# Acclimation and recovery dynamics of behavioral and coloration responses of a common fish (Squalius cephalus) to paracetamol exposure

Léa Lorrain-Soligon, Alexis Golven, Simon Agostini, Alexis Millot, Alexandre Bauer, Thierry Rigaud, Beatriz Decencière, Carine Puppo, Aurélie Goutte

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## To cite this version:

Léa Lorrain-Soligon, Alexis Golven, Simon Agostini, Alexis Millot, Alexandre Bauer, et al. Acclimation and recovery dynamics of behavioral and coloration responses of a common fish (Squalius cephalus) to paracetamol exposure. Chemosphere, 2025, 374, pp.144225. 10.1016/j.chemosphere.2025.144225. hal-04981941

HAL Id: hal-04981941

https://hal.science/hal-04981941v1

Submitted on 7 Mar 2025

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# Acclimation and recovery dynamics of behavioral and coloration responses of a common fish (*Squalius cephalus*) to paracetamol exposure

Léa Lorrain-Soligon¹,², Alexis Golven¹, Simon Agostini², Alexis Millot², Alexandre Bauer³, Thierry Rigaud³, Beatriz Decencière², Carine Puppo², Aurélie Goutte¹,⁴

¹Sorbonne Université, Université PSL, EPHE, CNRS, Milieux Environnementaux, Transferts et Interactions dans les hydrosystèmes et les Sols, METIS, Paris, France

²Centre de recherche en écologie expérimentale et prédictive (CEREEP-Ecotron IleDeFrance), Département de biologie, Ecole normale supérieure, CNRS-UAR 3194, PSL University, 77140 Saint-Pierre-lès-Nemours, France

³Biogéosciences (UMR-CNRS 6282), Université de Bourgogne, Dijon, France

⁴École Pratique des Hautes Études, PSL Research University, UMR 7619 METIS, Paris, France

## ORCIDs

LLS: 0000-0002-8723-7478

AB: 0000-0003-3162-9285

TR: 0000-0002-0163-6639

BD: 0000-0002-2847-7631

AuG: 0000-0001-8322-5843

*Correspondence: llorrain.lea@gmail.com

ID ORCID: 0000-0002-8723-7478

Full postal address : Sorbonne université, UMR 7619 METIS EPHE/SU/CNRS, 4 place Jussieu 75252 PARIS

## Acknowledgments

This work has benefited from technical and human resources provided by CEREEP-Ecotron IleDeFrance (CNRS/ENS UAR 3194) as well as financial support from the Regional Council of Ile-de-France under the DIM Program R2DS bearing the references I-05-098/R and 2015-1657. It has received a support under the program "Investissements d'Avenir" launched by the French government and implemented by ANR with the references ANR-10-EQPX-13-01 Planaqua, ANR-11-INBS-0001 AnaEE
France and ANR-10-IDEX-0001-02 PSL. The authors would like to thank the fishing federations of Essonne and Seine-et-Marne, AAPMA Val de Seine association and Eliott Salles for their help in capturing fish, and Jean-François le Galliard for providing the spectrophotometer and helping with its utilization.

**Conflict of interest statement**

The authors declare that they have no conflict of interest.

**Funding statement**

This study was funded by the "Agence Nationale de la Recherche (ANR)" through the ANR HELP (ANR-22-CE34-0014).

**Data archiving statement**

The data that supports the findings of this study will be made available by the corresponding author upon request.

**CRediT**

LLS and AuG conceptualized the study. LLS, AlG, SA, AM, BD, CP and AuG participated to fish sampling and fish tagging. LLS, AlG and SA participated to data collection. AB and TR participated to parasites differentiation. LLS and AlG analyzed videos, LLS analyzed data, and LLS and AuG wrote the initial draft. All authors have reviewed and edited the manuscript and approved the final version.
Graphical abstract

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**Abstract**

Freshwater ecosystems are increasingly exposed to pharmaceutical contamination, impacting non-target species. Concentrations can vary over time and location, allowing for potential acclimation or recovery effects. Additionally, parasites might interfere with the absorption and adverse outcomes pathways of pollutants. We examined the combined effects of paracetamol and parasite on the behavior and coloration of the European chub (Squalius cephalus), a ubiquitous fish species, from natural populations. Fish were exposed in mesocosms to acute doses of paracetamol (16 µg.g⁻¹ once a day over two days), followed by lower doses during a long-term exposure (1.6 µg.g⁻¹ once a week over three weeks), followed by a three-week recovery phase. Acute exposure induced marginal decreases in behavioral activity, and changes in dorsal brightness, hue and UV luminance. Interestingly, the long-term phase alone did not yield notable results on behavior and coloration. However, some effects of the acute exposure persisted during the long-term phase, highlighting that the expression of biological responses may be delayed in relation to past high exposure. Parasitism did not attenuate acute impacts, suggesting parasites may not help mitigate effects of paracetamol on behavior and coloration, but alone increased activity levels slightly. No effects of pollutant exposure, either of the acute or long-term phase, were observed after a recovery phase, indicating ability for recovery dynamics. Overall, our findings emphasize that pollutants effects can be highly transient, with rapid recovery when pollutant exposure ceased. Considering different exposure phases is crucial when assessing the ecological consequences of environmental contaminants.

**Key-words:** Acanthocephala; Acute; Biotic interactions; Long-term; Mesocosm; Paracetamol
# 1. Introduction

Despite their rich biodiversity and contribution to human well-being, freshwater ecosystems are threatened by anthropic pressures and global change, precipitating a global conservation crisis (Dudgeon et al., 2006; Reid et al., 2019). Pharmaceutical contaminations are now ubiquitous and numerous drug residues are detected in freshwater environments (Wilkinson et al., 2022), primarily released through domestic and industrial discharges (Li, 2014; Mezzelani et al., 2018). Even at trace environmental concentrations, pharmaceutical compounds are biologically active substances, which raises alarming environmental and sanitary issues (Arnold et al., 2014).

