# The dead can talk: Investigating trace element and organic pollutant exposure in mammalian roadkill under contrasting habitats

Noëlie Molbert, Fabrice Alliot, Aurélie Goutte, Maria Hansson

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Noëlie Molbert, Fabrice Alliot, Aurélie Goutte, Maria Hansson. The dead can talk: Investigating trace element and organic pollutant exposure in mammalian roadkill under contrasting habitats. Environmental Pollution, 2025, 367, pp.125648. 10.1016/j.envpol.2025.125648. hal-05086165

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# The dead can talk: Investigating trace element and organic pollutant exposure in mammalian roadkill under contrasting habitats☆

Noëlie Molbert<sup>a,*</sup> <sup>ID</sup>, Fabrice Alliot<sup>b,c</sup>, Aurélie Goutte<sup>b,c</sup>, Maria C. Hansson<sup>a ID</sup>

## ARTICLE INFO

**Keywords:**

Terrestrial ecosystems

Mammals

Hedgehogs

Phthalates

PCB

Essential and non-essential elements

## ABSTRACT

Urban environments are exposed to a substantial range of anthropic pressures, including chemical exposure. While trace metals and legacy pollutants have been well documented, the extent of wildlife exposure to emerging contaminants has received little attention, in terrestrial mammals. Concentrations of trace elements (Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Pb and Zn) and 48 organic pollutants (Polychlorinated Biphenyls: PCBs, Organochlorine Pesticides: OCPs, Polycyclic Aromatic Hydrocarbons: PAHs, phthalates and pyrethroid pesticides) were measured in tissues of European hedgehogs (*Erinaceus europaeus*) in southern Sweden. Road kills and carcasses collected in 2021 and 2022 by citizen science participants were used to characterize the level, tissue distribution and composition of mixtures in liver, spines and teeth samples. Phthalate diesters and PCBs were the most prevalent compounds in liver samples, with mean concentrations (±SD) of 1090 ± 681 and 284 ± 231 ng g⁻¹ of dry weight, respectively. Bioaccumulation patterns were observed for some organochlorine compounds but were hampered by the small sample size. Trace element concentrations and tissue distribution were strikingly equivalent between tissue type and age-classes (juveniles and adults) of hedgehogs, except for a handful of elements (e.g., Pb, Al, As), but contamination profiles differed between habitat types. While no adverse health effects are expected to occur from the body burdens measured for most of the target analytes, concerns might be expressed for the exposure to PCBs, phthalates and some non-essential trace metals (e.g., lead >15 µg g⁻¹ dw), which were found at levels above or approximating toxic thresholds in mammal tissues. The present study demonstrates the suitability of road kills as an alternative approach for environmental monitoring and the relevance of nondestructive biomonitoring to evaluate endogenous exposure in hedgehogs and possibly other terrestrial mammals and humans.

## 1. Introduction

Urban green spaces attract many species of wildlife with easier access to food, water or shelters that a surrounding fragmented or intensively farmed landscape around towns may no longer provide. However, wildlife faces the challenges of urbanization, forcing free-living organisms to cope with different environmental conditions than their counterparts in non-urban areas. Several pressing environmental factors, including widespread exposure to environmental pollutants, can have significant negative impacts on animal physiology, behavior, and health (Sumasgutner et al., 2023). Yet, environmental monitoring studies in terrestrial habitats highly influenced by anthropogenic factors have in

the past primarily been conducted using measurements of trace metals and legacy contaminants (e.g., Polychlorinated Biphenyls, Organochlorine Pesticides) in invertebrates and birds as ecological detectors (Chace & Walsh, 2006; Pollack et al., 2017). Of growing interest, wild urban mammals living in close vicinity to humans' dwellings could provide the environmental footprint and exposure effects derived from urban pollution overall (Dip et al., 2003; Rautio et al., 2010; Arıkan et al., 2018) and reasonably inform on human exposure to urban pollutants (Pollack et al., 2017). Since environmental monitoring is typically performed on tissues or fluids of sacrificed individuals, which represents an unsustainable option for endangered species or threatened populations, there is a need for non-lethal and non-invasive methods that impose

<footnote><sup>a</sup> Centre for Environmental and Climate Science, Lund University, Sweden</footnote>

<footnote><sup>b</sup> Sorbonne University, CNRS, EPHE, UMR METIS, F-75005, Paris, France</footnote>

<footnote><sup>c</sup> École Pratique des Hautes Etudes, PSL Research University, Paris, France</footnote>

<footnote>* This paper has been recommended for acceptance by Charles Wong.</footnote>

<footnote>* Corresponding author.
E-mail address: noelie.molbert@uconn.edu (N. Molbert).</footnote>
minimal stress on individuals and natural populations, while still
allowing investigations of pollution and its effects on species (D'Havé
et al., 2005a,b; D'Havé et al., 2006; D'Havé et al., 2009; Rasmussen
et al., 2019).

