Diffuse Pollution Conference Dulin SALINITY AND INORGANIC NITROGEN EFFECTS ON NITRIFICATION AND DENITRIFICATION RATES IN INTERTIDAL SEDIMENTS AND ROCKY BIOFILMS: DOURO RIVER ESTUARY, PORTUGAL Mglhães C. *, Moreir R.*, Wiee W.J.*,+ nd Bordlo A.A.* *Lortory of Hydroiology, Institute of Biomedicl Sciences; University of Porto, 99 Porto, Portugl + Deprtment of Mrine Sciences; University of Georgi, Athens, GA, USA (c_mglhães@yhoo.com, roscmpt@yhoo.com, wjwiee@erthlink.net ordlo@ics.up.pt), ABSTRACT The regultory effect of slinity nd inorgnic nitrogen on nitrifiction nd denitrifiction ws investigted in intertidl sndy sediments nd rocky iofilms of the Douro estury. A sesonl smpling progrm nd controlled experiments were conducted; nitrifiction nd denitrifiction rtes were mesured in slurries using the difluoromethne nd the cetylene inhiition techniques, respectively. Slinity did not show ny regultory control of denitrifiction for either environment, suggesting oth denitrifier communities contined dominntly euryhline cteri. However, cler stimultion of nitrifiction ctivity ws oserved when slinity incresed from to 1 psu (% nd %, for sediments nd rocky iofilms, respectively). Nitrte vilility clerly stimulted denitrifiction rtes in sndy sediments, wheres in rocky iofilms no regultory effect ws evident. N O:N rtio incresed rpidly with NO enrichment (% to 19% nd 1% to %, for sediments nd rocky iofilms, respectively). Ammonium enrichment experiments in sndy sediments showed stimultion of nitrifiction ctivity y % for the M of NH + tretment nd cler inhiitory effect when mended with M of NH +. Contrry, rocky iofilms nitrifiction ws stimulted in % when M of NH + ws dded. These results indicte tht slinity nd inorgnic nitrogen increses in the Douro estury could lter nitrifiction rtes nd lso denitrifiction rtes with consequent N O ccumultion. Keywords: Nitrogen Effects, Nitrifiction, Denitrifiction, Rocky Biofilms, Slinity Effects, Sndy Sediments. Introduction The progressive increse of nitrogen loding nd reduction of freshwter dischrge into esturine systems represent worldwide prolems (Jickells, 1998; De Jonge et l., ; Montgn et l., ). In the Douro River estury, the concentrtions of NO inputs from freshwter incresed drmticlly during the 199 s (INAG, ) nd dditionl nutrients will e dded once severl tretment plnts whith miniml nutrient removl egin opertion. On the other hnd, due to wter diversion for griculture within the wtershed nd climte chnge, the estury is fcing progressive decrese in freshwter input, which could consequently chnge slinity regimes. Benthic microil ctivity provides key role for nitrogen trnsformtions within the estury. Under oxic conditions, nitrifying cteri convert mmonium to NO nd susequently to NO. In the noxic zones, denitrifying cteri use NO or NO s electron cceptors, converting them into gseous (N or N O) forms. Both of these processes re considered importnt in reducing the lndderived N loding to costl wters (Jensen et l., 199, Seitzinger, ). Becuse enthic nitrifiction nd denitrifiction influence the fte of inorgnic nitrogen inputs into n estury, understnding of the environmentl fctors tht control those processes re of gret ecologicl importnce. Mny reltionships hve een oserved etween slinity nd inorgnic nitrogen vritions with sediment key nitrogen cycle processes (e.g. Ogilvie et l., 1997, Rysgrd et l., 1999), ut often, it is difficult to relte the oserved reltionships to single prmeter, since mny prmeters together exhiit vrition throughout n estury. In the present study sesonl smpling progrm nd controlled experiments were performed, in order to understnd the effect of the possile slinities chnges nd the inorgnic nitrogen increse (NO nd NH + ) on the nitrifiction