Hydrobiologia
DOI 10.1007/s10750-009-9746-4
PRIMARY RESEARCH PAPER
Effects of nutrient enrichment on C and N stable isotope
ratios of invertebrates, fish and their food resources
in boreal streams
Jenny Bergfur Æ Richard K. Johnson Æ
Leonard Sandin Æ Willem Goedkoop
Received: 20 August 2008 / Revised: 17 February 2009 / Accepted: 24 February 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Carbon and nitrogen stable isotopes are
frequently used to study energy sources and food web
structure in ecosystems, and more recently, to study
the effects of anthropogenic stress on aquatic eco-
systems. We investigated the effect of nutrient
enrichment on d13C and d15N in fine (FPOM), coarse
(CPOM) particulate organic matter, periphyton,
invertebrates and fish in nine boreal streams in
south-central Sweden. In addition, we analysed the
diet of benthic consumers using stable isotope data.
Increases in d15N of periphyton (R2 = 0.88), CPOM
(0.78), invertebrates (0.92) and fish (0.89) were
related to nutrient enrichment. In contrast, d13C
signatures did not change along the nutrient gradient.
Our results show that d15N has potential as a sensitive
indicator of nutrient enrichment in boreal streams.
Carbon and nitrogen isotopes failed to elucidate
putative diets of selected aquatic consumers. Indeed,
comparison of low- and high-impact sites showed
that d13C of many consumers were found outside the
Handling editor: M. Power
J. Bergfur
R. K. Johnson
L. Sandin
W. Goedkoop
Department of Aquatic Sciences and Assessment,
Swedish University of Agricultural Sciences,
P.O. Box 7050, 750 07 Uppsala, Sweden
J. Bergfur (&)
The Macaulay Land Use Research Institute,
Craigiebuckler, Aberdeen AB15 8QH, UK
e-mail: j.bergfur@macaulay.ac.uk
ranges of basal resource d13C. Moreover, ranges of
basal resource d13C and d15N overlapped at both low
and high sites, making discrimination between the
importance of allochthonous and autochthonous pro-
duction difficult. Our findings show that a frac-
tionation rate of 3.4% is not always be appropriate to
assess trophic interactions, suggesting that more
studies are needed on fractionation rates along
gradients of impairment.
Keywords Nutrients
FPOM
Periphyton

