Impact of Ammonium Nitrate on Growth and Survival of Six European
Amphibians
Manuel E. Ortiz,1 Adolfo Marco,2 Nelia Saiz,1 Miguel Lizana1
1
2
Department of Animal Biology, University of Salamanca, Campus Miguel de Unamuno, Salamanca 37007, Spain
Estación Biológica de Doñana, Spanish Council of Scientific Research, Apartado 1056, Sevilla 41013, Spain
Abstract. We conducted static experiments to assess the effects of ammonium nitrate fertilizer on embryos and larvae of
six European amphibians: sharp-ribbed salamander (Pleurodeles waltl), Iberian painted frog (Discoglossus galganoi), western spadefoot toad (Pelobates cultripes), common toad (Bufo
bufo), natterjack toad (Bufo calamita), and common tree frog
(Hyla arborea). Embryos were exposed to different and environmentally relevant concentrations of ammonium nitrate (0 to
200 mg NO3 /L) for 15 days. Hatching took place during the
experiments. H. arborea was extremely sensitive and had high
mortality after 8 days of exposure even at the lower fertilizer
levels. D. galganoi and B. bufo were also very sensitive and
had significant mortality after 15 days of exposure. The rest of
the species did not suffer lethal effects but suffered abnormalities or decreased growth at the highest fertilizer concentrations. Chemical fertilizers or manure could have contributed to
the observed decrease of B. bufo and D. galganoi in agricultural areas of the Iberian Peninsula during recent years. H.
arborea was the most sensitive species studied. The results of
our study showed that environmentally relevant levels of ammonium nitrate can induce mortality and might affect population dynamics of this species in agricultural environments.
Profound changes promoted by scientific and technological
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
the most widely used in the world (FMA, MAFFF, & SOAFD
1993); its application on crop fields has recently increased
(Agricultural Development and Advisory Service 1992;
Lanyon 1996). Moreover, the intensive farming of different
animal species and the subsequent production of ammonia-rich
residues have recently increased. Both the addition of chemical
Correspondence to: M. E. Ortiz; email: mortiz@usuarios.retecal.es
fertilizers and manure in the crops or the leaching of ammonium from farms are producing increased environmental nitrogen levels in aquatic ecosystems (United States Environmental
Protection Agency [US EPA] 1986, 1999; Vitousek et al.
1997). An excess of nitrogen in water bodies near crop fields or
livestock farms can be seriously harmful to aquatic wildlife
(e.g., Westin 1974; Camargo and Ward 1992). Amphibians
may be especially sensitive to water pollution because of their
permeable skin and gills (Blaustein et al. 1994). Decreased
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 (Hecnar 1995; Watt and Jarvis 1997). Environmentally
realistic ammonia or nitrate concentrations in water bodies are
likely to damage amphibian eggs and larvae in agricultural
regions (e.g., Cooke 1981; Marco et al. 1999; Rouse et al.
1999).
The aim of this work was to analyze the effect of ammonium
nitrate on embryonic and larval development of six amphibian
species usually found in farming areas of the Iberian Peninsula.
The natterjack toad (Bufo calamita) has disappeared from
several sites in the United Kingdom because of water and soil
acidification and habitat loss or degradation (Beebee et al.
1990; Denton et al. 1997). Populations of the common toad
(Bufo bufo) have also decreased in the United Kingdom (Cooke
1972; Hilton-Brown and Oldham 1991). The negative effects
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
population declines are currently unknown D. galganoi is the
least-common, quite rare in farming areas of central Spain, and
is probably the most frequently exposed species in this study to
ammonium nitrate because of its preference for shallow waters
(Martı́nez-Solano 2002).
To test the hypothesis that these six amphibian species are
sensitive to environmental levels of ammonium nitrate, we
studied the dose– effect relationship on embryos and young
larvae using static laboratory experiments.
Materials and Methods
Study Species
We collected sharp-ribbed salamander (P. waltl), western spadefoot
toad (P. cultripes), and common tree frog (H. arborea) eggs from
several ponds in Los Arribes del Duero (western Spain). Common toad
(B. bufo) and natterjack toad (B. calamita) eggs were collected from a
mountain stream in the Sierra de Gredos (central Spain), and Iberian
painted frog (D. galganoi) eggs were collected from a pond near
Doñana National Park (southwestern Spain). For each species, eggs
from at least three different clutches were collected in stages of early
development (stages 10 to 12; Gosner 1960; Harrison 1969) in areas
where they were abundant.
