Effects of a gadolinium-based contrast agent detected in wastewater on the clam Ruditapes decussatus: a multi-marker approach

Effects of a gadolinium-based contrast agent detected in wastewater on the clam Ruditapes decussatus: a multi-marker approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of a gadolinium-based contrast agent detected in wastewater on the clam Ruditapes decussatus: a multi-marker approach Aya AOUNI, Noureddine KHALLOUFI, Asma BOUSSELMI, Ateeqah GHAYTH ALZWAWY, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8632544/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The present study is an in-vivo evaluation of the toxicity of Dotarem (DOTA) for marine clam Ruditapes decussatus . Three concentrations of DOTA ( C1 = 12.5 µg.L − 1 , C2 = 25 µg.L − 1 , C3 = 50 µg.L − 1 ) were used for exposure on 7 days. Responses of R. decussatus after its exposure, were monitored using filtration rate, oxidative stress, lipo-peroxidation, neurotoxicity and histopathological markers. Four biomarkers were measured at the gills and digestive gland: two defense biomarkers catalase (CAT) and glutathione-S-transferase (GST), a cellular damage biomarker (MDA) and a neurotoxicity biomarker acetylcholinesterase (AChE). The filtration rate was significantly decreased by exposure to DOTA rising from 8.18 ± 4.22 mg.h − 1 .ind − 1 in the control to 2.03 ± 0.6 mg.h − 1 .ind − 1 in the clams after 7 days of exposure. The results showed that the activities of antioxidant enzymes (CAT, GST) and the cellular damage status (MDA) revealed concentration and organ-dependent responses for DOTA. Acetylcholinesterase activity (AChE) showed a highly significant decrease in the gills, independent of the DOTA exposure concentration, and in the digestive gland depending of dose exposure ( p < 0.0001). This contamination causes histopathological changes in both organs, marked by infiltrations, vacuolizations and cell necrosis. The intensity of these lesions depends on the concentration of this pollutant. Behaviour Dotarem Histopathology Mediterranean clam Multi-markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The Mediterranean Sea, recognized as a biodiversity hotspot, is home to approximately 7.5% of global marine biodiversity (Cantasano, 2024 ). However, it is heavily impacted by human activities (industrial, agricultural, urban, and domestic) that contribute to the contamination of various water bodies with numerous chemical pollutants, including emerging xenobiotics originating from medical and industrial sources. According to the United States Environmental Protection Agency (US-EPA), emerging contaminants are newly identified compounds lacking regulatory status, whose effects on human health and the environment are still poorly understood (Chavoshani et al., 2020 ). The study of the response of living organisms, populations, or communities to stressful conditions (eco-biological approach), or of the impact of contaminants on these organisms (ecotoxicological approach), as well as their biochemical reactions (Dellali et al., 2021 ), constitutes a central axis of ecosystem monitoring. Among the most used sentinel species in this field is the common clam, Ruditapes decussatus (Mansour et al., 2020 ), which is widely consumed by humans in various Mediterranean regions. Its broad distribution, sedentary lifestyle, and low cost make this species one of the most preferred bioindicator taxa for monitoring marine pollution (Velez et al., 2017 ). The use of an integrative approach including biomarker analysis, observation of histological changes in bivalve tissues, and behavioral responses has proven to be a highly relevant method for assessing contaminant impact and determining the health status of individuals and populations (Bousselmi et al., 2024 ). Accordingly, a multimarkers strategy has been implemented across different organs to highlight the effects of xenobiotics on aquatic biota. This strategy includes, in addition to behavioral markers (filtration rate) (Banaee et al., 2024 ), biochemical defense markers (catalase and glutathione S-transferase activities) (Dellali et al., 2021 ), markers of membrane lipid peroxidation (malondialdehyde) (Banaee et al., 2024 ), neurotoxicity biomarkers (acetylcholinesterase activity) (Banaee et al., 2024 ), and histological indicators (Stoyanova et al., 2020 ). Gadoteric acid, a contrast agent marketed under the name Dotarem®, is a gadolinium-based compound chelated with an organic acid. It is an emerging contaminant that have been found in different aquatic ecosystems, including surface, and drinking water (Dekker et al., 2024 ) in many parts of the world. Gadolinium, a metal belonging to the lanthanide family and classified among the rare earth elements, is characterized by its electromagnetic properties. It is used in several fields (Xia et al., 2011 ), notably in medicine (as a contrast agent in MRI, and in X-ray intensifying screens) (Ramirez and Munoz, 2024 ), as well as in industry (as a steel additive) (Islam and Tsnobiladze, 2024 ). The ionic form of gadolinium (Gd³⁺), which acts as an antagonist of the essential calcium ion (Ca²⁺), is responsible for its toxicity. This form (Gd³⁺) inhibits voltage-gated Ca²⁺ channels at micromolar concentrations (Nathalie et al., 2013 ) [13]. Chelated gadolinium is known to be non-metabolizable and, as it circulates in freshwater and marine environments, it can accumulate in the tissues of living organisms (Cesarini et al., 2024 ). Several clinical observations have reported adverse neurological effects, skin and kidney lesions, inflammatory responses, and fibrosis (both short- and long-term) in patients injected with gadolinium (Islam and Tsnobiladze, 2024 ). Presence of Gd³⁺ disrupts physiological mechanisms involving cellular calcium channels, affecting processes such as mitochondrial respiration, myocardial contractility, blood coagulation, and nerve transmission in mammalian cells (Nathalie et al., 2013 ). The accumulation of Gd³⁺ in various tissues, such as bones, liver, and lymph nodes, increases its toxicity (Amet and Deray, 2012 ). Furthermore, several studies have highlighted the potentially toxic effects of gadolinium on bivalves, emphasizing its accumulation and physiological consequences. In the clam Donax trunculus , gadolinium bioaccumulation is proportional to both concentration and exposure duration. This accumulation is associated with notable metabolic changes as well as the induction of oxidative stress, suggesting a biological response to the contaminant (Secco et al., 2024 ). Research on the mussel Mytilus galloprovincialis revealed that elevated water temperature amplifies gadolinium toxicity by intensifying lipid damage at the cellular level. This finding underscores a potential synergistic effect between emerging contaminants and climate warming (Figueiredo et al., 2022 ). A study on freshwater bivalves, Dreissena rostriformis bugensis and Corbicula fluminea , confirmed the ability of these species to bioaccumulate gadolinium in their digestive glands and gills, even when present in complexed forms (contrast agents), highlighting the persistence and bioavailability of gadolinium in the aquatic environment (Perrat et al., 2017 ). As part of monitoring studies on certain Tunisian watercourses, gadoteric acid has been detected in the waters of the Oued Guenniche, one of the tributaries of the Bizerte Lagoon. This study aims to examine the responses of the common clam Ruditapes decussatus following contamination by gadoteric acid (DOTA) under microcosm conditions. These responses were evaluated through: (i) measurement of filtration rate (behavioral), (ii) quantification of specific biomarkers (physiological), and (iii) tissue alterations in the gills, digestive glands, and gonads (histopathological). Materials and Methods Sampling, acclimation, rearing and exposure conditions of clams The clams used in this study were collected from the Bizerte Lagoon (northeastern Tunisia) near the city of Menzel Jemil (37°14’19’’N, 9°54’59’’E). These clams were transported to the laboratory in a cooler containing water from the collection site. In the laboratory, clams were acclimated in glass microcosms for 7 days under carefully controlled physicochemical conditions, temperature (T = 18 ± 2°C) and photoperiod (12h/12h), without feeding. The rearing water, sourced from Rimel beach (Mediterranean Sea), is renewed every 48 hours. Used Gadoteric acid (DOTA) in its commercial form, Dotarem® (0.5 mmol·mL⁻¹). Three tested concentrations: C1 = 12.5 µg·L⁻¹, C2 = 25 µg·L⁻¹, and C3 = 50 µg·L⁻¹. Each experimental condition, including a control, is replicated three times. A total of 65 individuals of homogeneous size (mean size = 38.6 ± 3.4 mm) were distributed into microcosms, with 5 individuals per aquarium containing 1.5 L of seawater, for an exposure duration of 7 days. Filtration Rate The filtration rate (FR) was measured using the method of Coughlan ( 1969 ), which is based on the decrease in the concentration of the dye neutral red in the water column under dark conditions. An individual clam was placed in a beaker containing 100 mL of a 1 g·L⁻¹ neutral red solution. Before introducing the clams into the solution, 10 mL of water was sampled from each beaker to assess the initial concentration (C 0 ). The clams were removed every 30 minutes over a period of 2 hours to evaluate the remaining concentration (C t ). A calibration curve was established using neutral red standards. Optical density at 530 nm was measured using a microplate reader. The filtration rate was calculated using the following equation: FR= [M/nt] log (C 0 /C t ) Where: FR : clearance rate (mg·ind⁻¹·h⁻¹) M : total volume of water (mL) nt : number of clams used at time t (in hours) C 0 : initial concentration of neutral red dye (1 g·L⁻¹) C t : final concentration of neutral red dye at time t Assays of biochemical markers At the end of the experiment, clams were dissected at 4°C. The gills and digestive glands of each animal were carefully removed and stored separately. Protein extraction was performed in phosphate buffer (0.1 M; pH 7.5). The homogenate was then centrifuged at 9000 rpm for 30 minutes at 4°C to obtain the S9 fraction, which was collected in Eppendorf tubes and stored at T= − 80°C for subsequent analyses. Total proteins content was determined using the Bradford method (1976) by measuring absorbance at 595 nm with a spectrophotometer. Catalase activity was measured following the method of Aebi ( 1985 ), which monitors the degradation of hydrogen peroxide by catalase, producing water and oxygen. Absorbance was recorded at 240 nm and activity expressed as µmol.min − 1 .mg − 1 of proteins. Glutathione S-transferase (GST) activity was quantified according to the method described by Habig et al. ( 1974 ). Optical densities were measured at 340 nm, and specific GST activity was expressed as µmol.min − 1 .mg − 1 of proteins. Malondialdehyde (MDA) content, an indicator of lipid peroxidation, was assessed using the method of Buege and Aust (1978), which quantifies thiobarbituric acid reactive substances (TBARS). Absorbance was measured at 530 nm, and results were expressed in µmol.mg − 1 of proteins. Finally, acetylcholinesterase (AChE) activity was evaluated following Ellman et al. ( 1961 ). The absorbance was measured at 412 nm, and activity expressed as µmol.min − 1 .mg − 1 of proteins. Histological Study To perform histological sections, the gills and digestive glands were removed and separated from other tissues. The histological preparation steps were the same as those described by Martoja and Martoja, but using a more modern automated technique, performed at the Department of Pathological Anatomy at the Regional Hospital of Menzel Bourguiba (Tunisia). The gills and digestive glands were first fixed in 5% formalin to preserve cellular structures. The samples were then dehydrated with a series of increasing concentrations of alcohol and toluene and treated with kerosene. They were then cut into 5 µm thick sections with a microtome, placed on slides, and stained with hematoxylin and eosin. The preparations were studied under a ZEISS optical microscope (G×40–100) and photographed with an Axio-Cam 105. Histopathological alterations were semi-quantitatively evaluated by ranking the severity of lesions (grades, 0 (absent), 1 (sometimes), 2 (frequent), 3 (very frequent), and 4 (always present)) as described by Riba et al. [26] and adapted in our study. The damage index is an average arithmetic value obtained from the semi-quantitative evaluation of the lesions for each tissue. Statistical Data Analysis Data from the various measurements are presented on boxplots (min-max, mean ± 2SD). Graphs and statistical analyses were performed using STATISTICA 8 software under Windows. The data was the first tested to evaluate the normality (Shapiro-Wilk test) and homogeneity of variances (Bartlett test). Variations in behavioral and biochemical responses of the clams were analyzed using one-way ANOVA compared to the control group. When significant differences were detected by ANOVA, post-hoc comparisons were conducted using Tukey’s Honestly Significant Difference (HSD) test. A probability level of p < 0.05 was considered statistically significant. Results Effect of DOTA contamination on filtration capacity of R. decussatus The exposure of the clam Ruditapes decussatus to different concentrations of gadoteric acid (C1 = 12.5 µg·L⁻¹, C2 = 25 µg·L⁻¹, C3 = 50 µg·L⁻¹) over a 7-day period revealed a very significant difference compared to the control group ( p = 0.0075) (Fig. 1 ). Indeed, the filtration rate decreased from 8.18 ± 4.22 mg·h − 1 .ind − 1 for the control group to 2.03 ± 0.6 mg·h − 1 .ind − 1 at concentration C2. However, at the highest concentration (C3), the filtration rate nearly returned to the levels observed in the control group (Fig. 1 ). Regarding the temporal dynamics, the filtration capacity exhibited a declining trend, decreasing from 14.10 ± 0.09 mg·h − 1 .ind − 1 after 30 minutes to 4.60 ± 0.01 mg·h − 1 .ind − 1 after 120 minutes in the control group. Exposure to 12.5 µg·L⁻¹ and 25 µg·L⁻¹ of gadoteric acid led to a slight increase in filtration rate over time. At the concentration