Assessing the applicability of stable isotope analysis to determine the contribution of landfills to vultures’ diet
Assessing the applicability of stable isotope analysis to determine the contribution of landfills to vultures' diet
Helena Tauler-Ametller 0 1
Antonio HernaÂ ndez-MatÂõas 0 1
Francesc PareÂ s 0 1
Joan Ll. Pretus 0 1
Joan Real 0 1
0 Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Facultat de Biologia and Institut de la Recerca de la Biodiversitat (IRBIO), Universitat de Barcelona , Barcelona, Catalunya , Spain , 2 Equip de Biologia de la ConservacioÂ , Universitat de Barcelona , Barcelona, Catalunya, Spain, 3 Departament de Ciències Ambientals , Facultat de Ciències, Universitat de Girona , Campus Montilivi, Girona, Catalunya , Spain
1 Editor: Antoni Margalida, University of Lleida , SPAIN
Human activities cause changes to occur in the environment that affect resource availability for wildlife. The increase in the human population of cities has led to a rise in the amount of waste deposited in landfills, installations that have become a new food resource for both pest and threatened species such as vultures. In this study we used stable isotope analysis (SIA) and conventional identification of food remains from Egyptian Vultures (Neophron percnopterus) to assess the applicability of SIA as a new tool for determining the composition of the diets of vultures, a group of avian scavengers that is threatened worldwide. We focused on an expanding Egyptian Vulture population in NE Iberian Peninsula to determine the part played by landfills and livestock in the diet of these species, and aimed to reduce the biases associated with conventional ways of identifying food remains. We compared proportions of diet composition obtained with isotope mixing models and conventional analysis for five main prey. The greatest agreement between the two methods was in the categories `landfills' and `birds' and the greatest differences between the results from the two methods were in the categories `livestock', `carnivores' and `wild herbivores'. Despite uncertainty associated to SIA, our results showed that stable isotope analysis can help to distinguish between animals that rely on waste and so present enriched levels of δ 13C than those that feed on the countryside. Indeed, a high proportion of food derived from landfills (nearly 50%) was detected in some breeding pairs. Furthermore we performed GLMM analyses that showed that high values of δ 13C in Egyptian Vulture feathers (a proxy of feeding in landfills) are related with high levels of humanization of territories. This method has the potential to be applied to other threatened vulture species for which there is a lack of information regarding resources they are consuming, being especially important as the main causes of vultures decline worldwide are related to the consumption and availability of food resources.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: Red Electrica the EspañaÐSA (http://
www.ree.es/en) financed the project, and
Universitat de Barcelona (http://www.ub.edu/web/
ub/ca/) supported HTA with a predoctoral grant
Competing interests: The authors have declared
that no competing interests exist.
Human activities have greatly transformed the Earth's land surface. Natural landscapes have
been altered irremediably by human use and management practices in many
human-dominated lands are constantly changing [
]. During this transformation of the landscape, humans
have modified the natural resources available to wild species with serious implications for their
population dynamics and distributions [2±4].
The increase in the human population in cities has led to an accumulation of large
quantities of waste, which is deposited in open-air landfills scattered throughout the countryside [
These sites provide plentiful and predictable food resources for many generalist or scavenger
species such as gulls, raven, fox and rats that are able to feed there. In some cases, these species
have undergone a rapid population increase as a result [6±10]. However, resources derived
from landfills are also used by other species that are not regarded as pests, some of which are
classified as threatened [
]. Although it is known that certain species of conservation
concern use landfills as feeding sites , few studies have quantified this use [
Over the last 20 years in the Iberian Peninsula the sight of large groups of vultures feeding
in landfills has become commonplace [
] and it has been suggested that these installations
are today a key source of food for certain vulture populations. Moreover, vultures also feed
in other resources that could act as a threat, as poisoned animals. The consumption of these
resources has caused in recent decades the crash of vulture populations in Asia and, more
recently, in many parts of Africa [
]. The generalized use of veterinary drugs on livestock,
pesticides, or intentional poisoning by poachers have pushed some raptor species that just two
decades ago were common worldwide to the brink of extinction [
]. Indeed, vultures are
considered today to be one of the most threatened groups of birds worldwide [
Despite these human-driven changes in resource distribution are carrying serious
behavioural and demographic responses of vultures populations, our knowledge of the effect of
these novel scenarios on vulture diets is still limited (but see [21±24]). This is partly due to
complexity to quantify the net contribution in terms of ingested or assimilated biomass in this
group of birds. In raptors, most conventional diet studies are based in the analysis of food
remains or pellets sampled at nests or resting sites [25±27]. These method allows the
identification of prey at a high taxonomic accuracy [
]. However, many vulture species such as
those of Gyps genus may ingest large amounts of meat from corpses from big sized animals
that may contribute scarcely to food sampled remains. In addition, species that use to feed on
smaller species, such as Egyptian vultures, commonly carry to nests pieces from large prey
while only a small fraction of it was feed; all these making challenging to stablish a
correspondence between sampled food remains and ingested biomass. Consequently, the potential biases
that have been recognized for conventional diet analysis, such as those linked to prey size or
], might be present in available quantitative assessments of diet composition
As an alternative, stable isotope analysis (SIA) can provide a representation of the food
digested and absorbed by an animal [
]. In this field, recent Bayesian isotopic mixing models
have been developed to generate potential dietary solutions for multiple dietary sources and to
account for uncertainty and variation in model estimates [
], and has been widely used to
infer the diet composition of a large range of organisms [32±35]. This method has been proven
useful for determining the part played in animals' diets of important food resources such as
wild herbivores or food from landfills [
]. In spite of this, SIA has been very little used to
study the diet of vultures (but see [
]). Thus, it would appear to be a promising tool for
investigating vulture diets under the changing scenarios in which vultures live and may be
useful as an indicator of on-going environmental changes.