Human infrastructures, including our networks of roads, hinder
wildlife movements and directly cause mortality of many species every
day (Bischof et al., 2016). Around 29 million mammals are likely killed
on the European roads every year (Grilo et al., 2020) and small mam-
mals that come out at night face the biggest threat from collisions.
Indeed, roadkill events are so frequent and widespread across regions
and landscapes that they offer unique and underutilized resource for
biological and ecological studies, in particular for rare and/or protected
species where destructive sampling is ethically unsuitable (Schwartz
et al., 2020). The use of citizen science participants as a sampling
method (e.g., by announcements through the press and social media)
makes it possible to investigate large areas and to a higher degree
determine when, where and what kind of animal species is killed.
Postmortem tissue samples of road killed animals therefore represent
invaluable non-invasive sources for chemical analyses to map contam-
inant exposure in the environment (Rautio et al., 2010; Arıkan et al.,
2018), while also alleviating concerns over the unethical trapping and
sedation of live animals. For instance, long-term roadkill carcasses
collection evidenced the declining levels of lead over time in European
otter (Lutra lutra) populations following regulations (Chadwick et al.,
2011) and alerted on the toxicity of secondary exposure to second
generation anticoagulant rodenticides on Tawny owl (Strix aluco;
Walker et al., 2008). Recognized as valuable "sentinels" for environ-
mental monitoring in terrestrial and aquatic ecosystems, wildlife car-
casses can advance our understanding of contaminant dispersal and
bioavailability, as well as inform about the effectiveness of risk miti-
gation measures (Schwartz et al., 2020; Coba-Males et al., 2023).

The main objective of the present study was to evaluate if road killed
animals could be a useful resource for determining incidence of various
persistent and emerging contaminants in terrestrial mammalian wildlife
living under contrasting habitats. We also aimed to assess if the identi-
fied levels of pollutants found in dead animals could provide informa-
tion about chemical risk factors for wildlife in urban environments. We
implemented a non-lethal biological monitoring of environmental
pollution in terrestrial ecosystems by investigating European hedgehog
(Erinaceus europaeus) carcasses to determine (i) contamination levels,
tissue-specific distribution and composition of exposure mixture as well
as (ii) assess whether urban mammal species are more at risks than their
rural counterparts. Hedgehogs may represent effective organisms for
environmental monitoring and assessment due to their biological and
ecological features. This small mammal is widely diffused, readily
available for sampling and encountered in a range of habitats, including
territories intensely cultivated and subjected to strong anthropogenic
interference. Given their life expectancy in the wild and their relatively
small-spaced home range, dead hedgehogs can be turned into a relevant
"tool" to expand our understanding of long-term effects of local pollu-
tion on wildlife, and humans. In Europe, wild populations of hedgehogs
have shrunk dramatically in recent years (Williams et al., 2018). The
underlying reasons for their decline mainly involved direct and/or in-
direct effects of chemical exposures (e.g., poisoning; The State of Brit-
ain's Hedgehogs, 2018), red uction of invertebrate prey availability and
habitat fragmentation, or a combination of all factors. Therefore, more
insights into the potential spread and bioaccumulation of anthropogenic
contaminants within the ecosystem, as well as their adverse biological
effects are essential to identify and formulate effective conservation and
management measures to promote biodiversity.

## 2. Materials and methods

### 2.1. *Sample collections*

European hedgehogs were collected with the help of citizen science

participants and local rescue centers in southern Sweden during sum-
mers of 2021 and 2022. The general public were informed by newspaper
articles and social media about the research project at Lund University.
Biological samples were collected from both road killed animals (N =
24) and individuals that had starved to death or died for unknown
reason within the same day (N = 27) (Fig. S1; Table S1). They were
placed into sealed zip-lock bags free of phthalates and then immediately
brought back to the lab where they were stored at -80 °C until further
analyses. Hedgehogs were divided into four groups based on their body
size (juvenile or adult) and habitat (rural or urban). Sampling localities
consisted of urban habitats located within medium-to large-sized towns
with >100,000 inhabitants (primarily in and around Lund, with some
from Malmö and Helsingborg), where managed parks and private gar-
dens or allotments as the only green spaces available. Rural-forest
habitats were characterized by crops and natural or semi-natural for-
ests, with only a few isolated houses, to contrast with urban sites.
Selected urban and rural habitats were sufficiently distant (i.e., a dis-
tance above the recorded dispersal distance of this species, >10 km) to
avoid occasional roamers from the surrounding areas (Doncaster et al.,
2008).

Spines (N = 51) and teeth (N = 26, lower incisors and molars) were
used to assess essential and non-essential element exposure. Differences
in sampling size among biological matrices were related to the avail-
ability of fresh and intact carcasses, with spines and teeth the most
abundant tissues on roadkill to carry out chemical analyses. Organic
contaminants were quantified in the liver (N = 12), as the primary organ
of bioaccumulation. When achievable, individuals were measured
(±0.5 cm), and sex was determined by visual discrimination. Spines,
teeth and the liver were collected postmortem from carcasses using
stainless steel instruments, strictly avoiding cross-contamination. Sam-
ples were stored in metal-free and polycarbonate tubes, and frozen at
-20 °C until subsequent analyses.