of 50 µg·L⁻¹, the filtration capacity initially increased during the first 30 minutes, then decreased by the end of the experiment, while remaining higher than the values observed under the other conditions (Fig. 2 ). Physiological responses of R. decussatus to DOTA exposure Catalase activity After 7 days of exposure of R. decussatus to gadoteric acid (DOTA), CAT activity remained generally stable in the gills across control and exposed groups, except at concentration C2 (25 µg·L⁻¹), which induced a significant increase in activity (p = 0.0173). In the digestive glands, the response shows more clear bell-shaped profile. The concentrations C1 and C2 triggered a significant increase in CAT activity compared to the control (p = 0.006), while concentration C3 (50 µg·L⁻¹) led to a decline in its activity (non-significant decrease relative to the control, yet it was significantly lower than that observed at concentrations C1 and C2) (Fig. 3 A). Glutathione S-Transferase Activity The variation in Glutathione S-transferase (GST) activity in the gills and digestive glands of Ruditapes decussatus exposed to different concentrations of gadoteric acid (DOTA) revealed a highly significant increase in this enzyme in both organs ( p < 0.0001) (Fig. 3 B) indicating a clear, dose-dependent induction of GST activity in response to DOTA exposure. In the gills, GST activity increased from 0.94 ± 0.19 µmol.min − 1 .mg − 1 of proteins on the control group to 1.41 ± 0.15 µmol.min − 1 .mg − 1 of proteins at concentration C3. Similarly, in the digestive glands, it rose from 0.82 ± 0.09 µmol.min − 1 .mg − 1 of proteins (control) to 1.22 ± 0.11 µmol.min − 1 .mg − 1 of proteins at the same concentration. Malondialdehyde Levels MDA levels showed a dose-dependent increase in both organs studied (Fig. 3 C). However, this increase was only highly significant ( p < 0.0001) in the gills at the highest concentration (C3, 50 µg·L⁻¹), rising from 19.07 ± 4.07 µmol.mg − 1 of proteins in the control group to 26.68 ± 1.7 µmol.mg − 1 of proteins in the C3 group. In contrast, in the digestive glands, the increase was progressive and highly significant, with MDA levels rising from 22.56 ± 1.07 µmol.mg − 1 of proteins in the control group to 33.61 ± 1.54 µmol.mg − 1 of proteins at concentration C3. Acetylcholinesterase Activity Acetylcholinesterase activity (AChE) showed a highly significant decrease in the gills ( p < 0.0001), independent of the DOTA exposure concentration (Fig. 4 ). The AChE activity decreased from 110.16 ± 13.06 µmol.min − 1 .mg − 1 of proteins in the untreated group to 45.65 ± 5.83 µmol.min − 1 .mg − 1 of proteins at the group treated with 50 µg·L⁻¹ of DOTA (C3). In the digestive gland, the AChE activity was also highly significant decrease and dose-dependent compared to the control group. This activity declined from 282.13 ± 12.38 µmol.min − 1 .mg − 1 of proteins in controls to 223.37 ± 14.13 µmol.min − 1 .mg − 1 of proteins on treated clams with C3 concentration of DOTA (Fig. 4 ). Histopathological Changes of the Gills In control animals, the gills exhibited a typical structure, consisting of two primary gill lamellae folded into secondary lamellae. Each lamella was composed of well-arranged gill filaments of nearly equal length and width. A gill filament consisted of an epithelium supported by two internal chitinous skeletal rods. The filament, crossed by a branchial sinus or hemolymphatic channel, showed three zones: frontal (distal), intermediate, and proximal (abfrontal). The distal and intermediate zones were covered with frontal, lateral, and fronto-lateral cilia. The epithelial cells were interspersed with mucus cells. Lamellae were connected by loose connective tissue containing lacunae that formed hemolymphatic vessels. The space between lamellae, referred to as water tubes, was separated by intermediate junctions or septa. These tubes communicated with the mantle cavity through occasional pores or ostia (Fig. 5 a). In gill tissues, the mean incidence of lesions increased progressively with rising concentrations of gadoteric acid. The highest mean histopathological index (3.93 ± 0.26) was recorded at the maximum concentration (C3) after 7 days of exposure, compared to an index of zero in untreated clams (Table 1 ). Table 1 Semi-quantitative intensity of histopathological alterations in the gills of Ruditapes decussatus exposed to gadoteric acid for 7 days. Incidence of lesions (adapted from Riba et al. [25]): (0) absent; (1) sometimes; (2) frequent; (3) very frequent; (4) always present. C: untreated; C1: 12,5 µg.L − 1 ; C2: 25 µg.L − 1 ; C3: 50 µg.L − 1 . Histopathological alterations Control (C1) (C2) (C3) Reducing filament size 0 2 3 4 Widening of the frontal area 0 2 3 4 Dilation of the hemolymphatic duct 0 2 3 4 Erosion of cilia 0 2 4 4 Hemocytic infiltration 0 1 3 4 Fusion of filaments 0 0 3 4 Rupture of the epithelium 0 0 3 4 Respiratory tissue damage 0 0 3 4 Alterations of connective tissue (dilation, degradation) 0 1 3 4 Filament hypoplasia 0 0 1 4 Cytoplasmic condensation 0 0 1 4 Caryorrhexis, karyolysis, pyknosis 0 0 1 4 Signs of apoptosis 0 0 1 4 Signs of neoplasia 0 0 0 3 Abnormal hemocytes 0 0 0 4 Mean lesions incidence 0,0 0,7 2,1 3,9 Exposure of clams to DOTA at 12.5 µg·L⁻¹ induced slight adaptive changes in the gills. Although the general architecture was preserved, a reduction in size and swelling of the frontal zone of the gill filaments were noted. Dilation of the hemolymphatic sinus was also observed at the apical region of the filaments. In addition, erosion of cilia and slight hemocyte infiltration into the hemolymphatic vessels were detected (Fig. 5 b). At the C2 concentration (25 µg·L⁻¹), the alterations were more significant and included adaptive, reversible, or irreversible changes. The respiratory tissue displayed multiple lesions with severe cilia erosion. Gill filaments were deformed, showing multiple fusions and areas of epithelial rupture (Fig. 5 c, circles). The underlying connective tissue was severely damaged, with marked dilation of hemolymphatic vessels due to cell loss and diffuse hemocyte infiltration. At the highest concentration of DOTA (50 µg·L⁻¹), the damage was severe, characterized by pronounced hypoplasia and loss of normal tissue structure through necrosis and cytoplasmic condensation. The filaments appeared shorter and wider, with extensive cilia erosion. In both respiratory epithelium and connective tissue, signs of cell death by necrosis or apoptosis were evident, with several nuclear abnormalities, including karyorrhexis, karyolysis, and pyknosis. Also, dissemination of neoplastic cells and abnormal hemocytes was noted (Fig. 5 d). Histopathological Changes in the Digestive Glands In control clams, the digestive diverticulum consists of highly coiled primary, secondary, and tertiary tubules. The tertiary tubules, which appear oval to round in cross-section, are the most abundant. The tubule wall, delineated by a basal membrane, is composed of a pseudostratified epithelium containing two cell types : eosinophilic and vesiculated digestive cells (CD), and basophilic secretory cells (CB), which are pyramidal in shape and interspersed at the base of the wall between digestive cells. The tubule lumen (L), whether closed or slightly open, may appear narrow or wide depending on the animal’s health status and metabolic activity. Digestive cells, whose nuclei are basally located, account for 60–80% of the tubule wall. The digestive tubules are surrounded by connective tissue containing hemocytes and fibrocytes (Fig. 6 a). Following exposure to DOTA, the mean incidence of histopathological alterations in the digestive glands of clams increased progressively with gadoteric acid concentration, reaching 3.8 ± 0.42 at the highest concentration (C3) after 7 days, compared to 0.1 ± 0.32 in the control group (Table 2 ). Table 2 Semi-quantitative intensity of histopathological alterations in the digestive glands of Ruditapes decussatus exposed to gadoteric acid for 7 days. Incidence of lesions (adapted from Riba et al. [25]): (0) absent; (1) sometimes; (2) frequent; (3) very frequent; (4) always present. C: untreated; C1: 12,5 µg.L − 1 ; C2: 25 µg.L − 1 ; C3: 50 µg.L − 1 . Histopathological alterations Control (C1) (C2) (C3) Hemocytic infiltration 0 3 3 4 Hypertrophy/hyperplasia of cells 0 2 3 3 Necrosis of digestive cells (DC) 0 2 3 4 Rupture/dislocation of the basement membrane 0 2 3 4 Tubular lumen dilation 1 2 3 4 Karyolysis, karyorrhexis, pyknosis 0 1 3 4 Gadoteric acid deposits in the wall 0 1 3 4 Cellular debris in the lumen 0 0 2 4 Tubular wall atrophy 0 1 2 4 Mitotic nuclei (digestive cells/hemocytes) 0 0 1 3 Mean lesions incidence 0.1 1.4 2.6 3.8 Exposure to 12.5 µg·L⁻¹ of DOTA (C1) induced marked hemocyte infiltration (HI), indicative of a strong inflammatory response. Hypertrophy and/or hyperplasia, particularly of the basophilic secretory cells, were observed. Some tubules exhibited wall damage due to necrosis (Nc) of digestive cells and rupture or dislocation of the basal membrane (Fig. 6 b). At 25 µg·L⁻¹ (C2), more severe lesions were evident, with greater lumen dilation, hemocyte infiltration, and areas of necrotic tissue. Signs of nuclear damage, including karyolysis, karyorrhexis, and pyknosis, were present. Hypertrophy/hyperplasia persisted, and irregularly shaped blue or purple deposits were observed in the tubule walls, which may represent mineralized gadolinium released following dechelation (Fig. 6 c). In clams exposed to 50 µg·L⁻¹ (C3), most digestive tubules showed atrophied walls and markedly dilated lumens due to extensive necrosis of digestive cells. Cellular debris accumulated in the tubule lumen (L). Pyknosis, karyorrhexis, and even mitotic figures were observed in both digestive cells and hemocytes, the latter also showing signs of genotoxicity. Hemocyte infiltration was pronounced. Blue or purple deposits were noted (probably mineralized gadolinium) in the tubule walls (Fig. 6 d). Histopathological Changes in the Gonads The gonad of Ruditapes decussatus is located within the visceral mass, surrounding the digestive tubules. It is composed of numerous acini delineated by a basal membrane (acinus wall). In the examined sections, one male individual was identified, while the rest were females, all at the maturity stage. The acini were filled with mature oocytes along with some immature ones. Mature oocytes, spherical to polygonal in shape, occupied the lumen, while developing oocytes were pedunculated, and the youngest were attached to the acinus wall via their pedicle. Mature oocytes contained a germinal vesicle or nucleus, often with a nucleolus, an abundant granular cytoplasm, and a thin outer vitelline membrane (Fig. 7 a). In the gonads, the mean incidence of histopathological alterations increased progressively with gadoteric acid concentration, reaching a maximum score of 4 at the highest concentration (C3) after 7 days of exposure (Table 3 ). Table 3 Semi-quantitative intensity of histopathological alterations in the gonads of Ruditapes decussatus exposed to gadoteric acid for 7 days. Incidence of lesions (adapted from Riba et al. [26]): (0) absent; (1) sometimes; (2) frequent; (3) very frequent; (4) always present. C: untreated; C1: 12,5 µg.L − 1 ; C2: 25 µg.L − 1 ; C3: 50 µg.L − 1 . Histopathological alterations Control (C1) (C2) (C3) Atretic oocytes 0 1 3 4 Degeneration of oocytes 0 0 2 4 Loss of spherical/polygonal shape of oocytes 0 0 2 4 Darkened cytoplasm (loss of eosinophilic staining) 0 0 2 4 Nuclear envelope dislocation 0 0 2 4 Chromatin condensation 0 0 2 4 Nucleolar fragmentation or absence 0 0 2 4 Disintegration of the acinar basement membrane 0 1 3 4 Gonadal fibrosis (collagen + fibroblasts) 0 0 1 4 Collagen fiber hypertrophy 0 0 1 4 Fibroblast proliferation 0 0 1 4 Mean lesions incidence 0.0 0.2 1.9 4.0 Clams exposed to 12.5 µg·L⁻¹ of DOTA (C1) showed slight changes, including some atretic oocytes and partial disintegration of the acinus wall (Fig. 7 b). At 25 µg·L⁻¹ (C2), more pronounced changes were evident, including numerous atretic or degenerating oocytes and extensive disintegration of the acinus wall. Atretic oocytes lost their typical spherical or polygonal shape, acquiring irregular forms. Their cytoplasm lost its eosinophilic staining and appeared darker. Nuclear alterations characteristic of atresia were observed, including dislocation of the nuclear envelope, chromatin condensation, and fragmentation or disappearance of the nucleolus (Fig. 7 c). At the highest concentration (50 µg·L⁻¹, C3), the gonads exhibited massive oocyte atresia and gonadal fibrosis, characterized by collagen fiber hypertrophy and increased fibroblast proliferation (Fig. 7 d). Discussion The monitoring of marine ecosystems is crucial for protecting biodiversity, ensuring food safety, and safeguarding public health in the face of emerging pollutants. It is imperative to intensify efforts to develop effective detection and management strategies to mitigate the impact of these contaminants on the environment and human populations (Ojija, 2024 ). Our investigation carried out in certain aquatic environments revealed the presence of gadoteric acid (unpublished data, for example in the Guenniche stream, one of the tributaries of the Bizerte lagoon). Gadoteric acid (DOTA) is a macrocyclic contrast agent, characterized by its high stability in vivo (Sieber et al., 2008 ). However, gadolinium in chelates can be exchanged with body cations such as copper, zinc, calcium or iron (Idee et al. 2006 , Dekker et al., 2024 ). The free ionic form (un-chelated gadolinium), known for its toxicity, can be associated with anions like CO 3 2− , PO 4 3− , which causes deposits in organs (Rogowska et al., 2018 ; Iyad et al., 2023 ) and then disturbs cellular functions. Researchers have indicated that Gd 3+ toxicity is linked to the blockage of specific Na + -Ca 2+ channels (Fretellier et al., 2015; Martino et al., 2021 ). This interference can lead to significant physiological disturbances in marine organisms, including clams (Trapasso et al., 2021 ; Coimbra et al., 2024 ; Moreira et al., 2025 ). Clams represent an excellent biological material and are widely used as bioindicators in environmental