Pharmaceuticals such as analgesics, antidiabetics, antibiotics and anticonvulsants are the most commonly found in riverine systems (Wilkinson et al., 2022). They have been detected in water bodies worldwide, often in concentrations that can affect non-target species. Concentrations range from 1000 ng.L⁻¹ for tramadol, propranolol and fluconazole, to up to 10 000 ng.L⁻¹ for paracetamol and metformin (Wilkinson et al., 2022). Based on both field and laboratory evidences, paracetamol are known to disrupt endocrine functions (Stahlschmidt-Allner et al., 1997), and induce neurotoxic effects (Iqubal et al., 2020; Vaudin et al., 2022) in vertebrates. Overall, pharmaceuticals can lead to behavior disruptions, which is particularly relevant in fish, for which it has been observed reduction of territoriality (Forsatkar et al., 2014), increased frozen time (Orozco-Hernández et al., 2022), and decreased boldness (risk taking behavior) and activity (Aich et al., 2024) or on the contrary increased boldness and activity (Brodin et al., 2017), which can all impact reaction to stimuli or stressors.
Escape from predation could also be achieved by rapid color change according to the
substrate, and it turns out that some pharmaceutics are known to alter coloration in
many fish species (Nilsson Sköld et al., 2013), due to a trade-off between the expression
of coloration and resistance to pollutants (Arellano-Aguilar and Macías Garcia, 2008).
Acetaminophen (also known as paracetamol), one of the most commonly used
analgesic (Wilkinson et al., 2022), is particularly interesting to study on this regard.

Indeed, paracetamol is a biologically active compound that primarily acts on
the cyclooxygenase (COX) pathway, inhibiting prostaglandin synthesis (Ayoub, 2021).
Disruption of this pathway can indirectly affect dopamine and serotonin systems, as
well as neuroendocrine signaling, which are critical regulators of behavior and stress
responses (Gądek-Michalska et al., 2013). Additionally, paracetamol can cause damage
to organisms through impacts on the antioxidant system (Wang et al., 2017). When
these damages occur, pigments may be allocated to detoxification mechanisms
(McGraw, 2005), rather than to coloration in exposed individuals (Arellano-Aguilar
and Macías Garcia, 2008; Lifshitz and St Clair, 2016). Paracetamol exposure can thus
lead to changes in behavior and coloration, which are essential for survival functions
such as food intake (Brodin et al., 2017; Corcoran et al., 2010), reproduction (Overturf
et al., 2015), as well as escape from predation (Martin et al., 2017). Paracetamol is water-
soluble, has a relatively short half-life in vertebrates due to rapid metabolism in the
liver, and is primarily excreted as glucuronide and sulfate conjugates (Mazaleuskaya
et al., 2015). However, in fish, differences in metabolic pathways can result in a greater
tendency to bioaccumulate than previously anticipated (Connors et al., 2013). As such,
even brief environmental exposures to paracetamol could lead to bioaccumulation,
particularly under repeated exposures.

Pharmaceuticals are used continuously and released to the environment,
resulting in long-term exposures of organisms. However, concentrations may vary in
space and time. For example, exposure to pollutants is higher downstream the
effluents of waste water treatment plants, and lower upstream of these outlets (Sim et
al., 2010). Over time, their concentrations can fluctuate due to factors such as seasonal
water flow changes, degradation rates, and variations in human consumption patterns
(Bavumiragira et al., 2022; Yang et al., 2021). Importantly, prior exposure to pollutants
can influence an organism's response to subsequent exposures, and individuals that
have been previously exposed to pharmaceuticals may develop some levels of
tolerance or altered sensitivity (Sun et al., 2014). Conversely, in poorly or randomly
exposed environments, individuals could express recovery from damages induced by
previous exposure in polluted water masses. Investigating whether effects are
permanent or reversible, i.e. whether adverse effects persist or lessen when exposure
to a chemical substance declines and stops, is a challenging and poorly-explored task
in ecotoxicology (Nunes et al., 2017; Sun et al., 2006; Sundin et al., 2019). In addition,
biotic interactions can play a significant role in modulating the effects of
pharmaceutical pollutants. Indeed, some parasites, such as acanthocephalan, can
mitigate the adverse effects of pollutants (Goutte and Molbert, 2022). These intestinal
parasites can accumulate pollutants (Molbert et al., 2020; Sures, 2006), mitigating their
adverse effects for their host. However, this benefit has mainly been investigated for
persistent pollutants. For example, mean concentrations of lead and cadmium in the
acanthocephalan *Pomphorhynchus laevis* were, respectively, 2700 and 400 times higher
than in the muscle of the host chub *Squalius cephalus* (Sures et al., 1994; Sures and
Taraschewski, 1995). But these effects have not been studied for less persistent
substances such as pharmaceutical substances, and potential benefits at the host
individual and phenotypical level have scarcely been assessed. Interestingly,
acanthocephalan parasites are known to influence host behavior and physiology
through their ability to manipulate fish enteric nervous system and the enteric
endocrine system (Bosi et al., 2022; Dezfuli et al., 2002), with possible links to stress
and behavior. Additionally, these parasites are carotenoid-sequestering organisms
(Balanean, 2010), potentially impacting their host's coloration by reducing carotenoid
availability for pigmentation. However, the direct effect they have on their fish
definitive host behavior and coloration is yet unknown. As such, integrating the
interaction between parasitism and exposure to paracetamol could reveal important
insights into biotic interactions that influence ecotoxicological outcomes, particularly
regarding behavior and coloration. , which is essential for developing a comprehensive
view of ecosystem health and intrinsic resilience.

