# Fate and impact at molecular level of diatrizoic acid and iohexol contrast agents in Dreissena polymorpha mollusks

Carine Arnaudguilhem, Gaëlle Daniele, Christelle Bonnefoy, Odile Dedourge-Geffard, Emmanuelle Vulliet, Alain Geffard, Sandra Mounicou

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## To cite this version:

Carine Arnaudguilhem, Gaëlle Daniele, Christelle Bonnefoy, Odile Dedourge-Geffard, Emmanuelle Vulliet, et al.. Fate and impact at molecular level of diatrizoic acid and iohexol contrast agents in Dreissena polymorpha mollusks. Environmental Science and Pollution Research, 2024, 31 (49), pp.59507-59520. 10.1007/s11356-024-35181-x. hal-04764488

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Distributed under a Creative Commons Attribution 4.0 International License
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Fate and impact at molecular level of diatrizoic acid and iohexol contrast agents in *Dreissena polymorpha* mollusks

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Carine ARNAUDGUILHEM<fnref n="1" />*, Gaëlle DANIELE<fnref n="2" />, Christelle BONNEFOY<fnref n="2" />, Odile DEDOURGE-GEFFARD<fnref n="3" />,
Emmanuelle VULLIET<fnref n="2" />, Alain GEFFARD<fnref n="3" />, Sandra MOUNICOU<fnref n="1" />

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**Corresponding author**

Carine Arnaudguilhem: carine.arnaudguilhem@univ-pau.fr +33540175069

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**Abstract**

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Iodinated contrast media (ICMs) used in X-ray imaging for medical diagnostics are released into wastewater and then encountered in river water at concentrations ranging from several dozen to hundreds of µg/L, and even thousands of µg/L in hospital effluents. ICMs are considered as emerging pollutants as their occurrence and impact on ecosystems and the environment are poorly documented. Even if they are considered inert for humans, aquatic organisms are continuously exposed to ICMs, and their potential deleterious effects are therefore questioned as we have recently demonstrated that they enter into organisms such as the zebra mussels. To answer this question, *Dreissena polymorpha* were exposed to two ICMs of different osmolality, diatrizoic acid and iohexol, at an environmental concentration (100 µg/L) for 21 days before a depuration phase of 4 days. The occurrence, fate and impact of both ICMs in these organisms were studied using a metallomic approach. Thus, iodine as well as endogenous copper and zinc were quantified and analyzed in cytosolic extracts of digestive glands, gills and gonads of mussels by size exclusion chromatography coupled to ICP MS. This work shows that a subcellular fractionation is necessary to distinguish variations in total elements content. The cytosolic iodoproteins chromatographic pattern was consistent for the three organs and confirmed the presence of ICMs in cytosols. Additionally, this exploratory work tends to show a weak biological effect of ICMs with a substantial variation of the profile of Cu-binding proteins mostly in the gill cytosol and to a lesser extent, in the digestive gland cytosol.

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**Keyword**

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<footnote n="1">¹ Université de Pau et des Pays de l'Adour, E2S UPPA, CNRS, IPREM, Pau, France</footnote>

<footnote n="2">² Universite Claude Bernard Lyon1, ISA, UMR 5280, CNRS, 5 rue de la Doua, 69100 Villeurbanne, FRANCE</footnote>

<footnote n="3">³ Université Reims Champagne Ardenne, UMR-I 02 SEBIO (Stress Environnementaux et BIOsurveillance des milieux aquatiques), UFR Sciences Exactes et Naturelles, Campus Moulin de Housse, BP 1039, 51687, Reims cedex 2, France</footnote>
## 37 Introduction

For many years, the contamination of water by pharmaceutical and personal care products has been demonstrated. Among these chemicals, iodinated contrast media (ICMs) are used in angiography to artificially enhance contrasts in tissues, organs, tumors and vascularization of lesions. The annual production, estimated at 5,000 tons (Fabbri et al., 2016), is increasing due to the growing number of people with access to healthcare. ICMs are designed to be as inert as possible, hardly metabolized and with a short half-time in the human body (Schobel and Gunzel, 1993). They are therefore rapidly excreted (80% within the first 24 h) and released into wastewater. Nevertheless, they are poorly eliminated in wastewater treatment plants (usually 10%) due to their high polarity (Mauch et al., 2023) with the exception of iopromide which can be degraded to 60-70% (Pal et al., 2014). Hence, ICMs are encountered at µg/L levels in surface water (Duirk et al., 2011; Xu et al., 2017) and at around 2400 µg/L in hospital effluents (Mendoza et al., 2015; Weissbrodt et al., 2009), thus becoming potential pollutants for aquatic fauna.

Mollusks, especially mussels, are frequently used to monitor freshwater contamination. Widely spread, they are important bioaccumulators due to their ability to filter large volumes of water. They can accumulate organic compounds such as polycyclic aromatic hydrocarbon (PAHs) (Teunen et al., 2021), polychlorinated biphenyl (PCBs) and polybrominated diphenyl ethers (PBDEs) (Bervoets et al., 2004), drugs (Bringolf et al., 2010; Rafiq et al., 2023) as well as inorganic compounds (cadmium (Cd), arsenic (As), copper (Cu) and zinc (Zn) (Bervoets et al., 2004; Voets et al., 2009). However, very few studies concern ICMs, which may be easily absorbed by living organisms due to their high polarity (Log Kow from -3.05 to 3.3). Arnnok et al. (2017) detected the presence of iopamidol in brain and gonads of fish with concentrations up to 1059 and 750 ng/g, respectively, whereas it was detected at trace levels in water (23 ng/L). Recently, Mauch et al. (2023) showed that the presence of mussels in combination with periphyton improved the removal of iopromide from wastewater, highlighting the uptake of ICM by the mollusk. Moreover, in a previous study, we showed that diatrizoic acid (DTZ) and iohexol (IO), two ICMs of different osmolality commonly detected in river water, were able to enter in mussels after exposure to environmental (100 µg/L) and high (up to 1000 µg/L) concentrations of ICMs (Daniele et al., 2023): their uptake can be monitored directly by determining the total iodine content after 4 or 7 days of exposure depending on the ICM, in different organs (digestive gland, gill and gonad). Furthermore, although both ICMs entered into the digestive gland, their bioaccumulation factors were very low, leading us to the hypothesis that the bivalve could use ICMs as a source of iodine (Daniele et al. 2023). Indeed, ICMs have an aromatic structure similar to that of thyroid hormones (thyroxine, triiodothyronine) and may therefore interfere with these natural hormones, potentially causing biological effects. Singh et al. (2019) suggested that ICMs can be considered as endocrine disrupting chemicals as they are able to bind to nuclear receptors in the endocrine system. As thyroid hormones play a major role in metabolism, growth, gene expression and reproduction (Soldin et al., 2008), any change in their homeostasis can negatively affect the development of the organism. A recent study by Bolten et al. (2023) supports this hypothesis showing that iopamidol was implicated in mitochondrial damage in zebrafish larvae. Therefore, ICMs could induce biological effects with a modification of iodine biochemistry at the molecular level and may affect, additionally, the homeostasis of essential and endogenous elements (i.e., Cu and Zn).