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Nevertheless, SIA is not exempt of limitations, as it requires accurate prior information
regarding the trophic ecology of the studied species and results usually present a wide range of
]. Incorporating prior information obtained from the analysis of stomach in
SIA has been shown to provide very reliable results . However, this method requires the
regurgitation of ingested food or the death of the animal, so its applicability is very limited
when working with species whose individuals are difficult to capture and cannot be sampled
repeatedly in time, an issue particularly concerning in endangered species. In addition, despite
the great popularity of SIA in ecological studies, formal comparisons between SIA and
conventional methods of diet analysis are still limited (but see [
]). In that context where more
available and feasible methodologies present some limitations, providing quantitative
assessments of diet composition of vultures based on the most easily applicable methods is crucial to
better understand their potentialities and the possible differences between these methods.
This study focused on the diet of the Egyptian Vulture, a species located at the top of the
food web and therefore a good indicator of mature ecosystems. This particularity, together
with the fact that it is sensitive to human activities, makes it a good indicator of environmental
changes [42±44]. We used both conventional diet analysis and SIA to infer the diet of this
endangered vulture, we assumed that if both methodologies agree, then both methods would
be providing a good approximation of resources consumed. Unlike the negative global trend
in the species [
], the study population in Catalonia (NE Iberian Peninsula) has grown over
the past 20 years, and has expanded and colonized new highly humanized areas from where it
was not known before [
]. This process has happened in parallel with the increase in open-air
landfills that has contributed to a higher probability of recruitment of breeding pairs in the
area as well as to a lesser probability of territory disappearance [
]. The main objective of
our study was to determine the part played by landfills in the diet of Egyptian Vulture breeding
pairs, using conventional identification of food remains and stable isotope analysis. We thus
hypothesized that breeding pairs found in humanized areas close to landfills are feeding on
these facilities and therefore that the contribution of resources from waste tips in their diet will
Study area and data collection
The study area lies in central eastern Catalonia (NE Iberian Peninsula, Spain) at altitudes of
200±1900 m a.s.l., and encompasses an area of cliffs running from the Prelitoral Mountains in
the south to the pre-Pyrenean Mountains in the north (Fig 1). It includes humanized zones
with high human population density (more than 150 hab/km2), as well as less populated rural
areas (13 hab/km2). Due to this dense human population there are nine landfills within the
study area that receive municipal waste, which, before being processed, is exposed and
available for different opportunistic species. In 2012±2015, all occupied Egyptian Vulture territories
were visited during the breeding season (March±August) by observers equipped with a
spotting scope (20±60x). Once nestlings were approximately 45±55 days old, they were caught with
the assistance of experienced climbers and three/four mantle feathers from each individual
were taken for sampling by SIA (2012±2015; n = 60 chicks from 19 territories). Food remains
for conventional diet analysis were collected in two moments during reproduction: (i) from
the nests when the nestlings' feathers were sampled and (ii) after the breeding season at the
same nests (2012±2014; n = 32 samples from 18 territories). Both two samples from each nest
and year were considered as one single statistical observation for diet analysis. In order to the
results of conventional and isotope analysis were comparable, only data of successful breeding
pairs was considered for analysis.