CPOM
Invertebrates
Fish
Boreal streams

d15N
d13C
Introduction
Stable isotope ratios of C and N have often been used
to follow changes in ecosystem structure and function
as well as to show which processes or components are
most sensitive to perturbation (Peterson & Fry, 1987).
Much early work with stable isotopes was focused on
nutrient cycling and food web relationships in aquatic
ecosystems (Ehleringer et al., 1986; Peterson & Fry,
1987; Finlay, 2001). One of the underlying reasons for
this focus is that aquatic primary producers, such as
algae, often have different d13C signatures compared
to terrestrial vegetation. As consumers tend to be
similar to the C isotopic composition of their diets,
differences in d13C ratios are often used to distinguish
and/or trace allochthonous and autochthonous C
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sources in aquatic ecosystems (Rounick & Winterbo-
urn, 1986; France, 1995a). Moreover, as the 15N
isotopic fraction increases with each trophic level
(Mulholland et al., 2000; Finlay, 2001; Post, 2002),
d15N isotopes are, on the other hand, useful to
differentiate trophic levels and food web dynamics.
More recently, stable isotopes have been suggested
as an important component in stream health assess-
ments in general (Bunn et al., 1999; Udy et al., 2006)
and specifically as an indicator of eutrophication in
aquatic ecosystems (Cabana & Rasmussen, 1996;
McClelland & Valiela, 1998; Cole et al., 2004). For
example, d15N has been shown to be a sensitive
indicator of nutrient enrichment in deposit-feeders
(Lake et al., 2001; McKinney et al., 2002) and
primary producers (McClelland & Valiela, 1998;
Cole et al., 2004). Elevated d15N in the nitrogen
source, N transformations, such as denitrification or
ammonia volatilization, or increased N-loading has
been identified as some of the main causes for
15
N increases in dissolved inorganic N and biota
(Pennock et al., 1996; Cole et al., 2004; Udy et al.,
2006). Similar to N dynamics, increases in d13C have
been shown to be related to eutrophication (Keeley &
Sandquist, 1992; Voss & Struck, 1997; Finlay, 2001).
In brief, increased primary production leads to
reduced discrimination of 13C in photosynthesis and
hence an increase in d13C. For example, changes in
d13C of Baltic Sea sediment have been related to
primary production (Voss et al., 2000). Most studies
of nutrient effects on ecosystem structure and func-
tion have focused on lakes and ponds (Lake et al.,
2001; McKinney et al., 2002; Vander Zanden et al.,
2005) or estuaries (McClelland & Valiela, 1998; Cole
et al., 2004). Although some authors have used stable
isotopes to address eutrophication in stream ecosys-
tems (Udy & Bunn, 2001; Ulseth & Hershey, 2005;
Udy et al., 2006), few have studied streams in boreal
regions (Harrington et al., 1998). Moreover, although
stable isotopes have been used to disentangle trophic
relationships in ecological research (Rounick et al.,
1982; Lake et al., 2001; Abreu et al., 2006), relatively
few studies have focused on how effects of human-
induced stress, such as eutrophication, may affect the
interpretation of trophic interactions in streams.
Findings that the functional role of species might
change with changes in environmental conditions has
implications for the concept of functional redundancy
in aquatic ecosystems (Wellnitz & Poff, 2001;
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Hydrobiologia
Northington & Hershey, 2006). For instance, changes
in N limitation can influence the feeding behaviour of
consumers (Fagan et al., 2002), and therefore it is
important to understand how functional shifts in
species-specific dietary changes may be altered by
human impact in order to increase our understanding
of ecosystem functioning.
In this study, we analysed stable isotope ratios
(d15N and d13C) of different fractions of organic
matter collected from stream habitats along a gradient
of increasing anthropogenic stress (i.e. agricultural
land use and nutrient enrichment of N and P) to better
understand how nutrient enrichment affects the pat-
terns and processes of organic matter transformation.
Specifically, we studied how d15N and d13C in three
different basal resources (fine particulate organic
matter, FPOM; coarse particulate organic matter,
CPOM; periphyton) and benthic macroinvertebrate
consumers and fish collected from nine boreal streams
vary with increased anthropogenic impact and nutri-
ent levels. Our study thus provides high-resolution
insight of the stable isotope patterns of basal food
resources and their consumers. We hypothesised that
nutrient enrichment would result in an increase in
d15N and d13C of basal food resources and conse-
quently be reflected in the aquatic consumers. Finally,
we used stable isotope data to evaluate if use of
dietary resources changed with nutrient enrichment.
Methods
Study sites
The study was conducted in nine mid-order (stream
orders ranging from 4 to 6, width ranging from 3 to
12 m) boreal streams in south-central Sweden
(Fig. 1). Sampling sites consisted of 25- to 50-m
stretches deemed representative of the stream sec-
tions. Catchment area of the streams varied from 45
to 843 km2, and the study sites were located at
altitudes between 4 and 158 m a.s.l. (Table 1). Land
use and vegetation cover within the catchments were
characterised by GIS, using a general overview map
(scale 1:100,000). Catchment land use/cover was
expressed as a proportion of the total area. Riparian
land use and vegetation, in a 5-m wide lateral zone on
both sides of the stream, were assessed in the field
using a standardised field protocol (Johnson &
Hydrobiologia
Fig. 1 a The study area in southern Sweden within the boreal region and (b) over view map of the nine study streams
Goedkoop, 2002; Wilander et al., 2003; Sandin &
Johnson, 2004). At each site microhabitat estimations
(e.g. mineral substrate classification) were obtained
according to the method outlined in Hering et al.
(2003). The streams were all circumneutral (pH
ranged from 6.5 to 7.7) and ranged from nutrient-poor
(8 lg TP/L and 17 lg NO3 ? NO2–N/L) to nutrient-
rich (93 lg TP/L and 1436 lg NO3 ? NO2–N/L).
For more information about stream characteristics see
Bergfur et al. (2007a, b).
Water chemistry samples were taken on two
occasions in autumn of 2002 and on three occasions
in 2003 (spring, summer and autumn). The water
samples were analysed for nutrients (e.g. TN,
NO3 ? NO2–N, NH4–N, TP and PO4–P) and other
relevant variables (e.g. pH, conductivity, alkalinity,
water colour and TOC content) according to certified
laboratory protocols (Johnson & Goedkoop, 2002;
Wilander et al., 2003).
Sampling for stable isotope analysis
Samples for carbon and nitrogen analyses included
CPOM, FPOM, periphyton, benthic invertebrate and
fish. All samples were collected during 2 weeks in
October 2003, except fish which were sampled in
September 2005.
CPOM was quantified (g/m2) at each site by
collecting the benthic detritial material in three
Surber samples (25 cm 9 25 cm). The substratum
was disturbed and the catch gently rinsed through a
1-mm sieve. The samples were freeze-dried and
weighed to nearest 0.1 g to quantify the amount of
CPOM in each stream. FPOM was sampled by
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Table 1 Selected stream characteristics of the nine study streams
Site Sandån Rastälven Sverkestaån Älgängsån Strömarån Penningbyån Husbyån Hågaån Sagån