### 2.2. *Chemical analyses*

Samples were lyophilized (Alpha1-4LD plus; Martin Christ) for 48h
and grounded with liquid nitrogen to obtain a fine powder.

**Metal analyses** – To remove external contamination, spines and teeth were washed with acetone and rinsed three times with Milli-Q® water (Millipore, Bedford, MA, USA). On average, 0.1–0.3 g of dried spines and teeth samples were weighted on the precision scale and transferred to digestion tubes (Teflon vessels CEM). Spines and teeth were digested at room temperature overnight by adding 7 mL of HNO₃ (ARISTAR, VWR) and 2.8 mL of H₂O₂ (30%). Digestion was completed by heating the samples in a microwave oven (CEM Mars 6) with the following method for both spines and teeth samples: 20 min ramp time to reach 200 °C and maintained at this temperature for 15 min before cooling. Once diges-
ted, the detection and quantification of silver (Ag), aluminum (Al),
arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu),
iron (Fe), magnesium (Mn), lead (Pb), and zinc (Zn) were performed by
inductively coupled plasma-mass spectrometry (ICP-MS; Aurora Elite,
Bruker) in Geochemistry laboratory of Geology Department, Lund Uni-
versity. The limit of detections (LOD) of all trace elements are presented
in the supplementary material (Table S2) and ranged from 0.001 µg g⁻¹
(Mn) to 0.06 µg g⁻¹ (Cu). Final concentrations in spines and teeth are
expressed as µg g⁻¹ of dry weight. All samples were run in batches that
included blanks containing acids only, to detect possible contamination
during the sample preparation.

**Organic micropollutant analyses** – A total of 48 organic pollutants, including 16 polycyclic aromatic hydrocarbons (PAHs; naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd] pyrene, dibenzo[a,h]anthracene, benzo[ghi]perylene), 7 phthalate esters (dimethyl phthalate [DMP], diethyl phthalate [DEP], n-butyl benzyl phthalate [BBP], di-n-butyl phthalate [DnBP], di-iso-butyl phthalate
[DiBP], di-2-ethylhexyl phthalate [DEHP], di-n-octyl phthalate [DnOP]), 7 pyrethroids (bifenthrin, permethrin, phenothrin, cyfluthrin, cypermethrin, fenvalerate, deltamethrin), 4 organochlorine pesticides (OCPs; pentachlorobenzene [PeCB], hexachlorobenzene [HCB], lindane [γ-HCB], and dichloro-diphenyl-dichloroethylene [p,p'-DDE]), 7 polychlorinated biphenyls (PCBs; PCB-28, PCB-52, PCB-101, PCB-118, PCB-138, PCB-153, PCB-180), 6 polybrominated diphenyl ethers (PBDEs; BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154) and DEET (N, N-diethyl-meta-toluamide) were quantified in liver samples following a previously validated analytical method (Molbert et al., 2019).

Freeze-dried and powdered liver samples (0.1 g) were analyzed with a gas chromatograph (GC; Agilent 7890 A) coupled to a 7000 B triple quadrupole mass spectrometer (MS/MS; Agilent Technologies). Target compounds in the gas phase were characterized using electron ionization (+70 eV), with the ionization source set at 250 °C. Molecules were separated on a Zebron SemiVolatile analytical column (30 m, 0.25 mm inner diameter × 0.25 µm film thickness; Phenomenex) connected to a Restek deactivated silica guard column (0.25 mm inner diameter). The oven temperature program was as follows: 70 °C for 4 min, then increased from 70 to 150 °C at 25 °C/min, 3 °C/min to 225 °C, and finally raised to 310 °C at 5 °C/min, where the temperature was held for 10 min. The MS/MS transfer line temperature was set at 250 °C. Peak detection and quantification were performed using multiple-reaction monitoring transitions for each analyte (see Molbert et al., 2019). Analyte concentrations below the limit of quantification (LOQ; Table S3) value were replaced by half of the LOQ. For each chemical family, concentrations are expressed as the sum of all congeners (i.e., ΣPCBs, ΣPAHs, ΣPhthalates, ΣOCPs, ΣPBDEs, ΣPyrethroids, ΣMetals) in ng g⁻¹ of dry weight (dw).

## 2.3. Statistical analyses

All statistical models were performed in R v.3.3.2 software (R Core Team, 2016) considering a statistical significance threshold of *p* < 0.05. For statistical purposes, concentrations below the LOQ were treated as half of the respective LOQ and nondetectable elements were treated as zero values (<LOD) to achieve the least biased estimate of reasonable maximum exposure. Tissue concentrations data, including organic pollutants and trace metals, were log-transformed to satisfy normality assumptions. Gender-dependent accumulation was not investigated since sex was only successfully determined for a subset of samples. We used the package “ggplot2” for data visualization (Wickham, 2016).