In this context, the goal of this study was to evaluate the impact of two ICMs (DTZ and IO) on a freshwater bivalve, *Dreissena polymorpha*. For that purpose, we carried out a comprehensive study using a metallomic approach to
investigate potential iodine interactions with cytosolic proteins, but also to analyze ICMs' impact on essential and endogenous elements such as Cu and Zn. Mussels were exposed in laboratory-controlled conditions during 21 days to both ICMs at 100 µg/L, corresponding to a high environmental level, followed by 4 days of depuration. Iodine, Cu and Zn speciation analysis were performed on three central organs in which iodine was preferentially found, i.e., gills as the major route of entrance for contaminants, digestive glands as the main detoxification organ and gonads involved in reproduction. After a subcellular fractionation, gills, digestive glands and gonads cytosols were analyzed by size exclusion chromatography - UV - inductively coupled plasma mass spectrometry (SEC-UV-ICP MS) to study (i) the potential interaction of ICMs with proteins and their potential differential expression, and (ii) the impact of ICMs exposure on the homeostasis of Cu and Zn bound to proteins and enzymes.

## Material and methods

### Chemical reagents

Diatrizoic acid (DTZ) and iohexol (IO) used for mussels' exposure (≥ 98.0 % purity), ammonium acetate, ammonia (ACS reagent, 28.0-30.0% NH₃), tetramethylammonium hydroxide (TMAH) and formic acid (FA) (purity >98%) were purchased from Sigma-Aldrich (St Quentin Fallavier, France) and methanol (HPLC grade, Scharlau) from Atlantic Labo (Bruges, France). Iodine, copper, zinc and tungsten (W) standards (1000 mg/L) were obtained from SCP sciences (Courtaboeuf, France). For exposure of mussels in tanks, stock solutions of DTZ and IO at 1000 mg/L were prepared in Cristalline Aurele drinking water (pH=7.3; Ca²⁺: 68 mg/L, Mg²⁺: 11 mg/L; Na⁺: 21 mg/L; K⁺: 2 mg/L, NO₃⁻ <1 mg/L). All other reagents used for analysis and samples dilution were prepared in Milli-Q water (resistivity ≥ 18.2 Ω cm) from a Milli-Q Elix water purification system (Millipore, Molsheim, France).

### Chronic exposure of Dreissena polymorpha

*Dreissena polymorpha* mollusks (20-22mm shell length) were collected from the Lac du Der (51290 Giffaumont-Champaubert, France, 48°33'035" N; 4°45'011" E) and were acclimated in the laboratory before experiment, during three weeks in darkness at 14 °C, in a Cristalline Aurele drinking water (renewed twice a week). They were fed twice a week with two microalgae (*Scenedesmus obliquus* and *Chlorella pyrenoidosa* from Greensea (Mèze, France) at a rate of 2 000 000 of microalgae per mussel).

Mussels (n=160 per exposure condition; a larger number of mussels than those required for this study was exposed for metabolomic study (not presented here)) were exposed in 4 L glass tanks (300Lx220Wx240H mm corresponding to a mussel density of 40 per liter) to 100 µg/L DTZ or IO during 21 days in static conditions, followed by 4 days of depuration. A control experiment with non-exposed mussels (n=160 in 4 L tank) was also conducted in the same conditions. As for acclimatization, all experiments were carried out in Cristalline Aurele drinking water, and mussels were fed every two days with microalgae. Water was renewed every two days and was spiked with 400 µL of a fresh stock solution of DTZ or IO at 1 g/L. An aliquot (5 mL) of water was collected before and after each water renewal to monitor the ICMs concentration. For each experiment (control, DTZ and IO exposures), three mussels were collected after 7, 14 and 21 days of exposure, and on day 22 and 25 during the depuration phase. Three control mussels were also collected at the beginning of the experiment (day 0). Gills, digestive glands and gonads were recovered in 1.5 mL Eppendorf tubes, weighted and stored at -80 °C until analysis.
117 Quantification of ICMs in water

118 Concentration of ICMs was monitored before and after each water renewal. DTZ and IO were quantified by LC-
119 MS/MS on a Xevo TQ-S triple-quadrupole mass spectrometer equipped with a StepWave ion guide and coupled
120 to a H-Class UPLC (Waters, Saint-Quentin-en-Yvelines, France), following a method described elsewhere
121 (Daniele, et al., 2023). Briefly, samples were analyzed on a Kinetex PS-C18 (100 × 2.1 mm; 2.6 µm) column after
122 a 10-fold dilution and addition of 50 µL phenacetine-ethoxy-1-¹³C as internal injection standard. Separation was
123 conducted following a gradient composed of H₂O with 0.1% formic acid and acetonitrile with 0.1% formic acid.
124 Detection was performed using electrospray in the positive mode and multiple reaction monitoring following two
125 transitions (m/z 614.8 → m/z 233.1 and m/z 614.8 → m/z 361.0 for DTZ, m/z 822.0 → m/z 603.0 and m/z 822.0 →
126 m/z 375.1 for IO).