Altitude (m a.s.l.) 158 134 84 14 28 10 12 4 7.4

Mean pH 6.5 ± 0.17 7.1 ± 0.06 6.7 ± 0.09 7.4 ± 0.14 7.6 ± 0.04 7.3 ± 0.17 7.3 ± 0.21 7.7 ± 0.16 7.5 ± 0.15
(6.2–6.7) (6.9–7.1) (6.6–6.8) (7.2–7.5) (7.6–7.7) (7.1–7.5) (7.0–7.5) (7.4–7.8) (7.4–7.7)
Mean alkalinity 0.06 ± 0.02 0.13 ± 0.01 0.12 ± 0.01 0.74 ± 0.54 1.97 ± 0.14 1.80 ± 0.16 2.16 ± 0.14 3.60 ± 1.22 1.96 ± 0.47
(meq/l) (0.04–0.09) (0.12–0.15) (0.11–0.13) (0.33–1.49) (1.74–2.09) (1.62–2.03) (2.034–2.37) (1.71–4.87) (1.51–2.65)
Mean NO2 ? 17 ± 13 19 ± 5.9 17 ± 9.1 68 ± 54 83 ± 26 22 ± 14 81 ± 158 1359 ± 964 1436 ± 1490
NO3-N (lg/l) (3–36) (12–26) (5–30) (21–142) (62–126) (3–41) (3–363) (150–2662) (41–3712)
Mean TP (lg/l) 11 ± 2.3 7.8 ± 2.8 13 ± 4.0 17 ± 4.5 24 ± 6.5 27 ± 5.4 52 ± 14 74 ± 38 93 ± 44
(8–14) (5–12) (9–18) (11–21) (16–32) (23–36) (33–67) (29–119) (47–157)
Stream order 4 5 5 4 4 4 5 4 6
Width (m) 7 12 10 6 3 6.5 5.5 6 6
2
Catchment area (km ) 45 247 386 68 55 99 156 122 843
Mixed coniferous forest 78 71 70 74 72 53 48 50 46
in catchment (%)
Arable land in catchment 0.12 1.0 3.1 0.56 6.3 13 19 24 36
(%)
Mean ± standard deviation and ranges in brackets. Sites are ordered by degree of impact according to loadings of the first PCA axis. For more detailed information see Bergfur
et al. (2007a, b)
Hydrobiologia
Hydrobiologia

Table 2 Trophic level classification of invertebrates according to Moog (1995), Schmedtje & Colling (1996) and the AQEM Consortium (2005) and of fish according to

Ruffe [Gymnocephalus cernuus (Linnaeus)], Bullhead (Cottus gobio Linnaeus)

collecting water samples and retaining the suspended
FPOM on pre-combusted (550°C) GF/C glass filters
(47 mm; Whatman International Ltd, Maidstone,

(Curtis 1834), Hydropsyche pellucidula (Curtis 1834), Hydropsyche

fario Linnaeus), Burbot [Lota lota (Linnaeus)] Perch ([15–20 cm),

England). Although FPOM is usually defined as

Eel [Anguilla anguilla (Linnaeus)], Pike (Esox lucius Linnaeus)

Heptagenia sulphurea (Müller 1776), Oligochaeta, Sphaeriidae,
Asellus aquaticus (Linnaeus 1758), Baetis spp., Heptagenia spp.,

Perch (\15 cm) (Perca fluviatilis Linnaeus), Trout (Salmo trutta

0.45–1,000 lm particles (Wallace & Grubaugh,

Gammarus pulex (Linnaeus 1758), Hydropsyche angustipennis

Roach [Rutilus rutilus (Linnaeus)], Bleak [Alburnus alburnus

1996), our use of GF/C filters resulted in FPOM
samples consisting of suspended seston [1.2 lm.