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Fig 1. Distribution of breeding pairs (yellow circles) and landfills (green symbols) located inside our study area
and considered for diet analysis. Grey gradient represent altitudes from 0 m (white) to 3000 m (black). Two
important cities are also represented.
Food components were identified to the lowest possible taxonomic level by consulting
collections in the Barcelona Museum of Natural Sciences, the Centre de Recursos de
Biodiversitat Animal (Biology Faculty, Barcelona University), the collection of one of the authors
(J. Real) and a reference guide [
]. Counts of items were carried out following [
Numbers of invertebrates and small- and medium-sized vertebrates were recorded as a minimum
number of individuals. Carnivores and ungulates were counted as the number of bones or
skeletal fragments as it was impossible to determine with precision the number of whole
individual specimens for most species. The three distal parts of the limbs, i.e. the
tarsus/carpus, metatarsus/metacarpus and digits associated with other smaller bones, were recorded as
one skeletal fragment. The same applied to skull fragments, which usually consisted of
several connected bones. Food components were categorized into six groups: landfill, livestock,
wild herbivores, carnivores, birds and others (mainly micro-mammals, reptiles, amphibians
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To differentiate between extensive livestock remains and landfill remains, cut bones or
bones with traces of butchery or cooking were assumed to have been obtained from landfills
], while the rest of the livestock remains were assumed to have been found as extensive
livestock carcasses in the field. We believe this assumption is plausible, first, because due to the
strict legislation and controls of intensive farms existing in Central Catalonia, it is very unlikely
to find carcasses of intensive livestock abandoned in the field or near farms. Second, because
despite there are some supplementary feeding points in our study area, most of them were no
longer active during our study period. Also, the few that were active were mostly intended to
the Bearded Vulture, so the remains that were provided were goat and sheep limbs. This type
of remains are difficult to carry to the nests by Egyptian vultures as they use the beak and,
consequently, the size of remains is not the optimal for this species. In addition, the period when
remains were provided in these feeding points for the Bearded Vulture was from November to
April or beginning of May (Catalan Government data, personal communication). In our study
population chicks are born at the end of May or June, so it is very unlikely that these remains
could be found in sampled nests. In the case of remains of Oryctolagus cuniculus and Sus scrofa
we classified them as landfill origin when they had signs of butchery, while the rest were
considered wild individuals. Regarding hair remains, white rabbits were considered domestic as
there are not white rabbits in the wild and hair samples of brown rabbits were classified using
a collection that allowed us to identify their origin according to their length (wild rabbits
present longer hair than domestic). In the case of pigs, in our study area there is not black Iberian
pig in farms so we assumed that black hair was from wild boar. When we had doubts about its
origin, we classified it as undetermined.
Stable isotope analysis
Nestling feathers were first cleaned in a solution of NaOH (0.25 M) and oven-dried at 40ÊC for
24h. Feathers were then ground into a fine powder and subsamples of 0.35 mg were loaded
into tin receptacles for combustion. Isotopic measurements were performed at the Scientific
and Technological Centres of the University of Barcelona using the methods described in [
Stable isotope ratios are reported as δ values and expressed in ½, according to the following
equation: δ X = [(Rsample/Rstandard) − 1] X 1000, where X is 13C or 15N and R is the
corresponding ratio 13C/12C or 15N/14N. Rstandard is the ratio of the international standards: Pee
Dee Belemnite (PDB) for 13C and atmospheric nitrogen (AIR) for 15N. Measurement
precisions for δ 13C and δ 15N were 0.15 ½ and 0.25½.
Isotope analysis can differentiate sources of food given that organisms reliant on plants that
use a C4 photosynthesis pathway have higher (more enriched) stable carbon isotope ratios
than those from food webs reliant on plants that use a C3 pathway [
]. This relationship is
useful in studies of livestock species since it can differentiate between cattle raised on intensive
farms that are fed with C4 plants (mainly corn) and cattle raised extensively feeding on
]. In our study area (Mediterranean and pre-Pyrenean mountains), the species that
live in extensive are those aimed to meat production and, as the climate allows it, they graze in
pastures during all the year without any supplement based in corn (personal communication
from farmers of our study area). More, most plant species that cover pasture lands
(Brachypodium, Festuca, Lolium, Agropyron, Koeleria, Oryzopsis, Molinia, Anthoxanthum, Holcus,
Dactylis) use a C3 pathway [
] so we assumed that any C4-type isotopic signature (reflected in
more enriched stable carbon isotope ratios) detected in Egyptian Vulture tissues will be due to
food obtained from the landfills where humans dispose of waste from cattle bred intensively.