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128 Cytosol extraction

129 Samples of gills, digestive glands and gonads (n=3 per sampling days), were ground in 100 mM ammonium
130 acetate, pH 7.4 (adjusted with ammonia) at 3,000 rpm using a G50 electric homogenizer (G-Biosciences, VWR,
131 France). The volume used for extraction was adapted to the sample size: 300 µL for gonads (average sample
132 weight: 33 mg) and 500 µL for digestive glands and gills (average sample weight: 62 mg and 54 mg, respectively).
133 Samples were kept in ice to prevent warm-up during grinding. An aliquot (400 µL) of the extract was
134 ultracentrifuged at 120 000 g at 4 °C during 45 min, on a HIMAC CS 120GX ultracentrifuge (Hitachi, Japan) to
135 recover cytosol. Cytosolic extracts, pellets, and remaining samples (homogenate) were separately collected in
136 tubes for total elements determination and speciation analysis.

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138 Alkaline extraction for iodine and metal total content determination

139 Iodine, Cu and Zn total contents were quantified in the cytosol, pellet and in the remaining homogenate. The last
140 two were extracted with 200 µL and 400 µL of 5% (TMAH) respectively, whereas cytosol (50µL aliquot) was
141 extracted with 10 µL of 25% TMAH to obtain a final TMAH concentration of 5%. Samples were placed on a water
142 bath at 90 °C during 3 h using a DigiPREP MS system (SCP Science, Courtaboeuf, France). After cooling down,
143 samples were centrifuged at 8,000 g for 15 min and the supernatant was diluted 50 times in H₂O to reduce the
144 TMAH concentration to 0.1%. An internal standard (W) was added to obtain a final concentration of 0.5 µg/L for
145 ICP MS analysis. An aliquot of 50 mg of a Certified Reference Material (ERM BB422 from LGC Standards,
146 Molsheim, France; certified iodine concentration: 1.4 ± 0.4 µg/g) was also extracted simultaneously with 500 µL
147 of 5% TMAH for quality control (average quantified iodine concentration 1.2 ± 0.1 µg/g).

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149 ICP MS total and speciation analysis

150 A 7700x ICP MS from Agilent Technology (Tokyo Japan) equipped with a Scott spray chamber (2 °C), a
151 Micromist nebulizer (400 µL/min) and nickel cones was used. Torch position and lenses voltages were optimized
152 daily and performances were checked with a tuning solution of 1 µg/L of Li, Y, Ba, Tl and Ce in 2% HNO₃. The
153 RF power of the argon plasma was set to 1550 W, the nebulization gas flow to ca 1.09 mL/min, and the collision
154 cell filled with H₂ gas (3.8 mL/min) to remove possible interferences. After tuning and before analysis, the system
155 was washed for 15 min with H₂O and 0.1% TMAH successively to lower the iodine background.
To determine total elemental concentration, alkaline extracts were analyzed by ICP MS using the conditions described above. Quantification was performed by internal calibration for ¹²⁷I, ⁶³Cu, ⁶⁵Cu, ⁶⁴Zn and ⁶⁶Zn isotopes, and ¹⁸⁴W was used as internal standard (0.09 s/isotope, 20 replicates, 100 sweeps/replicate). The 8-points calibration curve, ranging from 0 to 5 µg/L for iodine and from 0 to 50 µg/L for Cu and Zn, was prepared in 0.1% TMAH. The final concentrations (average concentration in the case of two isotopes) were normalized by the initial mass of the fresh organ weighted after dissection.

The analysis of iodine, Cu and Zn containing proteins was performed by Size Exclusion Chromatography coupled to a UV detector and to the ICP MS (SEC-UV-ICP MS). A 1260 Infinity Bio-inert liquid chromatography system equipped with a Diode Array and Multiple Wavelength Detector was used. Size Exclusion Chromatography was carried out with a Superdex 200 (10/300GL column 30 cm × 10 mm, 8.6 µm particle; GE Healthcare, Orsay, France). The column was calibrated with a mixture of proteins ranging from 1.3 kDa (cobalamin) to 670 kDa (thyroglobulin). Then, 100 µL of sample was injected (n=1/sample) and the elution was performed in isocratic mode using 0.7 mL/min of 100 mM ammonium acetate, pH 7.4 (adjusted with ammonia) during 60 min. Proteins elution from SEC was followed on-line at 280 nm. The elution of ¹²⁷I, ⁶³Cu and ⁶⁴Zn containing molecules was monitored by ICP MS with an integration time of 0.1 s/isotope.

Speciation of low molecular weight molecules (< 2 kDa) containing iodine was performed by Reversed Phase Liquid Chromatography (RPLC) on an Ultimate 3000 UPLC pump (Dionex, Paris, France) coupled to the ICP MS. A Nucleodur $\pi^2$ (125 × 2 mm, 3 µm) column (Macherey Nagel, Hoerdt, France) maintained at 20 °C was used. Cytosolic extracts (50µL) were centrifuged (14,000 g, 4 °C, 45 min) using a 2 kDa cut-off filter (Sartorius Vivacon Hydrosart, VWR, Fontenay sous Bois, France) to remove proteins fraction and 10 µL of the filtrate was then injected (n=1/sample) into the column. Separation was performed at 0.4 mL/min with 0.01% FA in H₂O (A) and MeOH (B) using the following gradient: 0 min: 95% A/5% B, 25 min: 100% B, 28 min: 95% A/5% B, 31 min: 95% A/5% B. The introduction of organic solvent into the ICP MS required to cool down the Scott chamber to -5 °C, the use of Pt cones, the introduction of 8% O₂ in the Ar sample gas (ca 0.75 mL/min) and the increase of plasma RF power to 1600 W. Acquisition was performed on ¹²⁷I, ⁶³Cu and ⁶⁴Zn isotopes with an integration time of 0.1 s/isotope.

## Data processing

Data were processed using Excel software. The concentrations in the whole organs were determined by summing the amount in each of the three fractions (pellet, cytosol and remaining homogenate).