(Linnaeus)], Minnow [Phoxinus phoxinus (Linnaeus)]

Periphyton was sampled from the light-exposed area
of three stones (preferably cobble). The inner area of
an open Plexiglas tube (9 cm diameter) was gently
brushed with a toothbrush, the periphyton was

Lamprey [Lampetra planeri (Bloch 1784)]

Taeniopteryx nebulosa (Linnaeus 1758)

siltalai Döhler 1963, Hydropsyche spp.

washed into 150 ml plastic containers using filtered

Rhyacophila nubila (Zetterstedt 1840)

water (GF/C) and the samples were transported in
coolers to the laboratory. In the laboratory, the
samples were centrifuged at 2,000 rpm (HERMLE
ZK 401 GmbH & Co, Gosheim, Germany), at 5°C for
10 min, the excess water decanted and the samples
frozen. Sample processing was done within 30 h after
collection. In one stream (Strömarån) too little
periphyton was collected from one of the three
samples resulting in only two replicate samples. Species
Invertebrates were collected by disturbing the
substrate and entrapping the dislodged material in a

Omnivores; benthic macro fauna/periphyton/macrophytes

hand-net with a 0.5-mm mesh size. The animals were

(turns to piscivores at later life stages) and piscivores

live sorted against a white background using 109 Shredder, grazer, filtraters, detritivores and predators
magnification, identified to lowest taxonomic unit
possible, usually to species, and thereafter frozen
(usually within 24 h). Invertebrates of the same

Predator; zooplankton/benthic meiofauna

(Feeds on CPOM, periphyton, seston)

taxonomic group were pooled to achieve sufficient

Shredder, grazer, filtraters, detritivores
Sundbom et al. (2003) and FishBase.se (http://artedi.nrm.se/fishbase_se/)

mass for analysis. As it was not possible to separate

the shell from the soft body tissue of molluscs (i.e. Predators; benthic macro fauna

Predators; benthic macro fauna

Sphaeriidae), our analyses include the isotopic com-
position of the entire animals (i.e. soft tissue and
shell). Previous studies on freshwater mussels indi-
Feeding mode/diet

cate that shells are more enriched in 13C than the soft
body tissue [i.e. Zebra mussels (Fry & Allen, 2003)
and freshwater pearl mussels (Geist et al., 2005)].
However, spherid clams are generally smaller and
have a shorter life history than zebra mussels and
freshwater pearl mussels, and to our knowledge it is
Predators (invertebrates ? fish)

not known if the isotopic signature differs between

the soft body tissue and the shell. Functional feeding
Predators (invertebrates)

groups were assigned according to Moog (1995),

Shmedtje & Colling (1996) and the AQEM Consor-
Primary consumers

tium (2005) (Table 2).

Trophic level

Fish were sampled by electrofishing, identified to

Omnivores

species and frozen before processing. Dorsal muscle

tissue was dissected from thawed fish and the samples
were re-frozen. Specimens of Eurasian minnow

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[Phoxinus phoxinus (Linnaeus)] were too small to
provide sufficient tissue mass for analysis; thus the
five specimens caught were pooled. Functional feed-
ing groups were assigned according to Sundbom et al.
(2003) and FishBase.se (http://artedi.nrm.se/fishbase_
se) (Table 2).
All samples were freeze-dried, ground in a mortar
and weighed to nearest 0.001 mg in tin capsules for
stable isotope (d13C and d15N) analyses. Samples
were analysed by the University of California, Davis
Stable Isotope Facility. The ratio between the heavy
(13C and 15N) and the light (12C and 14N) isotope was
measured, and isotopic d13C data are reported relative
to the Peedee Belemnite carbonate standard and d15N
data relative to the atmospheric N (N2) standard.
Analytical precision of reference material (NIST
1547 peach leaves) was 0.25 for d15N and 0.15 for
d13C. The delta notation indicates the difference
relative to the standard. A higher (more positive) d-
value indicates that the sample is enriched in the
heavy (13C or 15N) isotope (Fry, 2006).
Statistical analysis
Principal component analysis (PCA) on centred and
standardised variables was used to assess correla-
tively the structure of the environmental data and to
reduce the structure to a lower number of main
environmental gradients as linear combinations of the
environmental variables (Johnson & Wichern, 1988).
All variables were log10-transformed (except pH) to
more closely approximate the linear relationships
assumed in PCA. The number of meaningful princi-
pal components was determined by examining the
eigenvalues of the first few axes.
Basal resource (CPOM, periphyton and FPOM)
stable isotope ratios were compared using two-way
analysis of variance (ANOVA), with type of basal
resource and site used as factors. The two-way
ANOVAs were followed by either Tukey’s HSD or
Student’s t-test (e.g. d15N where only two basal
resources were compared: a = 0.05). All d13C and
d15N data were analysed for normality using the
Shapiro-Wilks test, and transformed to meet assump-
tions of normality; data that were not normally distrib-
uted after transformations, were rank-transformed.
Relationships between the main environmental
gradients (i.e. PCA loadings on the first and second
PC axes) and stable isotope composition of
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Hydrobiologia
periphyton, CPOM, FPOM, pooled invertebrates and
pooled fish data were analysed using linear regres-
sions. Comparisons of invertebrate stable isotope data
showed that the variations were larger within species
than within sites for both d13C and d15N (results not
shown). Thus, pooled means of invertebrate data at
each site were used in the regressions. For simplicity,
fish data were also pooled in the same way as
invertebrate data. All statistical analyses were per-
formed using JMP IN 5.1 (SAS Institute Inc.).
Results
Nutrient enrichment gradient
The first two principal components explained 56% of
the among-stream variance in water chemistry, land
use/cover and habitat composition (Fig. 2). Several
variables indicative of nutrient enrichment were
correlated with loadings of the first PC axis: the
proportion of arable and open land use in the
catchment area, amount of deciduous forest in the
riparian zone, TP and TN concentration, conductivity
and alkalinity. The second PC axis was interpreted as
representing habitat structure and composition; arable
land use in the riparian area and variability in in-
stream microhabitat composition (e.g. CPOM, wood
and coarse gravel) were correlated with this axis.
Hereafter, the first and second PC axes are referred to
as nutrient and habitat gradients, respectively.
The three sites with the highest PC loadings
(Sagån, Hågaån and Husbyån) were associated with
high-nutrient concentrations and agricultural land
use, while the three sites with the lowest scores
(Sandån, Rastälven and Sverkestaån) had low nutri-
ent concentrations and catchments dominated by
mixed-forest. Hereafter, these three high- and
low-nutrient streams are referred to as high- and
low-impact sites, respectively.
Stable isotope ratios of basal resources
d15N of periphyton and CPOM increased along the
nutrient gradient (R2 = 0.88, P \ 0.0001 and R2 =
0.78, P = 0.001, respectively) (Fig. 3A). No signif-
icant relationships were noted along the habitat
gradient. Additional regressions showed that the
d15N of periphyton and CPOM were significantly
Hydrobiologia