Experimental Procedures
Each species was tested at different times from winter 2001 to spring
2002. We conducted static experiments (Stephen 1975) in the laboratory at an environmental temperature and during the natural photoperiod. Within 24 hours after collection, the eggs were exposed to a
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
(legal maximum for water intended for human consumption; European
Council 1998); 100 (not in B. bufo and B. calamita experiments); and
200 mg NO3 -NO3NH4/L. United States Environmental Protection
Agency (US EPA) acute criteria for ambient ammonia is 36 mg N/L in
salmonids-absent waters (US EPA 1999). In our experiments, the
maximum concentration of ammonia— corresponding to 200 mg
NO3 -NO3NH4/L—was 45 mg N/L. This highest level has been found
in several agricultural ponds in the areas from which the eggs were
collected. We used 10 g NO3 -NO3NH4/L stock solution prepared
from ammonium nitrate salt (99% purity), which was pipetted into the
containers to get the experimental concentrations. We used dechlorinated tap water.
Water temperature and pH were checked daily. Water temperature
never varied more than 1.5°C within the same experiment, and its total
range—including all the experiments—remained between 15°C and
23°C. Overall water temperatures in each experiment were 16.3°C for
D. galganoi, 18.7°C for P. waltl, 18.9°C for P. cultripes, 21.0°C for B.
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.
Water in the tanks was renewed and nitrate levels were restored every
4 days. Previous studies conducted with similar methodology and
environmental conditions showed no significant deviations (>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 dechlorinated tap water and boiled for 5 minutes. P. waltl larvae were fed ad
libitum with Artemia larvae bred in the laboratory.
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
moment by the initial number of eggs. Abnormality rate was calculated
as a cumulative measure dividing the number of abnormal larvae (dead
or alive) at a given moment by the initial number of eggs. At the end
of each experiment, we measured total body length of the survivors
from mouth to tail tip, with the tail intact, using an electronic digital
caliper Stainless Hardened to the nearest 0.01 mm. Measurements
were always taken by the same investigator, and tadpoles were carefully placed straight on a plane surface before measuring. The measurer had long experience measuring tadpoles and knew previously
which treatment the measured tadpoles were from.
Analysis of Data
To determine the effect of ammonium nitrate on larval survival for
each amphibian species, we used repeated measure analysis of variance (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), and the categoric variable being nitrate concentration.
To determine which nitrate levels had a lethal effect or caused abnormalities in each species, we used post hoc (HDS Tukey) univariate
ANOVA. We also used univariate ANOVAs (and subsequent post-hoc
HDS Tukey tests) to determine for each species the effect of ammonium nitrate on larval size at the end of the experiment. For larval size
we considered the average values of each container.
Results
All species showed some degree of sensitivity. Differences in
mortality over time caused by ammonium nitrate were detected
in H. arborea and D. galganoi (Table 1). At day 8, H. arborea
was the most sensitive species and had high mortality (95%)
even at the lowest ammonium nitrate levels (F3,8 = 41.187,
p < 0.001). At day 15, higher concentrations of ammonium
nitrate also caused significant mortality in D. galganoi (F3,8 =
22.917, p < 0.001) larvae (Figure 1). Despite not showing a
response over time (Table 1), B. bufo was sensitive to 200 mg
NO3 /L after 15 days of exposure (F2,24 = 9.549, p = 0.001)
(Figure 1). The other three species did not suffer significant
mortality at any ammonium nitrate concentration.
Some larvae suffered edemas, bent tails, and lordosis. D.
galganoi, B. bufo, and B. calamita showed an increasing abnormalities 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). At day 4, B. bufo also
showed a high number of abnormal larvae at 200 mg NO3 /L
(Figure 2) (F2,24 = 7.735; p = 0.003). In spite of the low
abnormality rate showed by B. calamita after 4 days of exposure (Figure 2), a significant effect by ammonium nitrate was
detected at the highest level (F2,24 = 4.015; p = 0.031).
