The lowest-observedeffect dose (LOED, the lowest dded metl concentrtion significntly ffecting the process) in the spiked soils ws determined using t test (p 0.05) on the PNR between tretment nd control. The stndrd error of the difference between PNR vlues ws clculted from the stndrd errors of ech slope. All PNR vlues bove the LOED were lwys significntly lower thn the control. A logistic eqution [5] ws fitted to the PNR-log(metl concentrtion) dt (mesured metl concentrtions, not the dose) using the Mrqurdt method (proc NLIN, SAS 6., Cry, NC, USA). The EC50 (effect concentrtion cusing 50% reduction in the PNR) nd its 95% confidence intervl were derived from the pproprite prmeter of the logistic eqution nd its stndrd error, respectively. Significnt toxic effects on PNR in the field-contminted soils re determined s explined in the Results nd Discussion sections. RESULTS The soil nitrte concentrtion increses in liner fshion fter NH 4 ddition in ll soils, except t high metl ppliction, where nitrifiction is completely impired (e.g., soil 4, Fig. ). The soil nitrte content t dy 0 ws lrger in the control smples of soils,, nd 4 thn in corresponding metl-spiked soils (e.g., soil 4, Fig. ). This vribility is most likely the result of metl toxicity on N-minerliztion nd nitrifiction during the more thn nine months of storge fter metl spiking. No such trend ws, however, found for soil 3. Nitrifiction rte ws lwys mesured from the increse in soil nitrte during the 4-d test nd not bsed on the finl nitrte content fter 4 d. The highest differences in soil nitrte content t dy

4 4 Environ. Toxicol. Chem. 0, 00 E. Smolders et l. Fig.. The potentil nitrifiction rte (PNR) in the control nd spiked tretments of soil 4 t nturl soil ph ( ph ) nd t two djusted ph vlues ( ph 6.3; ph ). The soil ph refers to tht of the unspiked soil. 0 between contminted soils nd control soils mounted to mg NO 3 -N/kg dry weight (soil, difference between control nd highest rte). Soil (control) lso contined the highest nitrte concentrtions (30 mg NO 3 -N/kg dry wt) of ll uncontminted soils. The nitrifiction rte ws more difficult to quntify in tht soil, s soil nitrte concentrtion incresed only by bout 0 mg NO 3 -N/kg dry weight during the4dofincubtion. The nitrifiction rte ws reduced through the ddition of nitrte in preliminry experiments. The ddition of 80 mg NO 3 -N/kg dry weight (s KNO 3 in solution) to soil 4 reduced the PNR by 38%. In soil, PNR ws reduced by 34% fter ddition of 8 mg NO 3 -N/kg dry weight. Preliminry experiments showed tht the sensitivity of the test decreses if the nitrifiction test is mesured in 0- to 0- d intervl. This my be relted to the limittions in NH 4 substrte. Anlyses of the summed NH4-N nd NO3-N contents in soil showed significnt detectble mmonifiction strting from 6 d fter NH 4 ddition. In the 4-d test, less thn 56% of the dded NH 4 -N ws trnsformed into nitrte-n. Wilson [5] monitored nitrifiction in three soils for seven weeks fter dding 00 mg N/kg s NH 4 Cl. The soil nitrte content ws significntly lower t 00 mg /kg during weeks nd 3 fter substrte ddition, but this effect ws not observed t the end of the incubtion, when most of the substrte ws depleted in ll tretments. Nitrifiction rte is sensitive to metl stress, but the vribility in PNR between control soils my exceed the effects of metl stress. Figure shows the effect of dded on PNR for soil 4 t three soil ph vlues (ph ). The PNR of the soil t highest ph ws significntly reduced by 8% t the lowest rte (300 mg/kg dry wt). The PNR of the unspiked soil tht ws cidified to ph ws lso reduced by 4%, significntly lower thn the unspiked soil t ph, clerly illustrting the well-known effect of soil ph on nitrifiction [,8]. The metl ppliction, however, lso reduced soil ph, nd this ws most pronounced in the tretments of soil 3, where ph ws reduced from (control) to 5.3 t the highest rte. The decrese of the PNR cn prtly, but not completely, be relted to the decrese in the soil ph on metl ddition. Soil ph in soil 4 (not cidified) ws reduced from ph to 6.4 t the lowest rte (300 mg/kg), t which the PNR ws 8% lower thn in the control. The PNR in the control soil cidified to ph 6.3 ws 56% lower thn t ph (Fig. ). Significnt effects of on PNR were found t the lowest (soils 4) or second-lowest (soil ) rte t which PNR ws over 50% lower thn in control tretments (Tble 4). There ws dose response reltionship between PNR nd soil concentrtions in ll four soils (detils not shown). The estimted EC50 vlues re between 50 nd 350 mg /kg dry weight, or, on pore-wter bsis, between bout nd 00 mg /L. No indiction existed tht ws more toxic in the most cid soil thn in soils with ph (soils 3 nd 4). The medin effective concentrtion (EC50) vlues, expressed on pore-wter bsis, were highest in the most cid soils becuse totl EC50 vlues were similr cross soils nd pore-wter mrkedly increses with reducing soil ph. The response of the PNR to ws more vrible between soils nd rtes thn in the cse of. The highest