The isotopic signatures of prey items were obtained from the bibliography, considering
main prey determined by conventional analysis and collecting values of our study area or from
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For each source, it is indicated the mean of δ 13C and δ 15N and the standard deviation. The type of tissue sampled, the area and bibliographic reference of the study of
data collection are also specified.
other regions with similar climatic conditions when possible. Landfill isotopic values were
obtained from isotopic analysis of regurgitates composed of meat (mainly chicken and pork)
of Larus michaellis feeding on a landfill inside our study area where Egyptian Vultures have
been observed foraging [
]. These values were coherent with isotopic values of livestock
animals raised in intensive and fed with corn in other areas [55±57]. Extensive livestock
isotopic values were obtained from beef and lamb fed with C3 plants [
]; the wild herbivores
analysed were Oryctolagus cuniculus [
] and Sus scrofa [
]. To account for the diet fraction
containing carnivores, isotopic signatures of Vulpes vulpes were used [
]. Finally, the isotopic
signatures of Columba palumbus and other passerines (Corvidae, Sturnidae and Turdidae)
were taken from [
]. To obtain the isotopic value of categories that included more than one
prey item (livestock, wild herbivores and birds), the mean and variances of 13δC and 15δN
were estimated by assuming stratified random sampling and giving equal weight to each prey
type in a given category [
] (Table 1). The mixing model considered that all food sources
were taken into account, which means that there was no `others' category.
The Trophic Enrichment Factor (TEF) is necessary to perform stable isotope analysis, as
the isotope ratios of a consumer tissue are usually different from their diet due to biochemical
processes when proteins are digested, absorbed, and then re-arranged for new tissue synthesis.
This is the first-ever analysis of the diet of Egyptian Vultures using isotope analysis and also
the first of any vulture from the Accipitridae family. Thus, there are no reference values
available in the literature since Egyptian Vultures consume different resources or are taxonomically
distant from other species. To solve this shortcoming, we used the SIDER package from R
software, which estimates the TEF of species with unknown enrichment factors by taking into
account phylogeny, tissue type sampled, general type diet (in this case we treated our study
species as a carnivore), isotopic signature of the food source and measurement error [
perform the SIDER model, the values of δ 13C and 15 δ N for the food sources obtained from
the literature (see above) were used. TEF values obtained with SIDER model and used in
mixing models were 1.11 ½ ±1.12 for δ 13C and 3.33 ½ ±1.18 for δ 15N.
Once we had calculated the isotopic mean values and the standard deviations of the
nestlings' feathers, prey sources and TEF, we performed Bayesian isotopic mixing models using
the SIMMR package from R software [
] to estimate the relative contribution of each prey
type to the diet of the Egyptian Vulture. Models were built over four Markov chains with
10000 steps per chain with a burn in of 1000 iterations. Each nest and year was considered as a
single statistical observation by estimating the mean isotopic values of sampled siblings.
To assess the agreement between the conventional and stable isotope analyses we performed
three different test. First, the weighted Kappa statistic (Kw) was used to compare methods on
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an ordinal scale by ranking prey categories from higher to lower levels of consumption.
Secondly, an intra-class correlation coefficient (ICC) was used to test for the agreement of the two
methods in the quantitative prey consumption estimates. We performed a third analysis that
took into account uncertainty of prey estimations obtained with mixing models. We used
simulated values obtained with mixing models (selecting 1000 random vales for each nest) and
calculated the difference between each simulated value and the proportion of each prey obtained
with conventional methods for each territory and year. After, we calculated the proportion of
these difference values that were below 0 in order to study the distribution of the probability
that the estimates of prey consumption by mixing models would be greater than the estimates
by conventional methods. We assumed that if there were no differences between methods, the
distribution of this probability for each prey category would be symmetrical and would have
median values close to 0.5. As much as the distribution would be skewed towards 1 or 0 the
methods would be providing different estimates of consumption for the food resource in
question. Also, we reported mean differences between methods by subtracting the consumption
estimates obtained with conventional analyses from the predicted consumption obtained with
the SIA [
]. Only data from 2012±2014 was used to perform the comparisons as there was no
information for the conventional analysis from 2015.