Fig. 2 Principal
component analysis (PCA)
on water chemistry, land
use and habitat variables.
The first and second PC
axes explain 38.2%
(eigenvalue: 14.5) and
17.4% (eigenvalue: 6.60),
respectively, of the
variation in the data set.
Environmental variables
with loadings between 3
and -3 have been removed
for clarity. C = catchment
area, R = riparian zone

Fig. 3 Mean (±SE) of

d15N (A) and d13C (B) for
three basal resources
(periphyton, n = 3, except
Strömarån, n = 2; CPOM,
n = 3 and FPOM, n = 2),
invertebrates and fish.
Increasing impact from
nutrient enrichment was
extracted by principal
component analysis (see
text for details). Dashed
lines follow the standard
error bars

correlated with TN and TP (periphyton against TN:
R2 = 0.76, P = 0.0013 and periphyton against TP:
R2 = 0.91, P \ 0.0001; CPOM against TN: R2 =
0.72, P = 0.0024 and CPOM against TP: R2 = 0.83,
P = 0.0004). Due to the small amount of FPOM
collected from Sandån, Rastälven and Älgängsån we
were not able to analyse the FPOM d15N content at
these sites. However, changes in FPOM d15N along
the nutrient gradient were seemingly nonlinear. One-
way ANOVA followed by a Tukey-Kramer HSD
revealed significant among-site differences (F5,11 =
7.35, P = 0.0153); FPOM d15N increased in the
following order: Sagån \ Sverkestaån \ Strömarån
\ Penningbyån \ Hågaån \ Husbyån. No significant
relationships were found between the d15N of FPOM
and TN or TP (P [ 0.05), respectively.
Two-way ANOVA on d15N (H(x ? 1)) data of all
three basal resources was not possible since data for
FPOM were missing from three sites. However, two-
way ANOVA showed that d15N was significantly

