THE EFFECT OF ROUNDUP ON THE BIVALVE UNIO TUMIDUS MOLLUSK UTILIZING EX VIVO APPROACH

© 2020 V. V. Khoma et al.; Published by the Ivan Franko National University of Lviv on behalf of Біологічні Cтудії / Studia Biologica. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://www.budapestopenaccessinitiative.org and Creative Commons Attribution 4.0 License), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. UDC: 577.29:564.38:57.014

Glyphosate is the worldwide used herbicide of the first priority. However, its biochemical effects in the aquatic animals are studied scantly. The ex vivo approach has been recently proposed to provide the express evaluation of the adverse impact without the treating of the organisms. The aim of this study was to verify this approach for the assessment of the toxicity of glyphosate to the bivalve mollusk. The samples of the gills and digestive gland tissues of freshwater bivalve Unio tumidus mollusk were exposed to a range of the concentrations of glyphosate (commercial formulation Roundup MAX) at the concentrations 13.3, 26.7, 66.8 and 133.6 µg⋅L -1 during 2 h at 20 °C followed by 15 h at ~ 2-4 o C. The markers of oxidative injury (total antioxidant activity, end-products of lipid peroxidation (TBARS) and protein carbonyls (PC)), cellular low weight thiols GSH/GSSG and metallothionein (MT), and cholinesterase activity as the index of neurotoxicity were analyzed. We also assayed the index of cell vitality as the lysosomal membrane stability from the Neutral Red Retention (NRR) test. The results have shown that the lowest concentrations of glyphosate caused the most prominent changes of the indices: the decrease of MT concentration (by ~ two times) and cholinesterase activity. The total antioxidant activity was decreased substantially in all exposures correspon-

INTRODUCTION
Glyphosate (Gl), the modified aminoacid and the organophosphonate compound, N-(phosphonomethyl)glycine, is the most sprayed and most used herbicide in the world [4; 19]. Whereas it acts as the specific inhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase intrinsic for the plant and bacteria, blocking the synthesis of aromatic compounds, it was originally marketed as absolutely non-toxic for the animals [26]. However, its by-side effect on the non-targeted organisms were reported later: the loss of the immune defense of the animals and human due to the impact on the symbio tic microorganisms [25]. Besides, the strong chelating properties were found for Gl, that cause the immobilization of mineral nutrients by the organisms. Furthermore, in the manufacturing preparations, for example, the Roundup, the active substance was found to inhibit the cytochrome P450 enzymes. Plural effects of Gl and its commercial formulations on the reproductive system, developmental abnormalities and carcinogenic effects in the non-targeted organisms were reported [26]. The biochemical responses that serve for the early warning of ecotoxicity, are less known [18].
The in vitro approaches, such as the use of cell lines, primary cultures and reporter gene assays, have been successfully applied to test the toxicity of environmental chemicals [12]. The developed ex vivo approach seems to be more biologically relevant comparing to isolated cells due to the maintained natural inter-cellular interactions [14]. The experiments with the tissues of mollusks had started only recently [28; 17], despite these organisms are among the most frequently used in aquatic toxicology to detect adverse impacts in the environment.
The objective of this study was to verify express-method for the assessment of the early signs of the toxicity of Gl to the bivalve mollusk. The biomarkers of oxidative stress responses and the cytotoxicity were evaluated. The concentrations of Gl were corresponding to the environmentally realistic.