Finally, as δ 13C on animal tissues reflects the consumption of C3 or C4 plants and thus if
they had fed on intensive (ie landfills) or extensive livestock [
], we assessed whether isotopic
values of δ 13C of Egyptian vulture feathers were correlated with the availability of food and the
level of humanization in our study area. We performed Generalized Linear Mixed Models
(GLMM) to account for the potential non-independence of clustered observations from the
same territories and years. We performed four different models, all of them with δ 13C as a
response variable. The first two models considered food availability as explanatory variables,
using distance to landfill as a proxy of the access of vultures to landfills resources (Model 1)
and the number of cattle in extensive farming present in the territory (taken from the
municipal census) and potentially available to vultures (Model 2). The other two models used urban
surface (Model 3) and forest surface (Model 4) with an 8-km-radius buffer area around the
nest also as explanatory variables and as a proxy of the level of humanization of territories
(Table 2). We chose these variables as they can simply describe if territories are more urban or
rural and so, they could provide information of the typology and distribution of food in the
environment. For more details of the explanatory variables, see [
]. Territory and year were
included in all four models as random factors.
All models were compared with the null model that did not consider the explanatory
variable using a Likelihood Ratio Test and we report the level of significance but also AICc
of each model. GLMM were performed with the lmer function from the lme4 package of
There are indicated parameter estimates and standard errors (SE), AICc of each model, AICc of null model and also P Value obtained with the ANOVA performed
between null and full model.
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Using conventional analyses we identified 1483 prey items corresponding to at least 62
different species. According to this methodology, the diet of the study population was composed of
(mean ± SD) 30.46% (± 18.96) livestock, 19.85% (± 11.09) wild herbivores, 18.12% (± 11.64)
resources from landfills, 17.45% (± 10.70) birds, 9.33% (± 13.71) carnivores and 4.79% (± 3.66)
others. According to stable isotope mixing models the diet was composed of 15.52% (± 5.86)
livestock, 15.82% (± 11.87) wild herbivores, 26.04% (± 5.03) resources from landfills, 19.79%
(± 3.52) birds and 22.84% (± 11.88) carnivores (Fig 2). Isotope mixing models for diet
The arithmetic mean isotopic values (±SD) for 60 Egyptian Vulture nestlings were -21.97
(± 1.088) for δ13C and 10.08 (± 1.653) for δ15N. Isotope biplots (δ13C, δ15N) showed that
Egyptian Vulture nestlings lay within the space delineated by the main prey categories previously
corrected by TEFs (Fig 3).
Individual estimates for diet according to isotope mixing models predicted a high degree of
individual variability in regard to resources derived from landfills (Fig 4). Although there was
a large degree of overlap in confidence intervals, the results indicate that a number of breeding
pairs obtain an important proportion of their diet from landfills and that these pairs are likely
to consume fewer livestock resources. Livestock in general is represented in lower proportions
than landfills but still plays a significant role, especially in territories where the consumption of
prey from landfills is lower (S1 Fig).
Comparison of conventional methods and isotope mixing models
When ordering prey categories from higher to lower levels of consumption, the Kappa
test found good agreement for `landfills' (Kw = 0.564, P<0.001) and `birds' (Kw = 0.255,
P = 0.0129) but disagreement for the rest of prey sources. The ICC test only found agreement
Fig 2. Proportion of diet of Egyptian Vulture represented by main prey categories obtained by stable isotope analysis and conventional identification of food
remains. Medians, quartiles and outliers of each prey category are represented in the boxplot.
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Fig 3. Isotopic values (δ 13C and δ 15N) of Egyptian Vulture nestlings in Catalonia (n = 60) and main sources of food (mean ± SD). Same symbols correspond to
the isotopic values of nestlings from the same territory but different years.
between prey remains counts and SIAR estimates for landfills (ICC = 0.565, P<0.001) and
no significant agreement between methods for all other categories. According to the test that
incorporated uncertainties of mixing models, we obtained symmetrical distribution of
probabilities of differences below 0 and median values close to 0.5 for landfills (0.65) and birds
(0.48) categories, but asymmetrical distribution of probabilities and median values far from
0.5 for livestock (0.07), wild herbivores (0.21) and carnivores (0.89) (Fig 5a). This results
Fig 4. Percentage of the contribution of landfills in the diet of Egyptian Vulture nestlings estimated with Bayesian mixing
models (SIMMR). Data of each territorial pair but different year is represented with the same number. Boxes represent the
credible interval of 50% and error bars the credible interval of 95% obtained with SIMMR.