monitoring studies (Mezghani-Chaari et al. 2015 ). Their ability to bioaccumulate various pollutants in their tissues makes them valuable indicators (Moreira et al, 2025 ). In addition, many species are widely consumed by humans in various Mediterranean regions and then constitute a potential source of contaminants transfer to humans. Moreover, these organisms play an ecological role as natural water filters, contributing to the maintenance of aquatic ecosystem balance (Added et al., 2023 ). Gadolinium bioaccumulation has been observed in various organisms, including the human brain (Kanda et al, 2016 ; McDonald et al., 2015), rats and watercress leaves (Lindner et al., 2013 ), and bivalves (Perrat et al., 2017 ). Among macroinvertebrates, bivalves show the highest Gd bioaccumulation due to their filter-feeding behavior (Moreira et al., 2025 ). It appears that gadolinium accumulation, even in its chelated form, in bivalve, induces significant biochemical stress, neurotoxicity and a reduction in metabolic capacity, that disturb filtration capacity (Secco et al., 2023). Clam gills are particularly sensitive to environmental contaminants; the accumulation of gadolinium in these organs can interfere with their function, reducing water flow through the gill filaments and impairing filtration. Despite its high stability, exposure of Ruditapes decussatus to gadoteric acid (DOTA) revealed marked biological and histopathological effects. Our results showed a significant decrease in filtration capacity at 25 µg·L⁻¹, followed by an apparent return to control levels at 50 µg·L⁻¹. This non-monotonic response differs from the classical profile observed in bivalves exposed to pollutants, where a progressive inhibition of filtration is generally reported (Freitas et al., 2020 ). Such non-linear responses are increasingly recognized in ecotoxicology and complicate risk modeling. This type of response may reflect a hormetic phenomenon, a transient behavioral adaptation, or variability related to mucus secretion, as has been suggested for other filter-feeding mollusks. At the biochemical level, our results indicate a significant change in catalase (CAT), glutathione S-transferase (GST), malondialdehyde (MDA) and acetylcholinesterase (AChE) activity in the gills and digestive glands of clams exposed to DOTA. However, this change is more significant in the digestive tract than in gills, a result that corroborates the greater accumulation of DOTA in the digestive gland mentioned above. Gill and digestive cells, are directly exposed to the effect of DOTA and clams must fight against its accumulation through their mechanisms of detoxification, including the enzyme Glutathione S-transferases (GSTs). Activated xenobiotics in cytosol, in phase I, are conjugated with GSTs in phase II and are exported out of the cell. The significant induction of GST activity observed in gills and digestive glands indicates the activation of detoxification mechanisms, a response previously described in Mytilus galloprovincialis and Donax trunculus exposed to gadolinium or other rare earth elements (REEs) and metals (Pinto et al., 2019 ; Freitas et al., 2020 ; Andrade et al., 2023 ; Secco et al., 2024 ). While, Perrat et al. ( 2017 ) reported that Dreissena rostriformis bugensis , exposed to 10 µg.L − 1 of DOTA for 7 days, showed a marked increase in GST activity, Our results show that at a concentration of 12.5 µg.L-1, GST activity change remains non-significant, suggesting a different response depending on the species. Catalase activity exhibited a bell-shaped pattern, with stimulation at low and intermediate concentrations followed by a decrease at 50 µg·L⁻¹. A similar trend of response was observed in other marine clams and freshwater mussels that, in general, can augment their antioxidant defense capacity in the presence of Gd within a certain threshold in time and concentration of Gd, then this capacity is observed to decrease belong this threshold (Henriques et al., 2019 ; Hanana et al., 2017 ). In Donax truncatus exposed to gadolinium, Secco et al., ( 2024 ) showed an increase of antioxidant enzymes at the lowest concentrations of 10 µg/L and 50 µg/L and a decrease at the highest concentrations of 250 µg/L and 500 µg/L. This type of response, frequently observed in bivalves exposed to Gd and other metals, suggests an overload of antioxidant defenses (Banni et al., 2010 ; Marigómez et al., 2024 ; Ladhar-Chaabouni et al., 2012 ). In fact, catalase prevents the accumulation of hydrogen peroxide and protects cell organelles and tissues from damage caused by hydrogen peroxide, which is constantly produced by many metabolic reactions and various contaminants. However, although catalase activity often increases with H₂O₂ concentration, high concentrations of hydrogen peroxide immediately inhibit the catalase enzyme by altering the structure of its active site (Hadwan, 2018 ). This finding, suggests that the DOTA concentration of 50 µg·L⁻¹, leads to excessive accumulation of hydrogen peroxide, resulting in a negative effect on CAT function. H2O2 accumulation in cells, exceeding antioxidant capacity, causes oxidative stress, leading to cell damage and potentially cell death through apoptosis or necrosis. This stress can damage DNA, lipids, and proteins, trigger cell cycle arrest and activating stress responses. This result can explain the tissue damage observed in different organs, particularly in animals exposed to the highest concentration (C3), where necrosis and apoptosis patterns were observed. On the other hand, the reduction in the activity of oxidative stress enzymes can be partly explained by the study of Hanana et al. ( 2017 ) which indicates that exposure of the freshwater mussel, Dreissena polymorpha , to contrasting agent, leads to a downregulation in gene expressions of catalase (CAT) and glutathione-S-transferase (GST). Malondialdehyde (MDA) is one among final products of lipid peroxidation (LPO) in the cells, which can react with guanosine nucleotide and then cause DNA damage. Thus, MDA level is considered a reliable indicator of LPO caused by the rise in production of reactive oxygen species (ROS) and inefficiency of defense mechanisms (Sachdeva et al., 2014 ; Andrade et al., 2023 ). The dose-dependent increase in MDA levels observed further supports the establishment of oxidative stress, consistent with observations in marine animals exposed to gadolinium or other REETs (Andrade et al., 2023 ; Liu et al., 2023 ). Henriques et al. ( 2019 ) reported a significant increase of LPO in Mytilus galloprovincialis exposed to high concentrations (60 µg. L − 1 ) of gadolinium over 28 days. Pagano et al. ( 2016 ), show the same result in pluteus larvae of the sea urchin Paracentrotus lividus exposed to gadolinium for 48 hours after fertilization. Here, the increase in MDA, especially at C2 and C3, in parallel to the decrease in CAT at 50 µg·L⁻¹ in R. decussatus after 7 days of exposure to DOTA up then 25 µg·L⁻¹, suggest that defense mechanisms are unable to effectively counteract the excessive generated ROS (Pisoschi et al. 2021 ). Andrade et al. ( 2023 ) reported that LPO was occurred in C . fluminea at 10 µg·L⁻¹ for the same time of exposure (7 days), suggesting difference in tolerance and response between species exposed to Gd. As consequence, is the potential cell membrane and DNA damages, which are noted in others mussels exposed to Gd (Henriques et al. 2019 ; Trapasso et al. 2021 ). this hypothesis is supported by the high histopathological damages observed, in particular the increase in necrotic and/or apoptotic cells and possible presence of neoplastic cells. The strong inhibition of acetylcholinesterase (AChE) activity observed, regardless of concentration, showed that this enzyme is a sensitive biomarker of neurotoxicity in R . decussatus . This inhibition is more evident in gill than in digestive tract. Our result is in line with previous findings in others bivalves and macroinvertebrates exposed to pesticides, organic and particulate contaminants (Bebianno et al., 2004 ; English and Webster, 2012 ; De Marchi et al. 2018 ; Perić et al. 2017 ). However, in some studies, AChE showed an increase or unchanged trend. Cunha et al. ( 2022 ) showed that AChE presented an increase in M . galloprovincialis exposed to 10 µg·L⁻¹ of Gd for 14 days. Andrade et al. ( 2023 ) and Henriques et al. ( 2019 ) reported that low concentrations of Gd (15 µg·L⁻¹) di not result in inhibition of AChE activity in M . galloprovincialis yet at high concentrations (> 30 µg·L⁻¹) of Gd, AChE activity inhibition was observed. These findings highlight a different response depending on dose of Gd used and species. Liu et al. ( 2011 ) showed an increase of acetylcholine concentration, following the inhibition of AChE in R. philippinarum exposed to mercury (Hg). In bivalves (such as mussels, oysters, or clams), acetylcholinesterase (AChE) plays an important role in regulating muscular activity, particularly that of the valves and gill cilia involved in water filtration. Accumulation of acetylcholine, following AChE inhibition, leads to continuous stimulation of cholinergic receptors, overstimulation of muscles and cilia, followed by fatigue or paralysis. Such inhibition may partly explain the trend in evolution in filtration capacity observed in our study. Indeed, temporal dynamics of filtration capacity, exhibited a global declining trend, in particular at C2. At C3 filtration rate increased during the first 30 minutes, then decreased by the end of the experiment. Nevertheless, other hypothesis can be plausible: increase in filtration rate observed at high concentration of Gd may be related to a compensatory or hermetic response (hormesis) when bivalves activate mechanisms of detoxification, or increased gill movement to restore homeostasis or, cholinergic receptors may desensitize or down-regulate over time, reducing overstimulation and leading to increase filtration rate. The histopathological effects of gadoteric acid on the gills and digestive tract of Ruditapes decussatus clearly demonstrate dose-dependent toxicity and significant structural alterations affecting most tissues to varying degrees. Our study shows that at 12.5 µg.L − 1 , the clams maintain normal gill architecture but exhibit reduced filament size and slight malformations, indicating an initial stress response. At a concentration of 25 µg.L − 1 , partial cilia erosion appears, along with cellular degeneration and structural disorganization of the connective tissues, suggesting more severe damage and the activation of apoptotic mechanisms. At 50 µg.L − 1 nearly complete ciliary damage occurs, accompanied by pronounced hemocyte infiltration, highlighting the critical impact of high levels of gadoteric acid on gill integrity. Furthermore, DOTA contamination induces histopathological alterations in the gonadal gland, ranging from occasional cases of oocyte atresia and localized disintegration of the acinar basal membrane at the lowest concentration (C1), to massive oocyte atresia and gonadal fibrosis at the highest dose (C3). This fibrosis, characterized by hypertrophy of collagen fibers and proliferation of fibroblasts, was reported in animals exposed to Gd (Celiker et al. ( 2022 ). Similarly, according to Gonçalves and Bebianno ( 2023 ), exposure of Mytilus galloprovincialis to polystyrene nanoparticles induces gonadal fibrosis, characterized by collagen fiber hypertrophy and fibroblast proliferation. Such histological alterations can disrupt gonadal function and compromise the process of gametogenesis. Oocyte atresia is common in bivalves such as Cerastoderma edule (Chérel and Beninger, 2019 ) and Tapes philippinarum (Chérel and Beninger, 2017 ) and is characterized by oocyte degeneration at various developmental stages, often influenced by environmental stressors. According to Zhu et al. ( 2022 ), Mytilus spp. oocytes undergo significant morphological alterations under toxic stress, including the loss of their typical rounded shape in favor of irregular contours, and cytoplasmic darkening, clear signs of degeneration. In addition, marked nuclear alterations have been observed, such as nuclear envelope disintegration, chromatin condensation, and nucleolar fragmentation, all of which are key indicators of severe cellular damage. Histopathological alterations detected in gills (ciliary erosion, lamellar fusion, necrosis), digestive gland (Lumen dilation, inflammatory reaction, hemocyte infiltration, hypertrophy/hyperplasia, necrosis, pigment deposits probably linked to gadolinium mineralization), and gonads (oocyte atresia, fibrosis) are in agreement with lesions reported in bivalves exposed to heavy metals and rare earth elements (Rodríguez-Villalobos et al., 2025 ). The gonadal impact is particularly concerning, as it reflects potential impairment of reproductive capacity, similar to what has been demonstrated in R. decussatus exposed to triclosan (Added et al., 2023 ). Conclusion This work provides clear evidence that gadoteric acid, a macrocyclic gadolinium-based contrast agent traditionally regarded as environmentally inert, exerts measurable toxic effects on the clam Ruditapes decussatus . Exposure to environmentally relevant concentrations caused physiological impairment, oxidative and neurotoxic stress responses, and pronounced histopathological alterations in key target organs. The coordinated inhibition of acetylcholinesterase, disruption of antioxidant defenses, and tissue degeneration collectively indicate that gadoteric acid can compromise essential biological functions such as filtration, respiration, and reproduction. These results demonstrate that even stable gadolinium complexes are not fully benign in aquatic systems and may pose underestimated ecotoxicological risks. Consequently, gadolinium-based contrast agents should be integrated into environmental monitoring and risk assessment programs, particularly in coastal areas receiving hospital effluents. Further research is warranted to elucidate the mechanisms of gadolinium bioavailability and trophic transfer to refine their environmental safety evaluation. Declarations The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Aya AOUNI], [Asma BOUSSELMI] and [Noureddine KHALLOUFI]. The first draft of the manuscript was written by [Aya AOUNI]and [Mustapha BEJAOUI] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethics approval This is an observational study. 