### 2.3.1. Correlations of element concentrations between and within tissues

**Organic contaminants** – Differences in pollutant burden (Σ) in the liver between age-classes were analyzed using *t*-test and *Mann Whitney U test*. Linear models (LM) were used to explore the relationship between contaminant burden and body length. Model assumptions were checked by examining residual plots. Since individuals with intact liver were only collected in urban areas, habitat was not included as an explanatory variable.

**Trace elements** – The differences in trace metal concentrations between tissue type (spines and teeth) and according to age-classes were assessed using the analysis of variance (ANOVA) followed by Tukey's post hoc tests for pairwise comparisons if significant differences were found. For non-normally distributed data, nonparametric tests were applied to investigate differences in element concentrations among groups (Kruskal–Wallis tests followed by Dunn's tests). Linear correlation analysis (Pearson correlation coefficient) and linear regression analyses on log₁₀-transformed data were performed to explore the correlation of trace element concentrations between tissues and to investigate the suitability of using spines and teeth to monitor environmental contaminants in terrestrial ecosystems and to predict trace element contents in metabolically inert biological matrices. Linear models were built with tissue type and age-classes as explanatory variables and an interaction term between age-classes and tissue type was added to investigate

differential accumulation pattern with age.

### 2.3.2. Contamination profile signature between habitats

The hypothesis that individuals from distinct habitats (rural and urban) would cluster in the space defined by trace element concentrations in spine and teeth samples was assessed using bionts following the multivariate ordination technique principal component analysis (PCA). In all cases, the trace element data was standardized prior to analysis and the PCA were performed using the built-in prcomp function and the ‘FactoMineR’ and ‘Factoextra’ packages for visualization (Lê et al., 2008; Kassambara & Mundt, 2020).

## 3. Results

The concentrations of organic compounds and trace elements in different European hedgehog tissues, together with the detection frequencies, are shown in supplementary materials (Table S2; Table S3 & Table S4). These included 31 adults and 20 juveniles. Out of 51 hedgehog carcasses collected by citizen science participants and local rescue centers, 48% were identified as road kills. The cause of death could not be determined for the remaining individuals. Sexing was successfully performed on a handful of individuals (N = 21), with 67% of males.

### 3.1. Organic pollutant

#### 3.1.1. Liver concentrations and distribution

A total of 48 organic compounds were quantified in the liver, but only 27 were detected above the limits of quantification (>LOQ; Table S3). High-molecular weight compounds were the primary PAHs in the liver samples, although at relatively low levels, with detection frequencies ranging from 16% (Indeno_123_c4 pyrene) to 83% (Pyrene). The remaining PAHs compounds were below the LOQ (naphthalene, acenaphthylene, acenaphthene, fluorene, benzo[a]pyrene). For pyrethroids, only juvenile liver samples contained bifenthrin and permethrin above detection limits. Similarly, detectable concentrations of OCPs and PBDEs were encountered only in a fraction of the animals. For instance, 25% and 16% of the hedgehogs were positive for p,p'-DDE and PBDE-47, which were the only OCP and PBDE compounds detected in the liver of hedgehogs. Phthalates and PCBs had the highest detection frequencies of all organic compounds. Among these, DNBP, DEHP, DIBP, PCB-118, PCB-138, PCB-153 and PCB-180 were systematically detected in all animals.

The profiles of the target organic contaminants in the liver of European hedgehogs are shown in Fig. 1, only for chemical families with more than two compounds detected above the LOQ (e.g., PAHs, phthalates and PCBs), and the total concentrations (Σ) of organic contaminants are given in Table S4. Contamination profiles were largely similar between adult and juvenile hedgehogs (Fig. 1A-C). Pyrene concentrations represented up to 50% of total PAH exposure, with concentrations ranging from 0.80 (the limit of detection) to 12.8 ng g⁻¹ dw. Notably, the carcinogenic PAHs (benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, and dibenz(a, h)anthracene) represented 37 % of total PAHs. For PCBs, congeners 153 and 180 were the most abundant, with concentrations ranging from 0.64 to 196 ng g⁻¹ dw and from 2.30 to 261 ng g⁻¹ dw, respectively. Although pyrethroids and OCPs were poorly detected in liver samples, both permethrin and p,p'-DDE reached high concentrations in liver samples up to 594 ng g⁻¹ dw and 31.9 ng g⁻¹ dw, respectively. DNBP was the most abundant compound of all organic contaminants measured in the liver, with concentrations ranging from 151 to 1506 ng g⁻¹ dw, followed by DEP, DIBP and DEHP.