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Fig 5. Results of comparison of conventional methods and isotope analysis for the diet of Egyptian Vulture in
Catalonia during the period 2012±2014. (a) Probability of difference below 0 between stable isotope analysis and
conventional analysis for main prey of Egyptian Vulture. (b) Mean differences between stable isotope analysis and
conventional methods in estimates of the main prey of Egyptian Vulture. Standard errors of the differences are
supported findings obtained with Kw and ICC test, showing that landfills and birds were the
less biased categories and livestock, wild herbivores and carnivores presented more
disagreement between methods. Finally, regarding the mean differences obtained for each prey using
the different methods (Fig 5b) in accordance with all performed tests, there were fewest
differences for the categories `landfill' and `bird', followed by the `wild herbivores'. Most
disagreement was observed in the categories `livestock' and `carnivores'.
δ 13C, food availability and level of humanization
According to the GLMM analyses, there was no significant relationship between availability of
food expressed as distance to landfill and extensive livestock availability and δ 13C of feathers
of Egyptian Vulture chicks (Model 1 P = 0.0928 and Model 2 P = 0.3308). However, our results
revealed a positive relationship between δ 13C and the level of humanization reported with
urban surfaces (Model 3, P = 0.0009) being most urbanized areas those with higher levels of
δ 13C in Egyptian vulture feathers. Consequently, δ 13C was negative correlated with rural
areas reported with forest surface (Model 4, P = 0.0002) (Table 2).
In this study we assessed the role played by landfills in the diet of Egyptian Vultures, as well as
the usefulness of SIA as a tool for determining vulture diets. Both conventional analysis of
food remains and SIA are the most easily applicable methods to quantify diet composition of
vultures, though both of them are known to show some limitations [
]. So far, no
comparisons have ever been made between isotope and conventional analyses of a scavenger
species at the top of the food web. Here, we assumed that the agreement between methods for
certain food resources consumption would support the idea that the methods are reliably
approximating to the real consumption of those resources and, in this sense, we found that
both types of analyses provide similar contributions of food from landfills in this species'
overall diet. Importantly, our results also suggest that landfills are a major food resource for the
Egyptian Vulture, thus highlighting that human waste, besides its strong impact on
populations of generalist abundant species, may be also of great importance for populations of
endangered species able to exploit this type of resources.
10 / 18
Comparison of conventional methods and SIA
By comparing the most easily applicable methods to describe the diet of scavenger bird species
we provide valuable information to understand its potential biases and constraints, an issue
particularly concerning in our study group for which most available diet assessments where
done by conventional diet analysis [
]. We grouped food resources into five categories
and we considered three approaches to evaluate the agreement between methods. Our results
revealed that the two methods showed good agreement for two of the considered categories:
`landfills' and `birds'. Consequently, we suggest that that isotope analysis can be used as an
approximation of the consumption of food from landfills in vultures, a question of major
interest in our study. In contrast, the categories that were least in agreement were `livestock',
`carnivores' and `wild herbivores'. It is likely that conventional methods overestimate
`livestock' importance since cattle bones are usually large and easy-to-identify and remain in the
nest for long periods of time . In the case of wild herbivores, despite there is less
disagreement between methods, it is possible that there were also overestimated by conventional
methods due to the easy identification and long permanence of bones in the nest. On the other
hand, it is possible that carnivores contribute substantially to the diet of Egyptian vulture as
they are frequent roadkill [
]. However, these high levels of N detected in some chicks
could be due to the use of fertilizers or animal waste products that can increase the levels of
N in the environment and so arise N levels in Egyptian Vulture's prey . In spite of the
observed mismatches between methods, uncertainty in parameters estimates provided by SIA
were quite wide and, consequently, the percentages of diet provided by conventional methods
fell within the credible intervals provided by SIA for all categories (see Fig 5).
Thus, our results show that both methods present limitations to describe de diet of the
Egyptian Vulture mainly due to its wide range of prey consumed from different sources [
Nevertheless, the combination of both methods seems to provide complementary results. On
the one hand conventional methods allow the identification of prey at a high taxonomic
accuracy and this information of prey consumed is necessary to be incorporated in SIA. However,
the relationship between the type and amount of a given prey that is consumed and the type
and amount of a given prey that is quantified from nest remains has not been yet performed,
meaning that the magnitude and direction of potential biases in conventional analysis for
vulture species is unknown yet.