nd denitrifiction within intertidl sndy sediments nd rocky iofilms of the Douro River estury. METHODS Study re nd smpling The Douro River wtershed drin 17% of the Ierin Peninsul (Fig. 1). This drowned grnitic vlley estury is confined 1 km from the mouth y hydroelectric power dm. The estury verges 8 m in depth, hs semidiurnl tidl rnge of m, tempertures etween 8 C, slinities from to psu nd residence time etween. nd 1. dys (Vieir & Bordlo, ). Freshwter input into the estury during the postdm period (198) hs een reduced to ± m s 1 from ± m s 1 etween 19198. The estury wter column on verge contins µm NH +, µm NO nd 1 µm NO. Nitrte concentrtion in Crestum dm hs drmticlly incresed during the lst ten yers (Fig. ). In ddition, lrgely untreted sewge from pproximtely one million inhitnts is dischrged within its lower rech. This study ws conducted in two different intertidl environments within the lower estury (Fig. 1), intertidl rocky iofilms (Enteromorph sp. colonizing zones), nd intertidl sndy sediments. Both sites hve een chrcterized, in previous studies, in terms of totl orgnic mtter nd Chlorophyll concentrtions nd in the cse of sndy sediments lso in terms of mcrofun, microphythoenthos nd prticle size distriution, (Mglhães et l.,, Mglhães et l.,, Mglhães et l., ). 7
Diffuse Pollution Conference Dulin At ech site, monthly smples were tken etween Ferury to Octoer. Sediment cores (1 cores, cm dimeter nd 1 cm depth) nd rocky iofilms (out 1 ml, removed y scrping the rocks) nd respective wter column t the vicinity of ech sit e were collected lwys t low tide. Enrichment experiments were performing in dditionl rocky iofilms nd sediment collected during August ; esturine rckish wter nd freshwter from the Douro River were lso collected for nitrogen enrichments nd for slinity vrition experiments, respectively. All smples were kept cool nd trnsported in the drk to the lortory for processing no lter thn 1h fter collection. Figure 1Douro River estury nd loction of smpling sites. µm NO 1 9 8 7 1 R =.71 p <.1 199 199 199 199 1998 Figure Liner regression model etween yerly verged nitrte cencentrtion nd time (1991) in the Crestume Lever reservoir. Dt from the Portuguese Ministry of Environment (INAG, ). Experimentl Progrm Denitrifiction rtes were mesured in triplicte using the cetylene inhiition technique of Sorensen (1978). The slurries were prepred y dding 1 ml of esturine filtered wter (. µm) to ml serum ottle contining homogenized nd weighted sndy sediment (round g) or scrped rocky iofilm (round 1 g). Serum ottles were closed hermeticlly with utyl stoppers nd luminum crimp sels. The hedspce ws purged with helium nd smples were run without nd with cetylene (% vol:vol). All smples were incuted in the drk for h t constnt temperture ( ºC) nd stirring (7 rpm). At time zero, nd time one, fter hedspce equilirtion, 1 ml of gs smple were collected from ech serum ottle nd stored in 1 ml evcuted serum vil for lter nlysis of N O. Time one gs smple ws collected from ech serum ottle y dding simultneously 1 ml of M NCl solution (Joye et l., 199). Nitrifiction rtes were mesured in seprted slurries y dding ml of. µm filtered nd oxygen sturted esturine wter to ml serum ottles with homogenized nd weighted sndy sediment (round g) or rocky iofilm (round 1g), respectively. Smples were runed in triplicte without nd with difluoromethne (DFM) (1% vol:vol), ccording to Miller 7