#### 3.1.2. Inter- and intra-variability in liver concentrations

Hedgehogs from Southern Sweden were mainly exposed to phthalates (Σ7; 1090 ± 681 ng g⁻¹ dw; mean ± standard deviation) followed
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Contamination profiles in the liver of adult (N = 5) and juvenile (N = 7) hedgehogs collected in 2021 for (A) PAHs, (B) Phthalates and (C) PCBs. Compounds systematically below the LOQ are not represented.</figcaption></figure>

decreasingly by PCBs (Σ7, 284 ± 231 ng g⁻¹ dw), pyrethroids (Σ7, 113 ± 188 ng g⁻¹ dw) and PAHs (Σ16, 27.6 ± 14.1 ng g⁻¹ dw). When liver concentrations were further analyzed by separating hedgehogs according to their age class, PCB load in hedgehog liver significantly increased with body length (F(3,8) = 9.216, *p* = 0.002; Fig. 2C). In contrast to persistent pollutants, neither adults nor juveniles accumulated PAHs and phthalates in their liver (PAHs: F(3,8) = 3.954, *p* = 0.053; Phthalates: F(2,9) = 0.773, *p* > 0.05; Fig. 2A and B). Interestingly, a trend of reduced contaminant burden in older individuals was observed for PCBs. No such trend was observed for metabolizable compounds such as phthalates and PAHs. In fact, adults tended to be more contaminated than juveniles. In all of these cases, the overall difference between adult and juvenile hedgehogs appeared non-significant (−1.668 < *t* < 1.609; 0.138 < *p* < 0.218).

## 3.2. Trace element exposure

### 3.2.1. Trace element concentrations and distribution in teeth and spines

Measured concentrations (µg g⁻¹ dry weight) of essential (Cu, Cr, Co, Fe, Mn, Zn) and non-essential (Ag, Al, As, Cd, Pb) trace elements in spines and teeth of hedgehogs are presented in Table S2. Al, Cr, Fe, Mn and Zn were systematically reported in spines and teeth of both adult and juvenile hedgehogs, with Al, Fe and Zn the most abundant elements. Interestingly, Pb and Co were quantifiable in 100% of teeth samples against 88% in spines and accumulated preferentially in teeth at median concentrations around 1.5 and 6 times higher than in spines for Co and Pb, respectively. Although slight variations in chemical distribution occurred, element signatures – the relative proportions of each element – were overall strikingly similar between spines and teeth (Fig. S2). Element concentrations were significantly different between spines and teeth for some trace elements, but certainly not for all of them (Al, As, Co, Cu, Fe, Pb and Zn; Fig. 3A). Concentrations of Al, Co, Cu, Fe and Pb were the highest in spines (0.007 < *p* < 0.001), while Zn and As were predominant in teeth (*p* < 0.001 and *p* = 0.042, respectively). Details of element-specific variation between age-classes and tissue types (i.e., spines and teeth) are further presented in the supplementary materials (Table S2).

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Relationships between liver concentrations of (A) Phthalates, (B) PAHs and (C) PCBs (ng g⁻¹ dw) and body length (cm) in European hedgehogs (E. europaeus). Boxplots showing the distribution of PCB, phthalate and PAH concentrations (ng g⁻¹ dw) measured in the liver of adult (black) and juvenile (grey) hedgehogs. The box shows the interquartile range of the data (25th to 75th percentile) with the median value indicated with a thick black horizontal line. Black triangles represent mean values. Numbers below boxes represent sample size.</figcaption></figure>

The PCA ordination confirmed the dissimilarity in trace element patterns between biological matrices (Fig. 3B). Just under 50% of the total variation was accounted for by the first and second principal components collectively (PC1 and PC2, respectively). PC1 was characterized by Cd, Co, Cr and Pb, whereas Fe, Al, Mn and Cu mainly contributed to PC2. Elemental profiles in spines and teeth were distinct (PC1; $\chi^2$ = 22.36, *p* < 0.001), but only for adults despite the small overlap in multi-element concentrations (*p* = 0.002). Elemental composition in spines and teeth also separated out across PC2 ($\chi^2$ = 33.54, *p* < 0.001) between juveniles (*p* = 0.002) and adults (*p* = 0.009).

### 3.2.2. Predictability of trace element concentrations in spines and teeth

No significant relationships were observed between total trace
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** (A) Trace concentrations (µg g⁻¹ dw) in spines (grey bars) and teeth (green bars) of hedgehogs. Data are expressed as the mean ± SD. Asterisks indicate significant differences between tissues (*: *p* < 0.05; **: *p* < 0.0001). (B) PCA biplot showing trace element profiles between spines (green; N = 51) and teeth (grey; N = 26) collected from juvenile (filled diamonds) and adult (filled dots) hedgehogs (*E. europaeus*) in Sweden. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

element (Σ) concentrations in teeth and spines (LM: *F*_(1,24) = 1.283; *p* = 0.268), as well as for Ag, As, Cd, Co, Cr, Fe, Mn, Pb and Zn (Figs. S3 and S4 in the supplementary material for essential and non-essential trace elements respectively). Only Cu (LM: *F*_(1,22) = 4.647; *p* = 0.042; Fig. S5A) and Al (LM: *F*_(1,23) = 5.649; *p* = 0.026; Fig. S5B) concentrations were positively correlated between biological matrices, but with poor predictive power as evidenced by the percentage of variation accounted for by the models (R²; 0.37 and 0.28, respectively).