On the other hand, and as is usual in stable isotope studies of animal diets, our mixing
models were based on several assumptions. First, we used bibliographical isotopic values of prey
sources. A major constraint in isotopic analysis is to obtain representative samples of
consumed prey at the time that the food resource is used, since spatial and temporal variation in
isotopic values have been detected for some species [
]. Our study species shows a very
wide dietary range and we studied it for a fairly large area and time period, so it would have
been impossible to sample all potential food sources. Therefore, and according to our aims, we
considered the five main sources represented by dominant species according to conventional
analysis and selected those values in the literature with the highest geographic, ecological and
climatic similarities. In addition, the isotopic values of food resources obtained from other
areas corresponded to species or prey types that show low variation in isotopic values across
large geographic ranges [68±71], which suggest that the effect of having chosen those values
was small in our main results. Second, we estimated TEF values using SIDER models as we
had no values of a species phylogenetically close and ecologically similar to the one we are
interested in. As a result, uncertainty of TEF values was high. However, it is more correct to
use TEF values with large uncertainty than use values from other species with less uncertainty
but probably wrong . Overall, any potential biases in our stable isotope models were
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incorporated as much as possible and, furthermore the models provide us the level of
uncertainty in parameter estimates. In this sense, mixing models provided large credible intervals
around dietary proportions that should be taken into account when interpreting results.
Despite the difficulty of describing the diet of vultures, it is showed that SIA can help to
distinguish between animals that rely on waste and so present enriched levels of C than those that
feed on the countryside. However it is worth to mention that in our study area although there
are few supplementary feeding points, most of them are intended to Bearded Vulture so its
influence to Egyptian Vulture population is predicted to be low [43, Catalan Government data,
personal communication]. In this sense, in other areas where supplementary feeding points
are intended to Egyptian Vulture where the remains provided could come from intensive
farms, it would be difficult to distinguish between landfill and feeding points origin. Therefore,
our study provides an initial step towards further research on vultures' diet that should be
refined in the future to disentangle meaningful ecological and conservation questions.
Contribution of landfills to Egyptian Vulture diet
Diet studies have been described as powerful tools for monitoring food resources obtained by
raptors on large spatio-temporal scales since they are able to detect changes in resource
availability caused by new environmental scenarios [
]. Thus, our results are consistent with
these studies of other species as our diet analysis shows that an important part of the Egyptian
Vulture's diet is obtained from landfills, a novel food resource that has recently become
available in the environment. The diet of Egyptian Vulture has been described to be very variable
between different areas. In our case, pairs that feed most on landfills could obtain nearly a 50%
of their diet from these sources, but those pairs that consume most livestock this resources
represents less than 25% of their whole diet (S1 Fig). Despite the contribution of landfills to diet of
this species has been little quantified because most of the studies have focused on taxonomic
groups and not on the origin of these prey [
], it is known that waste can be important to
the diet of Egyptian Vulture in some areas [
]. In addition, the proportion of livestock in our
population is less than the livestock consumption described in other areas where cattle has
been defined as one of the main sources of food in the Egyptian Vulture [
], however it
is not the only exception described [
]. This result is relevant to conservation as vultures
are among the most endangered groups of birds at a global scale and that one of their major
threats comes from the type of food resources that they consume [
]. The main cause of
their decline is poisoning due to the consumption of dead animals that contain high levels of
pesticides or veterinary drugs such as diclofenac, which is causing a sharp reduction in vulture
populations throughout the world, especially in Gyps genus [
]. Despite it is a first
approximation, our results are important as they show that isotope analysis can provide
information about the use of livestock resources and the potential change to consuming novel
resources derived from landfills, which therefore allows us to assess the new threats to which
vultures are currently exposed.
Feeding on landfills can have several conservation implications at both individual and
population levels. At individual level, abundant and predictable food supplies should improve the
body condition and breeding performance of the individuals that feed at these sites ([
references therein). In this sense, it is worth mentioning that we only sampled chicks of
successful breeding pairs, so it could be possible that pairs that feed on landfills might be those
with higher breeding success. Further analysis of diet of pairs that fail would be necessary to
study if feeding of landfills could favour reproduction in our area. However, landfills can also
act as a threat as they represent a source of food that potentially contains pollutants and
poisons [76±79]. Moreover, the presence of the species in highly humanized areas where landfills
12 / 18
are found could also entail an increase in the risk of fatal casualties due to collisions with
power lines and other infrastructures [
]. Many of these threats are emerging as major causes
of mortality in large avian scavengers and may turn out to be catastrophic if they operate in
tandem with other threats such as poisoning [
]. At a population level, landfills can
promote the settlement of individuals attracted by the availability of food  and improve
survival rates and recruitment [
], but may also reduce fecundity [
]. Nevertheless, the great
predictability and availability of food associated with landfills has also led to rapid population
increase of some species and several studies have focused on the ecological and social
consequences of this overabundance [
Moreover, results of GLMM analysis pointed out that the level of δ 13C is linked to the level
of humanization of territories. High levels of δ 13C are associated with intensive livestock and
so landfill consumption [
], therefore territories that present more δ 13C are more likely to
be located in humanized areas. However, we didn't find any relation with δ 13C and our
measurement of food availability, estimated as distance to landfill and number of cattle present in
the territory, possibly due to the limitations of having an accurate metric for food availability.