All SEC-UV-ICP MS and RPLC-ICP MS chromatograms and peak areas were normalized by the fresh sample weight. To note that the relative proportion of an element in the proteins fraction was determined without considering the MW range where ICMs elute.

All data (concentrations, amounts, relative percentages) were compared using One-way ANOVA followed by Tukey's Honest Significant test (Tukey's HSD) with a risk α of 5% (R software).

## Results and discussion
In our previous study (Daniele et al., 2023), we showed that iodine accumulated in gills, digestive glands and gonads after an acute exposure experiment (i.e., 100 and 1000 µg/L) for 4 and 7 days (Daniele, et al., 2023). Here, to go further, we performed a chronic exposure at 100 µg/L for 21 days to study the biological effects of ICMs uptake by determining the iodine speciation by SEC-ICP MS (notably its distribution among proteins), and the total iodine content in the different fractions.

To compare the uptake of both ICMs in organisms, the concentration of each molecule in control and exposure waters was checked every 2 days, before and after each renewal. In control, traces of both ICMs were detected at concentrations close to the limit of quantification (DTZ LOQ: 0.1 µg/L and IO LOQ: 0.8 µg/L): DTZ was quantified in 6 samples at concentrations above LOQ (average concentration: 0.45 ± 0.7 µg/L) and IO was detected only once at 1.5 µg/L. In exposure waters, the ICMs concentrations remained stable throughout the exposure suggesting that there is no abiotic loss: average DTZ concentration was 91 ± 7.5 µg/L (after change) and 88.1 ± 13.4 µg/L (before change) corresponding to 56.4 ± 4.6 and 54.6 ± 8.3 µg/L of iodine. The average IO concentration was 124.9 ± 24 µg/L and 120.8 ± 23.7 µg/L after and before change respectively (corresponding to 57.9 ± 11.1 and 56 ± 11 µg/L of iodine). Iodine concentrations were similar for both exposures conditions.

## Iodine distribution within digestive glands

### Total iodine concentration in digestive glands

According to our previous study (Daniele et al., 2023), the digestive gland (DG) appeared to be an organ of interest as it accumulates iodine the most. In whole organ (Fig 1a-left column), iodine concentration does not differ significantly between control and exposed mussels. Indeed, the maximum mean concentrations are 137 ± 43 µg/kg in control (day 22), against 171 ± 76 µg/kg and 160 ± 41 µg/kg in organisms exposed to 100 µg/L of DTZ and IO respectively (day 21). However, looking at the subcellular level (Fig 1a-right column), an increase of iodine concentration is observed from day 7 of exposure in the cytosol. For DTZ exposure, the iodine concentration remains quite stable until day 22, whereas for IO exposure, a continuous increase is observed until day 14 before stabilizing until the end of experiment. This suggests a slower uptake of IO than DTZ which was also noticed in our previous study. In the cytosol, the difference in iodine content is more significant with an average maximum concentration of 42 ± 7 µg/kg and 57 ± 6 µg/kg for DTZ and IO exposures respectively, compared to 39 ± 7 µg/kg for the control. Furthermore, during the depuration phase, we observe a decrease of iodine concentration suggesting an elimination of ICMs. In pellets, no difference was observed between control and exposed mussels (Fig 1a SI).
<figure><img src="image_3.png" /><figcaption>**Fig 1:** Iodine levels in whole organ (left column) and cytosol (right column) of a) digestive glands, b) gills and c) gonads of *Dreissena polymorpha* (n=3/sampling date). Normalized mass: amount of iodine (ng) normalized by the fresh initial mass of the organ (g). To note: outlier high concentrations were obtained for one control sample on day 22 in the entire gonad, and for one sample exposed to DTZ on day 25 in both matrices. Dark blue line: control on day 0; blue: control; red and green: DTZ and IO exposures respectively. * Indicates a significant difference with a risk of 5% between control and exposed organisms</figcaption></figure>

### Iodine speciation in cytosolic fraction of digestive glands

A representative iodine SEC-ICP MS chromatogram of digestive gland cytosol is shown in Fig 2a for control, DTZ and IO exposures on day 21. The UV signal at 280 nm shows the elution of proteins over the entire mass range of the column. Except for the iodine peak at 30 min corresponding to small molecules (< 1300 Da), iodine profiles are similar for control and exposed mussels. Four molecular weight (MW) fractions of iodine containing proteins at low abundance are detected: fraction 1 (High MW): >306 kDa (elution time 10.1-15.7 min), fraction 2: 124-31.5 kDa (elution time 18-21.5 min), fraction 3: 31.5-14 kDa (elution time 21.5-23.5 min) and fraction 4: 14-4.5 kDa (elution time 23.5-26.4 min). Due to the lack of literature data and to the weak resolution of the chromatography used, it is difficult to go further into the identity of the proteins binding iodine at this level of investigation. Nevertheless, in the HMW region, globulin binding iodine might be expected. Moreover, thyroglobulin (an iodoprotein) seems to play a key role in mussel development as one containing domain has been identified in the shell matrix of blue mussel larva (Carini et al., 2019). Considering the peak areas of the entire
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chromatograms (Fig 2a SI), iodinated proteins are slightly more abundant in controls than in exposed mussels with a significant difference only on day 25. Furthermore, there is no difference of the iodine distribution within the protein's fraction, i.e. excluding the dominant iodine peak at 30 min, suggesting the absence of ICMs metabolization after uptake (Fig 2b).

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Concerning the major peak at 30 min (co-eluting with the ICM standards), it is only detected in the exposed groups and it increases throughout exposure leading us to hypothesize the presence of ICMs. Nevertheless, 30 min is also the elution time of iodine hormones (i.e. thyroxine-like considering their MW), so their presence cannot be ruled out. For DTZ, this peak appears on day 7 with a quite stable intensity throughout exposure, then it decreases after 3 days of depuration. For IO, it increases until day 14 and stabilizes until the end of exposure. We note a significant difference between both ICMs on day 14 with an intensity 2 times more important for IO than DTZ. This peak represents 41 ± 9% of the total iodinated molecules for DTZ and 50 ± 8% for IO (average percentage from day 7 to day 21) and it decreases to 14 ± 5% for DTZ and 32 ± 2% for IO on day 25 (data not shown), showing a slower depuration of IO. These results confirm those obtained with the quantification of the total iodine in cytosol. It can be explained by the higher osmolality of diatrizoic acid (2100 mOsm/kg against 640 mOsm/kg for IO) allowing a better diffusion of the molecule through the capillary basal lamina, during both the contamination, and also the elimination phases (Lavigne et al., 2003).