Diffuse Pollution Conference Dulin et l. (1998). All smples were incuted in the drk for h t constnt temperture ( ºC) nd stirring (7 rpm). A time zero nd time one 11 ml of overlying wter smples were collected efore nd fter the incution period. These smples were centrifuged,. µm filtered nd kept frozen ( ºC) for lter NH + nd NO +NO nlysis. In order to study slinity nd nitrogen regultory effects on nitrifiction nd denitrifiction independently, dditionl experiments were performed during August t ech site. Different concentrtions of NO (, 1 nd µm using stndrd solution of KNO ) nd NH + (,, µm using stndrd solution of (NH ) SO ) where dded to the collected esturine wter ( µm NO, µm NH nd 1 psu), nd denitrifiction nd nitrifiction mesured in triplicte, s descried ove, for ech different nitrte nd mmonium tretment, respectively. The slinity effects were evluted y mending the Douro River freshwter ( µm NO, µm NH nd psu) with slts using the rtificil sewter formul (Cvnugh, 197), to chieve different slinities (, 1 nd psu) nd nitrifiction nd denitrifiction mesured s descried ove. Anlytic determintions N produced vi denitrifiction ws clculted s the difference etween the N O produced with cetylene nd the N O produced without cetylene. N O produced vi denitrifiction ws clculted y the N O ccumultion in smples treted without cetylene. N O ws quntified in triplicte using Shimdzu gs chromtogrph equipped with n electroncptured detector, ccording to Joye et l. (199). Nitrifiction rtes were clculted y the difference etween NH + production mesured in incutions without DFM nd with DFM. Ammonium ws quntified s descried in Grsshoff et l. (198) nd nitrte plus nitrite ssyed y n dpttion of the spongy cdmium reduction technique (Jones, 198). Results nd Discussion Vlues of monthly wter slinity, time zero men concentrtion of NO plus NO nd NH + nd reltive relese of N nd N O (N O:N rtio) t ech site for the sesonl smpling progrm re given in Tle 1. Tle 1 Monthly incution wter slinity, time zero NO + NO nd NH + concentrtion nd N O:N rtio (men±stndrd error of the men) for sndy sediment (site I) nd rocky iofilms (site II); n: not ville. Slinity (psu) NO + NO (µm) + NH (µm) N O:N (%) Site I Site II Site I Site II Site I Site II Site I Site II Fe.7 1.9.±1.1 1.7±.1 1.1±.7 7.±1.1.±..±. Mr 1.1. 1.9±.9 99.8±.1 1.9±..±1. 7.±.1 1.±1. Apr 1. 1. 9.9±. 9.±. 8.±1.9 7.±. 1.±.1.±. My..1 1.±.1 8.±1.8.9±.8 1.±..±. 9.±.7 Jun.8 1..±. 1.±1.1 1.±.7 97.±1.1.±..±. Jul..8 1.8±. 1.±1..±1.9 18.±..1±. 1.±.1 Aug 1.9 1.9.±. 1.1±1. 8.±1.1 1.9±. 1.±.1.±.8 Sep 1. 1..±. 7.±.7 n 11.±..8±..8±.1 Oct.7 1.9.±1.1 1.7±.1 1.1±.7 7.±1.1.8±..±. Denitrifiction rtes Cler different mgnitudes of denitrifiction rtes were found during the monthly smpling period in sndy sediments (Fig. ). Higher rtes of denitrifiction were oserved in August (ANOVA, p <.1) nd significntly lower rtes were registered in June, July nd Octoer (ANOVA, p <.). In sndy sediments, nitrte vilility nd slinity vritions t time zero incution wter were clerly relted to the mesured denitrifiction rtes (Fig. ). While positive reltionship ws found etween this process nd nitrte concentrtion (R =.87, p <.1, n = 9), denitrifiction rtes were negtively relted with slinity (R =. 9, p =., n = 9). The nitrte enrichment experiment corroortes with the sesonl progrm results since progressive increse in denitrifiction (ANOVA p <.1) with the incresing of nitrte vilility ws found (Fig. ). However, in the slinity controlled experiment, no significnt chnges in denitrifiction rtes etween ech tretment were oserved (ANOVA, p =.1) (Fig. ). These results, together with the fct tht during the surveys, nitrte concentrtion ws strongly relted to the slinity of esturine wter (r =.81, p =.8, n = 9) (Fig. ), suggests tht slinity my not hve physiologicl effect on the denitrifying communities tht inhit sndy sediments. Thus, the significnt reltionship found etween slinity nd denitrifiction, occurred ecuse nitrte concentrtion is function of the slinity of the wter rther thn process directly dependent of given slinity. In contrst to the