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higher in periphyton than in CPOM (Fdf1 = 14.6,
P = 0.0005). Significant site effects on d15N were
also noted (Fdf8 = 15.0, P \ 0.0001), but no signif-
icant interaction effects were found.
d13C ratios of basal resources (Fig. 3B) showed no
significant relationships with the two PC axes, i.e. the
nutrient and habitat gradients, respectively. Differ-
ences in d13C ratios were noted among streams
(Fdf8 = 12.5, P \ 0.0001), and a significant interac-
tion effect was found between basal resources and sites
(Fdf16 = 6.66, P \ 0.0001). The observed interaction
indicates that there was no consistent pattern in basal
resource d13C along the nutrient gradient. However,
CPOM d13C was significantly, albeit weakly, corre-
lated with TP (R2 = 0.42, P = 0.0353). d13C of
periphyton was more depleted than that of CPOM
and FPOM (two-way ANOVA on rank-transformed
data, Fdf2 = 8.38, P = 0.0008), suggesting a substan-
tial internal fractionation inside the epilithic biofilms.
Stable isotope ratios of consumers
Elemental analyses were done on 13 invertebrate taxa
collected from the nine streams. Due to differences in
community composition we could not collect the
same species from all streams. The most common
species were Asellus aquaticus (Linnaeus 1758),
found at six, and hydropsychid caddisflies, found at
seven, of the nine sites.
Regression of invertebrate d15N against loadings of
the first PC axis revealed an increase along the
nutrient gradient and showed a strikingly good fit
(R2 = 0.92, P \ 0.0001) (Fig. 3A). Regression of
invertebrate d15N against TN and TP showed a
significant relationship between these variables (R2 =
0.83, P = 0.0004 and R2 = 0.963, P \ 0.0001,
respectively). In contrast, d13C was not related to
the nutrient gradient (P [ 0.05) (Fig. 3B). Further-
more, neither invertebrate d13C nor d15 N were related
to the habitat gradient (P [ 0.05).
In total, 11 fish species were caught but similar to
invertebrates we were not able to collect the same fish
species from all nine study streams. Common species
were perch (Perca fluviatilis Linnaeus), burbot
[Lota lota (Linnaeus)] and bullhead (Cottus gobio
Linnaeus) found at five of the nine sites. Regression
of mean fish d15N against loadings of the first PC
axis showed a clear increase in d15N along the
nutrient gradient (R2 = 0.89, P \ 0.0001) (Fig. 3A).
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Hydrobiologia
Significant relationships were found between fish
d15 N and TN (R2 = 0.651, P = 0.0053) and TP
(R2 = 0.852, P = 0.0002), respectively. No signifi-
cant relationship was found between fish d13C ratios
and the nutrient gradient (Fig. 3B), or between fish
isotope ratios and the habitat gradient.
Dietary changes along the nutrient gradient
Comparison of changes in trophic relationships with
nutrient enrichment was difficult as species assem-
blages differed between low and high impact sites.
Moreover, at both low- and high-impact sites the
d13C signal of many species was outside the ranges of
the basal resource d13C (Fig. 4). For instance, at low-
impact sites (i.e. Sandån and Sverkestaån) many of
the primary consumers and omnivores were more
depleted than the basal resources. In contrast, most
species at the higher trophic levels (i.e. fish) had
d13C signatures within the ranges of the basal
resources. Finally, the C isotopic signatures of the
basal resources at low-impact sites were generally
less depleted and more similar to each other than at
the high-impact sites.
A few common species were found at both low-
and high-impact sites. A. aquaticus was found to be
outside the ranges of the basal resources samples at
both low- and high-impact sites. Oligochaeta was also
found at both low- and high-impact sites; however, at
low-impact sites this group was not recognised as
feeding on the basal resources sampled, whereas at
high-impact sites the worms appeared to feed on
either periphyton (Sagån) or FPOM (Hågaån). Sphae-
rid clams, a filterer, were found at the three high-
impact sites. However, the d13C signatures of this
taxon indicated a diet on mainly CPOM at Sagån, a
combination of periphyton and FPOM at Hågan and
mainly periphyton at Husbyån. At Sverkestaån, the
clams were more depleted in 13C than any of the basal
resources sampled.
Using a trophic fractionation rate for d15N of 3.4%
(Minagawa & Wada, 1984; Post, 2002), suggests that
primary consumers were predominantly feeding on
CPOM at the high-impact sites (i.e. Hågaån and
Husbyån). At the low-impact sites primary consumers
fed on a mixture of CPOM/FPOM or a basal resource
highly depleted in 15N compared to the basal
resources sampled here (Fig. 5). Also, enrichment in
15
N with increasing trophic level (i.e. comparing
Hydrobiologia

Fig. 4 Mean (±SE) d13C of basal resources (FPOM, CPOM Rastälven and Sverkestaån). Different symbols correspond to
and periphyton), invertebrates and fish at high-impact sites each basal resource, invertebrate and fish species
(Sagån, Hågaån and Husbyån) and low-impact sites (Sandån,

Fig. 5 Mean (±SE) d15N of basal resources (FPOM, CPOM Sverkestaån). Different symbols correspond to each basal
and periphyton), invertebrates and fish at high- (Sagån, Hågaån resource, invertebrate and fish species
and Husbyån) and low-impact sites (Sandån, Rastälven and

invertebrates and fish) was apparent at both low- and and invertebrates, had nitrogen isotope signatures that
high-impact sites. Interestingly, eel (only found in one were highly depleted compared to other smaller fish
stream, Sagån), classified as a predator of both fish such as bullheads and ruffe.