All six species showed a negative effect of ammonium
nitrate on total length by the end of the experiment. Statistically
significant decreased larval size was detected at the highest
concentrations (Figure 3). D. galganoi larvae exposed to 100
mg NO3 /L also showed a significantly smaller size. P. cultripes tadpoles exposed to both 50 and 200 mg NO3 /L were
smaller than controls, although not those exposed to 100 mg
NO3 /L. A similar effect was observed in H. arborea; tadpole
Table 1 Results of repeated-measure ANOVAs comparing mortality over time of larvae of six amphibian species exposed to
ammonium nitratea
Species
Source of Variation
Mean Squares
df
P. waltl
Concentration
Error
Concentration
Error
Concentration
Error
Concentration
Error
Concentration
Error
Concentration
Error
0.102
0.045
0.263
0.037
0.012
0.060
0.018
0.050
0.043
0.039
1.336
0.054
3
8
3
8
3
8
2
24
2
24
3
8
D. galganoi
P. cultripes
B. bufo
B. calamita
H. arborea
F
p
2.280
0.156
7.074
0.012
0.202
0.892
0.360
0.701
1.090
0.352
24.856
<0.001
a
Dependent variable was mortality at 2, 4, 8, and 15 days (arcsin of square root transformed).
ANOVAs = analyses of variance.
Fig. 1. Mortality caused by ammonium nitrate in larvae of six amphibian species after 15 days of exposure. Pw = Pleurodeles waltl; Dg
= Discoglossus galganoi; Pc = Pelobates cultripes; Bb = Bufo bufo;
Bc = Bufo calamita; Ha = Hyla arborea
length was significantly lower than in controls only at 50 mg
NO3 /L. No differences between controls and tadpoles exposed to 100 mg NO3 /L were detected, and because survivors
of this species at 200 mg NO3 /L appeared in only one tank,
we could not use the post hoc tests at this level.
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; Hecnar 1995; Watt and Jarvis 1997; Jofre and Karasov 1999;
Marco and Blaustein 1999; Marco et al. 1999, 2001;
Schuytema and Nebeker 1999a, 1999b). We are unaware of the
existence of published data for the sensitivity of studied species
except B. bufo to nitrogenous fertilizers. Xu and Oldham
(1997) did not find lethal effects of ammonium nitrate in B.
bufo larvae exposed to 50 mg NO3 /L, whereas those exposed
to 100 mg NO3 /L showed a mortality rate of 21% after 30
days of exposure. Sensitivity of B. bufo to ammonium nitrate
was lower than that observed in our experiments (20% dead
after 15 days of exposure to 50 mg NO3 /L), so larvae used by
Xu and Oldham (1997) were more resistant than ours. However, other investigators have reported higher sensitivity in B.
bufo larvae. Berger (1989) found lethal effects of ammonium
nitrate on tadpoles exposed for 4 days to 15.7 mg NO3 /L. At
the same exposure time, the mortality observed in this study
was not significant even at 200 mg NO3 -NO3NH4/L. Baker
and Waights (1993) used sodium nitrate and reported 84.6%
deaths for B. bufo larvae exposed to 29 mg NO3 /L during 13
days, and 100% deaths for larvae exposed to 73 mg NO3 /L.
Tadpoles used by Baker and Waights (1993) were therefore far
more sensitive than ours, whose mortality rate was < 40% 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), whereas sodium ion does not appear to be an especially
hazardous substance. Schuytema and Nebeker (1999a) found
significant mortality of Pseudacris regilla larvae exposed to
sodium nitrate concentrations when the sodium level was considerably lower than that likely to be harmful (Padhye and Gate
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, & SOAFD 1993).
We observed a great interspecific variability 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)
found that 4-day LC50 values varied among four species from
75 to 174 mg NO3 - NO3NH4/L. Marco et al. (1999), using
potassium nitrate, found that nitrate levels higher than 15-day
LC50 for Ambystoma gracile or Rana pretiosa tadpoles did not
cause any effect on Bufo boreas or Hyla regilla. The results of
our study suggested that H. arborea may suffer lethal effects at
concentrations lower than the maximum allowed in water intended for human consumption (European Council 1998). D.
galganoi and B. bufo also showed high mortality, whereas
higher pollutant levels caused growth retardation in more tolerant species such as P. waltl, P. cultripes, and B. calamita.
