Predicted No-Effect Concentration of Roundup in the Composition with Pharmaceutical Chlorpromazine or Heating Causes Similar Biochemical Disturbances in Bivalve Mollusk Unio Tumidus

The ability of bioindicators to reect the specic impacts in complex exposures is unpredicted. This study aimed to track the particular effects of pesticide Roundup (Rn) and antipsychotic drug chlorpromazine (Cpz) on the mussel Unio tumidus at environmentally relevant mixtures. The mussels were treated by Rn (17 µg L −1 ), Cpz (18 µg L −1 ), mixture of Rn and Cpz at 18 o C (RnCpz) as well as by Rn at 25 o C (RnT). Digestive glands were examined after 14 days of exposure. The indexes of stress response (total antioxidant capacity, glutathione (GSH&GSSG), metallothioneins (MTSH and Zn-MT), protein carbonyls levels), and markers of metabolic and detoxication (CYP450 related (EROD), Glutathione S-transpherase (GST), cholinesterase, caspase-3, citrate synthase (CS) activities), lysosomal membrane integrity, and Zn level were analyzed. Mostly common responses of mussels were indicated as the increase of oxidative stress, MTSH (except Cpz-group), EROD and CS (except Rn-group) responses. Rn-group indicated almost no-effect or abnormal for expected symptoms effect. However, under the heating Rn caused the decrease of Zn accumulation and loss of lysosomal integrity. Cpz provoked major response diverseness: a decrease in Zn and GST levels and an increase in lysosomal integrity. Thus, complex exposures abolished the individual response traits. Summarising, the application of integrated indices has benets when evaluating the effects of complex exposures.

The ability of bioindicators to re ect the speci c impacts in complex exposures is unpredicted. This study aimed to track the particular effects of pesticide Roundup (Rn) and antipsychotic drug chlorpromazine (Cpz) on the mussel Unio tumidus at environmentally relevant mixtures. The mussels were treated by Rn (17 µg L −1 ), Cpz (18 µg L −1 ), mixture of Rn and Cpz at 18 o C (RnCpz) as well as by Rn at 25 o C (RnT).
Digestive glands were examined after 14 days of exposure. The indexes of stress response (total antioxidant capacity, glutathione (GSH&GSSG), metallothioneins (MTSH and Zn-MT), protein carbonyls levels), and markers of metabolic and detoxication (CYP450 related (EROD), Glutathione S-transpherase (GST), cholinesterase, caspase-3, citrate synthase (CS) activities), lysosomal membrane integrity, and Zn level were analyzed. Mostly common responses of mussels were indicated as the increase of oxidative stress, MTSH (except Cpz-group), EROD and CS (except Rn-group) responses. Rn-group indicated almost no-effect or abnormal for expected symptoms effect. However, under the heating Rn caused the decrease of Zn accumulation and loss of lysosomal integrity. Cpz provoked major response diverseness: a decrease in Zn and GST levels and an increase in lysosomal integrity. Thus, complex exposures abolished the individual response traits. Summarising, the application of integrated indices has bene ts when evaluating the effects of complex exposures.

