ImpactofAmmoniumNitrateonGrowthandSurvivalofSixEuropean
Amphibians
ManuelE.Ortiz,1 AdolfoMarco,2 NeliaSaiz,1 MiguelLizana1
1 DepartmentofAnimalBiology,UniversityofSalamanca,CampusMigueldeUnamuno,Salamanca37007,Spain
2 Estacio´nBiolo´gicadeDon˜ana,SpanishCouncilofScientificResearch,Apartado1056,Sevilla41013,Spain
Abstract. We conducted static experiments to assess the ef- fects of ammonium nitrate fertilizer on embryos and larvae of six European amphibians: sharp-ribbed salamander (Pleurode- les waltl), Iberian painted frog (Discoglossus galganoi), west- ern spadefoot toad (Pelobates cultripes), common toad (Bufo bufo), natterjack toad (Bufo calamita), and common tree frog (Hyla arborea). Embryos were exposed to different and envi- ronmentallyrelevantconcentrationsofammoniumnitrate(0to
fertilizers and manure in the crops or the leaching of ammo- nium from farms are producing increased environmental nitro- gen levels in aquatic ecosystems (United States Environmental Protection Agency [US EPA] 1986, 1999; Vitousek et al.
1997).Anexcessofnitrogeninwaterbodiesnearcropfieldsor livestock farms can be seriously harmful to aquatic wildlife (e.g.,Westin1974;CamargoandWard1992).Amphibians maybeespeciallysensitivetowaterpollutionbecauseoftheir
200mgNO3
/L)for15days.Hatchingtookplaceduringthe
permeableskinandgills(Blausteinetal.1994).Decreased
experiments.H.arboreawasextremelysensitiveandhadhigh
mortality after 8 days of exposure even at the lower fertilizer levels.D.galganoiandB.bufowerealsoverysensitiveand hadsignificantmortalityafter15daysofexposure.Therestof the species did not suffer lethal effects but suffered abnormal- ities or decreased growth at the highest fertilizer concentra- tions. Chemical fertilizers or manure could have contributed to the observed decrease of B. bufo and D. galganoi in agricul- tural areas of the Iberian Peninsula during recent years. H. arborea wasthemostsensitivespeciesstudied.Theresultsof our study showed that environmentally relevant levels of am- monium nitrate can induce mortality and might affect popula- tiondynamicsofthisspeciesinagriculturalenvironments.
Profoundchangespromotedbyscientific andtechnological advances in agricultural practices have occurred during the last decades. However, these advances have great costs in terms of environmental performance. Ecosystems in agricultural lands often become damaged by human activity. An important change in agricultural practices is the increase in the use of chemical fertilizers such as ammonium nitrate, which is one of themostwidelyusedintheworld(FMA,MAFFF,SOAFD
1993);itsapplicationoncropfields hasrecentlyincreased (Agricultural Development and Advisory Service 1992; Lanyon 1996). Moreover, the intensive farming of different animal species and the subsequent production of ammonia-rich residueshaverecentlyincreased.Boththeadditionofchemical
Correspondenceto:M.E.Ortiz;email:
water quality has been proposed as a cause for the global decline in amphibian populations (Blaustein and Wake 1990; Wake 1991; Boyer and Grue 1995). Fields are often fertilized in spring at the same time that amphibian eggs and larvae develop(Hecnar1995;WattandJarvis1997).Environmentally realistic ammonia or nitrate concentrations in water bodies are likely to damage amphibian eggs and larvae in agricultural regions(e.g.,Cooke1981;Marcoetal.1999;Rouseetal.
1999).
Theaimofthisworkwastoanalyzetheeffectofammonium nitrate on embryonic and larval development of six amphibian speciesusuallyfoundinfarmingareasoftheIberianPeninsula. The natterjack toad (Bufo calamita) has disappeared from several sites in the United Kingdom because of water and soil acidificationand habitat loss or degradation (Beebee et al.