Interspecific comparision in this study was made from experiments conducted at different times with up to 8°C of water
Table 2. Results of repeated-measure ANOVAs comparing abnormality rate over time of larvae of six amphibian species exposed to ammonium nitratea
Species
Source of Variation
Mean Squares
df
P. waltl
Concentration
Error
Concentration
Error
Concentration
Error
Concentration
Error
Concentration
Error
Concentration
Error
0.002
0.002
0.730
0.048
0.000
0.000
0.449
0.050
0.122
0.030
0.018
0.017
3
8
3
8
3
8
2
24
2
24
3
8
D. galganoi
P. cultripes
B. bufo
B. calamita
H. arborea
F
p
1.000
0.441
15.217
0.001
0.000
1.000
9.022
0.001
4.072
0.030
0.797
0.529
a
Dependent variable was abnormality rate at 2, 4, 8, and 15 days (arcsin of square root transformed) using time as a repeated measure.
ANOVAs = analyses of variance.
Fig. 2. Abnormality rates showed by three amphibian species after 2,
4, 8, and 15 days exposure to different ammonium nitrate levels. Light
grey bars correspond to controls, lined bars to 50 mg NO3 /L, and
dark grey bars to 200 mg NO3 /L. Signification of univariate ANOVAs to compare abnormality rates between treatments are shown (NS:
p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001)
temperature variation between experiments and with different
larval densities (from 12 to 20 larvae in the same water volume). Environmental variations can alter the response of larvae
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
in H. arborea would indicate a higher sensitivity to ammonium
nitrate because of the large difference noted with respect the
rest of species.
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/L did not produce abnormalities in embryos
of three species. However, we hardly can compare these results
with ours because both the nitrate concentrations and source
that we used were much more toxic than those used by Laposata and Dunson (1998). Jofre and Karasov (1999) found that
Rana calamitans embryos exposed to unionized ammonia
showed an increasing prevalence of abnormalities, which is
similar to what we found.
The negative effect of ammonium nitrate on growth rate has
been observed in all tested species. Our results are in accordance with those of Baker and Waights (1993), who obtained
a smaller size of B. bufo larvae exposed to 40 and 100 mg
Fig. 3. Mean (+ SE) larval size of six amphibian species after 15 days
of exposure to ammonium nitrate. Pw = Pleurodeles waltl; Dg =
Discoglossus galganoi; Pc = Pelobates cultripes; Bb = Bufo bufo; Bc
= Bufo calamita; Ha = Hyla arborea. Asterisks indicate results of
post-hoc test (HDS Tukey) to compare overall larval size among
controls and different concentrations of ammonium nitrate. Notice that
this comparison is not established for the highest level in H. arborea
because post-hoc tests could not be made. NS: p > 0.05; *p < 0.05;
**p < 0.01; ***p < 0.001
NaNO3/L compared with controls; however, Xu and Oldham
(1997) did not find any short-term effects of ammonium nitrate
on larval size with 100 mg NO3 /L, whereas tadpoles exposed
to 50 mg NO3 /L were larger than controls. We did not find
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 possibility for the ecotoxicologic experiments when water is renewed periodically, thus not allowing the growth of algae in
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
often enough to inhibit the development of algae. The smaller
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, but Xu and Oldham (1997) did not find any
difference among treatments in food consumption of B. bufo
larvae. The implications of the smaller size are difficult to
analyze because our experiments did not last until larval metamorphosis. 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 survival 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 (Gosner’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, and D. galganoi (unpublished data).
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
amphibian communities appears to be occurring (Barbadillo et
al. 1999). The most sensitive species, H. arborea, is disappearing form southern areas of the Iberian peninsula (Barbadillo et
al. 1999; Márquez 2002), and water pollution could be decisively contributing to its decline.
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 laboratory tests are likely to be hazardous in the field because of
synergism among nitrogenous fertilizers and other pollutants
(Hatch and Blaustein 2000; de Solla et al. 2002). In contrast,
some interspecific comparisons have found that the declining
species in the field are precisely those less sensitive to the
chemicals in the laboratory, and there is a great intraspecific
variation in the sensitivity to pollutants (Bridges and Semlitsch
2000), so it is difficult to assess the effects of water pollution
in the field from laboratory experiment. Moreover, ecotoxicologic studies in the field may provide different results than
those obtained in experimental laboratory analysis (Birge et al.
2000), so further studies about the implication of sensitivity of
these species to ammonium nitrate on their populations’ conservation must be conducted.
Acknowledgments.
We thank Joan M. Del Llano, Wouter de Vries,
and Gonzalo Alarcos for their help during the experiments. Gwyn
Jenkins helped with the translation of the manuscript. Two anonymous
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.
AP2001-2276 to M.E.O.).