Introduction
Bivalve molluscs are recognised bioindicators of surface water chemical pollution and climate changes.
Various integrative biomarkers have been utilised for characterising the health status of these organisms affected by abiotic stresses (Louis et al. 2020). However, speci c responses of molluscs to certain xenobiotics are not well understood and were examined generally in the exposures to single substances at high concentrations (Giménez and Nunes 2019), which are hardly expected in the environment unless The substances selected for the study, herbicide Roundup (Rn) and drug chlorpromazine (Cpz), are common aquatic pollutants. The Rn (a commercial form of glyphosate) belongs to the most frequently used pesticides globally as a weed killer (Matozzo et al. 2020). The levels of glyphosate in the freshwaters reach hundreds of µg L -1 (Bonansea et al. 2018). It has been suggested that one of the possible reasons for glyphosate toxicity could be its chelating properties towards divalent metal ions that alter the bioavailability of nutrients in plants and soil microorganisms (Mertens et al. 2018). Therefore, its impact on the metal metabolism in the non-targeted species can be expected. However, the signs of its toxicity for aquatic species are mostly reported as the oxidative stress responses, endocrine, immune and Another studied chemical Cpz is the rst generation neuroleptic medication known as dopamine D 2 receptor antagonist (Li et al. 2016). The Cpz investigation was motivated by its popularity. Among 197 substances analysed in hospital wastewater, chlorpromazine belongs to 15 particularly hazardous chemicals present at high concentrations (mg L −1 ) (Frédéric and Yves 2014). The persistence of Cpz in river water and strong adsorption on sediments have also been reported (Jiménez et al. 2016). According to Baresel et al. (2015), the mean Cpz concentration in Swedish sewage treatment plant e uents amounts to 11.3 ng L -1 . Recently, chlorpromazine has been found to have antiviral activity in vitro against the in uenza virus, HIV. Actually, it is listed among "the most promising molecules for inhibiting coronaviruses in human cells" (Stip et al. 2020). The drug is known to interact with the cell membrane dynamin and block clathrin-dependent endocytosis essential for coronavirus entry into the host cell (Plaze et al. 2020). Consequently, its input into the water runoff is expected to be enhanced. The biological effects of Cpz are associated with calcium homeostasis (Xu et al. 2010) and manganese toxicity (Nelson et al. 2018). Therefore, its biochemical effects expected to be related to the involvement in metals uptake and metabolism. High toxic pressure of Cpz has been con rmed on the sh plasma Based on our own experience (Khoma et al. , 2021 and aiming to provide environmentally expected limits of the impact, we used the low Rn concentration of 17 µg L -1 that corresponds to 6.1 µg L -1 or 36 nM of glyphosate. This concentration commensurate with approximately 0.2 of Predicted No Effect Concentration (PNEC), the estimate derived from multispecies data for long-term exposures (Maycock et al. 2012). Cpz concentration of 18 µg L -1 or 57 nM has been chosen to cause minor physiological effects in aquatic animals (Oliveira et al. 2015). The duration of exposure (14 days) and two temperatures (18 and 25 o C) were based on the previous experience (Khoma et al. 2021), the latter in relevance to the range of water temperatures in the sampling area at the Dniester River basin.
Due to the suspected ability of both substances to affect metal metabolism, we focused on the metallothionein-related thiols and their participation in zinc accumulation, the level of glutathione (GSH/GSSG), and lysosomal integrity. Also, we evaluated the oxidative stress and biotransformation activities (cytochrome P450-related ethoxyresoruphine O-diesterase and glutathione S-transferase), metabolic activity (citrate synthase), neurotoxicity (basing on the cholinesterase activity), and apoptosis (through the activity of caspase-3, the central executive enzyme). An integrative analysis of the biomarker responses was applied for the comparison of experimental groups.

Materials And Methods
Methodology used is given in detail in Supplementary materials.