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To confirm the presence of ICMs in the cytosol and to detect any other iodine small molecules, the extracts were analyzed by RPLC-ICP MS for better small molecules selectivity. Two reversed-phase columns were tested (Kinetex C18 100 × 2.1 mm, 2.6 µm and Nucleodur $\pi^2$ 125 × 2 mm, 3 µm) with a gradient of 0.01% FA in H$_2$O and MeOH or acetonitrile. The best separation was obtained with the Nucleodur $\pi^2$ column which combines hydrophobic and $\pi-\pi$ interactions allowing a better retention of aromatic compounds such as ICMs. A 33 min gradient with 0.01% FA in H$_2$O and MeOH was found to be optimal. In these conditions, IO eluted at 6 min (split peak) and DTZ at 13.5 min. An example of the obtained chromatograms of the filtrated cytosol (fraction < 2 kDa) is given in Fig 2c. To check any matrix effect and confirm the retention time of the ICM molecules, the filtrates were spiked with the ICM standards. The chromatograms confirm the only presence of DTZ and IO in exposed mussels and the absence of any other iodinated compounds.

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We can therefore conclude that both ICMs enter in the organisms and in the digestive gland cells, without interacting with proteins, and seem to remain in their native form since no metabolites were detected in the cytosol. Although this study was carried out on a small population of mussels (n=3 per condition) which may limit interpretation, the repetition of the samplings throughout exposures on days 7, 14, and 21 confirm the results.

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Their presence in digestive glands is not surprising as it is the major organ of detoxification in mollusks through intracellular digestion pathway by lysosomes. Furthermore, ICMs are rapidly excreted as soon as organisms are no longer exposed. Overall, these results confirm those obtained in our previous study (Daniele et al., 2023) where we detected the presence of DTZ and IO with a low bioaccumulation factor (BAF<1) in the digestive glands of mussels exposed to 10, 100 and 1000 µg/L of ICMs during 21 days compared to non-exposed ones.

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a) SEC-UV-ICP MS chromatograms

<figure><img src="image_4.png" /></figure>

b)

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

c) RPLC-ICP MS chromatograms

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Fig 2:** Iodine speciation in cytosols of *Dreissena polymorpha* digestive glands (n=3/sampling date)</figcaption></figure>

a) SEC-ICP MS chromatograms in control, DTZ and IO exposed mussels (day 21), b) relative percentages of iodoproteins throughout exposure (i.e chromatogram integration over the protein elution time only), c) RPLC-ICP MS chromatograms in control and exposed mussels (day 21). To note, the peak at 11 min is also observed in blank of 0.01% FA in H₂O and corresponds to a modification of the iodine ionization response with the gradient composition. Dark blue line: control on day 0; blue: control; red and green: DTZ and IO exposures respectively; orange: 280 nm UV signal; grey: control spiked with DTZ and IO. * indicates a significant difference with a risk of 5% between control and exposed organisms (red: DTZ; green: IO), and between DTZ and IO exposed organisms (black)

## Iodine distribution within gills

### Total iodine concentration in gills

As for digestive glands, no significant difference of the total iodine content was observed in entire gills (Fig 1b-left) regardless of the ICMs exposure. In whole organ, maximum average concentrations were of 263 ± 60, 292.5 ± 117 and 300 ± 107 µg/kg for control, DTZ and IO exposed mussels respectively over 21 days of exposure (the large error bars are due to both the biological diversity of the mussels and the small number of samples (n=3 per condition)). However, after 7 days of exposure to ICMs, we can observe an increase of the total iodine in the cytosol (Fig 1b-right) on day 7 for IO exposure and tends to decrease thereafter. For DTZ exposure, iodine concentration tends to increase throughout exposure but the difference with the control is not significant. Maximum concentration was observed on day 22 for DTZ (50.2 ± 28 µg/kg) and on day 7 for IO (61.8 ± 24 µg/kg) compared to 25.1 ± 17 µg/kg for controls. Iodine levels are about two times higher in the entire gills than in digestive glands, whereas they are similar in their cytosol. Furthermore, unlike other organs, pellets of gill extracts contain higher concentrations of iodine than cytosol, but without any difference between control and exposed organs (Fig 1b SI). This suggests a difference in iodine metabolism between gills and digestive glands, i.e. preferentially involving metallothioneins in digestive gland cytosol and Metal Rich Granule in gill pellets.
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*Iodine speciation in cytosolic fraction of gills*

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As shown in Fig 3a, SEC-ICP MS profiles of iodine containing proteins in gills are similar for the control and exposed mussels. They comprise a major peak of HMW proteins (>306 kDa, elution time 10.1-15.7 min) and two small peaks at 20.2 min (present in some samples) and 23.3 min corresponding to 35-95.6 kDa and 9.9-35 kDa proteins fractions, respectively. The abundance (Fig 2b SI) and relative distribution (Fig 3b) of iodine among the protein's fractions do not show any statistical difference, except on day 25, where we observe a significant decrease of the lowest MW proteins (9.9-35 kDa) for DTZ exposure. Beside the protein fraction, we also observe in the chromatogram, a peak at the ICMs elution time (30.1 min) in the exposed groups from day 7 to day 25 (with a significant difference between the two ICMs on days 21 and 22) and also in some control samples with a very low abundance. Moreover, in some samples (11 out of 48), low molecular weight iodine compounds elute at 35 min (chromatogram of IO exposure) and 44.5 min (data not shown). Their interaction with the stationary phase of the column suggests their ionic character. The presence of ICMs in exposed mussels was confirmed by RPLC-ICP MS (Fig 3d). For control samples, no DTZ or IO peaks were detected, either because their abundances are too low to be detected in RPLC-ICP MS, or the SEC-ICP MS peak at 30 min corresponds to another compound. Indeed, several small iodo-compounds can elute at the total volume of the column, both for controls and exposed samples, like hydrophilic compounds, molecules with MW<1.3 kDa such as thyroxin-like hormones found in hemolymph circulating in entire organisms.... Furthermore, no additional iodine peaks were detected in RPLC-ICP MS chromatogram, even for extracts containing small molecules eluting after the total column volume (30 min).