References
Agricultural Development and Advisory Service (1992) Survey of
fertiliser practice: Fertiliser use in farm crops in england and
Wales 1992. Agricultural Development and Advisory Service,
Edinburgh, UK
Baker J, Waights V (1993) The effect of sodium nitrate on the growth
and survival of toad tadpoles (Bufo bufo) in the laboratory. Herpetologica J 3:147–148
Barbadillo LJ, Lacomba JI, Pérez-Mellado V, Sancho V, López-Jurado
LF (1999) Anfibios y reptiles de la Peninsula Ibérica, Baleares y
Canarias. Editorial GeoPlaneta, Barcelona, Spain
Beebee TJC, Flower RJ, Stevenson AC, Patrick ST, Appleby PG,
Fletcher C, et al. (1990) Decline of the natterjack toad Bufo
calamita in Britain: Palaeoecological, documentary and experimental evidence for breeding site acidification. Biol Conserv
53:1–20
Berger L (1989) Disappearance of amphibian larvae in the agricultural
landscape. Ecol Int Bull 17:65–73
Berven KA (1990) Factors affecting population fluctuations in larval
and adult stages of the wood frog (Rana sylvatica). Ecology
71:1599 –1608
Birge WJ, Westerman AG, Spromberg JA (2000) Comparative toxicology and risk assessment of amphibians. In: Sparling DW,
Linder G, Bishop CA (eds) Ecotoxicology of amphibians and
reptiles. SETAC Press, Pensacola, FL, pp 727–791
Blaustein AR, Wake DB (1990) Declining amphibian populations: A
global phenomenon? Trends Ecol Evol 5:203–204
Blaustein AR, Wake DB, Sousa WP (1994) Amphibian declines:
Judging stability, persistence, and susceptibility of population to
local and global extinctions. Conserv Biol 8:60 –71
Boyer R, Grue CE (1995) The need for water quality criteria for frogs.
Environ Health Perspect 103:352–357
Bridges CM, Semlitsch, RD (2000) Variation in pesticide tolerance of
tadpoles among and within species of Ranidae and patterns of
amphibian decline. Conserv Biol 14:1490 –1499
Camargo JA, Ward JW (1992) Short-term toxicity of sodium nitrate
(NaNO3) to non- target freshwater invertebrates. Chemosphere
24:23–28
Cooke AS (1972) Indications of recent changes in status in the British
Isles of the frog (Rana temporaria) and the toad (Bufo bufo). J
Zool Lond 167:161–178
Cooke AS (1981) Tadpoles as indicators of harmful levels of pollution
in the field. Environ Pollut 25:123–133
de Solla SR, Pettit KE, Bishop CA, Cheng KM, Elliott JE (2002)
Effects of agricultural runoff on native amphibians in the Lower
Fraser River Valley, British Columbia, Canada. Environ Toxicol
Chem 21:353–360
Denton JS, Hitchings SP, Beebee TJC, Gent A (1997) A recovery
program for the natterjack toad (Bufo calamita) in Britain. Conserv Biol 11:1329 –1338
European Council (1998) The directive on quality of water intended
for human consumption 98/83/CE, Strasbourg, France
Fertiliser Manufacturers’ Association; Ministry of Agriculture, Fisheries, and Food; Scottish Office of Agriculture and Fisheries
Department (1993) The British survey of fertiliser practice: fertiliser use on farm crops 1992. HMSO, London, UK
Gosner KL (1960) A simplified table for staging anuran embryos and
larvae with notes on identification. Herpetologica 16:183–190
Harrison RG (1969) Harrison stages and description of the normal
development of the spotted salamander, Ambystoma punctatum
(Linn.). In: Harrison RG (ed) Organization and Development of
the Embryo. Yale University Press, New Haven, CT, pp 44 – 66.