In this study, we focus on the effects of paracetamol on the European chub
(*Squalius cephalus*), naturally infested by acanthocephalan parasites. We used a
ubiquitous fish species from natural populations, as well as an environmentally
relevant set up, with three phases of exposure to paracetamol (successive or not, see
methods) in mesocosms, representative of exposure in nature: acclimation (hereafter
"acute exposure", 16 µg.g⁻¹ paracetamol once a day for two days), long-term exposure
(1.6 µg.g⁻¹ paracetamol once a week for three weeks), and recovery (three weeks
without exposures). We aim to assess the impacts on both behavior (hiding duration,
speed, movements' complexity) and coloration (hue, UV and color saturation,
brightness, as well as rapid changes in these parameters). Such parameters are
particularly important for camouflage and thus predator avoidance. We hypothesize
that (1) paracetamol induces a more marked effect in acute exposure than in long-term
exposure (Fent et al., 2006), (2) individuals exposed in acute conditions to paracetamol
suffer lesser effects during long-term exposure, due to possible acclimation (Armario
et al., 2004), and (3) that this effect will be reduced in parasitized individuals (Molbert
et al., 2020). Additionally, (4) recovery should allow individuals to return to their
initial condition (Sun et al., 2006; Sundin et al., 2019). Lastly, we expect (5) effects of
acanthocephalan presence on their hosts (Connors and Nickol, 1991), particularly
regarding coloration due to the carotenoid based coloration of acanthocephalan
parasites (Kaldonski et al., 2009) and their ability to sequester carotenoids (Balanean,
2010).
## 2. Methods

### 2.1. Ethical Note

All experiments complied with European and French laws on animal experimentation.

All individuals were reared in reasonable population density and fed regularly (see below for rearing methods). Animal care protocols were approved by national ethics committee for animal experimentation under file number APAFIS #42968-2023032907492372. Wild fish were captured, manipulated and transported under authorization n°2023-DDT-SE-147.

### 2.2. Capture and housing conditions

152 European chub, *S. cephalus*, were captured at 8 points, along tributaries of the Seine River (On the École, Essonne, Loing and Orvanne Rivers) where the presence of intestinal parasites (*Pomphorhynchus sp.*, Acanthocephala) has been previously recorded. Electrofishing occurred on 5 different occasions (23/01/2024, 24/01/2024, 25/01/2024, 30/01/2024 and 31/01/2024), each site being only prospected once.

Individuals were then brought back to CEREEP-Ecotron facilities (Saint-Pierre-lès-Nemours, France, 48.282129 N, 2.672190 E, situated at a distance of less than 65 km from field capture sites). They were rapidly transferred in 100 L tanks filled with well-aerated pond water originating from a pond near CEREEP-Ecotron facilities under natural conditions of temperature and luminosity.

Chubs were anesthetized in diluted eugenol (dilution 1/2000e). Anesthetized chubs were carefully tagged with a passive integrated transponder device (PIT-tag
dimensions: 1.2×7 mm biolog-nano, Biolog-id, France, [ISO 11784 / 11785], 0.024 g,
diameter of the injector: 1.6 mm, smallest size currently available on the market)
inserted into the left flank intramuscularly. They were then weighted (±0.5 g, Ohaus,
Valor 2000W) and measured (from beginning of the head to the end of the tail ±
0.1 cm). All individuals had a body length > 10.0 cm (mean ± SE, 16.05 ± 0.42 cm) and
a mass at capture >10 g (56.40 ± 6.34 g). Afterward, they were left for recovery in 100
L tanks filled with well-aerated pond water of the same origin as previously stated,
until complete awakening of individuals (including normal swimming and response
to stimuli). Once fully recovered, they were transferred into five 12 000 L outdoor
mesoscoms (350 cm diameter, 120 cm height, Appendix A) filled with water of the
same origin under natural conditions of temperature and luminosity. The mesocosms
are equipped with anti-predatory nets and a floating raft to allow food provisioning,
shelter, and shade. Mechanical and biological filtration was installed in each tank to
stir the water and maintain its physico-chemical parameters (during the whole
duration of the experiment; temperature: 10.97°C ± 0.24, 175.46 µS.cm⁻¹ ± 1.54 SE,
dissolved oxygen: 10.72 mg.L⁻¹ ± 0.62 SE). Additionally, the bottom of the mesocosm
is lined with a fine mesh net (8×5 mm) which follows the shape of the mesocosm,
allowing a rapid and easy capture of fish. Fish were left acclimated until 10/02/2024
for individuals in the acute trial (see below) or 19/02/2024 for individuals in the acute
and then long-term trial (see below). During the whole duration of the acclimation and
experimental phase, fish were fed once every day with commercial fish pellets (Tetra).

**2.3. Experimental design**
Ten experimental groups (Table 1) were constituted following a full factorial design (see Table 1). The experiment was divided in 3 sub-experiments: 1) fish experimenting an acute exposure only: P (exposed to paracetamol during the acute phase), and C (exposed to a control during the acute phase); 2) fish experimenting an acute and then long-term exposure: CP (exposed to a control during the acute phase, and to paracetamol during the long-term phase), PP (paracetamol during acute and long-term phase), CC (control during the acute and long-term phase), PC (paracetamol during the acute phase, control during the long-term phase); and 3) fish experimenting an acute then long-term exposure, and then recovery phase: CPR, PPR, CCR and PCR (same treatment as previously, but individuals are then left in recovery). Behavioral and colorimetric measurements, as well as dissections, all happened at the end of exposure for each group.