dose of 00 mg /kg dry weight reduced the PNR by between 50 nd 80% compred to tht in control smples (not significnt for soil ). A 0 to 0% reduction ws observed in the PNR t 0 mg /kg dry weight (significnt only in soil 3) nd n insignificnt stimultion in soil. At mg /kg dry weight, significnt but smll reduction ws observed in PNR for soil 3, wheres no effects were found in the three other soils. The logistic dose response curves could not be fit to most of the tretments (Tble 4). Tble 4. The potentil nitrifiction rte (PNR) in - nd -spiked soils: the lowest-observed-effect dose (LOED) of nd the concentrtion t which the PNR is 50% lower thn in the control soil EC50 (95% CI) Soil PNR of control ( 95% CI) (mg N/kg d) b LOED (% inhibition) (mg/kg dry wt) Totl metl in soil (mg/kg dry wt, mesured concn.) Soil solution concn. (mg/l) 3 4 (3.5) 6.4 (0.) 8.8 (0.4). (0.9) 600 (9) 300 (58) 300 (9) 300 (8) (68) (4) 00 () 35 (4 563) 9 (9 9) 30 (4 383) 4 (8 33) c 304 (9,000) 05 ( 946) 84 (0 3) 99 (98 99) 0 (9 0) (.9 t 8% inhibition) Lowest-observed-effect dose is the lowest metl dose t which the PNR ws significntly (p 0.05) different from the control soil. Metl rtes were 300, 600, nd 3,000 mg /kg dry weight nd, 0, nd 00 mg /kg dry weight. b CI confidence intervl. c not determined.

5 Nitrifiction in metl-contminted soils Environ. Toxicol. Chem. 0, Fig. 3. The potentil nitrifiction rte (PNR) in uncontminted soils s function of soil ph. Arrow indictes the soil ph below which the PNR test cnnot be used since even zero PNR is within the 95% prediction intervl of uncontminted soils. The PNR ws mesured in the uncontminted soils to evlute its vribility. The PNR rnged from vlues not significntly different from 0 to mg N/kg/d. This lrge rnge of the PNR hmpers detection of toxic effects in field-collected soils. A reltionship between soil properties nd the PNR of uncontminted soil is possible wy to identify predicted reference vlue of the PNR. Stepwise multiple regression of the PNR to soil ph, crbon content, nd CEC showed tht ph is the dominnt fctor controlling the PNR nd tht mrginl but significnt effects of the crbon content nd CEC exist (Tble 3). Below ph 3.5, no significnt nitrifiction ws detected within 4 d. The PNR increse bout 3.5-fold between ph 5.0 nd.0. A qudrtic regression curve ws fitted to the PNR-pH dt yielding PNR 9. ph.0 ph (R 0.8) () This curve nd its 95% prediction intervl re given in Figure 3. A PNR vlue below the lower prediction intervl is defined here s vlue indicting toxicity. Figure 3 shows tht no toxicity cn be detected below ph becuse even zero PNR vlue is within the 95% prediction intervl. The reference PNR vlues cn lso be predicted using the multiple regression model bsed on C, ph, nd CEC (Tble 3). The prediction intervls of this model re smller thn those of the ph-bsed model. This model ws not used for identifying toxicity in the field-contminted soils becuse the C contents of these soils exceed the rnge on which the multiple regression model ws fitted. All PNRs re smll in the 5 contminted soils (Tble ). All these soils contin or t concentrtions well bove bckground. Soil 40 contins excessively high concentrtions, nd it is likely tht this soil hs been contminted by smelter shes. The PNR vlues, expressed s percentge of the PNR of n uncontminted soil t corresponding ph, re 0 to 44%, indicting tht the PNR is ffected in ll soils. In one soil (soil 30), significnt decrese ws observed in soil nitrte from 5 to 0 mg NO3-N/kg, in 4 d fter NH4 ddition. Significnt toxicity, defined s given previously, ws detected in out of the 0 soils with soil ph nd where toxicity cn be identified. The three other soils in which toxicity cn be detected but in which the PNR ws not significntly different from tht in uncontminted soils hve soil ph close to 0, tht is, the threshold ph below which no toxicity cn be identified. The PNRs in the 5 field-contminted soils re not significntly relted to totl or concentrtions, soil solution concentrtion (ll concentrtions tested fter logrithmic trnsformtion), or to soil ph, percentge C, or CEC. A significnt negtive reltionship ws found between the PNR nd the logrithm of the soil solution concentrtion (R 0.35, p 0.05). The PNR vlue, expressed s percentge of the PNR of n uncontminted soil t corresponding ph, were unrelted to ny of these prmeters. Toxicity in the field-contminted soils is most likely due to elevted nd not bsed on the following nlysis. In the metl-spiked soils, the PNR ws similrly reduced by 50 to 80% t the highest dose of 00 mg /kg dry weight (.8 mmol /kg dry wt) or the lowest dose of 300 mg /kg dry weight (4.6 mmol/kg dry wt). This indictes tht the dverse effects of nd to nitrifiction rte re not extremely different on weight or molr bsis. The verge : totl concentrtion rtio in the smelter ffected soils is 80 (minimum 9, wt bsis). The totl concentrtions in environmentl smples with concentrtions round the EC50 vlues (50 nd 350 mg /kg dry wt) rnge between nd 4 mg /kg dry