### 3.2.3. Trace element exposure profiles between habitat types

**Teeth** – The PCA ordination confirmed the dissimilarity in trace element patterns between habitats (Fig. 4A), with 60.5% of the total variation in the data accounted for by the first and second principal components collectively (PC1 and PC2, respectively). PC1 was characterized by Pb, Al, Co, Cr, Cu and Cd, whereas Fe, Mn and Ag mainly contributed to PC2. Only a handful of trace elements were found to have significant differences between habitats, with lower contaminant levels in the rural compartment (PC1: *F*_(1,23) = 7.003, R² = 0.35, *p* = 0.014) and were distinct between age-classes (*F*_(1,23) = 8.374, *p* = 0.008). However, elemental composition in spines and teeth did not separate out across PC2 (*F*_(2,23) = 2.412, *p* > 0.05).

**Spines** – The PCA ordination suggests that the degree of exposure to trace elements, essential and non-essential, is not dramatically increased in urban areas relative to their rural surroundings (Fig. 4B). Taken

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** PCA biplots showing trace element profiles between habitats (urban: orange dots; rural: grey dots) in (A) spines and (B) teeth of hedgehogs (N = 32, top panel; N = 13, lower panel) in Sweden. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

together, PC1 and PC2 captured 47.5% of the variability of the data. Cd, Pb and Co were positively associated with PC1, whereas PC2 was driven by Fe, Mn, Zn, Al, Cr and Cu. Trace element composition in hedgehogs from urban centers were not separated by principal components from individuals collected in rural areas, as evidence by the overlap in multi-element concentrations for adult and juvenile hedgehogs, and was confirmed upon statistical analysis (PC1: *F*_(1,47) = 0.303, *p* > 0.05; PC2: *F*_(1,47) = 0.130, *p* > 0.05).

## 4. Discussion

Our study documents the distribution of organic compounds and trace elements in ground-dwelling mammals from urban and rural habitats; and examines the efficiency of wildlife carcasses to assess the magnitude of environmental pollution. The urbanization of European hedgehogs has become a trend in many cities across Europe, where food
resources can sustain high population densities (Rasmussen et al., 2019). This finding is particularly relevant when considering that dietary intake can be a major route of exposure to contaminants. The insectivorous diet of hedgehogs, which mainly consists of invertebrates such as ground beetles, earthworms and slugs, make them particularly vulnerable to contaminants that accumulate in organisms living in close contact with soil (Fritsch et al., 2022), especially in urban areas where soils are often characterized by high concentrations of trace metals and organic compounds (Vane et al., 2014; Froger et al., 2021).

## 4.1. Organic pollutant exposure in liver

As far as we know, our work provides the first field evidence of exposure to PAHs, phthalates and pyrethroids in organs of urban terrestrial mammals. While no adverse health effects are expected to occur from the low body burdens measured for organochlorine and PAHs, concerns might be expressed for PCBs and phthalate exposure, which were the most abundant xenobiotics in hedgehog livers.

Multiple studies have linked urban activities to increased phthalate and PAH concentrations, with low molecular weight contaminants (DEHP, DiBP, DnBP, phenanthrene, fluoranthene, and pyrene) reported as the most abundant compounds in airborne particles and in the gaseous phase (Quintana-Belmares et al., 2018), street and indoor dust (Skrbić et al., 2016; Lim et al., 2021), and soils from city parks and residential areas (Morillo et al., 2007; Zeng et al., 2009). Their occurrence can be linked to their use and emissions given that phenanthrene, fluoranthene, and pyrene are typical markers of diesel vehicles (Devos et al., 2006). Similarly, the high contribution of DNBP and DEHP in the phthalate profile may be attributed to their use in industrial solvents and plastics for a range of consumer products (Maertens et al., 2004). Microplastics are a long-term source of phthalates and are commonly ingested by a range of small mammals in urban areas (Thrift et al., 2022), which likely enter the gut as a result of direct ingestion or *via* contaminated preys as evidenced along a terrestrial food web (Huerta Lwanga et al., 2017). Although experimental studies have demonstrated that low molecular weight phthalates can leach relatively quickly from polyethylene, polypropylene, and polystyrene (Hahladakis et al., 2018; Paluselli et al., 2019), to what extend sorbed compounds transfer from microplastic is currently unknown. Since *in situ* exposure to phthalates and PAHs have been scarcely investigated in terrestrial mammals, little information is available for comparison. PAH concentrations measured in our study were lower than those found in hair samples of wild carnivorous and herbivorous terrestrial mammals from Primorsky Krai, Russia (Iatrou et al., 2019). While PAH levels in hedgehogs could be representative of lower background concentrations in medium size towns of Southern Sweden, higher wildlife exposure to PAHs in Primorsky Krai is likely due to wood combustion as a result of annual wildfires in the region (Prikhodko et al., 2020). Although no data are available for urban mammals, mean DEHP concentrations in the liver of hedgehogs (118 ± 87.3 ng g⁻¹ dwt) were higher than values measured in polar bears (*Ursus maritimus*; <12–54 ng g⁻¹ wet weight) from Svalbard (Routti et al., 2021). Recently, the main metabolite of DEHP (MEHP) was measured in hair of wild boar (*Sus scrofa*) from medium to highly industrialized areas in Poland at mean concentration of 66.2 ± 58.7 ng g⁻¹ dw (Gonkowski et al., 2024). These results suggest that phthalate concentrations are rather high within urban centers, which could lead to adverse health effects such as higher occurrence of hepatocellular carcinoma and adenoma formations evidenced in animals exposed to phthalates, even at low levels (Tomar et al., 2013).