The contaminant used in the experiment was Paracetamol, supplied by LGC (LGC Standards, Dr. Ehrenstorfer, CAS 103-90-2). To ensure ecological relevance, we scaled the doses to approximate oral exposure by expected intake based on the body mass of our experimental fish, considering concentrations in water of 800 µg.L⁻¹ paracetamol during the acute phase and 80 µg.L⁻¹ paracetamol during the long-term phase (see below). With an average body mass of ~50 g, individuals were exposed to 16 µg.g⁻¹ (once a day during 2 days) and 1.6 µg.g⁻¹ (once a week during 3 weeks) paracetamol in the acute and long-term experiments, respectively. Two dilution were prepared and final concentrations were confirmed by LC/MS-MS analyses, for the acute (1570 µg.mL⁻¹ [~1600 µg.mL⁻¹], measured by LC/MS-MS) and long-term (158.6 µg.mL⁻¹ [~160 µg.mL⁻¹], LC/MS-MS) exposures. The concentrations measured by
LC/MS-MS correspond to environmentally plausible exposure scenarios when accounting for ingestion from contaminated prey or water sources over time.

Also the levels of paracetamol used in our experiment are environmentally relevant as maximum values of 227 µg.L⁻¹ were reported by Wilkinson et al. (2022), and are much below DL50 of 800 mg.L⁻¹ reported for the medaka fish (Yamamoto et al., 2007). Other studies using this pollutant in acute experiments have used concentrations in the water ranging from 8 µg.L⁻¹ to 80 000 µg.L⁻¹ (Matus et al., 2018; Pereira et al., 2018), while long-term exposure use concentrations ranging from 0.25 to 25 µg.L⁻¹ (Guiloski et al., 2017; Perussolo et al., 2019).

It is important to note that while waterborne exposure reflects the concentration in the aquatic medium, oral administration approximates the internal exposure due to feeding on contaminated sources, such as prey bioaccumulating the drug. In our design, oral exposure concentrations were scaled to match the environmental levels of waterborne contamination during typical feeding rates, bridging the gap between acute and long-term exposure scenarios, simulating both immediate and prolonged exposure pathways in polluted environments.

Two dilutions in milliQ water were prepared: 1600 µg.mL⁻¹ in the acute phase, and 160 µg.mL⁻¹ in the long-term phase. Control solution consisted only of milliQ water, either for acute or long-term exposure. Solutions were injected orally using a syringe in anesthetized chub, for both control chubs and individuals exposed to
paracetamol. The mean length of chubs did not differ among groups (P: 16.14 cm ±
0.96 SE; PP: 16.18 cm ± 1.18 SE; PPR: 15.63 cm ± 1.22 SE; PC: 16.11 cm ± 1.40 SE ; PCR:
16.12 cm ± 1.55 SE ; C: 16.00 cm ± 1.12 SE ; CP : 16.00 cm ± 1.53 SE ; CPR : 16.23 cm ±
1.47 SE ; CC : 16.10 cm ± 1.81 SE ; CCR : 15.93 cm cm ± 1.30 SE ; Linear Model:
SumSq=4.40, F-value=0.015, p-value=0.999).

2.3.1. Acute exposure, n=32

Acute solutions were injected orally in anesthetized chubs using a syringe, once a day for two consecutive days, from 10/02/2024 to 13/02/2024. Behavioral and colorimetric tests were performed 2 days after the last exposure, and dissections 3 days after the last exposure.

2.3.2. Acute and then long-term exposure, n=66

Acute solutions were injected orally using a syringe in anesthetized chubs, once a
day for two consecutive days on 19/02/2024 and 20/02/2024. Solutions used for the
long-term exposure were injected orally using a syringe in anesthetized chubs, once
a week during three consecutive weeks from 22/02/2024 to 11/03/2024. Behavioral
and colorimetric tests were performed the week just after the last exposure of the
long-term phase, and dissections from the week after behavioral and colorimetric
measurements.

2.3.3. Acute, long-term exposure, and then recovery, n=54
Acute solutions were injected orally using a syringe in anesthetized chubs, once a day for two consecutive days on 19/02/2024 and 20/02/2024. Solutions used for the long-term exposure were injected orally using a syringe in anesthetized chubs, once a week during three consecutive weeks from 22/02/2024 to 11/03/2024. Individuals were then left in mesocosms without disturbance during 3 weeks, until 02/04/2024 or 05/04/2024 depending on individuals. Behavioral and colorimetric tests were performed the days of the end of the recovery, and dissections from 1 to 5 days after these tests.

**2.4. Sample collection**

**2.4.1. Behavior**

We performed a comprehensive set of behavioral tests. Individuals were placed in an individual tank (63×44×42 cm) filled with 25 L of water from the same origin as their mesocosm, in a thermally controlled room (16°C) with artificial light originating from above. Fish were left acclimated for 10 min, and then video recorded for 15 min, using a camera placed above the tank (GoPro HERO 10; GoPro, San Mateo, CA, USA). Videos were analyzed with the software ToxTrac (Rodriguez et al., 2018). Using 'Tracking RealSpace' data, we determined the total duration of activity, number of activity windows (number of time individuals are active), total distance travelled, mean swimming speed, mean active swimming speed, and maximal swimming speed. Additionally, we evaluated DFAc (corrected Detrented Fluctuation Analysis) using the fractal package (version 2.0-4, (Constantine and Percival, 2017), on R 3.6.1), a robust method designed to estimate the degree to which time series are similar. Description of how DFAc were computed was presented in MacIntosh et al. (2013). DFA is
inversely related to the fractal dimension, a classical index of structural complexity (Mandelbrot, 1982), and thus smaller values reflect greater behavioral complexity. We performed a PCA in R 3.6.3 (R Core Team, 2020) on all the behavioral metrics for each study phase (see Figure 1), and extracted the coordinates on the first (PCA1) and second axis (PCA2), explaining most part of the variation, to analyze the variation of all variables together.

The resulting behavioral patterns provide insights into specific aspects of fish behavior. Swimming performance and distance provide insights into the physical capacity and energy expenditure of the fish (Beamish, 1978). Explorative behavior reflect the fish's ability to navigate and adapt to their environment (Baker et al., 2018). Movement complexity is useful for fish to enhance the probability of resource encounters (Bartumeus, 2007; Viswanathan et al., 2008) and prevent predators from anticipating the prey future position (Gazzola et al., 2021; Jones et al., 2011; Richardson et al., 2018).