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Evaluating the impact of gadolinium contamination on the marine bivalve Donax trunculus : Implications for environmental health . Environmental Toxicology and Pharmacology , 112, 104580. doi.org/10.1016/j.etap.2024.104580 Sieber, M. A., Lengsfeld P., Frenzel., Golfier S., Schmitt-Willich H., Siegmund F., Walter J., Weinmann H-J., Pietsch H. (2008). Preclinical investigation to compare different gadolinium-based contrast agents regarding their propensity to release gadolinium in vivo and to trigger nephrogenic systemic fibrosis-like lesions. European radiology , 18(10), 2164-2173. Stoyanova , S., Georgieva, E., Velcheva, I., Iliev, I., Vasileva, T., Bivolarski, V., Tomov, S., Nyeste, KJ, Antal, L. Yancheva, V. (2020). Évaluation multi-biomarqueurs dans le foie de la carpe commune ( Cyprinus carpio , Linnaeus 1758) après une exposition aiguë au chlorpyrifos. Water , 12 (6), 1837. https://doi.org/10.3390/W12061837 Trapasso, G., Chiesa, S., Freitas, R., Pereira, E. (2021). What do we know about the ecotoxicological implications of the rare earth element gadolinium in aquatic ecosystems?. Science of the Total Environment , 781 , 146273. https://doi.org/10.1016/j.scitotenv.2021.146273. Velez, C., Figueira, E., Soares, A., Freitas, R. (2017). Effects of increasing seawater temperature on economically important native and introduced clam species. Marine Environmental Research ,123, 62-70. https://doi.org/10.1016/j.marenvres.2016.11.010 Xia, Q., Feng, X., Huang, H., Du, L., Yang, X., Wang, K. (2011 ). Gadolinium-induced oxidative stress triggers endoplasmic reticulum stress in rat cortical neurons. Journal of Neurochem . 117, 38-47. https://doi.org/10.1111/j.1471-4159.2010.07162.x Zhu, J., Li, J., Chapman, E. C., Shi, H., Ciocan, C., Chen, K., Shi, X., Zhou, J., Sun, P., Zheng, Y., Rotchell, J. M. (2022). Gonadal Atresia, Estrogen-Responsive, and Apoptosis-Specific mRNA Expression in Marine Mussels from the East China Coast: A Preliminary Study. Bulletin of Environmental Contamination and Toxicology , 108, 1111–1117.https://doi.org/10.1007/s00128-022-03461-2 Additional Declarations No competing interests reported. Supplementary Files figureabstract.tif Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Feb, 2026 Reviews received at journal 13 Feb, 2026 Reviews received at journal 06 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers invited by journal 26 Jan, 2026 Editor assigned by journal 20 Jan, 2026 Submission checks completed at journal 19 Jan, 2026 First submitted to journal 18 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8632544","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":582071566,"identity":"0dfae4f0-80cd-4400-99be-4f3dfc93287f","order_by":0,"name":"Aya AOUNI","email":"","orcid":"","institution":"University of Carthage","correspondingAuthor":false,"prefix":"","firstName":"Aya","middleName":"","lastName":"AOUNI","suffix":""},{"id":582071567,"identity":"e421aeef-483e-4f75-ab35-f3b9ccf17986","order_by":1,"name":"Noureddine KHALLOUFI","email":"","orcid":"","institution":"University of Carthage","correspondingAuthor":false,"prefix":"","firstName":"Noureddine","middleName":"","lastName":"KHALLOUFI","suffix":""},{"id":582071568,"identity":"a54bb708-d391-4faf-9cb1-ee1a68fedb31","order_by":2,"name":"Asma BOUSSELMI","email":"","orcid":"","institution":"University of Carthage","correspondingAuthor":false,"prefix":"","firstName":"Asma","middleName":"","lastName":"BOUSSELMI","suffix":""},{"id":582071571,"identity":"db6aee5e-e50f-48e9-96d8-230d0205913c","order_by":3,"name":"Ateeqah GHAYTH ALZWAWY","email":"","orcid":"","institution":"University of Carthage","correspondingAuthor":false,"prefix":"","firstName":"Ateeqah","middleName":"GHAYTH","lastName":"ALZWAWY","suffix":""},{"id":582071572,"identity":"df2897a7-dd1d-48aa-94b8-ba5d4282eb77","order_by":4,"name":"Ezzeddine MAHMOUDI","email":"","orcid":"","institution":"University of Carthage","correspondingAuthor":false,"prefix":"","firstName":"Ezzeddine","middleName":"","lastName":"MAHMOUDI","suffix":""},{"id":582071581,"identity":"de6fe6a1-3325-4cc4-89bb-441493ba7b4d","order_by":5,"name":"Mustapha BEJAOUI","email":"data:image/png;base64,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","orcid":"","institution":"University of Carthage","correspondingAuthor":true,"prefix":"","firstName":"Mustapha","middleName":"","lastName":"BEJAOUI","suffix":""}],"badges":[],"createdAt":"2026-01-18 16:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8632544/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8632544/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101437822,"identity":"c34dc30e-d810-4813-b939-27c3bb476298","added_by":"auto","created_at":"2026-01-29 16:35:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61696,"visible":true,"origin":"","legend":"\u003cp\u003eFiltration rate (FR) (mg·h⁻¹·ind⁻¹) of untreated (Control) and treated clams with three concentrations of gadoteric acid (DOTA) (C1 = 12.5 µg.L\u003csup\u003e-1\u003c/sup\u003e, C2 = 25 µg.L\u003csup\u003e-1\u003c/sup\u003e, C3 = 50 µg.L\u003csup\u003e-1\u003c/sup\u003e) after 7 days of exposure. (a, b: significant differences at the 5% level, ANOVA followed by Tukey’s HSD test).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8632544/v1/fd9bae07093481c5339822d9.png"},{"id":101437821,"identity":"d7530fb4-4bc2-46a1-b2d4-7717e74d4321","added_by":"auto","created_at":"2026-01-29 16:35:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96474,"visible":true,"origin":"","legend":"\u003cp\u003eInstant change of filtration rate (FR) (mg·min⁻¹·ind⁻¹) of \u003cem\u003eRuditapes decussatus\u003c/em\u003e after 7-day exposure to DOTA (C = Control, DOTA1 = 12.5 µg.L\u003csup\u003e-1\u003c/sup\u003e, DOTA 2 = 25 µg.L\u003csup\u003e-1\u003c/sup\u003e, DOTA 3 = 50 µg.L\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8632544/v1/734fdf8eda2d5bfd171f8c98.png"},{"id":101751570,"identity":"c8d8f01f-adb0-4f54-9f7e-ac88bd987945","added_by":"auto","created_at":"2026-02-03 10:21:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":172039,"visible":true,"origin":"","legend":"\u003cp\u003eActivities of catalase (CAT) (A), glutathione S-transferase (GST) (B), and malondialdehyde (MDA) content (C) in the gills (G) and digestive glands (DG) of \u003cem\u003eRuditapes decussatus\u003c/em\u003e exposed to gadoteric acid (DOTA) in the laboratory for 7 days (C = Control, C1 = 12.5 µg.L\u003csup\u003e-1\u003c/sup\u003e, C2 = 25 µg.L\u003csup\u003e-1\u003c/sup\u003e, C3 = 50 µg.L\u003csup\u003e-1\u003c/sup\u003e). (ANOVA; a, b, a’, b’, c’: significant differences at p \u0026lt; 0.05: Tukey’s HSD test).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8632544/v1/3a54b2c1e770a3682939e542.png"},{"id":101437823,"identity":"6c49d58e-3825-4ba9-a768-e177c8a1a9f2","added_by":"auto","created_at":"2026-01-29 16:35:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101666,"visible":true,"origin":"","legend":"\u003cp\u003eAcetylcholinesterase activity (AChE) (µmol·min⁻¹·mg⁻¹ protein) in the gills (G) and digestive glands (DG) of \u003cem\u003eRuditapes decussatus\u003c/em\u003e exposed to gadoteric acid (DOTA) in the laboratory for 7 days (C = Control, C1 = 12.5 µg.L\u003csup\u003e-1\u003c/sup\u003e, C2 = 25 µg.L\u003csup\u003e-1\u003c/sup\u003e, C3 = 50 µg.L\u003csup\u003e-1\u003c/sup\u003e). (ANOVA; a, b, a’, b’, c’: significant differences at p \u0026lt; 0.05: Tukey’s HSD test).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8632544/v1/92e3c5f20c930b6eb949e86f.png"},{"id":101437828,"identity":"df5c5cde-c4fa-4113-9756-8224295d0569","added_by":"auto","created_at":"2026-01-29 16:35:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1811581,"visible":true,"origin":"","legend":"\u003cp\u003eMicrophotographs of gills of \u003cem\u003eRuditapes decussatus\u003c/em\u003e. Untreated clams (a), clams exposed to DOTA at 12.5 µg.L\u003csup\u003e-1\u003c/sup\u003e (b), 25 µg.L\u003csup\u003e-1\u003c/sup\u003e (c), and 50 µg.L\u003csup\u003e-1\u003c/sup\u003e (d). (Black arrow: cilia erosion; filled triangle: pyknotic nucleus; open triangle: neoplastic cells; star: connective tissue damage due to necrosis).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8632544/v1/83b67e84486228f48bed035d.png"},{"id":101437827,"identity":"6812c77c-41ba-4c87-b631-aad565827541","added_by":"auto","created_at":"2026-01-29 16:35:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1811607,"visible":true,"origin":"","legend":"\u003cp\u003eMicrophotographs of digestive tubules of \u003cem\u003eRuditapes decussatus\u003c/em\u003e. Untreated clams (a), clams exposed to DOTA at 12.5 µg.L\u003csup\u003e-1\u003c/sup\u003e (b), 25 µg.L\u003csup\u003e-1\u003c/sup\u003e (c), and 50 µg.L\u003csup\u003e-1\u003c/sup\u003e (d). (Bc\u0026nbsp;: basophilic cells, Ct: connective tissue, Dc: digestive cells, Hc: hemocytes, L: lumen. Hi: hemocyte infiltration, Hy: Hypertophy/hyperplasia, Nc: necrotic cells, Nt: necrotic tissue, Ma: male acinus).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8632544/v1/9fdb020f6d5dac3375290061.png"},{"id":101751624,"identity":"0da76e20-206a-4f28-b46a-217de8913a45","added_by":"auto","created_at":"2026-02-03 10:21:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1602291,"visible":true,"origin":"","legend":"\u003cp\u003eMicrophotographs of gonads of \u003cem\u003eRuditapes decussatus\u003c/em\u003e. Untreated clams (a), clams exposed to DOTA at 12.5 µg.L\u003csup\u003e-1\u003c/sup\u003e (b), 25 µg.L\u003csup\u003e-1\u003c/sup\u003e (c), and 50 µg.L\u003csup\u003e-1\u003c/sup\u003e (d). (Asterisque: degenerated oocyte by necrosis and manyatretic oocytes (Ao). Note high vacuolization with swelling of cytoplasmic organelles (mitochondria-like) and enlargement of the nuclear vesicle. Note also nucleolus lysis (Nl) and chromatin condensation).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8632544/v1/95c29402108b935f18b043ca.png"},{"id":101754999,"identity":"13fb195f-6913-4b6f-a3ce-29bf0ec29ba5","added_by":"auto","created_at":"2026-02-03 10:48:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9291199,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8632544/v1/4950c24d-608a-4738-bb54-af530f7737dd.pdf"},{"id":101437826,"identity":"3a1bdcae-c4d5-479f-beb3-35c1e7e70ddb","added_by":"auto","created_at":"2026-01-29 16:35:45","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":734530,"visible":true,"origin":"","legend":"","description":"","filename":"figureabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-8632544/v1/88cbc0209ed201b45436591a.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of a gadolinium-based contrast agent detected in wastewater on the clam Ruditapes decussatus: a multi-marker approach","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Mediterranean Sea, recognized as a biodiversity hotspot, is home to approximately 7.5% of global marine biodiversity (Cantasano, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, it is heavily impacted by human activities (industrial, agricultural, urban, and domestic) that contribute to the contamination of various water bodies with numerous chemical pollutants, including emerging xenobiotics originating from medical and industrial sources. According to the United States Environmental Protection Agency (US-EPA), emerging contaminants are newly identified compounds lacking regulatory status, whose effects on human health and the environment are still poorly understood (Chavoshani et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The study of the response of living organisms, populations, or communities to stressful conditions (eco-biological approach), or of the impact of contaminants on these organisms (ecotoxicological approach), as well as their biochemical reactions (Dellali et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), constitutes a central axis of ecosystem monitoring.\u003c/p\u003e \u003cp\u003eAmong the most used sentinel species in this field is the common clam, \u003cem\u003eRuditapes decussatus\u003c/em\u003e (Mansour et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which is widely consumed by humans in various Mediterranean regions. Its broad distribution, sedentary lifestyle, and low cost make this species one of the most preferred bioindicator taxa for monitoring marine pollution (Velez et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The use of an integrative approach including biomarker analysis, observation of histological changes in bivalve tissues, and behavioral responses has proven to be a highly relevant method for assessing contaminant impact and determining the health status of individuals and populations (Bousselmi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Accordingly, a multimarkers strategy has been implemented across different organs to highlight the effects of xenobiotics on aquatic biota. This strategy includes, in addition to behavioral markers (filtration rate) (Banaee et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), biochemical defense markers (catalase and glutathione S-transferase activities) (Dellali et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), markers of membrane lipid peroxidation (malondialdehyde) (Banaee et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), neurotoxicity biomarkers (acetylcholinesterase activity) (Banaee et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and histological indicators (Stoyanova et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGadoteric acid, a contrast agent marketed under the name Dotarem\u0026reg;, is a gadolinium-based compound chelated with an organic acid. It is an emerging contaminant that have been found in different aquatic ecosystems, including surface, and drinking water (Dekker et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) in many parts of the world. Gadolinium, a metal belonging to the lanthanide family and classified among the rare earth elements, is characterized by its electromagnetic properties. It is used in several fields (Xia