If we compare the total content of iodine in gill cytosols (Fig 1b-right) with the sum of the areas of all the peaks detected in SEC-ICP MS (Fig 3c), some differences can be observed on day 7. Indeed, total iodine content in cytosol after alkaline extraction is increased from control to DTZ and IO exposures on day 7, compared to the stable iodine level determined from SEC-ICP MS chromatograms for the three exposure conditions. This could be explained by the presence of iodate which is retained on the column stationary phase and therefore not detected in SEC-ICP MS analysis. Then, regulation of iodine might occur from the beginning of the exposure, especially with IO. The uptake of ICMs by mussels leading to the release of iodate from biomolecules might be hypothesized. This kind of regulation has already been observed in the case of exposure to high concentrations of essential elements by Voest et al. (2009). They compared the proportion of metals in the biological detoxified and sensitive fractions of Zebra mussels when there are exposed to high concentrations of essential elements (i.e., Cu and Zn) versus a toxic element (Cd). They showed that metal concentration in the potentially toxic sensitive fraction (organelles and heat denaturated proteins) is all the greater as the metal is toxic, suggesting the regulation of a fraction of essential elements above a threshold.
a) SEC-UV-ICP MS chromatograms

<figure><img src="image_10.png" /></figure>

b)

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

c)

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

d) RPLC-ICP MS chromatograms

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

**Fig 3:** Iodine speciation in cytosols of *Dreissena polymorpha* gills (n=3/sampling date). a) SEC-ICP MS chromatograms in control, DTZ and IO exposed mussels (day 21), b) relative percentages of iodoproteins throughout exposure, c) sum of iodine peaks areas detected in SEC-ICP MS, d) RPLC-ICP MS chromatograms in control and exposed mussels (day 21). To note, the peak at 11 min is also observed in blank of 0.01% FA in H₂O and corresponds to a modification of the iodine ionization response with the gradient composition. Dark blue line: control on day 0; blue: control; red and green: DTZ and IO exposures respectively; orange: 280 nm UV signal; grey: control spiked with DTZ and IO. * indicates a significant difference with a risk of 5% between control and exposed organisms (red: DTZ; green: IO), and between DTZ and IO exposed organisms (black)

353 Iodine distribution within gonads

354 Total iodine concentration in gonads

355 The total iodine content in gonads is presented in Fig 1c for the whole organ and the cytosol (left and right columns, respectively) after exposure to both ICMs. No significant difference is observed between control and exposed mussels, either for the whole organ or for the cytosolic fraction. Concentrations are lower than in the two other organs: in the whole organ, maximum average concentrations of 211.5 ± 284 µg/kg (85.2 ± 22 µg/kg if we exclude a control with an outlier concentration), 117.16 ± 85 µg/kg and 95.9 ± 29 µg/kg were quantified for control, DTZ and IO exposed mussels respectively. In the cytosol, average concentrations reach 27.8 ± 5 µg/kg for controls, 38.3 ± 35 µg/kg for DTZ (one sample with a very high concentration on day 25) and 34.1 ± 13.5 µg/kg for IO exposure conditions. Therefore, these data suggest that ICMs exposure might not impact the gonad iodine biochemistry.
Iodine speciation in cytosolic fraction of gonads

Representative iodine SEC-ICP MS chromatograms are presented in Fig 4a. The data highlight the presence of DTZ and IO in the cytosol with the main (ca 70-80%) iodine peak detected at the elution time of ICM standards (30 min). The presence of both ICMs is also confirmed by RPLC-ICP MS analysis (Fig 4c) although the DTZ peak is weakly intense (the absence of memory effect on the column was checked before sample injection by injection of 0.01% FA in H₂O). ICMs are detected from day 7 and are quite stable throughout exposure (Fig 2c SI, MW < 1.3 kDa fraction). They decrease more or less rapidly during the depuration phase: DTZ is almost no longer detected on day 25 (except for the sample with high total iodine concentration leading to the high error bar), whereas IO is excreted more slowly, since it is still detected on day 25 but in low abundance. Beside the ICMs peak, four MW iodoproteins are distinguished: fraction 1 HMW proteins > 306 kDa (elution time 10.1-15.7 min); fraction 2: 34-100 kDa (18.5-21.3 min); fraction 3: 14-34 kDa (21.3-23.5 min) and fraction 4: 4.8-14 kDa (23.5-26.3 min). The similar iodine distribution among these 4 fractions (Fig 4b) under the different exposure conditions (no significant difference except for fraction 3 on day 21 under IO exposure), suggests that they do not interact with cytosolic proteins as supported by the results of iodine total content.

<figure><img src="image_17.png" /><figcaption>**Fig 4:** Iodine speciation in cytosols of *Dreissena polymorpha* gonads (n=3/sampling date). a) SEC-ICP MS chromatograms in control, DTZ and IO exposed mussels (day 21), b) relative percentages of iodoproteins throughout exposure, c) RPLC-ICP MS chromatograms in control, control spiked with 10 µg/L of DTZ and IO, and exposed mussels on day 21. To note, the peak at 11 min is also observed in blank of 0.01% FA in H₂O and corresponds to a modification of the iodine ionization response with the gradient composition. Dark blue line: control on day 0; blue: control; red and green: DTZ and IO exposures respectively; orange: 280 nm UV signal;</figcaption></figure>
387

grey: control spiked with DTZ and IO. * indicates a significant difference with a risk of 5% between control and exposed organisms

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### Impact on homeostasis of copper and zinc

391

It is well known that metal contamination can disturb the interactions between proteins and essential metals.