### 2.4.2. Coloration measurement

Fish are known to change their coloration according to the substrate (Nilsson Sköld et al., 2013). As pollutants are known to impede coloration of individuals (Lifshitz and St Clair, 2016; Tartu et al., 2023), our objective was both to understand if paracetamol can impede chubs coloration, and to understand if paracetamol and parasites can influence the speed of this change in coloration (coloration lability, i.e. background matching). To do so, individuals were left for 30 minutes in a black tank (75×55×45 cm) filled with 30L water originating from their mesocosm, to allow darkening of animals. After 30
minutes (T0), fish were taken outside of the water under controlled lighting conditions.
Coloration of each individual was assessed using a spectrophotometer (Ocean Optics
PX-2 Pulsed Xenon lamp) following the recommendations of Badiane et al. (2022,
2017). Each individual was briefly dried using a paper towel in order to remove
residual water which can bias measures. For each individual, two measurements were
taken on the dorsal area, at T0. The fish was then placed in a white tank (75×55×45 cm)
filled with 30L water of their mesocosm. Imaging procedures were repeated after 30
seconds in this condition (T30s), this duration being previously identified to allow
changes in coloration in fish (LLS personal observations).

We processed spectral data in R 3.6.3 (R Core Team, 2020) using the package *pavo* 2.0 (Maia et al., 2019), according to the methodology of Badiane et al. (2022). We were thus able to extract total brightness, hue, UV saturation and luminance, Violet-Blue saturation, Green saturation and Yellow-Orange saturation. For a given individual, each value was the average of the two measurements. We performed a PCA in R 3.6.3 (R Core Team, 2020) on all the coloration metrics for each study phase (acute, long-term and recovery) and for each time of measurement (T0 and T30s, see Figure 1), and extracted the coordinates on the first (PCA1) and second axis (PCA2), explaining most part of the variation, to analyze the variation of all variables together.
All these coloration metrics allow us to have an overall idea of how much energy an individual can invest in coloration, and as such, how it can influence their fitness (Badiane et al., 2022; Tartu et al., 2023).
### 2.4.3. *Acanthocephalan parasites*

After these measurements fish were euthanized with eugenol overdose and dissected. Chub intestine and body cavity were examined for the presence of acanthocephalan parasites.

## 2.5. Statistical analyses

All statistical analyses were performed using R 3.6.3 (R Core Team, 2020) and Rstudio v1.1.419. We computed Linear Mixed Models (LMMs), or quasipoisson Generalized Linear Mixed Models (GLMMs) from the *lme4* (Bates et al., 2015) and the *MASS* package (Ripley et al., 2013). For LMMs, normality was checked (shapiro test) and data were log10 or square root (sqrt) transformed when residuals did not follow the normality assumption. For all computed tests, models accuracy was also tested using the *check_model* function from the *performance* package (Lüdecke et al., 2020). For all tests, the fish site of capture and mesocosm were included as random effects. Significance level was set at 0.05, effects were considered marginally significant at $\alpha<0.10$, and best variables were retained using a top-down selection. Acanthocephalan presence was used for statistical analyses.

### 2.5.1. *Acute exposure*

After acute exposure only, the two experimental groups (C and P) did not differ in acanthocephalan number (Gamma GLMM, $X^2=0.813$, p-value=0.367; C: mean number of parasites=1.20 ± 0.56, min=0, max=6; P: mean number of parasites=0.76 ± 0.34, min=0, max=5).
The effects of treatment (acute exposure), acanthocephalan presence and their interaction were tested on behavior PCA1, behavior PCA2 (sqrt transformed), dorsal coloration PCA1 and PCA2 at T0 and T30s (all sqrt transformed), using LMMs.

### 2.5.2. Long-term exposure and recovery

After long-term exposure, the four experimental groups (CC, CP, PC and PP) did not differ in acanthocephalan number (Gamma GLMM, $X^2=0.634$, p-value=0.889; CC: mean number of parasites=2.00 ± 0.80, min=0, max=11; CP: mean number of parasites=1.53 ± 0.37, min=0, max=5; PC: mean number of parasites=1.53 ± 0.65, min=0, max=10; PP: mean number of parasites=1.65 ± 0.53, min=0, max=8).

After long-term exposure, the effects of treatment (acute exposure and long-term exposure separately), acanthocephalan presence and their interaction (triple interaction: acute exposure * long-term exposure * acanthocephalan presence, when non-significant double interaction were then considered) were tested on behavior PCA1 and PCA2 (sqrt transformed), dorsal coloration PCA1 (sqrt transformed) and PCA2 at T0, dorsal coloration PCA1 and PCA2 at T30s (both sqrt transformed) using LMMs. One individual was excluded from the analysis as it presented more acanthocephalan parasites than other individuals (52 acanthocephalan parasites, while the second more infected individual presented 11 parasites).

After recovery, the four experimental groups (CC, CP, PC and PP) did not differ in acanthocephalan number (Gamma GLMM, $X^2=4.318$, p-value=0.229; CC: mean number of parasites=2.38 ± 0.69, min=0, max=7; CP: mean number of parasites=2.56±
1.41, min=0, max=20; PC: mean number of parasites=4.71 ± 2.16, min=0, max=27; PP: mean number of parasites=4.77 ± 2.77, min=0, max=37).