Hatch AC, Blaustein AR (2000) Combined effects of UV-B, nitrate
and low pH reduce the survival and activity level of larval Cas-
cades Frogs (Rana cascadae). Arch Environ Contam Toxicol
39:494 – 499
Hecnar SJ (1995) Acute and chronic toxicity of ammonium nitrate
fertilizer to amphibians from southern Ontario. Environ Toxicol
Chem 14:2131–2137
Hilton-Brown D, Oldham RS (1991) The status of the widespread
amphibians and reptiles in Britain, 1990, and changes during the
1980s. Nature Conservancy Council Contract Survey. London,
UK
Jofre MB, Karasov WH (1999) Direct effects of ammonia on three
species of North American anuran amphibians. Environ Toxicol
Chem 18:1806 –1812
Lanyon LE (1996) Does nitrogen cycle?. Agric Consult February:17
Laposata MM, Dunson WA (1998) Effects of Boron and Nitrate on
Hatching Success of Amphibian Eggs. Arch Environ Contam
Toxicol 35:615– 619
Marco A, Blaustein AR (1999) The effects of nitrite on behavior and
metamorphosis in Cascades frogs (Rana cascadae). Environ Toxicol Chem 18:946 –949
Marco A, Cash D, Belden L, Blaustein AR (2001) Sensitivity to urea
fertilization in three amphibian species. Arch Environ Contam
Toxicol 40:406 – 409
Marco A, Quilchano C, Blaustein AR (1999) Sensitivity to nitrate and
nitrite in pond-breeding amphibians from the Pacific Northwest,
USA. Environ Toxicol Chem 18(12):2836 –2839
Márquez R (2002) Hyla arborea. In: Pleguezuelos JM, Márquez R,
Lizana M (eds) Atlas y Libro Rojo de los Anfibios y Reptiles de
España. Directión General de Conservación de la Naturaleza,
Madrid, Spain, pp 103–105
Martı́nez-Solano I (2002) Discoglossus galganoi: In: Pleguezuelos
JM, Márquez R, Lizana M (eds) Atlas y Libro Rojo de los
Anfibios y Reptiles de España. Dirección General de Conservación de la Naturaleza, Madrid, Spain, pp 85– 86
Morales JJ, Lizana M, Martin-Sánchez R, López-González J (1997)
Nuevos datos sobre la distribución de anfibios en la provincia de
Salamanca. Bol Asoc Herp Esp 8:12–13
Padhye AD, Ghate HV (1992) Sodium chloride and potassium chloride tolerance of different stages of the frog, Microhyla ornata.
Herpetologica J 2:18 –23
Rouse JD, Bishop CA, Struger J (1999) Nitrogen pollution: an assessment of its threat to amphibian survival. Environ Health Persp
107:799 – 803
Schuytema GS, Nebekar AV (1999a) Comparative effects of ammonium and nitrate compounds, on Pacific treefrog and African
clawed frog embryos. Arch Environ Contam Toxicol 36:200 –206
Schuytema GS, Nebeker AV (1999b) effects of ammonium nitrate,
sodium nitrate and urea on red-legged frogs, Pacific treefrogs, and
African clawed frogs. Bull Environ Contam Toxicol 63:357–364
Semlitsch RD, Scott DE, Pechmann JHK (1988) Time and size at
metamorphosis related to adult fitness in Ambystoma talpoideum.
Ecology 69:184 –192
Smith DC (1987) Adult recruitment in chorus frogs: Effects of size and
date at metamorphosis. Ecology 68:344 –350
Stephen CE (1975) Methods for acute toxicity tests with fish, macroinvertebrates and amphibians. EPA-660/3-75-009. U.S. Environmental Protection Agency, Corvallis, OR, USA
US EPA (1986) Quality criteria for water 1986. EPA 440/5-86-001,
Office of Water Regulations and Standards, Washington, DC,
USA
US EPA (1999) Water quality criteria; notice of availability; 1999
update of ambient quality criteria for ammonia. EPA-822-R-99014. U.S. Environmental Protection Agency. Federal Register,
64:71974 –71980. Washington, DC, USA
Vitousek PM, Aber J, Howarth RW, Likens GE, Matson PA, Schindler
DW, Schlesinger WH, Tilman GD (1997) Human alteration of the
global nitrogen cycle: causes and consequences. Issues in Ecology
1:1– 6
Wake DB (1991) Declining amphibian populations. Science 253:422–
424
Watt PJ, Jarvis P (1997) Survival analysis in palmate newts exposed to
ammonium nitrate agricultural fertilizer. Ecotoxicology 6:355–
362
Westin DT (1974) Nitrate and nitrite toxicity to salmonoid fishes. Prog
Fish-Cult 36:86 – 89
Xu Q, Oldham RS (1997) Lethal and sublethal effects of nitrogen
fertilizer ammonium nitrate on common toad (Bufo bufo) tadpoles.
Arch Eviron Contam Toxicol 32:298 –303