Experimental groups
Adult bivalve molluscs Unio tumidus Philipson, 1788 (Unionidae) (~6 years old, ~8.5 cm length, and 60-70 g weight) were collected in a river site assumed to be a reference ). Specimens were transported to the laboratory and preacclimated to the laboratory conditions for up to seven days after the capture in the aerated, dechlorinated, softened tap water and fed 500 mg of Tropical SuperVit Basic contained beta-1.3/1.6-glucan twice a week. After that, molluscs were distributed randomly to ve groups. The rst group was exposed to the aquarium water only and was considered control (C). The Rn-and RnT-groups were exposed to organophosphonate pesticide Roundup MAX (17 µg L −1 , corresponding to 6.1 µg L −1 or 36 nM of glyphosate) at the temperatures 18 o C and 25 o C, respectively. The temperature was increased gradually in the RnT-group during 24 h. The Cpz-group was exposed to 18.0 µg L -1 or 56 nM of Cpz, and the RnCpz-group -to a mixture of Rn and Cpz at 18 o C. The duration of exposure was 14 days. Water was changed and chemicals replenished every two days. Throughout the experiment, molluscs were fed with the same regularity. No mortality was detected during the experimental exposures.
After exposures, molluscs were dissected on ice. For lisosomal membrane stability and cholinesterase activity, the samples were utilised immediately. For all analyses except metallothioneins and caspase-3, tissues were homogenized (10% w/v) in 0.1 M phosphate buffer, pH 7.4, containing 100 mM KCl and 1 mM EDTA, as well as 0.1 mM phenylmethylsulfonyl uoride (PMSF) for proteolysis inhibition.
Homogenates were centrifuged at 6000g for 10 min, and the resulting supernatant was kept at -40 o C. For the analysis of cholinesterase, the 10% w/v homogenate was prepared in the same buffer without PMSF. The protein concentration was analysed in the 6000g supernatant according to the method of Lowry et al. (1951), using bovine serum albumin as the protein standard. Low weight (approximately 7 kDa) fractions with high absorbance at 254 nm and high D 254 /D 280 density ratio were identi ed as putative MTs-containing peak and pooled (to the total of 10 mL) for the Zn determination.

Assays for metallothionein and glutathione
Total glutathione and oxidized glutathione (GSSG) concentrations were quanti ed by the glutathione reductase recycling assay (Gri th 1980) in the protein-free extract of homogenate using DTNB.
Concentration was expressed as nmol g -1 FW. The concentration of the reduced glutathione (GSH) was calculated as the difference between the total glutathione and GSSG concentrations. The redox-index of glutathione (RI GSH) as the ratio of concentrations GSH/GSSG was calculated.

Oxidative stress and toxicity assays
Total antioxidant capacity (TAC) was determined as ABTS radical scavenging activity (Re et al. 1999). ABTS •+ radical cations (ABTS*) were generated from 2,2'-Azino-di-[3-ethylbenzthiazoline sulphonate] (ABTS) by potassium persulfate. The ascorbic acid was used as the reference compound. The reduction in absorbance of ABTS* solution was recorded at 734 nm. The result was compared with control (only ABTS * solution).
Protein carbonyls (PC) were determined as an index of protein oxidation in the sediment of 10% w/v homogenate in sulfosalicylic acid after its centrifugation by reaction with 2,4-dinitrophenylhydrazine (DNPH) (Reznick and Packer 1994 . After xation, the formaldehyde impregnated tissue fragments were removed and frozen (up to one week). Dye was extracted in acid alcohol (1% acetic acid in 50% ethyl alcohol) and analyzed spectrophotometrically at 550-nm.
The ability of exposures to induce cytochrome P450 (CYP450) activity was quanti ed as 7ethoxyresoru n O-deethylase (EROD) activity in the supernatant of 10% w/v homogenate by measuring the formation of resoru n at 572 nm (Klotz et al. 1984). The reaction was initiated by the addition of 0.5 mM NADPH. EROD activity was calculated using a molar extinction coe cient of 73.2 10 3 М -1 ·сm -1 and referred to the soluble protein concentration.

Cellular soluble thiols and zinc accumulation in the digestive gland
The level of GSH increased in all exposures except Rn, particularly, by 1.8 times, in the RnCpz-group. In the latter group, the level of GSSG was also increased. As a result, the RI GSH increased compared to control in all exposed groups except the Rn-group by 36.0-48.8% (Fig. 1a-c). MT concentration increased by 33.6-57.7% in all Rn-contained exposures, mostly in the RnT-group, whereas Cpz did not change the level of MT (Fig. 1d). Zn-t concentration decreased in the RnT-and Cpz-groups compared to control value by 15.5 and 24.3% correspondingly, whereas the level of Zn-MT increased in all groups, particularly bỹ 53% in the RnT-and Cpz-groups ( Fig. 1e and f).