weight (ssuming : weight rtio of 80). In tht concentrtion rnge, hs little effect on PNR in the spiked soils. Similr conclusions cn be drwn on the bsis of soil solution nd concentrtions in the metl-spiked nd field-contminted soils (detils not shown). Totl concentrtions in the field-contminted soils where toxicity ws found were 60 to 34,00 mg /kg dry weight, nd most smples contined more thn the EC50s of the -spiked soils ( mg/kg dry wt). Inhibition of the PNR in metl-spiked soils is, however, likely to be higher thn in soils where metls hve equilibrted for much longer period. This my be relted to the limited rection time of metls in the freshly spiked soils or to the potentil dverse effects of the slts in these soils tht hve not been leched prior to testing. It hs been demonstrted tht the EC50 of (totl metl in soil) for reproduction of Folsomi cndid is twofold higher in soil tht hs been leched fter Cl spiking thn in soil tht ws not leched fter spiking [6]. Metl concentrtions in pore wter my be better bsis to compre toxicity between spiked soils nd field-contminted soils []. The concentrtions in the pore wter of the fieldcontminted soils, where toxicity ws identified, rnge between.4 nd 4 mg /L. These concentrtions should be compred with pore-wter concentrtions of metl-spiked soils t similr ph vlues s the field-contminted soils (ph 6. nd one soil t ph.0). Pore-wter in the control soils 3 nd 4 were 0.6 to 0. mg/l (ph ) nd incresed t the first rte to 9 mg/l (soil 3, ph 6.0) or.9 mg/l (soil 4, ph 6.4), t which bout 80% inhibition of the PNR in both soils ws observed. This nlysis indictes tht the pore-wter concentrtions of the field-contminted soils re in the rnge where toxic effects in metl-spiked soils were identified. DISCUSSION Even under controlled lbortory conditions, the PNR proved to be prmeter too vrible to effectively identify toxic effects in field-contminted soils. As n exmple, the predicted PNR (95% prediction intervl) t ph 6. is mg N/kg/d. The lower boundry of the prediction intervl is 8% of the expected vlue (toxicity cn be identified only if more thn % inhibition occurs). The - nd -spiked soils (soils 4) hve been subjected

6 44 Environ. Toxicol. Chem. 0, 00 E. Smolders et l. to 30 different biossys (microbiologicl tests, invertebrte tests, nd phytotoxicity tests). The PNR ws the most sensitive biossy; however, the PNR ws lso the most vrible endpoint between control soils (J. Bierkens et l., unpublished results). An overll positive correltion ws observed between vribility of the endpoint mong control soils nd the metl sensitivity of the test (J. Bierkens et l., unpublished results). The lck of consistent response in uncontminted soils strongly reduces the power of the PNR s biossy for screening metl-contminted soils. This power could be incresed if uncontminted smples re found with properties s close s possible to tht of the test smples. Correspondence in soil ph is miniml prerequisite. The use of ph buffer to test ll soils t identicl ph will likely increse the sensitivity of the ssy but decreses its ecologicl relevnce since metl vilbility is strongly ph dependent. An increse of the power of the PNR ssy by including better or more control smples effectively mens tht dverse effects of metls could then be identified t lower levels of metl contmintion. However, the ecologicl significnce of this sttisticl pproch then becomes lso more questionble. Without elborting on the potentil ecologicl effects of reduced nitrifiction in soil, it should lredy be reclled tht the effect of metls on the PNR is smll compred with the effect of soil properties (minly ph) on the PNR in otherwise uncontminted soils (Fig. 3 nd Suvé et l. [6]). Moreover, n dverse effect on the potentil nitrifiction rte mesured under lbortory conditions does not infer similr dverse effect on the nitrifiction in the field. The potentil nitrifiction rte, s tested here, reflects the nitrifiction rte t sturting substrte concentrtions (V mx ). This sitution rrely occurs in most erobic soils since it is known tht NH 4 -N usully rep- resents minor frction of the totl minerl N in soils ( NH 4 NO 3 ); tht is, the nitrifiction potentil usully exceeds the mmonifiction rte in soil. No field dt re vilble indicting tht field nitrifiction rtes re impired becuse of metls. The PNR ssy should therefore be considered s test tht indictes the existence of stress fctor in soil. However, no bsis exists by which to predict ecologicl effects of reduced PNR in soil. Vlidtion of this ssy with field nitrifiction rte is t lest required before the PNR ssy dt cn be dopted for the derivtion of soil qulity guidelines. Acknowledgement We re indebted to the Openbre Afvlstoffenmtschppij voor het Vlmse Gewest nd the Fund for Scientific Reserch, Flnders. Sttisticl dvice from Wim Coucke is gretly pprecited. REFERENCES. Bewley RJF, Stotzky G Effects of cdmium nd simulted cid-rin on mmonifiction nd nitrifiction in soil. 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