MATERIALS AND METHODS
Adult Unio tumidus Philipson, 1788 (Unionidae) mollusks (~ 6 years old, 8±1 cm length, and 42±5 g weight) were collected at the confirmed as relatively undisturbed in our previous studies site [15]. The bivalves were collected in autumn season. After period of acclimation in aerated dechlorinated softened tap water (14 days), mollusks were dissected. The samples of digestive gland and gills were maintained at temperature -40 °C till exposure (no more than a week). The modified ex vivo approach of El Haj et al. [17] was used. Selection of Gl concentration in the present study was based on environmentally relevant concentrations of this herbicide. For example, according to environmental qua lity standards of Brazilia, the maximum value allowed for Class I waters for glyphosate is 65 µg/L [17]. Environmentally realistic concentrations of Gl in Europe corresponding to dozens and hundreds of µg⋅L -1 [7]. We treated isolated tissues of U. tumidus mussels with organophosphonate pesticide glyphosate (Gl, formulation Samples were placed into the experimental solution of Gl in Ringer's solution for mollusks (in g⋅L -1 , 8 NaCl, 0.2 KCl, 1.4 Na 2 HPO 4 and 0.272 KH 2 PO 4 (pH 7.4)). Gill tissue was utilized for glutathione (GSH/GSSG) and lysosomal stability (NRR) assays due to its direct contact with water. Gills of mollusks are highly sensitive target for neurotoxic effects, therefore we utilized them for determining of cholinesterase (ChE) activity [5].
The digestive gland was analyzed for oxidative injury as the main metabolic active tissue.
Methodology used for detection of end-products of lipid peroxidation (thiobarbituric acid-reactive substances, TBARS) and protein carbonyls (PC), metallothioneins (MTs), GSH and GSSG concentrations, ChE activity was described in our previous works [15 ; 16]. Lipid peroxidation (LPO) was determined by TBARS production [21] and referred to fresh weight of tissue (FW). A molar extinction coefficient of 1.56·10 5 M -1 ·cm -1 was used. Protein carbonyl (PC) content, as an index of protein oxidation, was measured by reaction with 2,4-dinitrophenylhydazine (DNPH) [24]. The absorbance was determined by spectrophotometry at 375 nm, and amount of PC was calculated by using a molar extinction coefficient of 2.2·10 4 M -1 ·cm -1 . The results were expressed as nmol PC·per g of FW. Total glutathione (GSH) and oxidized glutathione (GSSG) concentrations were quantified by glutathione reductase recycling assay [3]. To estimate GSSG level, protein free sample was treated with 2-vinylpyridine prior to assay. The rate of 5-thionitrobenzoic acid formation from 5,5-dithiobis-2-nitrobenzoate (DTNB) was detected spectrophotometrically at 412 nm. Cholinesterase (ChE, EC 3.1.1.7) activity was determined according to the colorimetric method of Ellman et al [9] with utilizing acetylcholine iodide as substrate. The rate of thionitrobenzoate production evaluated at 412 nm was used to estimate hydrolysis. Enzyme activity was calculated using a molar extinction coefficient of 13.6·10 3 М -1 ·сm -1 and referred to FW.
Total antioxidant/ABTS radical scavenging activity of tissues was determined according to Re et al. [23]. ABTS •+ radicals were pre-generated by potassium persulfate. The ascorbic acid was used as the reference compound. The reduction in absorbance was recorded at 734 nm. Obtained result was compared with control (ABTS solution).
Index of cell vitality as lysosomal membrane stability was detected from the Neutral Red Retention (NRR) test, as it was described by El Haj et al [17]. Briefly, tissues were incubated with neutral red for 2 h to allow for uptake of supravital dye into the lysosomes of viable cells. Then samples were washed with saline and fixed in formaldehyde (0.5% in 1% CaCl 2 ) for 1 h, then freezed during 30 h. After that, the dye was extracted in acid alcohol (1% acetic acid in 50% ethyl alcohol) and measured at 550 nm. All measurements were carried out in 12 samples from 6 specimens in each group. The results were expressed as means ± standard deviation (SD). The reliability of deviation of two series was calculated using the Student's t-test. Difference between groups was considered reliable at P<0.05. All statistical calculations were performed with Statistica v 12.0 and Excel for Windows-2000.