Pyrethroid insecticides have been widely introduced into the environment as agricultural and industrial by-products, especially in urban settings (allotments, residential gardens and urban parks). By ingesting baits or consuming contaminated prey items, which include plant materials and fruits (Nottingham et al., 2019), hedgehogs are likely exposed to a range of pesticides. Recently, a range of pesticides were screened in European hedgehogs (Schanzer et al., 2021; Rasmussen

et al., 2024a), including rodenticides, fungicides, nematicides, insecticides and herbicides. Only a handful of residues were detected in liver samples, including p,p'-DDE and permethrin at occurrences matching those reported in our study. When compounds were detected, both p,p'-DDE and permethrin were found at higher concentrations in the liver compared with Schanzer et al. (2021). Although permethrin shows moderate to high acute oral toxicity (Boland & Angles, 2010), the concentrations measured in hedgehog livers remain below toxic values specified for small mammals (LD₅₀ from 430 to 4000 µg g⁻¹; Toynton et al., 2009). However, caution should be exercised given that permethrin was only detected in young hedgehogs and that *in vivo* experiments demonstrated a higher sensitivity in early life exposure (Drago et al., 2014).

Polychlorinated biphenyls were the most abundant organochlorine pollutants in hedgehogs. The concentrations and predominance of PCB-180 and PCB-153 in liver samples were consistent with previous studies (D'Havé et al., 2009; Rasmussen et al., 2024b). Although PCB concentrations in the liver of both adult and juvenile hedgehogs were high (38.8 ± 31.4 ng g⁻¹ wet weight [wwt] and 81.5 ± 49.4 ng g⁻¹ wwt, respectively) considering their manufacturing ban more than 30 years ago, they did not exceed the critical hepatic concentration for mammals associated with animal's death (4–5 µg g⁻¹ wwt; Kannan et al., 2000). PCBs are a group of chemicals that are not easily broken down, which explains their persistency in the environment. Given their marked lipophilicity, they preferably accumulate in fatty tissues and biomagnify within food webs (Law et al., 2003). For hibernating and lactating animals, lipids remobilization associated with metabolic energy and fetus' growth releases the accumulated contaminants into the animal's circulation. Previous works on terrestrial and marine mammal species, such as bears, porpoises and seals, reveal that juveniles were carrying higher burden of PCBs than their mothers (Debier et al., 2003; Bytingsvik et al., 2012; Williams et al., 2020). This passive transfer to offsprings occurs during either gestation or lactation through the umbilical cord or *via* lipid-rich milk. Although the fat percentage in the mother's milk of European hedgehogs is only 10% (20–50% in polar bears and seals; Baker, 1990; Hedberg et al., 2011), PCB-180 was found at higher concentrations in juveniles compared to adult hedgehogs in our study. However, it is important to specify that our data included a limited number of individuals, so further studies are therefore needed to confirm these results. This maternal pollutant offloading of toxic chemicals may not only result in substantially higher contaminant levels in offsprings relative to adults but could also lead to adverse health effects, since this transfer occurs during critical development periods.

So far, only one study has investigated the occurrence of PBDEs in the liver of hedgehogs, with concentrations ranging from 1.0 to 1177 ng g⁻¹ wwt (Σ PBDEs; D'Havé et al., 2005a). The differences in concentrations of PBDE congeners in hedgehog livers between studies might be explained by the shift in the overall industrial use and production of PBDEs, with a replacement of penta- and octa-BDE with deca-BDE following regulations (Directive 2003/11). While PBDE congeners were below detection limits in our samples, analyses of terrestrial herbivorous mammals in Sweden also revealed low PBDE levels (Sellström et al., 1993), although one might expect species-specific differences due to trophic position dissimilarities between herbivorous and omnivorous species.