et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), notably in medicine (as a contrast agent in MRI, and in X-ray intensifying screens) (Ramirez and Munoz, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), as well as in industry (as a steel additive) (Islam and Tsnobiladze, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The ionic form of gadolinium (Gd\u0026sup3;⁺), which acts as an antagonist of the essential calcium ion (Ca\u0026sup2;⁺), is responsible for its toxicity. This form (Gd\u0026sup3;⁺) inhibits voltage-gated Ca\u0026sup2;⁺ channels at micromolar concentrations (Nathalie et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) [13]. Chelated gadolinium is known to be non-metabolizable and, as it circulates in freshwater and marine environments, it can accumulate in the tissues of living organisms (Cesarini et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Several clinical observations have reported adverse neurological effects, skin and kidney lesions, inflammatory responses, and fibrosis (both short- and long-term) in patients injected with gadolinium (Islam and Tsnobiladze, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Presence of Gd\u0026sup3;⁺ disrupts physiological mechanisms involving cellular calcium channels, affecting processes such as mitochondrial respiration, myocardial contractility, blood coagulation, and nerve transmission in mammalian cells (Nathalie et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The accumulation of Gd\u0026sup3;⁺ in various tissues, such as bones, liver, and lymph nodes, increases its toxicity (Amet and Deray, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, several studies have highlighted the potentially toxic effects of gadolinium on bivalves, emphasizing its accumulation and physiological consequences. In the clam \u003cem\u003eDonax trunculus\u003c/em\u003e, gadolinium bioaccumulation is proportional to both concentration and exposure duration. This accumulation is associated with notable metabolic changes as well as the induction of oxidative stress, suggesting a biological response to the contaminant (Secco et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Research on the mussel \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e revealed that elevated water temperature amplifies gadolinium toxicity by intensifying lipid damage at the cellular level. This finding underscores a potential synergistic effect between emerging contaminants and climate warming (Figueiredo et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A study on freshwater bivalves, \u003cem\u003eDreissena rostriformis bugensis\u003c/em\u003e and \u003cem\u003eCorbicula fluminea\u003c/em\u003e, confirmed the ability of these species to bioaccumulate gadolinium in their digestive glands and gills, even when present in complexed forms (contrast agents), highlighting the persistence and bioavailability of gadolinium in the aquatic environment (Perrat et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs part of monitoring studies on certain Tunisian watercourses, gadoteric acid has been detected in the waters of the Oued Guenniche, one of the tributaries of the Bizerte Lagoon. This study aims to examine the responses of the common clam \u003cem\u003eRuditapes decussatus\u003c/em\u003e following contamination by gadoteric acid (DOTA) under microcosm conditions. These responses were evaluated through: (i) measurement of filtration rate (behavioral), (ii) quantification of specific biomarkers (physiological), and (iii) tissue alterations in the gills, digestive glands, and gonads (histopathological).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSampling, acclimation, rearing and exposure conditions of clams\u003c/h2\u003e \u003cp\u003eThe clams used in this study were collected from the Bizerte Lagoon (northeastern Tunisia) near the city of Menzel Jemil (37\u0026deg;14\u0026rsquo;19\u0026rsquo;\u0026rsquo;N, 9\u0026deg;54\u0026rsquo;59\u0026rsquo;\u0026rsquo;E). These clams were transported to the laboratory in a cooler containing water from the collection site. In the laboratory, clams were acclimated in glass microcosms for 7 days under carefully controlled physicochemical conditions, temperature (T\u0026thinsp;=\u0026thinsp;18\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and photoperiod (12h/12h), without feeding. The rearing water, sourced from Rimel beach (Mediterranean Sea), is renewed every 48 hours.\u003c/p\u003e \u003cp\u003eUsed Gadoteric acid (DOTA) in its commercial form, Dotarem\u0026reg; (0.5 mmol\u0026middot;mL⁻\u0026sup1;). Three tested concentrations: C1\u0026thinsp;=\u0026thinsp;12.5 \u0026micro;g\u0026middot;L⁻\u0026sup1;, C2\u0026thinsp;=\u0026thinsp;25 \u0026micro;g\u0026middot;L⁻\u0026sup1;, and C3\u0026thinsp;=\u0026thinsp;50 \u0026micro;g\u0026middot;L⁻\u0026sup1;. Each experimental condition, including a control, is replicated three times. A total of 65 individuals of homogeneous size (mean size\u0026thinsp;=\u0026thinsp;38.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 mm) were distributed into microcosms, with 5 individuals per aquarium containing 1.5 L of seawater, for an exposure duration of 7 days.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFiltration Rate\u003c/h3\u003e\n\u003cp\u003eThe filtration rate (FR) was measured using the method of Coughlan (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1969\u003c/span\u003e), which is based on the decrease in the concentration of the dye neutral red in the water column under dark conditions. An individual clam was placed in a beaker containing 100 mL of a 1 g\u0026middot;L⁻\u0026sup1; neutral red solution. Before introducing the clams into the solution, 10 mL of water was sampled from each beaker to assess the initial concentration (C\u003csub\u003e0\u003c/sub\u003e). The clams were removed every 30 minutes over a period of 2 hours to evaluate the remaining concentration (C\u003csub\u003et\u003c/sub\u003e). A calibration curve was established using neutral red standards. Optical density at 530 nm was measured using a microplate reader. The filtration rate was calculated using the following equation:\u003c/p\u003e \u003cp\u003eFR= [M/nt] log (C\u003csub\u003e0\u003c/sub\u003e/C\u003csub\u003et\u003c/sub\u003e)\u003c/p\u003e \u003cp\u003eWhere:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eFR\u003c/b\u003e: clearance rate (mg\u0026middot;ind⁻\u0026sup1;\u0026middot;h⁻\u0026sup1;)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eM\u003c/b\u003e: total volume of water (mL)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003ent\u003c/b\u003e: number of clams used at time \u003cem\u003et\u003c/em\u003e (in hours)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eC\u003c/b\u003e \u003csub\u003e \u003cb\u003e0\u003c/b\u003e \u003c/sub\u003e: initial concentration of neutral red dye (1 g\u0026middot;L⁻\u0026sup1;)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eC\u003c/b\u003e \u003csub\u003e \u003cb\u003et\u003c/b\u003e \u003c/sub\u003e: final concentration of neutral red dye at time \u003cem\u003et\u003c/em\u003e\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e\n\u003ch3\u003eAssays of biochemical markers\u003c/h3\u003e\n\u003cp\u003eAt the end of the experiment, clams were dissected at 4\u0026deg;C. The gills and digestive glands of each animal were carefully removed and stored separately. Protein extraction was performed in phosphate buffer (0.1 M; pH 7.5). The homogenate was then centrifuged at 9000 rpm for 30 minutes at 4\u0026deg;C to obtain the S9 fraction, which was collected in Eppendorf tubes and stored at T= \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analyses. Total proteins content was determined using the Bradford method (1976) by measuring absorbance at 595 nm with a spectrophotometer. Catalase activity was measured following the method of Aebi (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), which monitors the degradation of hydrogen peroxide by catalase, producing water and oxygen. Absorbance was recorded at 240 nm and activity expressed as \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins. Glutathione S-transferase (GST) activity was quantified according to the method described by Habig et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). Optical densities were measured at 340 nm, and specific GST activity was expressed as \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins. Malondialdehyde (MDA) content, an indicator of lipid peroxidation, was assessed using the method of Buege and Aust (1978), which quantifies thiobarbituric acid reactive substances (TBARS). Absorbance was measured at 530 nm, and results were expressed in \u0026micro;mol.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins. Finally, acetylcholinesterase (AChE) activity was evaluated following Ellman et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1961\u003c/span\u003e). The absorbance was measured at 412 nm, and activity expressed as \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins.\u003c/p\u003e\n\u003ch3\u003eHistological Study\u003c/h3\u003e\n\u003cp\u003eTo perform histological sections, the gills and digestive glands were removed and separated from other tissues. The histological preparation steps were the same as those described by Martoja and Martoja, but using a more modern automated technique, performed at the Department of Pathological Anatomy at the Regional Hospital of Menzel Bourguiba (Tunisia). The gills and digestive glands were first fixed in 5% formalin to preserve cellular structures. The samples were then dehydrated with a series of increasing concentrations of alcohol and toluene and treated with kerosene. They were then cut into 5 \u0026micro;m thick sections with a microtome, placed on slides, and stained with hematoxylin and eosin. The preparations were studied under a ZEISS optical microscope (G\u0026times;40\u0026ndash;100) and photographed with an Axio-Cam 105. Histopathological alterations were semi-quantitatively evaluated by ranking the severity of lesions (grades, 0 (absent), 1 (sometimes), 2 (frequent), 3 (very frequent), and 4 (always present)) as described by Riba et al. [26] and adapted in our study. The damage index is an average arithmetic value obtained from the semi-quantitative evaluation of the lesions for each tissue.\u003c/p\u003e\n\u003ch3\u003eStatistical Data Analysis\u003c/h3\u003e\n\u003cp\u003eData from the various measurements are presented on boxplots (min-max, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;2SD). Graphs and statistical analyses were performed using STATISTICA 8 software under Windows. The data was the first tested to evaluate the normality (Shapiro-Wilk test) and homogeneity of variances (Bartlett test). Variations in behavioral and biochemical responses of the clams were analyzed using one-way ANOVA compared to the control group. When significant differences were detected by ANOVA, post-hoc comparisons were conducted using Tukey\u0026rsquo;s Honestly Significant Difference (HSD) test. A probability level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffect of DOTA contamination on filtration capacity of\u003c/b\u003e \u003cb\u003eR. decussatus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe exposure of the clam \u003cem\u003eRuditapes decussatus\u003c/em\u003e to different concentrations of gadoteric acid (C1\u0026thinsp;=\u0026thinsp;12.5 \u0026micro;g\u0026middot;L⁻\u0026sup1;, C2\u0026thinsp;=\u0026thinsp;25 \u0026micro;g\u0026middot;L⁻\u0026sup1;, C3\u0026thinsp;=\u0026thinsp;50 \u0026micro;g\u0026middot;L⁻\u0026sup1;) over a 7-day period revealed a very significant difference compared to the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0075) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Indeed, the filtration rate decreased from 8.18\u0026thinsp;\u0026plusmn;\u0026thinsp;4.22 mg\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.ind\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the control group to 2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mg\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.ind\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at concentration C2. However, at the highest concentration (C3), the filtration rate nearly returned to the levels observed in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding the temporal dynamics, the filtration capacity exhibited a declining trend, decreasing from 14.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mg\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.ind\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after 30 minutes to 4.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.ind\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after 120 minutes in the control group. Exposure to 12.5 \u0026micro;g\u0026middot;L⁻\u0026sup1; and 25 \u0026micro;g\u0026middot;L⁻\u0026sup1; of gadoteric acid led to a slight increase in filtration rate over time. At the concentration of 50 \u0026micro;g\u0026middot;L⁻\u0026sup1;, the filtration capacity initially increased during the first 30 minutes, then decreased by the end of the experiment, while remaining higher than the values observed under the other conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhysiological responses of\u003c/b\u003e \u003cb\u003eR. decussatus\u003c/b\u003e \u003cb\u003eto DOTA exposure\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003eCatalase activity\u003c/h3\u003e\n\u003cp\u003eAfter 7 days of exposure of R. decussatus to gadoteric acid (DOTA), CAT activity remained generally stable in the gills across control and exposed groups, except at concentration C2 (25 \u0026micro;g\u0026middot;L⁻\u0026sup1;), which induced a significant increase in activity (p\u0026thinsp;=\u0026thinsp;0.0173). In the digestive glands, the response shows more clear bell-shaped profile. The concentrations C1 and C2 triggered a significant increase in CAT activity compared to the control (p\u0026thinsp;=\u0026thinsp;0.006), while concentration C3 (50 \u0026micro;g\u0026middot;L⁻\u0026sup1;) led to a decline in its activity (non-significant decrease relative to the control, yet it was significantly lower than that observed at concentrations C1 and C2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\n\u003ch3\u003eGlutathione S-Transferase Activity\u003c/h3\u003e\n\u003cp\u003eThe variation in Glutathione S-transferase (GST) activity in the gills and digestive glands of \u003cem\u003eRuditapes decussatus\u003c/em\u003e exposed to different concentrations of gadoteric acid (DOTA) revealed a highly significant increase in this enzyme in both organs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) indicating a clear, dose-dependent induction of GST activity in response to DOTA exposure. In the gills, GST activity increased from 0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins on the control group to 1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins at concentration C3. Similarly, in the digestive glands, it rose from 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins (control) to 1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins at the same concentration.