After recovery, the same effects were tested on behavior PCA1 and PCA2 (sqrt
transformed), dorsal coloration PCA1 and PCA2 at T0, dorsal coloration PCA1 and
PCA2 at T30s (both sqrt transformed) using LMMs.
**3. Results**

**3.1. Acute exposure**

Behavioral PCA1 after the acute phase was positively linked to total distance travelled, mean speed, mean active speed, and overall activity (total active time, activity duration, Figure 1A). Behavioral PCA2 was positively linked to maximal speed and negatively linked to behavioral complexity and behavioral windows (Figure 1A). Dorsal coloration PCA1 at T0 was positively correlated to orange and UV saturation, and negatively correlated to blue and green saturation (Figure 1A). Dorsal coloration PCA2 at T0 was positively linked to high brightness, UV luminance, but low hue (Figure 1B). Dorsal coloration PCA1 at T30s was positively linked to change in UV luminance, and negatively linked to change in green saturation (Figure 1C). Dorsal coloration PCA2 at T30s was positively linked to change in hue and negatively to change in UV saturation (Figure 1C).

Behavioral PCA1 was marginally higher for individuals presenting acanthocephalan (Figure 2A, Appendix B), implying marginally higher to total distance travelled, mean speed, mean active speed, and overall activity for parasitized individuals. Behavioral PCA2 was only marginally higher for individuals exposed to paracetamol during the acute phase (Figure 2B, Appendix B), implying higher maximal speed, and lower behavioral complexity and behavioral windows for individuals exposed to paracetamol during the acute phase. Dorsal coloration PCA1 at T0 was neither influenced by treatment, acanthocephalan presence, or their interaction (Figure 2C, Appendix B). Dorsal coloration PCA2 at T0 was lower for individuals exposed to paracetamol during the acute phase, and for individuals
infested with acanthocephalan (Figure 2D, Appendix B), indicating higher hue but lower brightness and UV luminance for these individuals. Dorsal coloration PCA1 at T30s was neither influenced by treatment, acanthocephalan presence, or their interaction (Figure 2E, Appendix B). Dorsal coloration PCA2 at T30s was marginally influenced by the interaction between treatment and acanthocephalan presence, and overall lower for individuals exposed to paracetamol (Figure 2F, Appendix B). Indeed, dorsal coloration PCA2 at T30s was not influenced by the presence of acanthocephalan parasites in control individuals, while it was lower for infested individuals, as compared to those uninfested in the paracetamol-treated group (Appendix C), implying higher UV saturation and lower hue for these individuals. Similarly, dorsal coloration PCA2 at T30s was not influenced by the paracetamol treatment in individuals without acanthocephalan, but was lower for infested individuals that were exposed to paracetamol compared to control ones (Appendix C), implying higher change in UV saturation and lower change in hue for these individuals.

## 3.2. Long-term exposure

After the long-term exposure, behavioral PCA1 was positively correlated with higher total distance travelled, mean speed, and activity (Figure 1D). Behavioral PCA2 was positively linked to high maximal speed, and negatively linked to behavioral complexity, behavioral windows, (Figure 1D). Dorsal coloration PCA1 at T0 was positively linked to higher Yellow-Orange saturation, brightness, and UV luminance, but negatively linked to Green saturation and Violet-Blue saturation (Figure 1E). Dorsal coloration PCA2 at T0 was positively linked to higher Hue but lower UV
saturation (Figure 1E). Dorsal coloration PCA1 at T30s was positively linked to increase change in UV luminance and brightness but lower changes in blue saturation (Figure 1F). Dorsal coloration PCA2 at T30s was positively linked to higher changes in UV saturation and green saturation, but lower changes in yellow-orange saturation.

Behavioral PCA1 values were only marginally higher for individuals exposed to paracetamol during the acute phase, regardless of their treatment during long-term exposure (PP and PC individuals, Figure 3A, Appendix B), implying higher total distance travelled, mean speed and activity for these individuals. Behavioral PCA2 was neither influenced by acute and long-term exposure, or acanthocephalan presence (Figure 3B, Appendix B). Dorsal coloration PCA1 at T0 was marginally lower for individuals exposed to paracetamol during the acute phase regardless of their treatment during long-term exposure (PP and PC individuals, Figure 3C, Appendix B), implying lower Yellow-Orange saturation, brightness, and UV luminance, but higher Green saturation and Violet-Blue saturation. Dorsal coloration PCA2 at T0 was influenced by the interaction between acute and long-term exposure (Figure 3D, Appendix B). Indeed, for individuals exposed to the control during the long-term phase (CC and PC individuals), dorsal coloration PCA2 at T0 was similar if they were exposed to the control or to paracetamol during acute exposure, but when individuals are exposed to paracetamol during long-term exposure (CP and PP individuals), it was lower for individuals exposed to paracetamol during the acute exposure (lower hue and higher UV saturation for PP individuals) as compared to control individuals during acute exposure (CP) (Appendix C). It was also similar for control individuals
during the acute exposure (CC and CP individuals), but when individuals are exposed
to paracetamol during the acute exposure (PP and PC), it was lower for individuals
exposed to paracetamol during the long-term exposure (lower hue and higher UV
saturation for PP individuals) as compared to controls during the long-term exposure
(PC) (Appendix C). Dorsal coloration PCA1 at T30s was only marginally higher for
individuals with acanthocephalan (Figure 3E, Appendix B), implying higher changes
in UV luminance and brightness, and lower changes in blue saturation for these
individuals. Dorsal coloration PCA2 at T30s was marginally higher for individuals
exposed to paracetamol during the acute exposure, regardless of their treatment
during acute exposure (PP and PC individuals; Figure 3F, Appendix B), implying
higher changes in UV saturation and green saturation, but lower changes in yellow-
orange saturation.