1990;Dentonetal.1997).Populationsofthecommontoad
(Bufobufo)havealsodecreasedintheUnitedKingdom(Cooke
1972;Hilton-BrownandOldham1991).Thenegativeeffects of nitrate on this species have already been shown (Baker and Waights 1993; Xu and Oldham 1997). Some of the Iberian populations of common toad inhabiting farming regions have decreased during recent years (Morales et al. 1997, personal observation). Studies of sharp-ribbed salamander (Pleurodeles waltl), Iberian painted frog (Discoglossus galganoi), western spadefoot toad (Pelobates cultripes), and common tree frog (Hyla arborea) conservation are scarce, and reports about their populationdeclinesarecurrentlyunknown D.galganoiisthe least-common, quite rare in farming areas of central Spain, and isprobablythemostfrequentlyexposedspeciesinthisstudyto ammonium nitrate because of its preference for shallow waters (Mart´ınez-Solano2002).
To test the hypothesis that these six amphibian species are sensitive to environmental levels of ammonium nitrate, we
studiedthedose–effectrelationshiponembryosandyoung larvaeusingstaticlaboratoryexperiments.
MaterialsandMethods
StudySpecies
We collected sharp-ribbed salamander (P. waltl), western spadefoot toad (P. cultripes), and common tree frog (H. arborea) eggs from severalpondsinLosArribesdelDuero(westernSpain).Commontoad (B. bufo) and natterjack toad (B. calamita) eggs were collected from a mountain stream in the Sierra de Gredos (central Spain), and Iberian paintedfrog(D.galganoi)eggswerecollectedfromapondnear Don˜anaNationalPark(southwesternSpain).Foreachspecies,eggs from at least three different clutches were collected in stages of early development (stages 10 to 12; Gosner 1960; Harrison 1969) in areas wheretheywereabundant.
ExperimentalProcedures
Eachspecieswastestedatdifferenttimesfromwinter2001tospring
2002. We conducted static experiments (Stephen 1975) in the labora- tory at an environmental temperature and during the natural photope- riod.Within24hoursaftercollection,theeggswereexposedtoa series of ammonium nitrate dilutions (three treatment levels except both Bufo species, which were exposed to two treatment levels) and one control (no contaminant added) for 15 days. Test were conducted in 3.5 L-tanks containing 2 L solution. Each treatment level was replicated three times (nine times in the B. bufo and B. calamita experiments). The tanks for each experiment were randomly assigned to one of the treatments. We used nominal concentrations of 0; 50 (legalmaximumforwaterintendedforhumanconsumption;European Council1998);100(notinB.bufoandB.calamitaexperiments);and
200mgNO3 -NO3NH4/L. UnitedStatesEnvironmentalProtection
Larval mortality and the presence of abnormalities were monitored, and dead larvae were removed every 24 hours. Mortality rate was calculated dividing the accumulated number of dead larvae at a given momentbytheinitialnumberofeggs.Abnormalityratewascalculated asacumulativemeasuredividingthenumberofabnormallarvae(dead oralive)atagivenmomentbytheinitialnumberofeggs.Attheend of each experiment, we measured total body length of the survivors from mouth to tail tip, with the tail intact, using an electronic digital caliperStainlessHardenedtothenearest0.01mm.Measurements were always taken by the same investigator, and tadpoles were care- fully placed straight on a plane surface before measuring. The mea- surer had long experience measuring tadpoles and knew previously whichtreatmentthemeasuredtadpoleswerefrom.
AnalysisofData
To determine the effect of ammonium nitrate on larval survival for each amphibian species, we used repeated measure analysis of vari- ance (ANOVA) with the dependent variable being the proportion of dead and abnormal larvae at 2, 4, 8, or 15 days (arcsin of square root transformed),andthecategoricvariablebeingnitrateconcentration. To determine which nitrate levels had a lethal effect or caused abnor- malities in each species, we used post hoc (HDS Tukey) univariate ANOVA.WealsousedunivariateANOVAs(andsubsequentpost-hoc HDS Tukey tests) to determine for each species the effect of ammo- nium nitrate on larval size at the end of the experiment. For larval size weconsideredtheaveragevaluesofeachcontainer.