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMalondialdehyde Levels\u003c/h2\u003e \u003cp\u003eMDA levels showed a dose-dependent increase in both organs studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, this increase was only highly significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the gills at the highest concentration (C3, 50 \u0026micro;g\u0026middot;L⁻\u0026sup1;), rising from 19.07\u0026thinsp;\u0026plusmn;\u0026thinsp;4.07 \u0026micro;mol.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins in the control group to 26.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 \u0026micro;mol.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins in the C3 group. In contrast, in the digestive glands, the increase was progressive and highly significant, with MDA levels rising from 22.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 \u0026micro;mol.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins in the control group to 33.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54 \u0026micro;mol.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins at concentration C3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAcetylcholinesterase Activity\u003c/h2\u003e \u003cp\u003eAcetylcholinesterase activity (AChE) showed a highly significant decrease in the gills (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), independent of the DOTA exposure concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The AChE activity decreased from 110.16\u0026thinsp;\u0026plusmn;\u0026thinsp;13.06 \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins in the untreated group to 45.65\u0026thinsp;\u0026plusmn;\u0026thinsp;5.83 \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins at the group treated with 50 \u0026micro;g\u0026middot;L⁻\u0026sup1; of DOTA (C3).\u003c/p\u003e \u003cp\u003eIn the digestive gland, the AChE activity was also highly significant decrease and dose-dependent compared to the control group. This activity declined from 282.13\u0026thinsp;\u0026plusmn;\u0026thinsp;12.38 \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins in controls to 223.37\u0026thinsp;\u0026plusmn;\u0026thinsp;14.13 \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of proteins on treated clams with C3 concentration of DOTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Changes of the Gills\u003c/h2\u003e \u003cp\u003eIn control animals, the gills exhibited a typical structure, consisting of two primary gill lamellae folded into secondary lamellae. Each lamella was composed of well-arranged gill filaments of nearly equal length and width. A gill filament consisted of an epithelium supported by two internal chitinous skeletal rods. The filament, crossed by a branchial sinus or hemolymphatic channel, showed three zones: frontal (distal), intermediate, and proximal (abfrontal). The distal and intermediate zones were covered with frontal, lateral, and fronto-lateral cilia. The epithelial cells were interspersed with mucus cells. Lamellae were connected by loose connective tissue containing lacunae that formed hemolymphatic vessels. The space between lamellae, referred to as water tubes, was separated by intermediate junctions or septa. These tubes communicated with the mantle cavity through occasional pores or ostia (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eIn gill tissues, the mean incidence of lesions increased progressively with rising concentrations of gadoteric acid. The highest mean histopathological index (3.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26) was recorded at the maximum concentration (C3) after 7 days of exposure, compared to an index of zero in untreated clams (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSemi-quantitative intensity of histopathological alterations in the gills of \u003cem\u003eRuditapes decussatus\u003c/em\u003e exposed to gadoteric acid for 7 days. Incidence of lesions\u003c/p\u003e \u003cdiv class=\"Credit\"\u003e\u003cp\u003e(adapted from Riba et al. [25]): (0) absent; (1) sometimes; (2) frequent; (3) very frequent; (4) always present. C: untreated; C1: 12,5 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; C2: 25 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; C3: 50 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistopathological alterations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(C1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(C2)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(C3)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReducing filament size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWidening of the frontal area\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDilation of the hemolymphatic duct\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eErosion of cilia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemocytic infiltration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFusion of filaments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRupture of the epithelium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRespiratory tissue damage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlterations of connective tissue (dilation, degradation)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilament hypoplasia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCytoplasmic condensation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaryorrhexis, karyolysis, pyknosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSigns of apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSigns of neoplasia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbnormal hemocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean lesions incidence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0,0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0,7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2,1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3,9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eExposure of clams to DOTA at 12.5 \u0026micro;g\u0026middot;L⁻\u0026sup1; induced slight adaptive changes in the gills. Although the general architecture was preserved, a reduction in size and swelling of the frontal zone of the gill filaments were noted. Dilation of the hemolymphatic sinus was also observed at the apical region of the filaments. In addition, erosion of cilia and slight hemocyte infiltration into the hemolymphatic vessels were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eAt the C2 concentration (25 \u0026micro;g\u0026middot;L⁻\u0026sup1;), the alterations were more significant and included adaptive, reversible, or irreversible changes. The respiratory tissue displayed multiple lesions with severe cilia erosion. Gill filaments were deformed, showing multiple fusions and areas of epithelial rupture (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, circles). The underlying connective tissue was severely damaged, with marked dilation of hemolymphatic vessels due to cell loss and diffuse hemocyte infiltration.\u003c/p\u003e \u003cp\u003eAt the highest concentration of DOTA (50 \u0026micro;g\u0026middot;L⁻\u0026sup1;), the damage was severe, characterized by pronounced hypoplasia and loss of normal tissue structure through necrosis and cytoplasmic condensation. The filaments appeared shorter and wider, with extensive cilia erosion. In both respiratory epithelium and connective tissue, signs of cell death by necrosis or apoptosis were evident, with several nuclear abnormalities, including karyorrhexis, karyolysis, and pyknosis. Also, dissemination of neoplastic cells and abnormal hemocytes was noted (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Changes in the Digestive Glands\u003c/h2\u003e \u003cp\u003eIn control clams, the digestive diverticulum consists of highly coiled primary, secondary, and tertiary tubules. The tertiary tubules, which appear oval to round in cross-section, are the most abundant. The tubule wall, delineated by a basal membrane, is composed of a pseudostratified epithelium containing two cell types : eosinophilic and vesiculated digestive cells (CD), and basophilic secretory cells (CB), which are pyramidal in shape and interspersed at the base of the wall between digestive cells. The tubule lumen (L), whether closed or slightly open, may appear narrow or wide depending on the animal\u0026rsquo;s health status and metabolic activity. Digestive cells, whose nuclei are basally located, account for 60\u0026ndash;80% of the tubule wall. The digestive tubules are surrounded by connective tissue containing hemocytes and fibrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eFollowing exposure to DOTA, the mean incidence of histopathological alterations in the digestive glands of clams increased progressively with gadoteric acid concentration, reaching 3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42 at the highest concentration (C3) after 7 days, compared to 0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 in the control group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSemi-quantitative intensity of histopathological alterations in the digestive glands of \u003cem\u003eRuditapes decussatus\u003c/em\u003e exposed to gadoteric acid for 7 days. Incidence of lesions\u003c/p\u003e \u003cdiv class=\"Credit\"\u003e\u003cp\u003e(adapted from Riba et al. [25]): (0) absent; (1) sometimes; (2) frequent; (3) very frequent; (4) always present. C: untreated; C1: 12,5 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; C2: 25 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; C3: 50 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistopathological alterations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(C1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(C2)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(C3)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemocytic infiltration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypertrophy/hyperplasia of cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNecrosis of digestive cells (DC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRupture/dislocation of the basement membrane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubular lumen dilation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKaryolysis, karyorrhexis, pyknosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGadoteric acid deposits in the wall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellular debris in the lumen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubular wall atrophy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMitotic nuclei (digestive cells/hemocytes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean lesions incidence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1.4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2.6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eExposure to 12.5 \u0026micro;g\u0026middot;L⁻\u0026sup1; of DOTA (C1) induced marked hemocyte infiltration (HI), indicative of a strong inflammatory response. Hypertrophy and/or hyperplasia, particularly of the basophilic secretory cells, were observed. Some tubules exhibited wall damage due to necrosis (Nc) of digestive cells and rupture or dislocation of the basal membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). At 25 \u0026micro;g\u0026middot;L⁻\u0026sup1; (C2), more severe lesions were evident, with greater lumen dilation, hemocyte infiltration, and areas of necrotic tissue. Signs of nuclear damage, including karyolysis, karyorrhexis, and pyknosis, were present. Hypertrophy/hyperplasia persisted, and irregularly shaped blue or purple deposits were observed in the tubule walls, which may represent mineralized gadolinium released following dechelation (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eIn clams exposed to 50 \u0026micro;g\u0026middot;L⁻\u0026sup1; (C3), most digestive tubules showed atrophied walls and markedly dilated lumens due to extensive necrosis of digestive cells. Cellular debris accumulated in the tubule lumen (L). Pyknosis, karyorrhexis, and even mitotic figures were observed in both digestive cells and hemocytes, the latter also showing signs of genotoxicity. Hemocyte infiltration was pronounced. Blue or purple deposits were noted (probably mineralized gadolinium) in the tubule walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Changes in the Gonads\u003c/h2\u003e \u003cp\u003eThe gonad of \u003cem\u003eRuditapes decussatus\u003c/em\u003e is located within the visceral mass, surrounding the digestive tubules. It is composed of numerous acini delineated by a basal membrane (acinus wall). In the examined sections, one male individual was identified, while the rest were females, all at the maturity stage. The acini were filled with mature oocytes along with some immature ones. Mature oocytes, spherical to polygonal in shape, occupied the lumen, while developing oocytes were pedunculated, and the youngest were attached to the acinus wall via their pedicle. Mature oocytes contained a germinal vesicle or nucleus, often with a nucleolus, an abundant granular cytoplasm, and a thin outer vitelline membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eIn the gonads, the mean incidence of