123

Fish in our study was sampled two years later than
the basal resources and invertebrates, thus caution is
advised when interpreting the fish data. However, fish
are long-lived and relatively large organism and have
been shown to have relatively constant isotopic
signatures over time, less variable than smaller
organisms like invertebrates and algae (Cabana &
Rasmussen, 1996). Moreover, previous studies have
shown no seasonal effects on stable isotope signa-
tures (France, 1997), therefore we assume that the
response of fish N isotope signatures to nutrient
enrichment is likely similar between the two years in
this study.
Discussion
Our results showed that d15N of fish, invertebrates,
periphyton and CPOM were positively correlated
with nutrient enrichment. Only FPOM concentration
was not significantly related to the nutrient enrich-
ment gradient. In this study, we have chosen to
evaluate changes along a complex gradient (the PC
axis) as well as against single nutrient variables (TN
and TP) so as not to increase the likelihood of
spurious correlations. Our results show that d15N
changes with not only increasing nutrient concentra-
tions, but also with changes in land-use. Recent
studies have shown that d15N increases along nutrient
and agricultural gradients (Harrington et al., 1998;
Vander Zanden et al., 2005; Udy et al., 2006), and
increased d15N has even been used to track fertilisers
from surrounding landscapes in aquatic environments
(Exner & Spalding, 1994; Pennock et al., 1996).
Indeed, our finding of increased d15N in CPOM and
periphyton most likely originates from increased
d15N in runoff from agricultural land use. As stream
ecosystems often rely on nutrient inputs of terrestrial
origin, it is not surprisingly that both land use and
nutrients have been argued to be the best predictors of
d15N in aquatic biota (Vander Zanden et al., 2005;
Udy et al., 2006).
Previous studies in these streams have shown
functional measures, such as leaf-litter breakdown, to
be poorer measures of ecosystem integrity than more
traditional use of biotic invertebrate indices (Bergfur,
2007; Bergfur et al., 2007a, b). Our study also
showed that d15N was better related to the nutrient
gradient than leaf-litter breakdown rates. The relative
123
Hydrobiologia
ease of analysing stable isotopes compared to leaf-
litter breakdown, which takes several months, espe-
cially in low-nutrient gradients such as these Swedish
systems, and low risk of losing samples due to
adverse weather conditions, further supports the use
of d15N as an indicator of changes in nutrient cycling
as suggested by Udy and Bunn (2001) and Udy et al.
(2006). In addition, the strong patterns across multi-
ple organism groups and trophic levels, coupled with
the fact that only minor taxonomic skills are neces-
sary lends further support to the use of d15N in
bioassessment.
Changes in d15N could indicate changes in nutrient
delivery to aquatic ecosystems that could be missed
by single water chemistry measurements (Cole et al.,
2004). However, most studies have focused on
changes in d15N as an effect of changes in nutrient
loadings from clear-cutting (Udy & Bunn, 2001),
sewage treatment plants (Cole et al., 2004; Ulseth &
Hershey, 2005) or agricultural land use (Harrington
et al., 1998). Therefore, more work is needed to
evaluate how other types of disturbances, such as
acidification or alterations in habitat, affect isotope
values in aquatic ecosystems. Collectively, our find-
ings on d15N dynamics in medium-sized boreal
streams lend further support to the use of d15N as a
sensitive indicator of nutrient enrichment, and in
particular support the use of N isotope ratios as a
robust tool in bioassessment (Udy & Bunn, 2001;
Udy et al., 2006). However, more studies are
needed, covering broader gradients in nutrient
concentration to determine potential threshold values
of d15N in order to further develop its use as a tool
bioassessment.
In contrast to the positive relationship between
d15N and the nutrient gradient, d13C ratios of the
basal resources and the benthic consumers showed no
relationship with the nutrient gradient as implied by
Finlay (2001), Keeley and Sandquist (1992) and Voss
and Struck (1997). This finding implies that any
effect of nutrient enrichment on photosynthetic rates
in these streams was probably small and had no effect
on the discrimination between heavy and light C
isotopes in primary producers. Interestingly, the d13C
signatures of different invertebrate taxa in Sagån
were highly variable and strongly depleted (Fig. 4),
possibly due anoxia in an upstream reservoir and the
C-fractionation by methanotrophic bacteria (see
Grey & Deines, 2005).
Hydrobiologia
Dietary changes along the nutrient gradient
We found few differences between the d13C signa-
tures of periphyton, CPOM or FPOM at low-impact
sites, implying that stable C isotope ratios were of
limited use for discriminating between allochthonous
and autochthonous production. However, epilithic