## 4.2. Trace element exposure

Overall, levels shown in the present work are in agreement with previous studies on a range of terrestrial mammals (Alleva et al., 2006) and, in particular, with those carried out in hedgehog tissues (Hernández et al., 1985; D'Havé et al., 2005a; D'Havé et al., 2006; Vermeulen et al., 2009; Rautio et al., 2010; Baptista et al., 2023; Valverde et al., 2024). Local geological features and/or anthropogenic sources, such as mining, farming, and industrial processes, could explain the few discrepancies in element composition between studies. Only Co,
Zn and Pb accumulated more intensively in teeth than in spines, and inversely for Al, Fe, As and Cu. Tissue-specific concentrations could be attributed to physiological regulations and/or differential affinity of trace elements for the selected matrices. For instance, lead acts as calcium analog and is therefore preferentially accumulated in tissues that are actively calcifying, with 90% of the total body burden of Pb found in bones and teeth (Talmage & Walton, 1991). Spines undergo a growth process during which trace elements are incorporated from blood vessels during keratinization (Reeve, 1994), whereas their incorporation in calcified structures happens in their mineral phase (i.e., mineralization). Importantly, while dental tissues are hard and physically stable, hedgehogs undergo periodic partial molts (Reeve, 1994), which may affect element concentrations in spines. Teeth may therefore allow a more reliable retrospective investigation of chronic and past exposure to trace elements, although accumulation varies depending on tooth-related factors (type, development and dental anomalies) (Kamberi et al., 2012). In general, data on tissue concentrations did not support a predictable relationship between the chemical signal recorded in their metabolically stable structures and soft tissues (Vermeulen et al., 2009; Valverde et al., 2024), which are important target sites for a number of pollutants. This result suggests a decoupling of the trace element incorporation into hair and spines versus labile tissues. Yet, external tissues may accumulate substantial concentrations of Al, As, Cr, Cu, Ni and Pb such that smaller differences in hair or spines may represent relatively larger differences in internal tissues (D'Havé et al., 2006).

Adult hedgehogs picked up in urban habitats had contamination profile characterized by Pb, Al, Co, Cr, Cu and Cd compared to their rural counterparts. While naturally present in the environment, trace elements such as lead, cadmium or zinc in urban soils and soils along roads are mainly released from traffic emission and fossil fuel combustion (Trombulak & Frissell, 2000; D'Havé et al., 2005b). Data from the literature clearly indicates a relationship between some element concentrations and the degree of urbanization (Valverde et al., 2024). Predation on earthworms, which bioaccumulate toxic elements such as Pb, Cu, Cd, may constitute a major pathway for the entry of trace elements into hegdehog's bodies and the food web in general (Richardson et al., 2020; Tözsér et al., 2022).

Toxic effects likely occur in small mammals, but the significance of increased element concentrations for mammalian and wildlife species is largely unknown because toxicologic data on free-living wildlife species remain scarce. Few studies have evaluated endogenous concentrations of biologically relevant trace metal in mammalian samples (Chmielnicka et al., 1982; Eisler, 1993; Wegst-Uhrich et al., 2015) and, to our knowledge, basal concentrations of essential trace metals in teeth and spines of European hedgehogs have not yet been described. In addition, interpreting the effects of inert tissue concentrations on animal's health based on threshold levels in soft tissue are challenging given the lack of predictable relationship between the chemical signal recorded in metabolically stable structures and soft tissues. Recently, biliary hyperplasia was evidenced in the liver of hedgehogs with elevated trace element concentrations (Baptista et al., 2023). Among the most toxic trace elements, Pb was measured at concentrations up to 4 times the values reported in hedgehogs from polluted areas (Pankakoski et al., 1993; Vermeulen et al., 2009), and some hedgehogs' levels even surpassed 50 µg g⁻¹ dw. Eight hedgehogs had Pb concentrations in their teeth above the critical renal value of 15 µg g⁻¹ dw, considered as indicative of Pb toxicosis in mammals (Ma, 1996). Since trace element concentrations are generally lower in inert matrices compared to internal organs (see Rasmussen et al., 2024b), individuals with high Pb levels stored in their teeth likely experienced detrimental health effects.

## 5. Conclusion

The present study has demonstrated the potential of roadkill and carcasses to estimate the magnitude of environmental pollution. Inert

tissues of hedgehogs collected from separate habitats confirmed that they accumulate substantial concentrations of trace elements and that their composition is representative of locally occurring elements, including toxic ones. Although teeth may be preferred for metal exposure assessment, as they captured a wider range of elements over time and reflect information of long-term exposure, spines are more easily sampled given their availability on road kills and carcasses in general. Even in relatively low to moderate urbanized areas, road kills also reveal that urban mammals are exposed to high levels of organic contaminants, mainly phthalates and PCBs, reaching concentrations which may reflect adverse effects in organisms. It therefore appears that hedgehogs may serve as relevant indicators of environmental pollution and reveal hazards of chemical exposure in urban environments, especially for human health concerns.

## CRediT authorship contribution statement

**Noëlie Molbert:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. **Fabrice Alliot:** Writing – review & editing, Methodology, Investigation. **Aurélie Goutte:** Writing – review & editing, Investigation. **Maria C. Hansson:** Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization.

## Funding sources

This work was financially supported by the Swedish Research council Formas grant number 2019-01892.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Maria Hansson reports financial support was provided by Swedish Research Council Formas. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors are grateful to Sofia Mebrahtu Wisén at Lund University for helping with the trace element analyses. We thank Dorte Kjeldmand at the Wildlife refuge centre KFV Skåne-Sydväst in Skanör for aiding in the collections of hedgehogs. We also deeply thank the general public for all the invaluable help notifying us about dead hedgehogs in and around the town of Lund and southwest Skåne, this study would not have been possible to carry out without this engagement.

## Appendix B. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2025.125648.

## Data availability

Data will be made available on request.

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