Indexes of stress and toxicity
The evaluation of oxidative stress responses indicated substantial activation of the TAC by 23.4-51.3% in all exposures (Fig. 2a). The level of protein oxidation products (PC) increased compared to control in all exposures except the Rn-group (Fig. 2b). The highest increase by 32.5% was recorded in RnT-group.
The monooxygenase-related biotransformation function (CYP450) attested from the EROD activity was greatly enhanced by 2.4-3 times in all treated groups except the Rn-group (Fig. 2c). The changes of GST activity compared to control occurred in single treatments, i.e., increase by 26.2% in the Rn-group and decrease by 21.9% in the Cpz-group. In contrast, no changes were evident in the joint treatments in the RnT-and RnCpz-groups (Fig. 2d).
For the indexes of toxicity, some differences were also found (Fig. 3). ChE activity increased compared to control value in the Rn-, RnCpz-and Cpz-groups and did not differ from control in the RnT-group (Fig. 3a).
The lysosomal integrity decreased in the RnT-group, increased in the Cpz-group and did not change in the rest of the groups (Fig. 3b).
The activity of the central executive enzyme of apoptosis, caspase-3, was substantially increased in the molluscs' exposures to Rn and RnCpz (by 36.4%) and was not changed in other treatment groups (Fig.  3c). The metabolic intensity was indicated by the citrate synthase activity. It enhanced compared to control in all exposures, except the Rn-group, particularly in the Cpz-related groups, by 36.5-43.0% (Fig.  3d).

Data integration
The Pearson correlation analysis revealed multiple associations among the studied indices in U. tumidus across all experimental groups (SI Tabl. B1). The greatest number of correlations was found for the indexes of oxidative stress and GSH. In opposite, MT and NRR did not show any correlations. The application of PCA to the datasets to identify relations between indices (Fig. 4a) shows a good distribution of the variables with a factor loading higher than |0.37|. The set of Zn-MT, TAC (ABTS*), PC, EROD, GSH, RI GSH and CS belonged positively to the rst principal component (PC1) and Zn-t located opposite. The set of caspase-3, ChE and GST was related positively to PC2 and opposite to CS with factor loading higher than |0.42|. Generally, 53.6% of the variance was accounted for the rst three factors.
According to Canonical discriminant analysis, all specimens in the C-, RnCpz-and Cpz-groups and 75% of specimens in the Rn-and RnT-groups were classi ed correctly. Fig. 4b shows that the C-and Rn-groups jointly (group centroids -7.55 and -3.96, correspondingly) are well separated in the space of discriminant variables along the axis 1 compared to located oppositely the Cpz-group with the centroid value 7.33. Subjected to combined exposures, the RnT-and RnCpz-groups were placed between these opposite positions along axis 1 and located similarly with centroids values 2.97 and 2.51 along axis 2.
The calculated values of IBR/n in relation to baseline (control) value equaled 0.54, 0.69, 1.71 and 1.84, respectively for the Rn-RnT-, RnCpz-and Cpz-groups. The highest IBR/n values were observed in the RnCpz-and Cpz-group, indicating the particular increase in the A values for most indexes (Fig. 5). The input of Zn-MT was the highest similarly in all groups. The A values in the Cpz group were opposite to other groups for plural indexes. On the other hand, the changes compared to control in the Rn-group were lesser for all indices except Zn-MT and GSH.

Discussion
This study aimed to indicate whether the combine exposure or heating can distort the speci c responses of bivalve molluscs to aquatic pollutants at lower possible environmental concentrations. Discriminant analysis indicated the lesser distinction from control for Rn-group, con rming the effect of concentration equalled 0.2 PNEC as almost negligible. The highest difference was detected for the Cpz-group (Fig. 4b,   5). Interestingly, the concerted exposures (RnT-and RnCpz-groups) produced similar integrative responses, distinct from each exposure to individual substance. However, a number of peculiar manifestations were indicated in each group.