results for intertidl sndy sediments, there ws no sesonl signl for denitrifiction with regrd to either slinity or nitrogen concentrtion in the intertidl rocky iofilms (Fig. ). These results re in concordnce with the sence of cler slinity regultory effect in the slinitycontrolled experiments, where no sttisticl differences etween denitrifiction rtes in the three slinities tested (ANOVA, p =.1) were found (Fig. d). The nitrte enrichment experiments showed wek stimultion of denitrifiction ctivity etween zero nd 1 µm of NO dditions (Fig. c). However, the lck of sttisticl differences etween the different tretments performed, (ANOVA, p =.), indictes tht nitrte concentrtion effect on denitrifiction rtes ws not evident. The pprent sence of cler nitrte regultory effect for rocky iofilms suggests tht this denitrifier community ws not nitrte limited. As in the cse of the intertidl 7
Diffuse Pollution Conference Dulin sndy sediments, nitrte vilility hs lso een identified s primry fctor in regulting enthic denitrifiction (e.g. Ogilvie et l., 1997). On the other hnd, the sence of cler physiologiclly slinity effect on the denitrifiction rtes, contrry to wht ws oserved y Rysgrd et l. (1999), indictes tht euryhline denitrifying cteri inhit oth studied environments. Slinity (ups 1 1 7 Slinity NO 7 1 µm NO M NO Slinity (ups 1 1 Slinity NO 1 1 8 µ M NO NO nmol N g wet weight1 h 1 1 nmol N g wet weight 1 h 1 1 1 Fe Mr Apr My Jun Jul Aug Sept Oct Fe Mr Apr My Jun Jul Aug Sept Oct Figure Sesonl vrition of denitrifiction rtes nd esturine wter slinity nd time zero nitrte concentrtion (error rs = stndrd error of the men of three replictes). ) sndy sediments; ) rocky iofilms. In order to evlute the mgnitude of the N O ccumultion during the denitrifiction process, the reltive relese of N nd N O (N O:N rtio) ws determined (Tle 1). N O to N rtios in sndy sediments were consistent with literture vlues (Seitzinger, 1988). However for the rocky iofilms, the otined N O:N rtios were much wider, with the higher vlue oserved for the higher nitrte concentrtion (Tle 1). For oth sites, nitrte enrichment experiments showed reltively higher N O production with n incresed of the mount of nitrte dded, which grees with other reports (Seitzinger et l., 198). In the intertidl sndy sediments, N O:N rtio rose from.% t zero µm of NO ddition to 18.8% t 1 µm of NO ddition, mintining this vlue for the µm of NO ddition. In the cse of rocky iofilms, the N O:N rtio incresed ws progressive with the increse of nitrte dded (11.8% to 1.9% nd.%, respectively for µm, 1 µm nd µm of NO ). These results demonstrte progressive decrese in denitrifiction efficiency with the increse in nitrte concentrtion, since progressive ccumultion of N O ws oserved. The increse of nitrous oxide production is of much concern since it is considered potent greenhouse gs nd promoter of ozone depletion in the tmosphere (Dickinson & Ciceron, 198). nmol N g wet weight 1 h 1 nmol N g wet weight 1 h 1 1 1 8 7 1 8 nmol N g wet weight 1 h 1 7 1 1 1 µm NO µm NO nmol N g wet weight 1 h 1 1 1 psu 1 psu psu psu 1 psu psu ups 1 ups ups ups 1 ups ups Figure Denitrifiction rtes in sndy sediments (, ) nd rocky iofilms (c, d) t different slinities nd nitrte tretments (error rs = stndrd error of the men of three replictes). For nitrte tretments (, c), esturine rckish wter ws mended with µm of NO, 1 µm of NO nd µm of NO. For the slinity tretments (, d), Douro River freshwter ws mended with different concentrtions of slts to rech psu, 1 psu nd psu. NITRIFICATION RATES Nitrifiction rtes in sndy sediments showed pek in June (ANOVA, p <.1) with lower vlues nd slight vritions for the other months (Fig. ). The sesonl pttern of mmonium concentrtion in time zero esturine incution wter (Fig. ) ws significntly nd linerly relted with the nitrifiction rtes mesured (R =., p =., n = 8). Also liner nd positive reltionship etween slinity nd nitrifiction rtes ws registered (R =.8, p <., n = 9). Controlled experiments showed n independently regultory effect of slinity nd mmonium in the nitrifiction ctivity of sndy sediments (Fig, ). A nitrifiction stimultion of % nd 17% in the incutions performed t 1 psu nd t psu, respectively, compred to the incution t psu (Fig. ) ws oserved. In the mmonium enrichment experiment, c d 7