Results
All species showed some degree of sensitivity. Differences in mortalityovertimecausedbyammoniumnitrateweredetected in H. arborea and D. galganoi (Table 1). At day 8, H. arborea wasthemostsensitivespeciesandhadhighmortality(95%)
Agency(USEPA)acutecriteriaforambientammoniais36mgN/Lin
salmonids-absent waters (US EPA 1999). In our experiments, the
evenatthelowestammoniumnitratelevels(F3,8
=41.187,
maximum concentration of ammonia—corresponding to 200 mg NO3 -NO3NH4/L—was45mgN/L.Thishighestlevelhasbeenfound in several agricultural ponds in the areas from which the eggs were collected.Weused10gNO3 -NO3NH4/L stocksolutionprepared from ammonium nitrate salt (99% purity), which was pipetted into the containers to get the experimental concentrations. We used dechlori- natedtapwater.
Water temperature and pH were checked daily. Water temperature nevervariedmorethan1.5°Cwithinthesameexperiment,anditstotal range—includingalltheexperiments—remainedbetween15°Cand
23°C. Overall water temperatures in each experiment were 16.3°C for D.galganoi,18.7°CforP.waltl,18.9°CforP.cultripes,21.0°CforB. bufo, 21.1°C for B. calamita, and 22.2°C for H. arborea. pH varied between 7.20 and 7.80. No statistical differences among treatments within the same experiment were detected in water temperature or pH. Waterinthetankswasrenewedandnitratelevelswererestoredevery
4 days. Previous studies conducted with similar methodology and environmentalconditionsshowednosignificantdeviations(25%) from the original nitrate concentrations within a 7-days period (Marco et al. 1999). At the beginning of the experiments, eggs from each clutch were divided among the tanks, each one containing the same number of eggs from the different clutches. We assigned 20 eggs to each tank except for the D. galganoi (15 eggs/tank) and P. waltl (12 eggs/tank) experiments. After hatching, anuran larvae were fed ad libitum with lettuce that had been previously washed with dechlori- nated tap water and boiled for 5 minutes. P. waltl larvae were fed ad libitumwithArtemialarvaebredinthelaboratory.
p0.001).Atday15,higherconcentrationsofammonium
nitratealsocausedsignificantmortalityinD.galganoi(F3,8 =
22.917,p0.001)larvae(Figure1).Despitenotshowinga
response over time (Table 1), B. bufo was sensitive to 200 mg NO3 /Lafter15daysofexposure(F2,24 =9.549,p=0.001) (Figure 1). The other three species did not suffer significant mortalityatanyammoniumnitrateconcentration.
Some larvae suffered edemas,bent tails,and lordosis. D. galganoi,B.bufo,andB.calamitashowedanincreasingab- normalities occurrence over time (Table 2). At day 2, D. galganoi showed high abnormality rate (Figure 2) at 200 mg NO3 /L(F3,8 =9.733;p=0.005).Atday4,B.bufoalso showedahighnumberofabnormallarvaeat200mgNO3 /L (Figure2)(F2,24 =7.735;p=0.003).Inspiteofthelow abnormality rate showed by B. calamita after 4 days of expo- sure(Figure2),asignificant effectbyammoniumnitratewas detectedatthehighestlevel(F2,24 =4.015;p=0.031).
All six species showed a negative effect of ammonium nitrateontotallengthbytheendoftheexperiment.Statistically significant decreasedlarvalsizewasdetectedatthehighest concentrations (Figure 3). D. galganoi larvae exposed to 100 mgNO3 /Lalsoshowedasignificantlysmallersize.P.cul- tripestadpolesexposedtoboth50and200mgNO3 /Lwere smaller than controls, although not those exposed to 100 mg NO3 /L.AsimilareffectwasobservedinH.arborea;tadpole
Table1 Resultsofrepeated-measureANOVAscomparingmortalityovertimeoflarvaeofsixamphibianspeciesexposedto ammoniumnitratea
Species / SourceofVariation / MeanSquares / df / F / pP.waltl / Concentration / 0.102 / 3 / 2.280 / 0.156
Error / 0.045 / 8
D.galganoi / Concentration / 0.263 / 3 / 7.074 / 0.012
Error / 0.037 / 8
P.cultripes / Concentration / 0.012 / 3 / 0.202 / 0.892
Error / 0.060 / 8
B.bufo / Concentration / 0.018 / 2 / 0.360 / 0.701
Error / 0.050 / 24
B.calamita / Concentration / 0.043 / 2 / 1.090 / 0.352
Error / 0.039 / 24
H.arborea / Concentration / 1.336 / 3 / 24.856 / 0.001
Error / 0.054 / 8
aDependentvariablewasmortalityat2,4,8,and15days(arcsinofsquareroottransformed). ANOVAs=analysesofvariance.