Can Zinc-related parameters serve as speci c biomarkers of effect for low concentrations of Roundup and chlorpromazine?
The involvement of Zn in cellular defence and regulation seems to be the common mechanism that can determine the resistance to adverse impacts. ). Indeed, we found a decrease in the Zn uptake in the RnT-and Cpz-groups (Fig. 1a), whereas Rn per se did not change Zn t. Importantly, at twice higher concentration, than in this study, Rn also decreased the Zn t level in the digestive gland of U. tumidus only in the mixture at elevated temperature (Khoma et al. 2021). Similarly, distortion of the response to Rn by warming was shown by Amid et al. (2018) in the tropical staghorn coral Acropora formosa. The speci city of the effect of Cpz on the molluscs is supported by the presence of a dopamine D 1 -like receptor in the albumen gland of air-breathing freshwater snail Helisoma duryi (Planorbidae) (Mukai et al. 2004). Nevertheless, this effect of Cpz was abolished in the complex exposure of U. tumidus to Rn and Cpz. Thus, we con rm the ability of Rn and Cpz impact the Zn accumulation in the tissues, although for Rn only under the heating.
The elevated accumulation of Zn in MTs was the shared manifestation (Fig. 1f). MT plays a critical role in Zn homeostasis under various conditions but Zn chelating in the thiolate clusters of MT depends on the redox state of thiols (Krężel and Maret 2016). Adverse effects of different nature decrease the level of labile Zn and metalation of MT in molluscs even when the total level of this protein is increased (Khoma et al. 2021). In the present study, the elevation of Zn-MT was well coordinated with the TAC and GSH responses (Fig. 4a). Consequently, this common response can indicate the enhancing of antioxidant activity and redox state of thiols. In opposite, the loss of MT metalation is frequently accompanied by plural signs of toxicity. For example, in the exposure to 33.6 µg/L of Rn, the depletion of Zn-MT was accompanied by the decrease of the ChE and GST activities, lysosomal integrity, and increase in the GSSG level (Khoma et al. 2021). In the present study, the signs of toxicity were almost absent. This attested the low molecular weight thiols as the successful biochemical drivers that provide the redox power in the exposures of low-level toxicity and provide the hormetic-like response.

Enzymes of biotransformation as the probable targets for Roundup and chlorpromazine
Data concerning the biotransformation of Rn and Cpz by the enzymes of phases I and II are scant and contradictory. Phosphonate-based pesticide formulations are known to cause the reduction in CYP450 enzyme levels in mammals (Mesnage and Antoniou 2017). However, the exposure of rats to Rn in the drinking water caused a decrease of EROD activity in male, but an increase in the female specimens In the present study, the increase of EROD activity was not a speci c for Cpz feature. The strong correlation between EROD and a set of indices of antioxidant and metabolic activities and Zn-MT attested this increase as a part of a well-coordinated, probably hermetic-like defensive response to low concentrations of xenobiotics (Fig. 4a).
The GST activity did not correlate with EROD in this and other studies on aquatic animals from polluted areas or exposed to several substances (Domingues et al. 2010). The decrease or absence of GST response has been reported in the exposures of molluscs to higher Rn or glyphosate concentrations (Matozzo et al. 2019;Khoma et al. 2021). In the present study, GST activation can be explained by the low-strength Rn impact. GST activation has been also indicated in the testes of rats exposed to Rn (Liz Oliveira Cavalli et al. 2013). For the Cpz, the decrease in GST activity was one of the most particular reactions observed in this study. The inhibition of GST has also been shown for the effect of Cpz in vitro (Türkan et al. 2020). Moreover, among four tested drugs, Cpz has been found to be the best inhibitor for the GST enzyme (Oliveira et al. 2015). Hence, the two manifestations con rmed in our study, Zn and GST levels' decrease, might be effect-speci c for this drug. However, both of them were abolished in the combined RnCpz-exposure.
In the present study, the GST activity was coordinated with those of the ChE and caspase-3 (SI Tabl. B1, , whereas the upregulation of ChE in molluscs is seldom detected and discussed (Domingues et al. 2010). In this study, upregulation of ChE can be a sign of the non-toxic level of exposure to Rn, that is consistent with other manifestations in Rn-group. In the vertebrates, the simultaneous increase in ChE and caspase-3 activities has been indicated (Hu et al. 2009), and ChE has been proposed as a marker for apoptosis (Zhang and Greenberg 2012). This common regularity has been detected for the neurons of insect Locusta migratoria, where the addition of the ChE inhibitors reduced apoptotic cell death (Knorr et al. 2020). In the freshwater mussels Unionidae, simultaneous increase of ChE and caspase-3 activities has been indicated under the effect of pesticides, pharmaceuticals and heating (Khoma et al. 2021). Nevertheless, the common regularity in alterations of GST, ChE and caspase-3 can not be explained unambiguously basing on the current study data. Hence, we found that EROD activation was a constituent of the common, probably hermetic-like, stress response, while the set of GST, ChE and caspase-3 activities was more exposure-speci c.