Obtaining this metrics is particularly challenging in our study species, since breeding pairs of
Egyptian Vultures make long daily movements to feed at predictable feeding sources located
over 100 km away  and so, despite having territories located far from landfills, these pairs
could be feeding on these installations.
In our study case, feeding on landfills in addition to the reduced mortality of individuals
from our study area [
] could have prompted the Egyptian Vulture population increase that
has taken place over the last 30 years in Catalonia. This is a paradoxical situation in which an
endangered species has been favoured by human waste when, typically, modern human
activities are in fact detrimental to biodiversity. Similar processes could be occurring in other
threatened species but could hitherto have been overlooked. However, a new scenario is expected to
occur when landfills are closed in compliance with European legislation [
], together with a
resolution by the European Parliament (European Parliament resolution of 9 July 2015 on
resource efficiency: moving towards a circular economy), urges the European Commission to
reduce levels of residual waste to close to zero by 2020. In this context it is thus expected that
the amount of waste available to vultures in coming years will fall and the effect of a landfill
closure to vulture populations has been little studied [
]. Thus, this study provides a first
approximation of a new tool that should be refined in order to monitor the potential diet
change caused by this new resource scenario that will help design conservation measures for
endangered vultures in the future.
S1 Fig. Percentage of the contribution of livestock in the diet of Egyptian Vulture nestlings
estimated with Bayesian mixing models (SIMMR). Data of each territorial pair but different
year is represented with the same number.
S1 Table. Mean ± SD ½ values of δ13C and δ15N obtained for the Egyptian Vulture nest
lings included in Bayesian mixing models. It is specified the number of territory and the
number of years that each territory was sampled (n).
S2 Table. Contribution of different food components to the diet of Egyptian Vulture nestlings obtained with conventional diet analysis.
13 / 18
We would like to thank the Grup de Suport de Muntanya belonging to the Cos d'Agents Rurals
(Departament d'Agricultura, Generalitat de Catalunya) for help with the fieldwork and for
climbing the nest-cliffs. We are also grateful for the support received from the Servei de
Biodiversitat de la Generalitat de Catalunya, the natural parks of Sant LlorencË del Munt and
Muntanya de Montserrat (DiputacioÂ de Barcelona) and La Garrotxa Volcanic Zone Natural Park
(Generalitat de Catalunya). We would also like to thank F. Fontelles for helping with the
conventional diet analysis and the Barcelona Museum of Natural Sciences for allowing us to use
their reference collections. Thanks are also due to I. PeragoÂn and the Scientific and
Technological Centres of the University of Barcelona for the Stable Isotope Analysis and to J. Resano for
his help with the statistical analysis. We are also grateful to Mercedes Gil from Red EleÂctrica de
España SA and the support received from Grup de Naturalistes d'Osona-ICHN (J. Baucells, C.
Martorell). Finally, we are grateful to J. A. DonaÂzar, S. Oppel, two anonymous reviewers and
editor A. Margalida for their help in improving the manuscript. All applicable institutional
and/or national guidelines for the care and use of animals were followed. We are also grateful
to Mercedes Gil from Red EleÂctrica de España SA.
Conceptualization: Helena Tauler-Ametller, Antonio HernaÂndez-MatÂõas, Joan Ll. Pretus,
Formal analysis: Helena Tauler-Ametller, Antonio HernaÂndez-MatÂõas.
Funding acquisition: Joan Real.
Investigation: Helena Tauler-Ametller, Antonio HernaÂndez-MatÂõas, Francesc PareÂs, Joan
Methodology: Helena Tauler-Ametller, Antonio HernaÂndez-MatÂõas, Joan Ll. Pretus, Joan
Project administration: Joan Real.
Resources: Francesc PareÂs.
Supervision: Antonio HernaÂndez-MatÂõas, Joan Ll. Pretus, Joan Real.
Writing ± original draft: Helena Tauler-Ametller.
Writing ± review & editing: Helena Tauler-Ametller, Antonio HernaÂndez-MatÂõas, Joan Ll.
Pretus, Joan Real.
14 / 18
15 / 18
16 / 18
17 / 18
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