RESULTS AND DISCUSSION
High sensitivity of responses in the isolated tissues to effect of Roundup was revealed. Total antioxidant activity in the digestive gland was most affected (Fig. 1A). It was reduced by about 30 % comparing to control at all exposures. However, in the studied time limits, that was not realized in the enhanced level of the end-products of the oxidative injury (TBARS and PC) detecting early stage of injury (Fig. 1B, C). Only exposure to 66.8 µg⋅L -1 of Gl increased significantly (P<0.04), but not prominently level of PC in digestive gland.
The concentration of GSH in the gills was decreased by the highest concentrations of Gl by 1.6 times. It was accompanied by the increased oxidation of this thiol (by two times) with the reducing of the Redox Index of GSH (GSH/GSSG) from 3.5 to 1.7 at the highest concentration of Gl ( Fig. 2A, B).
The metallothioneins of the digestive gland were most targeted at low concentrations of Gl with the decreasing by 1.8 times (Fig. 2C). Similarly, the cholinesterase activity in the gills was also changed at low concentration of Gl with the decrease by 21% (Fig. 3A). The determining of lysosomal membrane stability in the gills gives the unusual results: it was increased four times at the exposure to the highest concentration (Fig. 3B). Transorb® formulated product) revealing the ex vivo cytotoxicity at the exposures to 24 and 2,400 µg⋅L -1 of Gl in the tissues of the bivalves. Despite Gl is one of the most used herbicide worldwide, its biological effects are poorly investigated [18]. The methodology applied in our study confirms the suitability of ex vivo approach and strengthens it due to application of low environmentally realistic concentrations and widening a set of the biomarkers. The oxidative effect of Gl was proved. Similar to utilized concentrations of Gl caused the same response in vivo in freshwater amphipod Gammarus pulex during the acute exposure (decreased level of GSH and increased level of TBARS) [22]. In our study, the metallothioneins appeared to be most vulnerable thiol at low concentration of Gl followed by the GSH, that was depleted at higher concentrations of Gl. Probably, a depletion of the metallothionein and GSH can explain a decrease in total antioxidant scavenging activity, whereas GSH and metallothioneins enable to neutralize free radicals, especially the reactive oxygen species (ROS) such as the superoxide, hydroperoxyl, hydroxyl (OH • ) radicals, having electron acceptor ability [12]. An increased production of the Gl-mediated oxyradicals was suspected in exposures [2]. In present study, an impact on the cholinergic system was shown with a decrease of cholinesterase activity. Despite Gl is a organophosphorus compound that do-not belong to the organophosphates, typical inhibitors of cholinesterase, like chlorpyrifos [5; 6], its neurotoxic effect was evident. It was also indicated in the exposures of G. pulex [22] and Mytilus galloprovincialis [18].
We detected increased retention of the dye in the lysosomes under the exposures to higher Gl concentrations. This response is opposite to typical permeabilization of lysosomal membranes under the toxic exposures [17]. We explain this manifestation by strong chelating ability of Gl or other compounds of the formulation [20]. Presence of surfactants in commercial formulations modulated effect of Gl for juvenile oysters (Crassostrea gigas) [25]. Roundup MAX formulation contains surfactants POEA (polyethoxylated tallow amines). As a wetting agents, they can cause membrane permeability [8]. However, some surfactants, like poloxamer 188, cause an increase in membrane stability, opposite to sodium dodecyl sulphate lowering the interactions between phospholipid molecules due to its incorporation in cellular membranes [27]. The strengthe ning of the lysosomal membranes was also shown for human fibroblasts with cholesterol-loaded lysosomes [1]. In our study, cholesterol content was able to change the membrane stability more than three times. In ex vivo study of El Haj et al. [17], NRR test had shown a decrease of retention under 50 and 5,000 µg•L -1 concentrations of the Roundup Transorb. Regarding an increased release of the dye from lysosomes at lower concentration of Gl, it was also observed by El Haj et al. [17]. That might be explained by extremely long time of exposition of the dissected tissue under 15 h treatment at 20 °C. However, the absence of the effect at 500 µg•L -1 of substance raises skepticism concerning reasons for this loss of vitality in the study [17].

CONCLUSION
In this study, ex vivo approach was used to detect early biological effects of Gl in low environmentally realistic concentrations. However, long-term exposure of the organism to environmentally realistic concentrations of Gl can cause particular responses, including the adaptation to this xenobiotic. Further validation is needed to compare of the results of ex vivo and in vivo experiments.

Conflict of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Animal Rights: This article does not contain any studies with animal subjects performed by the any of the authors.