How can we attest the severity of injury?
The analysis of each studied biomarker con rmed that we used low enough concentrations of xenobiotics, which did not provoke obvious toxicity. The mobilisation of antioxidants and alterations of metabolic activity correspond to low dose stimulation or hormetic response (Lefcort et al. 2008). This depending on the severity of impact. In any case, GSH and MTSH (last except Cpz-group) activation can support the redox balance and provide the Zn-chelating in the present study's model exposures.
In this study, the joint exposure to Rn and heating can be quali ed as the most disturbing. Only in this group, the lysosomal integrity was decreased. Similar observation has been indicated previously at higher Generally, the calculated IBR-indices gave the possibility to analyze both speci c and common traits of responses, and detect their severity (Fig. 5).

Conclusions
The applied low subchronic exposures to common aquatic pollutants allowed indicating hormetic-like mobilisation of antioxidant and metabolic responses in the bivalve molluscs with certain speci city in the Roundup-and chlorpromazine-exposed groups. Complex exposures abolished this speci city, and heating enhanced the toxicity of almost no-effect concentration of Roundup to molluscs. Discriminant analysis and IBR calculation of the battery of biomarkers highlighted the bene ts of multi-marker expertise in identifying of response strength and speci city.

Declarations
Ethical Approval and Consent to Participate All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Consent to Publish
Not applicable.

Authors Contributions
Oksana Stoliar contributed to conception and design, acquisition, analysis, interpretation, drafted manuscript, and gave nal approval. Levonas Manusadžianas contributed to conception, design, critically revised manuscript, and gave nal approval. Lesya Gnatyshyna contributed to design, analysis and interpretation and gave nal approval; Vira Khoma contibuted to experimental analysis, statistic preparation and gave nal approval; Viktoria Martinyuk contibuted to sampling, analysis and gave nal approval; Tetyana Matskiv contibuted to analysis, draft preparation and gave nal approval; Vitaliy Baranovsky contributed to statistic preparation, acquisition and gave nal approval, Mykola Gladiuk contibuted to sampling, draft preparation and gave nal approval; Brigita Gylytė contributed to draft preparation and gave nal approval.  The results of Principal Component Analysis applied to reveal the relations between measured parameters (a) and scatter plots of the canonical values on the rst and second canonical discriminant axes to discriminate the groups of molluscs (b). a: The abbreviations see in the text. B: control group (1); groups exposed to Roundup (2), Roundup and heating (3), Roundup and chlorpromazine (4), and chlorpromazine (5) during 14 days