Fig. 1.Mortality caused by ammonium nitrate in larvae of six am- phibianspeciesafter15daysofexposure.Pw=Pleurodeleswaltl;Dg
=Discoglossusgalganoi;Pc=Pelobatescultripes;Bb=Bufobufo;
Bc=Bufocalamita;Ha=Hylaarborea
lengthwassignificantly lowerthanincontrolsonlyat50mg NO3 /L. No differences between controls and tadpoles ex- posedto100mgNO3 /Lweredetected,andbecausesurvivors ofthisspeciesat200mgNO3 /Lappearedinonlyonetank, wecouldnotusetheposthoctestsatthislevel.
Discussion
This study suggests that ammonium nitrate can be seriously hazardous for amphibian survival as has been suggested by previous studies (Berger 1989; Baker and Waights 1993; Hec- nar 1995; Watt and Jarvis 1997; Jofre and Karasov 1999; Marco and Blaustein 1999; Marco et al. 1999, 2001; SchuytemaandNebeker1999a,1999b).Weareunawareofthe existenceofpublisheddataforthesensitivityofstudiedspecies except B. bufo to nitrogenous fertilizers. Xu and Oldham (1997)didnotfindlethaleffectsofammoniumnitrateinB. bufo larvae exposed to 50 mg NO3 /L, whereas those exposed to100mgNO3 /Lshowedamortalityrateof21%after30 days of exposure. Sensitivity of B. bufo to ammonium nitrate was lower than that observed in our experiments (20% dead after15daysofexposureto50mgNO3 /L),solarvaeusedby
Xu and Oldham (1997) were more resistant than ours. How- ever, other investigators have reported higher sensitivity in B. bufo larvae. Berger (1989) found lethal effects of ammonium nitrateontadpolesexposedfor4daysto15.7mgNO3 /L.At the same exposure time, the mortality observed in this study wasnotsignificantevenat200mgNO3 -NO3NH4/L.Baker and Waights (1993) used sodium nitrate and reported 84.6% deathsforB.bufolarvaeexposedto29mgNO3 /Lduring13 days,and100%deathsforlarvaeexposedto73mgNO3 /L. Tadpoles used by Baker and Waights (1993) were therefore far moresensitivethanours,whosemortalityratewas40%after
15 days of exposure to 200 mg NO3 /L. It is especially relevant if it is remembered that ammonium nitrate toxicity could be caused mainly by ammonia ion as shown by Baker and Waights (1993) and Schuytema and Nebeker (1999a,
1999b),whereassodiumiondoesnotappeartobeanespecially hazardous substance. Schuytema and Nebeker (1999a) found significantmortalityofPseudacrisregillalarvaeexposedto sodium nitrate concentrations when the sodium level was con- siderablylowerthanthatlikelytobeharmful(PadhyeandGate
1992). Differences between ammonium and sodium nitrate should be analyzed, but we are just assessing the effects of ammonium nitrate as a much more widely used substance (FMA,MAFF,SOAFD1993).