3.3. <u>Recovery</u>

After recovery, behavioral PCA1, behavioral PCA2, dorsal PCA1 and PCA2 at T0 and
T30s were neither influenced by acute exposure, long-term exposure, acanthocephalan
biomass, or their interaction (Figures 4 A, B, C, D, E, F, Appendix B).
## 4. Discussion

This study highlights differential responses of fish behavior and coloration according to duration and intensity of exposure to paracetamol, and comparatively low effects in fish where parasites presence was confirmed. However, the parasitized individuals exhibited increased behavioral activity and speed, as well as lower brightness, UV luminance, and higher hue. During an acute phase (16 µg.g⁻¹ paracetamol once a day for two days), chubs exposed to paracetamol showed marginal decreases in behavioral activity, and changes in dorsal coloration, particularly regarding brightness, hue and UV luminance. Interestingly, the long-term exposure phase alone (1.6 µg.g⁻¹ paracetamol once a week during three weeks), did not yield notable results on behavior and coloration. However, and most importantly, some effects of the acute exposure persisted during the long-term phase. No effects of pollutant exposure, either of the acute or long-term phase, were observed after a recovery phase, indicating ability for recovery dynamics.

### 4.1. Acute effects

An acute exposure to paracetamol had a noticeable impact on behavior and coloration, as shown in other studies (Cedron et al., 2020; Matus et al., 2018). Indeed, exposed fish showed decreased behavioral complexity, and maximal swimming speed, but higher activity windows, probably linked to an initial stress response and neurotoxic disruption (Pereira et al., 2018). Exposed individuals also expressed lower brightness, UV luminance, and increased hue, when confronted to a black background during 30
minutes. Lower brightness, UV luminance, and increased hue might all allow camouflage on dark substrates, supporting the assumption that pollutant exposures are enhancing coloration by black melanin (Lifshitz and St Clair, 2016). Pollutants are also emphasized to modify hue and saturation of individuals (Larramendy, 2017; Ujhegyi and Bókony, 2020). Conversely, after 30 seconds on a white background, when coloration is expected to adaptively lighten, exposed individuals expressed a lower change in hue and in UV saturation. Therefore, while baseline coloration might be adapted to dark substrate, chubs can no longer mobilize pigments to achieve rapid color change. Such process imply either blocking processes of melanin dispersion, or the breakdown or dispersion of existing melanin (Kindermann and Hero, 2016). Our results emphasize that, as a stress response, individuals might mobilize more energy into baseline coloration than into behavioral escape. This response is strengthened by acanthocephalan presence, suggesting that energetic costs of parasitism might mainly influence rapid adjustments rather than longer term responses.

Overall during the acute phase, the presence of acanthocephalans did not modify these effects, suggesting that, during acute paracetamol exposure, parasitism might not attenuate adverse effects of pollutants, while such attenuation had been shown for oxidative stress (Brázová et al., 2021; Molbert et al., 2021, 2020). Our results suggest that the effects on behavior and coloration are less easily mitigated by acanthocephalan infection than physiological parameters. A threshold above which acanthocephalan does no longer have benefits effects might also exist, because of high toxicity and comparatively low accumulation benefits (Goutte and Molbert, 2022).
## 4.2. Long-term effects

After the long-term exposure to paracetamol, no behavioral effects were observed as the result of long-term exposure alone, which suggest that prolonged exposure do not necessarily lead to behavioral disturbances. A similar pattern was found in other studies considering cholinesterase (Pereira et al., 2018), a proxy of neuronal activity. If pharmaceutics, including paracetamol, are often found to induce neurotoxic disruptions (Iqubal et al., 2020; Vaudin et al., 2022), this might mainly be in response to higher concentrations. No change in baseline coloration or change in rapid change coloration can be attributed to long-term exposure alone, as shown in another study considering fluoxetine and conducted across 5 years (Aich et al., 2024), which highlights varying responses across time and possible acclimation. However, in our experimental setup, this pattern is still influenced, after long-term exposure, by acute exposure.

## 4.3. Acclimation and recovery

After the long-term phase, behavior was still influenced by individuals' acute exposure. Indeed, individuals exposed to paracetamol during acute exposure (groups PP and PC) expressed higher speed, activity, and total number of activity windows at the end of the long-term exposure, as compared to groups exposed to a control during acute exposure (groups CP and CC). Interestingly, while it might be a prolongation of the effect found just after acute exposure (group P), it is noteworthy that these effects are not found just after acute exposure, suggesting on the contrary a potential
compensatory effect (Berman et al., 2009). However, the effects of acute exposure on coloration after the long-term phase (groups PP and PC) were similar to those found just after acute exposure (group P). We evidenced lower brightness and UV luminance (as compared to groups exposed to a control during acute exposure, groups CP and CC), when confronted to a black background during 30 minutes, and individuals struggling to lighten their coloration effectively, showing reduced brightness and UV saturation shifts, when confronted to a white background. The acute phase did not meaningfully prepare the fish for sustained exposure, but exerted long term effects on individuals (Hawkey et al., 2020), particularly regarding coloration. Our findings suggest that acute exposure to substances like paracetamol could lead to lasting changes in behavior and coloration, which might have specific consequences for natural populations, particularly in terms of conspicuousness to predators. Additionally, the minimal influence of behavioral responses after long-term exposure at low doses suggests that the acute exposure does not fully prepare individuals for prolonged environmental challenges, potentially leaving them less resilient in fluctuating or long-term stress scenarios common in natural ecosystems. While moderate, the long-term effect of pollutants could accumulate in populations under frequent or widespread exposure, subtly shaping ecological interactions and fitness outcomes over time.

Despite the long-lasting effect of acute exposure, we highlighted that, during the recovery phase, behavioral and physiological parameters largely converged across treatments, indicating a recovery from prior pollutant exposure (three weeks after last exposure). Comparisons in all behavioral and coloration parameters showed no
significant differences between groups, suggesting that recovery processes effectively counteract prior exposure effects (Nunes et al., 2017), also highlighting the lability of the effects on both behavior and coloration. While another study had shown that 14-day recovery period after exposure of chub to tramadol, another analgesic drug, does not allow the behavioral variables to return to normal (Santos et al., 2021), we highlighted that this effect might either be drug dependent, or dependent to exposure and/or recovery duration. The restoration of normal behavior and color change responses has significant implications for survival, as it suggests that fish can eventually restore physical capacity (Beamish, 1978), ability to navigate and adapt to their environment (Baker et al., 2018), as well as response to potential predation, as well as effective camouflage strategies, reducing predation risk in fluctuating environments (Duarte et al., 2017). Overall, we demonstrated that the impact of pharmaceutical pollution on aquatic organisms can be mitigated over time.