µm N H Diffuse Pollution Conference Dulin stimultion of the nitrifiction ctivity of % ws oserved for the µm of NH + tretment, ut cler inhiitory effect of the process ws registered when µm of NH + ws dded (Fig. ), suggesting tht mmonium vilility stimultes the nitrifying ctivity up to certin point. According to the sesonl dt, significntly positive nd liner reltionship ws found etween nitrifiction rtes nd mmonium concentrtion within rnge of 1.1 1. µm, suggesting tht the sndy sediment nitrifying cteri were stimulted, t lest within such rnge. 1 nmol NH + g wet weight 1 h 1 1 1 8 + µmn H M NH + + nmol NH + g wet weight 1 h 1 18 1 1 1 1 8 Fe Mr Apr My Jun Jul Aug Sept Oct Figure Sesonl vrition of nitrifiction nd time zero NH + concentrtion (drk spots = null rtes; error rs = stndrd error of the men of three replictes). ) sndy sediments; ) rocky iofilms. Correltion nlysis did not show ny reltionship etween sesonl nitrifiction rtes in rocky iofilms nd sesonl mmonium nd slinity vritions. Nevertheless, when in the control experiments mmonium concentrtion nd slinity were isolted, regultory effect of these two prmeters independently ws demonstrted (Fig. c, d), with cler stimultion of nitrifiction ctivity (%) t the highest mmonium concentrtion tested (Fig c). With respect to the controlled slinity experiment, stimultion of the nitrifiers ctivity y % ws oserved for the intermedited slinity, wheres n lmost totl inhiition of the process ws registered for psu (Fig. d). In contrst, recent studies hve shown tht progressive slinity increse reduced the nitrifiction ctivity (Rysgrd et l., 1999, Cmpos et l., ). On the other hnd, some culture experiments showed tht esturine isoltes of mmonium oxidizers exhiited optimum nitrifiction rtes t intermedite slinities (1 psu) (Jones & Hood, 198) or even etween psu (McFrlne & Herert, 198) with susequent reduction of the ctivity or inctivtion for higher slinities. The lck of evidence for mmonium concentrtion nd/or slinity s regultory prmeters for rocky iofilms during the sesonl surveys, suggests tht other prmeters could hve n importnt role in regulting the nitrifier ctivity in these iofilms. Joye & Holliught (199) suggested tht sulfide could e n importnt regultory fctor for nitrifiction. Similr to the results of this study, severl uthors hve demonstrted the strong mmonium regultory effect of nitrifiction ctivity (e.g Berounsky & Nixon, 199; Butturini et l., ). However, in this study is interesting to stress the differences etween the responses of ech studied environment to the mmonium tretments performed. While rocky iofilms nitrifiction ws gretly stimulted t the higher concentrtion of mmonium, sndy sediments showed cler inhiitory effect for such high concentrtion of mmonium (Fig., c). All these results from the sesonl progrm nd the controlled experiments suggests tht slinity nd mmonium concentrtion hve different ut importnt regultory effect on the nitrifier communities tht inhit sndy sediments nd rocky iofilms of the intertidl res of the Douro estury. While rocky iofilms nitrifier communities showed much higher tolernce to high levels of mmonium concentrtions compred with the sndy sediments, they do not tolerte s high slinities s the sndy sediments nitrifier community. 18 1 1 1 1 8 M NH + 77