We observed a great interspecificvariability on sensitivity to ammonium nitrate. A similar conclusion has been drawn by other investigators studying the sensitivity of other amphibian species to nitrogenous fertilizers. For example, Hecnar (1995) foundthat4-dayLC50valuesvariedamongfourspeciesfrom
75to174mgNO3 -NO3NH4/L.Marcoetal.(1999),using potassium nitrate, found that nitrate levels higher than 15-day LC50forAmbystomagracileorRanapretiosatadpolesdidnot cause any effect on Bufo boreas or Hyla regilla. The results of ourstudysuggestedthatH.arboreamaysufferlethaleffectsat concentrations lower than the maximum allowed in water in- tended for human consumption (European Council 1998). D. galganoi and B. bufo also showed high mortality, whereas higher pollutant levels caused growth retardation in more tol- erant species such as P. waltl, P. cultripes, and B. calamita. Interspecific comparisioninthisstudywasmadefromexper- imentsconductedatdifferenttimeswithupto8°Cofwater
Table2. Resultsofrepeated-measureANOVAscomparingabnormalityrateovertimeoflarvaeofsixamphibianspeciesexposedtoammo- niumnitratea
Species / SourceofVariation / MeanSquares / df / F / pP.waltl / Concentration / 0.002 / 3 / 1.000 / 0.441
Error / 0.002 / 8
D.galganoi / Concentration / 0.730 / 3 / 15.217 / 0.001
Error / 0.048 / 8
P.cultripes / Concentration / 0.000 / 3 / 0.000 / 1.000
Error / 0.000 / 8
B.bufo / Concentration / 0.449 / 2 / 9.022 / 0.001
Error / 0.050 / 24
B.calamita / Concentration / 0.122 / 2 / 4.072 / 0.030
Error / 0.030 / 24
H.arborea / Concentration / 0.018 / 3 / 0.797 / 0.529
Error / 0.017 / 8
aDependentvariablewasabnormalityrateat2,4,8,and15days(arcsinofsquareroottransformed)usingtimeasarepeatedmeasure. ANOVAs=analysesofvariance.
Fig.2. Abnormalityratesshowedbythreeamphibianspeciesafter2,
4, 8, and 15 days exposure to different ammonium nitrate levels. Light greybarscorrespondtocontrols,linedbarsto50mgNO3 /L,and darkgreybarsto200mgNO3 /L.Signification ofunivariateANO- VAs to compare abnormality rates between treatments are shown (NS: p0.05;*p0.05;**p0.01;***p0.001)
temperature variation between experiments and with different larval densities (from 12 to 20 larvae in the same water vol- ume).Environmentalvariationscanaltertheresponseoflarvae during the experiment, so we must consider that differences in sensitivity could also be consequence of the variation in the experimental design. Nevertheless, the greater effect observed inH.arboreawouldindicateahighersensitivitytoammonium nitrate because of the large difference noted with respect the restofspecies.
Few studies have dealt with the relationship between nitrate exposure and quantitative analysis of abnormalities. Laposata and Dunson (1998) found that nitrate concentrations up to 40 mg NO3 -NO3Na/Ldid not produce abnormalities in embryos ofthreespecies.However,wehardlycancomparetheseresults with ours because both the nitrate concentrations and source that we used were much more toxic than those used by Lapo- sata and Dunson (1998). Jofre and Karasov (1999) found that Rana calamitans embryos exposed to unionized ammonia showed an increasing prevalence of abnormalities, which is similartowhatwefound.