392

Although ICMs do not seem to impact homeostasis of iodine, except in gills, they may interfere with essential elements such as Cu and Zn, which we also monitored.

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394

*In digestive glands*, we observe a decrease of Cu concentration in the exposed groups compared with the control, in the whole organ (Fig 3a-left SI) on days 21 and 25 and in their cytosol on day 25 (Fig 5a). The average concentrations in cytosol are 1656 ± 341 µg/kg in control, 1164 ± 155 µg/kg for DTZ and 990 ± 267 µg/kg for IO exposures. For Zn (Fig 3a-right SI), no significant difference is observed during exposure to both ICMs (average concentration 3319 ± 825 µg/kg) in whole digestive gland. However, these data have to be taken with caution as Zn can precipitate in alkaline media. Impact of iodine supplementation on the homeostasis of Cu and Zn has already been observed. Indeed, Penglase et al. (2013) showed that cod larvae (*Gadus morhua*) fed with rotifers enriched with high level of iodine had around 16% more Cu than control ones; however, no significant difference was observed for Zn. Conversely, lower contents of Cu were observed in common carp and gilthead sea bream fed with iodine-rich seaweed and selenized-yeast but without any explanation of the underlying mechanism (Barbosa et al., 2020).

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In the cytosol, metals are mostly bound to proteins. Indeed, as shown in Fig 5b-left, Cu is mainly associated with the low molecular weight MTLP fraction (2.5-12 kDa, around 60% of the total proteins), followed by proteins of 12-28.2 kDa (around 15%) and then with proteins >28.2 kDa (around 15%) and with small molecules <2.5 kDa (around 10%). The Cu distribution patterns among proteins (Fig 5c-left) are similar for all exposure times, with the exception of DTZ exposure, where we notice a small decrease of the 12-28.2 kDa proteins from day 14 to day 25. As seen earlier, some iodoproteins are found in this 12-28.2 kDa fraction (with I/Cu peak apex overlapping).

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411

Given the weak response here, we might state that some iodine might disturb Cu regulation in the 12-28.2 kDa proteins fraction leading to the sequestration of Cu in the MTLP fraction which tends to increase simultaneously.

412

413

The role of the metallothioneins in Cu and other metals sequestration and in detoxification processes in the digestive gland of scallops exposed to high level of Cu has already been reported (Sánchez-Marín et al., 2023).

414

415

The Zn-proteins profiles (Fig 5b-right) of the digestive glands of exposed and control mussels are similar. Around 15% of Zn is linked to proteins > 125 kDa, 35% to proteins of 25-125 kDa, 20% to 11.8-25 kDa proteins, 22% to 5.4-11.8 kDa proteins (MTLP fraction) and 8% to small molecules (<5.4 kDa, i.e Zn bound to small proteins/peptides or to some organic ligands). Their distribution is quite similar for both ICMs (Fig 5c-right). Zn bound to medium and high MW (25-670 kDa) decrease on days 7 and 14 and at the same time, Zn bound to small molecules (<5.4 kDa) increased on day 14. For IO, we also observe a decrease in the Zn-MTLP on day 14. This may suggest the implementation of a Zn regulation process due to ICMs exposure.

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<figure><img src="image_18.png" /><figcaption>**Fig 5:** Cu (left column) and Zn (right column) distribution in cytosols of *Dreissena polymorpha* digestive glands (n=3/sampling date). a) Cu and Zn levels in cytosols (normalized mass: amount of iodine (ng) normalized by the initial mass of the organ (g)), b) representative SEC-UV-ICP MS chromatogram in mussel exposed to 100 µg/L of DTZ on day 21, c) relative percentages of Cu and Zn containing proteins. Dark blue line: control on day 0; blue: control; red and green: DTZ and IO exposures respectively; orange: 280 nm UV signal. * indicates a significant difference with a risk of 5% between control and exposed organisms (red: DTZ; green: IO), and between DTZ and IO exposed organisms (black)</figcaption></figure>

*In gills*, uptake of both ICMs has an impact on the homeostasis of Cu and Zn. Indeed, Cu concentrations decrease in the cytosol of exposed groups (Fig 6a-left), while the level of Cu linked to the metal-sensitive enzymes/proteins fraction (i.e., 28.2-670 kDa) increases from day 7 to 22 and simultaneously the level of Cu bound to MTLP decreases (Fig 6c-left). This suggests the presence of free Cu (not detectable by SEC-ICP MS) in the control mussels. This free Cu would be handled by metal-sensitive proteins fraction rather than by the detoxification proteins fraction, either for regulation purpose or for a defense mechanism set-up during ICMs exposure. Conversely, no significant difference is observed in the total Zn concentration in the whole organ and in the cytosol (Fig 3b-right SI and Fig 6a-right). However, interestingly, by looking at the distribution of Cu and Zn within the protein fractions, the same behavior is observed (Fig 6b and 6c) with an increase of both elements in HMW protein fractions and a decrease in the lower ones. Indeed, the levels of Cu and Zn bound to proteins in 28.2-670 kDa and in 25-125 kDa fractions respectively are statistically increased throughout the exposure while both elements are decreased in the MTLP and in the 11.8-25 kDa fraction for Zn. This trend in the regulation of Cu and Zn is all the more highlighted in gill than in digestive gland cytosol. This shows that the uptake of ICMs leads to a modification of Cu and Zn biochemistry of the gill cytosol. These two fractions correspond to the MW range where Cu/Zn-superoxide dismutase (SOD) could be found. Indeed, Cu/Zn-SOD with apparent molecular weight of 130 kDa has
been characterized in cytosolic extract of digestive glands and gills of the blue mussel *Mytilus edulis* in chemically contaminated water (Manduzio et al. 2003) while a partial Cu/Zn-SOD of 130 amino acids was reported for *Dreissena polymorpha* (Geret et al., 2004). Cu/Zn-SOD is of particular interest because of its involvement in the oxidative stress regulation and its potential up-regulation in the case of toxic metal exposure (Barillet et al., 2011; Lerebours et al., 2010). Our data might suggest that ICMs would induce light prooxidant conditions with ROS production leading to an activation of antioxidant defenses. Further investigations would be needed to measure for example enzymes activity.