## 4.4. Acanthocephalans effect

Parasitized individuals generally exhibited increased behavioral activity and speed, as well as lower brightness, UV luminance, and higher hue, suggesting that the parasites might influence stress responses and/or energy allocation (Connors and Nickol, 1991). Because of the carotenoid-based coloration of acanthocephalan parasites (Kaldonski et al., 2009) and their ability to sequester carotenoids (Balanean, 2010), one may predict impairment in parasitized chubs to exhibit yellow-orange coloration. However, in our study, parasitism mainly induce subtle changes in brightness, UV luminance, and hue, which may impact individuals ability to camouflage under fluctuating environments
(Duarte et al., 2017). Parasites alone might thus influence fish behavior and coloration, despite having no influence on the overall impact of paracetamol on fish, contrarily to what is found for more persistent pollutants (Goutte and Molbert, 2022; Molbert et al., 2021). Nevertheless, some effects can be different according to the phase of exposure, highlighting individual dependent effects (Franceschi et al., 2008), or seasonality in the effects of acanthocephalans (Benesh et al., 2009; Sures, 2014). However, interpretation of these results in terms of parasite implications is limited due to the low number of parasites found in individuals (as compared to individuals originating from other sites, A. Bauer pers. Obs.), and because of the environmental origin of infections. Host physiology, immune status, and environmental conditions may all contribute to variations in parasite load, making it challenging to disentangle direct causal relationships (Fleury et al., 2009; Patz et al., 2000), but we believe that selection fish originating from the same sites in each group may reduce these effects. Still, to address these limitations, future studies should control the infection for individual, as well as explore the broader implications of such interactions across different types of contaminants and ecological settings.

**5. Conclusion**

Paracetamol exposure initially led to noticeable changes in behavior and coloration, mainly after acute exposure, and which are still express after long-term exposure, highlighting that a previous exposure might influence ulterior exposure to pollutants. Our results suggest that parasitism may not help mitigate effects of paracetamol on behavior and coloration. Most importantly, we found no long-term impacts of
paracetamol after the recovery phase, indicating that recovery over time can occur,
and emphasizing the importance of considering different exposure phases when
assessing the ecological consequences of environmental contaminants.
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Caption to Figures

**Figure 1** - PCA for ADG) behavioral metrics, BEH) coloration metrics after 30 minutes on a black background (T0) and CFI) after 30 seconds on a white background (T30s), for the ABC) acute, DEF) long-term, and GHI) recovery phases. The coordinates on the first (PCA1) and second axis (PCA2), explaining most part of the variation, were extracted to analyze the variation of all variables together.

**Figure 2** - After the acute exposure to paracetamol, effect of treatment (individuals exposed to control or paracetamol) and acanthocephalan presence on A) behavior PCA1, B) behavior PCA2, C) dorsal coloration PCA1 at T0, D) dorsal coloration PCA2 at T0, E) dorsal coloration PCA1 at T30s, F) dorsal coloration PCA2 at T30s. Variables changing with each PCA are given on each axis and in Figure 1ABC. Mean ± SE.

**Figure 3** - After the acute and long-term exposure to paracetamol, effect of treatment and acanthocephalan presence on A) behavior PCA1, B) behavior PCA2, C) dorsal coloration PCA1 at T0, D) dorsal coloration PCA2 at T0, E) dorsal coloration PCA1 at T30s, F) dorsal coloration PCA2 at T30s. Variables changing with each PCA are given on each axis and in Figure 1DEF. CP: individuals exposed to a control during the acute phase, and to paracetamol during the long-term phase; PP: paracetamol during acute and long-term phases; CC: control during acute and long-term phases; PC: paracetamol during the acute phase, and control during the long-term phase. Mean ± SE.

**Figure 4** - After the acute and long-term exposure to paracetamol followed by recovery, effect of treatment and acanthocephalan presence on A) behavior PCA1, B) behavior PCA2, C) dorsal coloration PCA1 at T0, D) dorsal coloration PCA2 at T0, E) dorsal coloration PCA1 at T30s, F) dorsal coloration PCA2 at T30s. Variables changing with each PCA are given on each axis and in Figure 1GHI. CPR: individuals exposed to a control during the acute phase, and to paracetamol during the long-term phase; PPR: paracetamol during acute and long-term phases; CCR: control during acute and long-term phases; PCR: paracetamol during the acute phase, and control during the long-term phase; all followed by recovery. Mean ± SE.
<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure 4</figcaption></figure>
Caption to tables

**Table 1** - Experimental design. “C”= exposure to control, “P”= exposure to paracetamol. During acute exposure, paracetamol was applied at 16 µg.g⁻¹, once a day during two days. During long-term exposure, paracetamol was applied at 1.6 µg.g⁻¹, once a week over three weeks.
Caption to appendices

Appendix A - (A) Side view and (B) aerial view of the mesocosms used in this experiment

Appendix B - Statistical results for the effect of treatment, acanthocephalan biomass, and their interaction, after acute, acute and long-term, or acute, long-term and recovery exposure. After long-term exposure and after recovery, the effects of treatments were separately tested for acute exposure and long-term exposure. We thus tested for the effect of acute exposure, long-term exposure, acanthocephalan biomass and their interaction (triple interaction acute exposure\* long-term exposure \* acanthocephalan biomass).

Appendix C - Post hoc analyses for the interaction between treatment and acanthocephalan presence and between acute and long-term exposure.