The negative effect of ammonium nitrate on growth rate has been observed in all tested species. Our results are in accor- dancewiththoseofBakerandWaights(1993),whoobtained asmallersizeofB.bufolarvaeexposedto40and100mg
Fig.3. Mean(+SE)larvalsizeofsixamphibianspeciesafter15days ofexposuretoammoniumnitrate.Pw=Pleurodeleswaltl;Dg= Discoglossusgalganoi;Pc=Pelobatescultripes;Bb=Bufobufo;Bc
= Bufocalamita;Ha= Hylaarborea.Asterisksindicateresultsof post-hoc test (HDS Tukey) to compare overall larval size among controlsanddifferentconcentrationsofammoniumnitrate.Noticethat this comparison is not established for the highest level in H. arborea becausepost-hoctestscouldnotbemade.NS:p0.05;*p0.05;
**p0.01;***p0.001
NaNO3/Lcomparedwithcontrols;however,XuandOldham (1997) did not findany short-term effects of ammonium nitrate on larval size with 100 mg NO3 /L, whereas tadpoles exposed to50mgNO3 /Lwerelargerthancontrols.Wedidnotfind any effect on B. bufo larval size at 50 and 100 mg NO3 /L; however, the larval size tendencies with respect to nitrate levels, as observed by Xu and Oldham (1997) were similar to our results with P. cultripes and H. arborea. Harmless field nitrate levels could be advantageous for amphibian larvae by favoring the growth of larval food such as algae and other aquatic vegetation. Xu and Oldham (1997) rejected that possi- bility for the ecotoxicologic experiments when water is re- newedperiodically,thusnotallowingthegrowthofalgaein the experimental containers. Equally, we cannot attribute the observed effects in P. cultripes or H. arborea larval size to the food increase because water renewal frequency (4 days) was oftenenoughtoinhibitthedevelopmentofalgae.Thesmaller
size observed in tadpoles exposed to ammonium nitrate could be a consequence of decreased food ingestion as a result of activity loss. We did not measure the feeding activity in our experiments,butXuandOldham(1997)didnotfind any difference among treatments in food consumption of B. bufo larvae.Theimplicationsofthesmallersizearedifficult to analyze because our experiments did not last until larval meta- morphosis. A larger size at metamorphosis has been related to higher survival rate of postmetamorphic individuals (Smith
1987; Semlitsch et al. 1988; Berven 1990). We can hardly conclude anything, but our results could indicate a lower sur- vival probability of the larvae affected by the ammonium nitrate.
An important question in assessing sensitivity to ammonium nitrate could be the life stage at the beginning of the exposure. B. bufo larvae used by Xu and Oldham (1997) were more tolerant than ours. These investigators began their experiments with individuals at a late larval stage (Gosner’s 32 to 35; Gosner 1960), whereas ours were at the gastrula stage (Gos- ner’s 10 to 12) at the beginning of the tests. Higher sensitivity of earlier larval stages has been reported for several amphibian species such as Triturus helveticus (Watt and Jarvis 1997), P. cultripes,andD.galganoi(unpublisheddata).
Studies of conservation of amphibian species used in this study are scarce at the moment, but declines in some of their populations have been reported in some European regions (Beebee et al. 1990; Hilton-Brown and Oldham 1991; Denton et al. 1997). Population decreases of B. bufo and D. galganoi have also been reported in farming areas of Central Spain (Morales et al. 1997), where a general trend to decreased amphibiancommunitiesappearstobeoccurring(Barbadillo et al. 1999). The most sensitive species, H. arborea, is disappear- ing form southern areas of the Iberian peninsula (Barbadillo et al. 1999; Ma´rquez 2002), and water pollution could be deci- sivelycontributingtoitsdecline.
Water pollution by nitrogenous fertilizers might thus be contributing to the disappearance of the most sensitive species. However, it has been reported that harmless levels in labora- torytestsarelikelytobehazardousinthefield becauseof synergism among nitrogenous fertilizers and other pollutants (Hatch and Blaustein 2000; de Solla et al. 2002). In contrast, someinterspecific comparisonshavefoundthatthedeclining speciesinthefield arepreciselythoselesssensitivetothe chemicals in the laboratory, and there is a great intraspecific variationinthesensitivitytopollutants(BridgesandSemlitsch
2000),soitisdifficulttoassesstheeffectsofwaterpollution inthefield fromlaboratoryexperiment.Moreover,ecotoxico- logicstudiesinthefield mayprovidedifferentresultsthan thoseobtainedinexperimentallaboratoryanalysis(Birgeetal.
2000), so further studies about the implication of sensitivity of these species to ammonium nitrate on their populations’ con- servationmustbeconducted.
Acknowledgments. We thank Joan M. Del Llano, Wouter de Vries, and Gonzalo Alarcos for their help during the experiments. Gwyn Jenkinshelpedwiththetranslationofthemanuscript.Twoanonymous reviewers provided useful information about the manuscript. Funding was provided by projects CICYT-FEDER No. IFD97-1468, CICYT No. BMC2000-1139, and Ministry of Education of Spain (Grant No. AP2001-2276toM.E.O.).
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