biofilms in our high-impact sites tended to be more
depleted in 13C compared to those found in low-
impact sites (roughly -31 to -36 and -27 to -32,
respectively). These results suggest that the accumu-
lation of more epilithic biomass at the more nutrient-
rich sites leads to C-constraints within the epiphytic
matrix, as diffusion across the benthic boundary layer
is slow and assimilation of C-fractionated occurs
within grazer communities (Hill & Middleton, 2006).
Although stable C isotopes have frequently been used
to determine the origin of different food sources in
stream ecosystems (Rounick et al., 1982, Finlay,
2001), ratios often overlap between terrestrial plant
detritus and algae, confounding their use (France,
1995a, 1996). Moreover, although we did find that
CPOM differed from periphyton and FPOM at high-
impact sites, most species were found outside the
ranges of the basal resources complicating interpre-
tation. These results show that, even though we
analysed three different basal food resources, we
were not able to cover the feeding selectivity of the
consumers, i.e. on specific algal types or microfauna
in biofilms or specific fractions of suspended partic-
ulate organic matter (SPOM).
Given the limitations of using C isotope ratios in
aquatic ecosystems, recent focus has shifted to the
use of d15N to determine the relative importance of
allochthonous and autochthonous inputs to aquatic
ecosystems (France, 1995b; Zah et al., 2001). In
particular, terrestrial autotrophs generally have lower
d15N signatures than freshwater autotrophs (France,
1995b; Mulholland et al., 2000) and our finding that
periphyton was more enriched in 15N than CPOM
supports to this conjecture. However, determining
whether the invertebrates fed primarily on periphy-
ton, CPOM or FPOM was difficult since invertebrates
that feed on two or more food sources have d15N and
d13C intermediate of their diet (Fry, 2006). Differ-
ences in d15N signatures and shifts or inherent
differences in community composition also made it
difficult to unequivocally show how diet changed
with increased nutrient enrichment. Recently, a
number of studies have shown that species’ function
varies with changes in environmental conditions
(Wellnitz & Poff, 2001; Abreu et al., 2006;
Northington & Hershey, 2006). For instance, N
limitation could induce consumers to preferentially
feed on more nutrient-rich food sources (Fagan et al.,
2002). Thus, further studies on how nutrient enrich-
ment affects food webs in boreal streams are needed,
if possible, focussing on ecosystems with similar
species communities.
Both basal resources and invertebrates were sam-
pled during the time of abscission, a time when the
streams undergo a marked change in the main food
resources available, i.e. from autochthonous to allo-
chthonous. Studies have shown seasonal variations in
stable isotope ratios in macroinvertebrates in both
streams and lakes (Zah et al., 2001; Bohman, 2005).
This could also explain why the C isotopic signatures
of consumers were outside the ranges of the basal
resource samples, i.e. our sampling effort did not
capture the range of temporal variability. Vanderklift
and Ponsard (2003) also showed how different taxo-
nomic classes and form of nitrogenous waste produced
by the organisms could influence fractionation. Previ-
ous studies have shown that 15N fractionation can vary
from -0.7 to 9.2% between adjacent trophic levels
(e.g. Vander Zanden & Rasmussen, 2001), although a
fractionation of 3.4% across trophic levels has gen-
erally been adopted (Minagawa & Wada, 1984; Post,
2002). Further studies are thus needed to unravel the
relationship between the degree of impact on ecosys-
tems and isotopic fractionation and how this would
affect stream food web studies.
In conclusion, our study adds to the growing body
of literature showing the effects of nutrients and land
use on, in particular, stable N isotopes in aquatic
ecosystems. As stable isotope analyses are relatively
inexpensive and as no elaborate sampling procedures
or taxonomic expertise are needed, the approach
shows promise as a tool in bioassessment schemes.
However, discerning diet of aquatic consumers was
less direct. Large overlaps in basal resource isotope
signatures and uncertainties regarding fractionation
rates in different species added to these difficulties.
Acknowledgements This work was partly supported by the
EU-funded projects STAR (5th framework programme,
contract no. EVK1-CT-2001-00089) and Euro-limpacs (6th
Framework Programme, contract no. GOCE-CT-2003-505540)
and The Macaulay Institute and the Scottish Government Rural
123

and Environment Research Directorate (Waters Objective,
Programme 3). We would like to thank the many people at the
Department of Environmental Assessment at SLU involved in
the collection and analysis of data used in this study, especially
to Sofie Lücke for preparations of samples for elemental
analysis.
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