histopathological alterations increased progressively with gadoteric acid concentration, reaching a maximum score of 4 at the highest concentration (C3) after 7 days of exposure (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSemi-quantitative intensity of histopathological alterations in the gonads of \u003cem\u003eRuditapes decussatus\u003c/em\u003e exposed to gadoteric acid for 7 days. Incidence of lesions\u003c/p\u003e \u003cdiv class=\"Credit\"\u003e\u003cp\u003e(adapted from Riba et al. [26]): (0) absent; (1) sometimes; (2) frequent; (3) very frequent; (4) always present. C: untreated; C1: 12,5 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; C2: 25 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; C3: 50 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistopathological alterations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(C1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(C2)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(C3)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtretic oocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDegeneration of oocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLoss of spherical/polygonal shape of oocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDarkened cytoplasm (loss of eosinophilic staining)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNuclear envelope dislocation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChromatin condensation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNucleolar fragmentation or absence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisintegration of the acinar basement membrane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGonadal fibrosis (collagen\u0026thinsp;+\u0026thinsp;fibroblasts)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCollagen fiber hypertrophy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFibroblast proliferation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean lesions incidence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1.9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e4.0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eClams exposed to 12.5 \u0026micro;g\u0026middot;L⁻\u0026sup1; of DOTA (C1) showed slight changes, including some atretic oocytes and partial disintegration of the acinus wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). At 25 \u0026micro;g\u0026middot;L⁻\u0026sup1; (C2), more pronounced changes were evident, including numerous atretic or degenerating oocytes and extensive disintegration of the acinus wall. Atretic oocytes lost their typical spherical or polygonal shape, acquiring irregular forms. Their cytoplasm lost its eosinophilic staining and appeared darker. Nuclear alterations characteristic of atresia were observed, including dislocation of the nuclear envelope, chromatin condensation, and fragmentation or disappearance of the nucleolus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eAt the highest concentration (50 \u0026micro;g\u0026middot;L⁻\u0026sup1;, C3), the gonads exhibited massive oocyte atresia and gonadal fibrosis, characterized by collagen fiber hypertrophy and increased fibroblast proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe monitoring of marine ecosystems is crucial for protecting biodiversity, ensuring food safety, and safeguarding public health in the face of emerging pollutants. It is imperative to intensify efforts to develop effective detection and management strategies to mitigate the impact of these contaminants on the environment and human populations (Ojija, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur investigation carried out in certain aquatic environments revealed the presence of gadoteric acid (unpublished data, for example in the Guenniche stream, one of the tributaries of the Bizerte lagoon). Gadoteric acid (DOTA) is a macrocyclic contrast agent, characterized by its high stability \u003cem\u003ein vivo\u003c/em\u003e (Sieber et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, gadolinium in chelates can be exchanged with body cations such as copper, zinc, calcium or iron (Idee et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Dekker et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The free ionic form (un-chelated gadolinium), known for its toxicity, can be associated with anions like CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, which causes deposits in organs (Rogowska et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Iyad et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and then disturbs cellular functions. Researchers have indicated that Gd\u003csup\u003e3+\u003c/sup\u003e toxicity is linked to the blockage of specific Na\u003csup\u003e+\u003c/sup\u003e-Ca\u003csup\u003e2+\u003c/sup\u003e channels (Fretellier et al., 2015; Martino et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This interference can lead to significant physiological disturbances in marine organisms, including clams (Trapasso et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Coimbra et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Moreira et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eClams represent an excellent biological material and are widely used as bioindicators in environmental monitoring studies (Mezghani-Chaari et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Their ability to bioaccumulate various pollutants in their tissues makes them valuable indicators (Moreira et al, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In addition, many species are widely consumed by humans in various Mediterranean regions and then constitute a potential source of contaminants transfer to humans. Moreover, these organisms play an ecological role as natural water filters, contributing to the maintenance of aquatic ecosystem balance (Added et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGadolinium bioaccumulation has been observed in various organisms, including the human brain (Kanda et al, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; McDonald et al., 2015), rats and watercress leaves (Lindner et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and bivalves (Perrat et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Among macroinvertebrates, bivalves show the highest Gd bioaccumulation due to their filter-feeding behavior (Moreira et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It appears that gadolinium accumulation, even in its chelated form, in bivalve, induces significant biochemical stress, neurotoxicity and a reduction in metabolic capacity, that disturb filtration capacity (Secco et al., 2023). Clam gills are particularly sensitive to environmental contaminants; the accumulation of gadolinium in these organs can interfere with their function, reducing water flow through the gill filaments and impairing filtration. Despite its high stability, exposure of \u003cem\u003eRuditapes decussatus\u003c/em\u003e to gadoteric acid (DOTA) revealed marked biological and histopathological effects. Our results showed a significant decrease in filtration capacity at 25 \u0026micro;g\u0026middot;L⁻\u0026sup1;, followed by an apparent return to control levels at 50 \u0026micro;g\u0026middot;L⁻\u0026sup1;. This non-monotonic response differs from the classical profile observed in bivalves exposed to pollutants, where a progressive inhibition of filtration is generally reported (Freitas et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Such non-linear responses are increasingly recognized in ecotoxicology and complicate risk modeling. This type of response may reflect a hormetic phenomenon, a transient behavioral adaptation, or variability related to mucus secretion, as has been suggested for other filter-feeding mollusks.\u003c/p\u003e \u003cp\u003eAt the biochemical level, our results indicate a significant change in catalase (CAT), glutathione S-transferase (GST), malondialdehyde (MDA) and acetylcholinesterase (AChE) activity in the gills and digestive glands of clams exposed to DOTA. However, this change is more significant in the digestive tract than in gills, a result that corroborates the greater accumulation of DOTA in the digestive gland mentioned above.\u003c/p\u003e \u003cp\u003eGill and digestive cells, are directly exposed to the effect of DOTA and clams must fight against its accumulation through their mechanisms of detoxification, including the enzyme Glutathione S-transferases (GSTs). Activated xenobiotics in cytosol, in phase I, are conjugated with GSTs in phase II and are exported out of the cell. The significant induction of GST activity observed in gills and digestive glands indicates the activation of detoxification mechanisms, a response previously described in \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e and \u003cem\u003eDonax trunculus\u003c/em\u003e exposed to gadolinium or other rare earth elements (REEs) and metals (Pinto et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Freitas et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Andrade et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Secco et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). While, Perrat et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported that \u003cem\u003eDreissena rostriformis bugensis\u003c/em\u003e, exposed to 10 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of DOTA for 7 days, showed a marked increase in GST activity, Our results show that at a concentration of 12.5 \u0026micro;g.L-1, GST activity change remains non-significant, suggesting a different response depending on the species.\u003c/p\u003e \u003cp\u003eCatalase activity exhibited a bell-shaped pattern, with stimulation at low and intermediate concentrations followed by a decrease at 50 \u0026micro;g\u0026middot;L⁻\u0026sup1;. A similar trend of response was observed in other marine clams and freshwater mussels that, in general, can augment their antioxidant defense capacity in the presence of Gd within a certain threshold in time and concentration of Gd, then this capacity is observed to decrease belong this threshold (Henriques et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hanana et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In \u003cem\u003eDonax truncatus\u003c/em\u003e exposed to gadolinium, Secco et al., (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) showed an increase of antioxidant enzymes at the lowest concentrations of 10 \u0026micro;g/L and 50 \u0026micro;g/L and a decrease at the highest concentrations of 250 \u0026micro;g/L and 500 \u0026micro;g/L. This type of response, frequently observed in bivalves exposed to Gd and other metals, suggests an overload of antioxidant defenses (Banni et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Marig\u0026oacute;mez et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ladhar-Chaabouni et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In fact, catalase prevents the accumulation of hydrogen peroxide and protects cell organelles and tissues from damage caused by hydrogen peroxide, which is constantly produced by many metabolic reactions and various contaminants. However, although catalase activity often increases with H₂O₂ concentration, high concentrations of hydrogen peroxide immediately inhibit the catalase enzyme by altering the structure of its active site (Hadwan, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This finding, suggests that the DOTA concentration of 50 \u0026micro;g\u0026middot;L⁻\u0026sup1;, leads to excessive accumulation of hydrogen peroxide, resulting in a negative effect on CAT function. H2O2 accumulation in cells, exceeding antioxidant capacity, causes oxidative stress, leading to cell damage and potentially cell death through apoptosis or necrosis. This stress can damage DNA, lipids, and proteins, trigger cell cycle arrest and activating stress responses. This result can explain the tissue damage observed in different organs, particularly in animals exposed to the highest concentration (C3), where necrosis and apoptosis patterns were observed. On the other hand, the reduction in the activity of oxidative stress enzymes can be partly explained by the study of Hanana et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) which indicates that exposure of the freshwater mussel, \u003cem\u003eDreissena polymorpha\u003c/em\u003e, to contrasting agent, leads to a downregulation in gene expressions of catalase (CAT) and glutathione-S-transferase (GST).\u003c/p\u003e \u003cp\u003eMalondialdehyde (MDA) is one among final products of lipid peroxidation (LPO) in the cells, which can react with guanosine nucleotide and then cause DNA damage. Thus, MDA level is considered a reliable indicator of LPO caused by the rise in production of reactive oxygen species (ROS) and inefficiency of defense mechanisms (Sachdeva et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Andrade et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The dose-dependent increase in MDA levels observed further supports the establishment of oxidative stress, consistent with observations in marine animals exposed to gadolinium or other REETs (Andrade et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Henriques et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported a significant increase of LPO in \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e exposed to high concentrations (60 \u0026micro;g. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of gadolinium over 28 days. Pagano et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), show the same result in pluteus larvae of the sea urchin \u003cem\u003eParacentrotus lividus\u003c/em\u003e exposed to gadolinium for 48 hours after fertilization. Here, the increase in MDA, especially at C2 and C3, in parallel to the decrease in CAT at 50 \u0026micro;g\u0026middot;L⁻\u0026sup1; in \u003cem\u003eR. decussatus\u003c/em\u003e after 7 days of exposure to DOTA up then 25 \u0026micro;g\u0026middot;L⁻\u0026sup1;, suggest that defense mechanisms are unable to effectively counteract the excessive generated ROS (Pisoschi et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Andrade et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported that LPO was occurred in \u003cem\u003eC\u003c/em\u003e. \u003cem\u003efluminea\u003c/em\u003e at 10 \u0026micro;g\u0026middot;L⁻\u0026sup1; for the same time of exposure (7 days), suggesting difference in tolerance and response between species exposed to Gd. As consequence, is the potential cell membrane and DNA damages, which are noted in others mussels exposed to Gd (Henriques et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Trapasso et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). this hypothesis is supported by the high histopathological damages observed, in particular the increase in necrotic and/or apoptotic cells and possible presence of neoplastic cells.