Diffuse Pollution Conference Dulin nmol NH + g wet weight 1 h 1 nmol NH + g wet weight 1 h 1 1 1 8 7 1 nmol NH + g wet weight 1 h 1 µm NH + µm NH + nmol NH + g wet weight 1 h 1 8 1 1 psu 1 psu psu psu 1 psu psu ups 1 ups ups ups 1 ups ups c d Figure Nitrifiction rtes in sndy sediments (, ) nd rocky iofilms (c, d) t different slinities nd mmonium tretments (error rs = stndrd error of the men of three replictes). For mmonium tretments (, c), esturine rckish wter ws mended with µm of NH +, µm of NH + nd µm of NH +. For the slinity tretments (, d), Douro River freshwter ws mended with different concentrtions of slts to rech psu, 1 psu nd psu. CONCLUSIONS Denitrifying cteri of intertidl sndy sediments nd rocky iofilms were not physiologiclly influenced y the presence of se slts, indicting tht euryhline denitrifying cteri inhit oth environments. Incresing nitrte concentrtion clerly stimulted denitrifiction rtes in sndy sediments; however in rocky iofilms this stimultion ws not so evident, suggesting tht these denitrifying communities re not nitrte limited. For oth sites, it ws demonstrted decrese in denitrifiction efficiency with the increse in nitrte concentrtion, with progressive ccumultion of N O. While oth communities showed optiml nitrifiction ctivity t intermedite slinities, sndy sediments nitrifiers tolerted etter the incresing in slinity thn the nitrifier communities from rocky iofilms. Ammonium concentrtion regultory effect in the nitrifiction rtes of oth environments ws demonstrted, nd the different responses oserved etween sites, suggests tht rocky iofilms nitrifier communities re more tolernt to higher mmonium concentrtions. ACKNOWLEDGMENTS The uthors re grteful to Prof. S. Joye for technicl dvice nd ssistnce in N O smples processing. This study ws prtil supported y the Portuguese Foundtion for Science nd Technology thought grnt to C. Mglhães. REFERENCES Cmpos J. L., MosquerCorrl A., Méndez R. nd Lem J. M. (). Nitrifiction in sline wstewter with high mmoni concentrtion in n ctivted sludge unit. Wter Res.,,. Cvnugh G. M. (197). Formule nd Methods VI. The Mrine Biologicl Lortory, Woods Hole, MA. de Jonge V. N., Elliott M. nd Orive E. (). Cuses, historicl development, effects nd future chllenges of common environmentl prolem: eutrophiction. Hydroiol., 7/7,119. Dickinson R. E. nd Cicerone R. J. (198). Future glol wrming from tmospheric trce gses. Nture, 71, 17 17. I.N.A.G. (). http://www.ing.pt Jensen K. M., Jensen M.H. nd Kristensen E. (199). Nitrifiction nd denitrifiction in Wdden Se sediments (Königshfen, Islnd of Sylt, Germny) s mesured y nitrogen isotope piring nd isotope dilution. Aqut. Micro. Ecol., 11, 181191. Jones M. N. (198). Nitrte reduction y shking with cdmium: lterntive to cdmium columns. Wter. Res., 18,. Jickells T. D. (1998). Nutrient iogeochemistry of the costl zone. Science, 81, 17. Jones R. D. nd Hood M. A. (198). Effects of temperture, ph, slinity, nd inorgnic nitrogen on the rte of mmonium oxidtion y nitrifiers isolted from wetlnd environments. Micro. Ecol.,, 97. Joye S. B. nd Holliugh J. T. (199). Influence of sulfide inhiitio of nitrifiction on nitrogen regenertion in sediments. Science, 7,. Joye S. B., Smith S. V., Holliugh J. T. nd Perl H. W. (199). Estimt ing denitrifiction rtes in esturine sediments: A comprison of stoichiometric nd cetylene sed methods. Biogeochemistry, : 1971. McFrlne G. nd Herert R. A. (198). Effect of oxygen tension, slinity, temperture nd orgnic mtter concentrtion on the growth nd nitrifying ctivity of n esturine strin of Nitrosomons. FEMS Microiol. Lett.,,17111. 78
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