<figure><img src="image_19.png" /><figcaption>**Fig 6:** Cu (left column) and Zn (right column) distribution in cytosols of *Dreissena polymorpha* gills (n=3/sampling date). a) Cu and Zn levels in cytosols (normalized mass: amount of iodine (ng) normalized by the initial mass of the organ (g)), b) representative SEC-UV-ICP MS chromatogram in mussel exposed to 100 µg/L of DTZ on day 21, c) relative percentages of Cu and Zn containing proteins. Dark blue line: control on day 0; blue: control; red and green: DTZ and IO exposures respectively; orange: 280 nm UV signal. * indicates a significant difference with a risk of 5% between control and exposed organisms (red: DTZ; IO: green), and between DTZ and IO exposed organisms (black)</figcaption></figure>

*In gonads*, Cu and Zn concentrations are not affected by the absorption of ICMs (Fig 3c SI and Fig 7a), as no significant difference between levels is noticed throughout exposure in the entire organ and in the cytosol. From the SEC-ICP MS chromatogram, five Cu-proteins fractions are detected: the 2.5 -12 kDa fraction (MTLP) of highest abundance, followed by < 2.5kDa (LMW complexes), 28.2-670 kDa, 12-28.2 kDa, and finally the high MW > 670 kDa of low abundance (Fig 7b-left). Their distributions are similar (Fig 7c-left) showing the absence of proteins overexpression.
Concerning Zn-proteins pattern, five groups are identified (Fig 7b-right and 7c-right): the most intense are Zn-binding protein fractions in the medium MW range (i.e. 25-125 kDa and 11.8-25 kDa), followed by the MTLP fraction (5.4-11.8 kDa), the HMW fraction (125-670 kDa) and finally the < 5.4 kDa and > 670 kDa fractions of very low abundance. The distribution of the Zn-binding proteins is not impacted by ICMs exposure, with the exception of IO exposure for day 14 which leads to a Zn decrease in the MTLP fraction and an increase in the 25-125 kDa fraction simultaneously, followed by an increase of the HMW fraction (> 125 kDa) on day 21.

Cu distribution

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

Zn distribution

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

**Fig 7:** Cu (left column) and Zn (right column) distribution in cytosols of *Dreissena polymorpha* gonads (n=3/sampling date). a) Cu and Zn levels in cytosols (normalized mass: amount of iodine (ng) normalized by the initial mass of the organ (g)), b) representative SEC-UV-ICP MS chromatogram in mussel exposed to 100 µg/L of DTZ on day 21, c) relative percentages of Cu and Zn containing proteins. Dark blue line: control on day 0; blue: control; red and green: DTZ and IO exposures respectively; orange: 280 nm UV signal. * indicates a significant difference with a risk of 5% between control and exposed organisms (red: DTZ; IO: green), and between DTZ and IO exposed organisms (black)

Conclusion

The quantification of total iodine in the three whole organs did not allow to reveal the presence of ICMs. Only a refine analysis of the cytosolic fractions of the organ highlighted ICMs uptake. Indeed, SEC-ICP MS and RPLC-ICP MS analysis showed the presence of ICMs in the three respective cytosolic extracts from the beginning of exposure, and their rapid excretion after exposure, especially for digestive glands and gonads. Due to their
difference of osmolality, iohexol enters and is excreted more slowly than diatrizoic acid. However, none of them seem to interfere with iodoproteins, since we do not observe significant difference between the control and exposed iodoproteins profiles. Nevertheless, exposure to ICMs tends to modify the homeostasis of Cu and Zn in gills and digestive glands, resulting in a small decrease in total Cu content in the organs and their cytosol, and a modification of the distribution of Cu and Zn containing proteins. In gills, we observe an overexpression of the medium and high MW Cu and Zn containing proteins and a decrease of MTLP for both ICMs, suggesting the implementation of an oxidative stress regulation. In digestive glands, the modification of Cu-protein profiles upon exposure to DTZ led to hypothesize that iodine might disturb Cu regulation in the MW protein (12-28.2 kDa). Zinc regulation is less impacted by ICMs exposure, rather characterized by up-regulation of Zn-binding proteins of MW < 5.4 kDa while Zn-binding proteins of MW > 25 kDa decrease. Overall, ICMs appear to have very weak effects on cytosol biochemistry but measurement of key enzymes (involved in defense mechanisms) should be performed to verify this assumption.

504

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## Data availability statements

591

Data supporting the findings of this study are available within the paper and its Supplementary Information files.

592

Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.

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## Statements & Declarations

596

### Funding sources

597

This work was carried out as part of the “ACTIONS” research project funded by the French National Research Program for Environmental and Occupational Health of Anses (2018/1/222). We thank Aquitaine Region and Feder for funding of the ICP MS instrument through the AQUITRACE project (convention number 20131206001–13010973).

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600

601

### Competing Interests
602 The authors declare that they have no known competing financial interests or personal relationships that could

603 have appeared to influence the work reported in this paper.

604 Author Contributions

605 Carine ARNAUDGUILHEM: Conceptualization, Methodology, Investigation, Writing – Original Draft

606 Gaëlle DANIELE: Funding acquisition, Writing – Review and editing, Project administration

607 Christelle BONNEFOY: Resources, Writing – Review and editing

608 Odile DEDOURGE-GEFFARD: Resources

609 Emmanuelle VULLIET: Funding acquisition, Writing – Review and editing, Project administration

610 Alain GEFFARD: Resources, Writing–Review and editing

611 Sandra MOUNICOU: Conceptualization, Methodology, Writing–Review and editing

612 Ethics Approval

613 Not applicable

614 Consent to Participate

615 Not applicable

616 Consent to Publish

617 Not applicable

618 Statement on welfare of animals

619 Experiments concerning *Dreissena polymorpha* are not submitted for approval by an Ethical Committee. However,

620 the experimental design was defined to use the less as possible of organisms and optimised with others studies.

621