\u003c/p\u003e \u003cp\u003eThe strong inhibition of acetylcholinesterase (AChE) activity observed, regardless of concentration, showed that this enzyme is a sensitive biomarker of neurotoxicity in \u003cem\u003eR\u003c/em\u003e. \u003cem\u003edecussatus\u003c/em\u003e. This inhibition is more evident in gill than in digestive tract. Our result is in line with previous findings in others bivalves and macroinvertebrates exposed to pesticides, organic and particulate contaminants (Bebianno et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; English and Webster, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; De Marchi et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Perić et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, in some studies, AChE showed an increase or unchanged trend. Cunha et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) showed that AChE presented an increase in \u003cem\u003eM\u003c/em\u003e. \u003cem\u003egalloprovincialis\u003c/em\u003e exposed to 10 \u0026micro;g\u0026middot;L⁻\u0026sup1; of Gd for 14 days. Andrade et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Henriques et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported that low concentrations of Gd (15 \u0026micro;g\u0026middot;L⁻\u0026sup1;) di not result in inhibition of AChE activity in \u003cem\u003eM\u003c/em\u003e. \u003cem\u003egalloprovincialis\u003c/em\u003e yet at high concentrations (\u0026gt;\u0026thinsp;30 \u0026micro;g\u0026middot;L⁻\u0026sup1;) of Gd, AChE activity inhibition was observed. These findings highlight a different response depending on dose of Gd used and species. Liu et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) showed an increase of acetylcholine concentration, following the inhibition of AChE in \u003cem\u003eR. philippinarum\u003c/em\u003e exposed to mercury (Hg). In bivalves (such as mussels, oysters, or clams), acetylcholinesterase (AChE) plays an important role in regulating muscular activity, particularly that of the valves and gill cilia involved in water filtration. Accumulation of acetylcholine, following AChE inhibition, leads to continuous stimulation of cholinergic receptors, overstimulation of muscles and cilia, followed by fatigue or paralysis. Such inhibition may partly explain the trend in evolution in filtration capacity observed in our study. Indeed, temporal dynamics of filtration capacity, exhibited a global declining trend, in particular at C2. At C3 filtration rate increased during the first 30 minutes, then decreased by the end of the experiment. Nevertheless, other hypothesis can be plausible: increase in filtration rate observed at high concentration of Gd may be related to a compensatory or hermetic response (hormesis) when bivalves activate mechanisms of detoxification, or increased gill movement to restore homeostasis or, cholinergic receptors may desensitize or down-regulate over time, reducing overstimulation and leading to increase filtration rate.\u003c/p\u003e \u003cp\u003eThe histopathological effects of gadoteric acid on the gills and digestive tract of \u003cem\u003eRuditapes decussatus\u003c/em\u003e clearly demonstrate dose-dependent toxicity and significant structural alterations affecting most tissues to varying degrees. Our study shows that at 12.5 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the clams maintain normal gill architecture but exhibit reduced filament size and slight malformations, indicating an initial stress response. At a concentration of 25 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, partial cilia erosion appears, along with cellular degeneration and structural disorganization of the connective tissues, suggesting more severe damage and the activation of apoptotic mechanisms. At 50 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nearly complete ciliary damage occurs, accompanied by pronounced hemocyte infiltration, highlighting the critical impact of high levels of gadoteric acid on gill integrity.\u003c/p\u003e \u003cp\u003eFurthermore, DOTA contamination induces histopathological alterations in the gonadal gland, ranging from occasional cases of oocyte atresia and localized disintegration of the acinar basal membrane at the lowest concentration (C1), to massive oocyte atresia and gonadal fibrosis at the highest dose (C3). This fibrosis, characterized by hypertrophy of collagen fibers and proliferation of fibroblasts, was reported in animals exposed to Gd (Celiker et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, according to Gon\u0026ccedil;alves and Bebianno (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), exposure of \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e to polystyrene nanoparticles induces gonadal fibrosis, characterized by collagen fiber hypertrophy and fibroblast proliferation. Such histological alterations can disrupt gonadal function and compromise the process of gametogenesis.\u003c/p\u003e \u003cp\u003eOocyte atresia is common in bivalves such as \u003cem\u003eCerastoderma edule\u003c/em\u003e (Ch\u0026eacute;rel and Beninger, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and \u003cem\u003eTapes philippinarum\u003c/em\u003e (Ch\u0026eacute;rel and Beninger, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and is characterized by oocyte degeneration at various developmental stages, often influenced by environmental stressors. According to Zhu et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), \u003cem\u003eMytilus\u003c/em\u003e spp. oocytes undergo significant morphological alterations under toxic stress, including the loss of their typical rounded shape in favor of irregular contours, and cytoplasmic darkening, clear signs of degeneration. In addition, marked nuclear alterations have been observed, such as nuclear envelope disintegration, chromatin condensation, and nucleolar fragmentation, all of which are key indicators of severe cellular damage.\u003c/p\u003e \u003cp\u003eHistopathological alterations detected in gills (ciliary erosion, lamellar fusion, necrosis), digestive gland (Lumen dilation, inflammatory reaction, hemocyte infiltration, hypertrophy/hyperplasia, necrosis, pigment deposits probably linked to gadolinium mineralization), and gonads (oocyte atresia, fibrosis) are in agreement with lesions reported in bivalves exposed to heavy metals and rare earth elements (Rodr\u0026iacute;guez-Villalobos et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The gonadal impact is particularly concerning, as it reflects potential impairment of reproductive capacity, similar to what has been demonstrated in \u003cem\u003eR. decussatus\u003c/em\u003e exposed to triclosan (Added et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis work provides clear evidence that gadoteric acid, a macrocyclic gadolinium-based contrast agent traditionally regarded as environmentally inert, exerts measurable toxic effects on the clam \u003cem\u003eRuditapes decussatus\u003c/em\u003e. Exposure to environmentally relevant concentrations caused physiological impairment, oxidative and neurotoxic stress responses, and pronounced histopathological alterations in key target organs. The coordinated inhibition of acetylcholinesterase, disruption of antioxidant defenses, and tissue degeneration collectively indicate that gadoteric acid can compromise essential biological functions such as filtration, respiration, and reproduction. These results demonstrate that even stable gadolinium complexes are not fully benign in aquatic systems and may pose underestimated ecotoxicological risks. Consequently, gadolinium-based contrast agents should be integrated into environmental monitoring and risk assessment programs, particularly in coastal areas receiving hospital effluents. Further research is warranted to elucidate the mechanisms of gadolinium bioavailability and trophic transfer to refine their environmental safety evaluation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Aya AOUNI], [Asma BOUSSELMI] and [Noureddine KHALLOUFI]. The first draft of the manuscript was written by [Aya AOUNI]and [Mustapha BEJAOUI] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis is an observational study. The Faculty of Sciences of Bizerte Research Ethics Committee has confirmed that no ethical approval is required.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the entire team, especially Mr. Ahmed NHILI, of the laboratory of pathological anatomy at the regional hospital of Menzel Bourguiba (Tunisia) for their contribution in preparing the histological sections.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eAdded, A., Khalloufi, N., Khazri A., Harrath, A.H., Mansour, L., Nahdi, S., Boufahja, F., Aldahmash, W., Alrefaei, A.F., et Dellali, M. 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(2022).\u003c/strong\u003e Gonadal Atresia, Estrogen-Responsive, and Apoptosis-Specific mRNA Expression in Marine Mussels from the East China Coast: A Preliminary Study. \u003cem\u003eBulletin of Environmental Contamination and Toxicology\u003c/em\u003e, 108, 1111\u0026ndash;1117.https://doi.org/10.1007/s00128-022-03461-2\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-geochemistry-and-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"egah","sideBox":"Learn more about [Environmental Geochemistry and Health](https://www.springer.com/journal/10653)","snPcode":"10653","submissionUrl":"https://submission.nature.com/new-submission/10653/3","title":"Environmental Geochemistry and Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Behaviour, Dotarem, Histopathology, Mediterranean clam, Multi-markers","lastPublishedDoi":"10.21203/rs.3.rs-8632544/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8632544/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study is an \u003cem\u003ein-vivo\u003c/em\u003e evaluation of the toxicity of Dotarem (DOTA) for marine clam \u003cem\u003eRuditapes decussatus\u003c/em\u003e. Three concentrations of DOTA \u003cb\u003e(\u003c/b\u003eC1\u0026thinsp;=\u0026thinsp;12.5 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C2\u0026thinsp;=\u0026thinsp;25 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C3\u0026thinsp;=\u0026thinsp;50 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were used for exposure on 7 days. Responses of \u003cem\u003eR. decussatus\u003c/em\u003e after its exposure, were monitored using filtration rate, oxidative stress, lipo-peroxidation, neurotoxicity and histopathological markers. Four biomarkers were measured at the gills and digestive gland: two defense biomarkers catalase (CAT) and glutathione-S-transferase (GST), a cellular damage biomarker (MDA) and a neurotoxicity biomarker acetylcholinesterase (AChE).\u003c/p\u003e \u003cp\u003eThe filtration rate was significantly decreased by exposure to DOTA rising from 8.18\u0026thinsp;\u0026plusmn;\u0026thinsp;4.22 mg.h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.ind\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the control to 2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mg.h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.ind\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the clams after 7 days of exposure. The results showed that the activities of antioxidant enzymes (CAT, GST) and the cellular damage status (MDA) revealed concentration and organ-dependent responses for DOTA. Acetylcholinesterase activity (AChE) showed a highly significant decrease in the gills, independent of the DOTA exposure concentration, and in the digestive gland depending of dose exposure (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This contamination causes histopathological changes in both organs, marked by infiltrations, vacuolizations and cell necrosis. The intensity of these lesions depends on the concentration of this pollutant.\u003c/p\u003e","manuscriptTitle":"Effects of a gadolinium-based contrast agent detected in wastewater on the clam Ruditapes decussatus: a multi-marker approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 16:35:37","doi":"10.21203/rs.3.rs-8632544/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-24T22:07:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-13T16:43:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-06T11:42:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220324563555622724843403954404673665248","date":"2026-02-03T06:52:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87827329325935878468655469696759274839","date":"2026-01-26T21:20:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-26T21:18:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T23:19:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-19T18:28:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Geochemistry and Health","date":"2026-01-18T16:11:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-geochemistry-and-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"egah","sideBox":"Learn more about [Environmental Geochemistry and Health](https://www.springer.com/journal/10653)","snPcode":"10653","submissionUrl":"https://submission.nature.com/new-submission/10653/3","title":"Environmental Geochemistry and Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d6e0962d-1125-4f77-aabd-f1922d30f96d","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T20:23:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 16:35:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8632544","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8632544","identity":"rs-8632544","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}